AU2021368151B2

AU2021368151B2 - Escherichia coli compositions and methods thereof

Info

Publication number: AU2021368151B2
Application number: AU2021368151A
Authority: AU
Inventors: Annaliesa Sybil Anderson; Laurent Oliver CHORRO; Robert George Konrad DONALD; Jacqueline Marie LYPOWY; Rosalind PAN
Original assignee: Pfizer Inc
Current assignee: Pfizer Inc
Priority date: 2020-10-27
Filing date: 2021-10-25
Publication date: 2024-09-05
Anticipated expiration: 2041-10-25
Also published as: CO2023005372A2; AU2021368151A1; WO2022090893A3; IL302362A; AU2021368151A8; MX2023004912A; TW202227467A; JP7664386B2; WO2022090893A2; PE20231934A1; JP2023546615A; KR20230096033A; CA3199610A1; EP4237428A2

Abstract

This invention relates to compositions that include a polypeptide derived from

Description

ESCHERICHIA COLI COMPOSITIONS AND METHODS THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefits of U.S. Provisional Application No. 63/254,195, filed October 11, 2021, U.S. Provisional Application No. 63/144,058, filed February 1, 2021, and U.S. Provisional Application No. 63/106,077, filed October 27, 2020. The entire content of each of the foregoing applications is incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING This application is being filed electronically via EFS-Web and includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled "PC72671_ST25.txt" created on October 26, 2020 and having a size of 160 KB. The sequence listing contained in this .txt file is part of the specification and is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION The present invention relates to new immunogenic compositions for use in a vaccine for eliciting immune responses against E. coli and Klebsiella pneumoniae serotypes in a subject.

BACKGROUND OF THE INVENTION Escherichiacoli is one of the most common human bacterial pathogens with clinical presentations that include blood stream infections, urinary tract infections (catheter and non catheter associated; surgical site infections, pneumonia, and serious food poisoning related diarrhea. They are classified serologically by differences in the structure of the lipopolysaccharide-associated O-antigen (>180 known serotypes), the capsule polysaccharide K-antigen (> 100 serotypes), and the flagellar H-antigen (>50 serotypes). Urinary tract infections (UTIs) most often present as a cystitis that in some individuals can recur repeatedly following resolution. Left untreated, they can progress to pylonephritis and blood stream infections. E. coli infections are associated with high levels of antibiotic resistance

[Al-Hasan MN, et al. The Journal of antimicrobial chemotherapy 2009; 64:169-74] with many strains being resistant to multiple antibiotics including antibiotics of last resort such as carbapenems and polymyxins [Zowawi HM, et al. Nature reviews Urology 2015; 12:570-84]. In particular, O25b serotype multilocus sequence type (MLST) 131 has emerged as a worldwide pandemic clone, causing predominantly community-onset infections with high rates of resistance to extended-spectrum cephalosporins (ESBLs) and fluoroquinolones [Rogers BA, et al. The Journal of antimicrobial chemotherapy 2011; 66:1-14; Nicolas-Chanoine M-H, et al.

Clinical Microbiology Reviews 2014; 27:543-74]. E. coli BSI and UTI infecting strains are also known as invasive Extra-intestinal Pathogenic E. coli (ExPEC) or uropathogenic E. coli (UPEC). Second to E. coli, Klebsiella spp. (including K. pneumoniae and K. oxytoca) are the next most common Gram-negative pathogens associated with invasive infections including UTIs, pneumonia, intra-abdominal infection, and bloodstream infection (BSI) [Nicolas-Chanoine M-H, et al. Clinical Microbiology Reviews 2014; 27:543-74; Podschun R, et al. Clin Microbiol Rev 1998; 11:589-603; Yinnon AM, et al. QJM : monthly journal of the Association of Physicians 1996; 89:933-41; Anderson DJ, et al. PLoS One 2014; 9:e91713]. Klebsielia maintain a profound ability to acquire antibiotic resistance through horizontally transmissible ESBL and .0 carbapenem resistance conferring genes [Chen L, et al. Trends Microbiol 2014; 22:686-96; Iredell J, et al. Bmj 2016; 352:h6420]. Accordingly, during the last decade the prevalence of ESBL-resistant Klebsiela producing extended-spectrum p-lactamases (ESBL) has increased dramatically globally. Klebsieia spp. can express up to 8 different O-types and >80 K-types. While there are a multitude of K-antigens associated with virulent Klebsiela strains, only four 0 .5 antigen serotypes account for > 80% of Klebsielia clinical isolates irrespective of sample site (blood, urine, sputum), infection status (invasive versus non-invasive) or the nature of acquisition (community vs nosocomial) [Follador R, et al. Microbial Genomics 2016; 2:e000073]. The increased rate of invasive multidrug-resistant (MDR) E. coli and Klebsiea infections in the vulnerable newborn population and the elderly underscores the need for vaccine-based approaches as an alternative to standard-of-care antibiotics which are becoming less effective.

SUMMARY OF THE INVENTION To meet one or more of these or other needs, the present invention relates to compositions and methods of use thereof for producing new immunogenic compositions for use in a vaccine for eliciting immune responses against E. coli and Klebsie//a pneumoniae serotypes .5 in a subject. In some embodiments of the methods provided herein, the subject is a mammal, preferably a human. In some particular embodiments, the human is a child, such as an infant. In some other particular embodiments, the human is a woman, particularly a pregnant woman. The composition may be administered to the subject with or without administration of an adjuvant. The effective amount administered to the subject is an amount that is sufficient to elicit an immune response against an E. coli or K. pneumoniae antigen in the subject. Subjects that can be selected for treatment include those that are at risk for developing an E. color K. pneumoniae infection because of exposure or the possibility of exposure to E. coli or K. pneumoniae. Because humans may be infected with E. color K. pneumoniae by the age of 2, the entire birth cohort is included as a relevant population for immunization. This could be done, for example, by beginning an immunization regimen anytime from birth to 6 months of age, from 6 months of age to 5 years of age, in pregnant women (or women of child-bearing age) to protect their infants by passive transfer of antibody, infants still in utero, and subjects greater than 50 years of age. In one aspect, the invention relates to a composition comprising a FimH polypeptide comprising amino acids having a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 27, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, and SEQ ID NO: 113, or any combination thereof. In some embodiments, the composition further includes a saccharide selected from any saccharide having a Formula in Table 1, preferably Formula 01A, Formula 01B, Formula 02, Formula 06, and Formula 025B, wherein n is an integer from 1 to 100, preferably 31 to 100. In one aspect, the invention relates to a composition that includes a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, and SEQ ID NO: 29, or any combination thereof. In another aspect, the invention relates to a composition that includes a polypeptide having at least n consecutive amino acids from any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, and SEQ ID NO: 29, wherein n is 7 or more (eg. 8, 10, 12, 14, 16, 18, 20 or more). In some embodiments, the composition further includes a saccharide selected from any one Formula in Table 1, preferably Formula 01A, Formula 01B, Formula 02, Formula 06, and Formula 025B, wherein n is an integer from 1 to 100, preferably 31 to 100.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A-1H- depict amino acid sequences, including amino acid sequences for exemplary polypeptides derived from E. coli or fragments thereof; and amino acid sequences for exemplary wzzB sequences. FIG. 2A-2T - depict maps of exemplary expression vectors. FIG. 3 - depicts results from expression and purification. FIG. 4 - depicts results from expression and purification. FIG. 5 - depicts results from expression. FIG. 6A-6C - depict pSB02083 and pSB02158 SEC pools and affinities, including yields.

FIG. 7- depicts results from expression of pSB2198 FimH dscG Lock Mutant construct. FIG. 8 - depicts results from expression of pSB2307 FimH dscG wild type. FIG. 9A-9C - depict structures of O-antigens synthesized by the polymerase-dependent pathway with four or less residues in the backbone. FIG. 10A-10B - FIG. 10A depicts structures of O-antigens synthesized by the polymerase dependent pathway with five or six residues in the backbone; FIG. 10B depicts O-antigens believed to be synthesized by the ABC-transporter-dependent pathway. FIG. 11 - depicts computational mutagenesis scanning of Phel with other amino acids having aliphatic hydrophobic sidechains, e.g. Ile, Leu and Val, that may stabilize the FimH protein and accommodate mannose binding. FIG. 12A-12B-depict plasmids: a pUC replicon plasmid, 500-700x copies per cell, Chain length regulator (FIG. 12A); and P15a replicon plasmid,10-12x copies per cell, O-antigen operon (FIG. 12B). FIG. 13A-13B - depict modulation of O-antigen chain length in serotype 025a and 025b strains by plasmid-based expression of heterologous wzzB and fepE chain length regulators. Genetic complementation of LPS expression in plasmid transformants of wzzB knockout strains 025K5H1 (025a) and GAR2401 (025b) is shown. On the left side of FIG. 13A, LPS profiles of plasmid transformants of 025a O25K5HAwzzB are shown; and on the right, analogous profiles of 025b GAR 2401AwzzB transformants. An immunoblot of a replicate gel probed with 025 specific sera (Statens Serum Institut) is shown in FIG. 13B. 025aAwxxB (Knock out) background associated with Lanes 1-7; 025b 2401 AwzzB (Knock out) background associated with Lanes 8-15. FIG. 14 - depicts long chain O-antigen expression conferred by E. coli and Salmonella fepE plasmids in hostO25K5H1AwzzB. FIG. 15 - depicts that Salmonella fepE expression generates Long O-antigen LPS in a variety of clinical isolates. FIG. 16A-16B- depict plasmid-mediated Arabinose-inducible Expression of 025b Long 0 antigen LPS in 025b O-antigen knock-out host strain. Results from an SPS PAGE are shown in FIG. 16A and results from an 025 Immuno-Blot are shown in FIG. 16B, wherein Lane 1 is from Clone 1, no arabinose; Lane 2 is from Clone 1, 0.2% arabinose; Lane 3 is from Clone 9, no Arabinose; Lane 4 is from Clone 9, 0.2% Arabinose; Lane 5 is from 055 E. coli LPS Standard; and Lane 6 is from 0111 E. coli LPS Standard, in both FIG. 16A and in FIG. 16B. FIG. 17 - depicts plasmid-mediated Arabinose-inducible Expression of Long O-antigen LPS in common host strain. FIG. 18 - depicts expression of 025 O-antigen LPS in Exploratory Bioprocess strains.

FIG. 19A-19B - depict SEC profiles and properties of short (FIG. 19A, Strain 1 025b wt 2831) and long 025b 0-antigens (FIG. 19B, Strain 2025b 240lAwzzB / LT2 FepE) purified from strains GAR2831 and '2401AwzzB / fepE. FIG. 20A-20B - depict vaccination schedules in rabbits: (FIG. 20A) Information regarding vaccination schedule for rabbit study 1 VAC-2017-PRL-EC-0723; (FIG. 20B) vaccination schedule for rabbit study 2 VAC-2018-PRL-EC-077. FIG. 21A-21C - depict 025b Glycoconjugate IgG responses, wherein -0- represents results from Prebleed; -0- Bleed 1 (6 wk); -A- Bleed 2 (8 wk); -*- Bleed 3 (12 wk). FIG. 21A depicts results from Rabbit 1-3 (Medium Activation); FIG. 21B depicts results from Rabbit 2-3 (Low Activation); FIG. 21C depicts results from Rabbit 3-1 (High Activation). FIG. 22A-22F - depict IgG responses to 025b Long0-antigen Glycoconjugate, i.e., Low activation 025b-CRM 19 7conjugate (FIG. 22D-22F, wherein -0- represents results from Prebleed from Rabbit 2-1, -0- Week 12 Antisera from Rabbit 2-1) vs unconjugated polysaccharide, i.e., free 025b polysaccharide (FIG. 22A-22C, wherein -0- represents results from Prebleed from Rabbit A-1, -0- Week 6 Antisera from Rabbit A-1, -A- Week 8 Antisera from Rabbit A-1). Note that MFIs are plotted on log scale to highlight differences between pre immune and immune antibodies in the <1000 MFI range. FIG. 22A depicts results from Rabbit A-1 (Unconjugated Poly); FIG. 22B depicts results from Rabbit A-3 (Unconjugated Poly); FIG. 22C depicts results from Rabbit A-4 (Unconjugated Poly); FIG. 22D depicts results from Rabbit 2-1 (low activation); FIG. 22E depicts results from Rabbit 2-2 (low activation); and FIG. 22F depicts results from Rabbit 2-3 (low activation). FIG. 23A-23C - depict surface expression of native vs long 025b 0-antigen detected with 025b antisera. FIG. 23A depicts results wherein -0- represents results from 025b 2831 vs PD3 antisera; -0- represents results from 025b 2831 wt vs prebleed; -A- represents results from 025b 2831 / fepE vs PD3 antisera; -V- represents results from 025b 2831 / fepE vs prebleed. FIG. 23B depicts results wherein -0- represents results from 025b 2401 vs PD3 antisera; -0- represents results from 025b 2401 vs prebleed; -A- represents results from 025b 2401 / fepE vs PD3 antisera; -V- represents results from025b 2401 / fepE vs prebleed. FIG. 23C depicts results wherein -0- represents results from E. coli K12 vs PD3 antisera; and -0- represents results from E. coli K12 vs prebleed. FIG. 24 - depicts generalized structures of the carbohydrate backbone of the outer core oligosaccharides of the five known chemotypes. All glycoses are in the a-anomeric configuration unless otherwise indicated. The genes whose products catalyse formation of each linkage are indicated in dashed arrows. An asterisk denotes the residue of the core oligosaccharide to which attachment of 0-antigen occurs.

FIG. 25 - depicts that unconjugated free 025b polysaccharide is not immunogenic (dLIA), wherein -0- represents results from Week 18 (1wk = PD4) Antisera from 4-1; -0- represents results from Week 18 (1wk = PD4) Antisera from 4-2; -A- represents results from Week 18 (1wk = PD4) Antisera from 5-1; -V- represents results from Week 18 (1wk = PD4) Antisera from 5-2; -*- represents results from Week 18 (1wk = PD4) Antisera from 6-1; -A- represents results from Week 18 (1wk = PD4) Antisera from 6-2. FIG. 26A-26C- depict graphs illustrating the specificity of BRC Rabbit 025b RAC conjugate immune sera OPA titers. FIG. 26A shows OPA titers of Rabbit 2-3 pre-immune serum (-0-) and post-immune serum wk 13 (-E-). FIG. 26B shows OPA titers of Rabbit 1-2 pre-immune serum (-0-) and post-immune serum wk 19 (-E-). FIG. 26C shows Rabbit 1-2 wk 19 OPA Titer Specificity, in which OPA activity of Rabbit 1-2 immune serum is blocked by pre-incubation with 100g/mLof purified unconjugated 025b long O-antigen polysaccharide, wherein -0 represents results from Rabbit 1-2 immune serum wk 19; and -V- represents results from Rabbit 1-2 wk 19 w/R1 Long-OAg. FIG. 27A-27C - FIG. 27A depicts an illustration of an exemplary administration schedule. FIG. 27B and FIG. 27C show graphs depicting O-antigen 025b IgG levels elicited by unconjugated 025b long O-antigen polysaccharide (FIG. 27B, 025b Free Poly (2pg)) and derived 025b RAC/DMSO long O-antigen glycoconjugate (FIG. 27C,025b-CRM19 7 RAC Long (2pg)), wherein-...- (dotted line) represents Naive CD1 025b IgG level. FIG. 28A-28B - depict graphs showing OPA immunogenicity of RAC, eTEC 025b long glycoconjugates, and single end glycoconjugates post dose 2 (FIG. 28A) and post dose 3 (FIG. 28B), wherein -O- represents results from single end short 2 pg; -0- single end long 2pg; -A- RAC/DMSO long 2pg; -V- eTEC long 2pg; * Background control (n=20). tResponder rates are % mice with titers > 2x unvaccinated baseline. FIG. 29 - depicts graph showing OPA immunogenicity of eTEC chemistry and modified levels of polysaccharide activation. tResponder rates are % mice with titers > 2x unvaccinated baseline. FIG. 30A-30B- depict an illustration of an exemplary administration schedule (FIG. 30A); and a graph depicting protection of mice immunized with doses of E. coli eTEC conjugates from lethal challenge with 025b isolate (FIG. 30B), wherein -0- represents eTEC Long Chain 17% activation; -A- eTEC represents Long Chain 10% activation; -7- represents eTEC Long Chain 4% activation; -0- represents 025b Polysaccharide; -0- represents unvaccinated controls. FIG. 31 - depicts a schematic illustrating an exemplary preparation of single-ended conjugates, wherein the conjugation process involves selective activation of 2-Keto-3-deoxyoctanoic acid

(KDO) with a disulfide amine linker, upon unmasking of a thiol functional group. The KDO is then conjugated to bromo activated CRM 197 protein as depicted in FIG. 31 (Preparation of Single-Ended Conjugates). FIG. 32A-32B - depict an exemplary process flow diagram for the activation (FIG. 32A) and conjugation (FIG. 32B) processes used in the preparation of E. coliglycoconjugate to CRM 197

. FIG. 33 - depicts structures of the repeat unit (RU) of E. coli and K. pneumoniae polymannan 0 antigens. Legend: Trimeric E. coli 08 and K. pneumoniae 05 are identical, as are the terameric E. coli 09A/ K. pneumoniae 03a and pentameric E. coli 09/ K. pneumoniae 03. Differentiation of the K. pneumoniae 03 subtypes at the level of biosynthetic enzyme sequences is described in Guachalla LM et al. (Scientific Reports 2017; 7:6635). FIG. 34A-34B - depict E. coli serotype 08 immune sera is bactericidal against an invasive K. pneumoniae serotype 05 strai. Legend: Rabbit immune sera elicited by an E. coli serotype 08 O-antigen CRM 197 conjugate was evaluated in bactericidal assays with an E. coli08 strain (FIG. 34A) and a K. pneumoniae 05 strain (FIG. 34B). Potent opsonophagocytic assay (OPA) activity against an E. coli08 strain was observed after two vaccine doses (week 15) that was absent following preadsorption with unconjugated 08 polysaccharide (08-OAg), or with matched pre-immune sera (week 0). The same rabbit immune serum showed antigen-specific serum bactericidal activity (SBA) against the K. pneumoniae 05 strain. BRC - baby rabbit complement, hC - lgG/lgM depleted human sera as complement source. FIG. 35A-35B - depict E. coliserotpye 09 O-antugen immune sera is bactericidal against an invasive K. pneumoniae 03 isolate. Legend: Rabbit immune sera elicited by an E. coli serotype 09a O-antigen CRM 197 conjugate was evaluated in opsonophagocytic assays (OPAs) with an E. coli09a strain (FIG. 35A) and a K. pneumoniae 03b strain (FIG. 35B). OPA activity against the E. coli09 strain was observed after two vaccine doses (week 15) that was absent following preadsorption with unconjugated 09 polysaccharide (09-OAg), or with matched pre-immune sera (week 0). The same rabbit immune serum also showed potent antigen-specific serum bactericidal activity (SBA) against the K. pneumoniae 03b strain. BRC, baby rabbit complement; hC, lgG/lgM depleted human sera used as complement source. FIG. 36A-36B - depict that the E. coli FimHLD Antigen requires a potent adjuvant to elicit neutralizing antibodies. FIG. 36A depicts the dosing schedule of Study VAC-2019-PRL-EC 1369: between 12 and 20 CD-1 mice per group were vaccinated with 3 pg or 30 pg doses of E. coli FimHLD antigen with or without 20 pg QS21/PS80 or 50 pg AIPO 4 adjuvant. FIG. 36B depicts the titers after dose 3 of individual mice vaccinated with wild-type FimHLD or FimHLD lock mutant antigens which are shown as closed symbols or open symbols, respectively. p values from t-tests (unpaired Welch's correction) of log transformed neutralization titer data are indicated.

FIG. 37A-37B- demonstrates that FimH-DSG variants are more immunogenic than FimHLD constructs. FIG. 37A. depicts the dosing schedule of Study VAC-2019-PRL-EC-1438: 20 CD-1 mice per group were vaccinated with 10 pg or 30 pg of E. coli FimHLD or FimH-DSG antigen variants with 20 pg QS21/PS80. FIG. 37B - depicts the titers after dose 3 of individual mice vaccinated with wild-type FimHLD or FimHLD lock mutant antigenswhich are shown as closed symbols or open symbols, respectively. p values from t-tests (unpaired Welch's correction) of log transformed neutralization titer data are indicated. FIG. 38 - depicts the schedule and dosing for a FimH-DSG and O-antigen combination and adjuvant formulation study (Study VAC-2020-PRL-EC-1679). FIG. 39A-39B - depict Study VAC-2020-PRL-EC-1679 which demonstrates the influence of adjuvant and FimH-DSG and 4-valent O-antigen combinations on FimH neutralization. Yeast mannan assay binding neutralization titers at PD2 (FIG. 39A) and PD3 (FIG. 39B) timepoints. Closed symbols reflect mice vaccinated with O-antigens or FimH-DSG antigens only; open symbols are mice in groups vaccinated with FimH-DSG O-antigen combinations as labeled on x-axis. FIG. 40A-40B - depict results of bladder assay with mouse serum pools corroborate yeast mannan assay data with individual mouse sera. Bladder cell binding inhibition curves from pooled mouse sera (n=10). Details are same as in the legend to FIG. 39A-39B. Rabbit anti FimH positive control serum titrations are also shown, as an invariant assay reference standard. FIG. 41 - depicts the influence of adjuvant and FimH-DSG on O-Antigen specific serum IgG levels. Closed and open symbols represent PD2 and PD3 timepoint values, respectively. Ten pre-vaccinated CD-1 mice per group were pooled to determine the baseline IgG level for each antigen. The GMT of each pre-vaccinated pool was < the lower limit of quantitation (0.15 pg/mL, dotted line), determined from standard curve bias analysis. Responder rates are indicated as the percentage of mice per group that show greater than a 5-fold increase in titer over baseline. Differences in antibody titers between groups were analyzed by unpaired t test with Welch's correction **P < 0.05. FIG. 42 - depicts the influence of adjuvant and FimH-DSG on O-antigen-specific serum OPA titers. Details are same as for FIG. 41. Dotted lines reflect baseline titers from mouse pre vaccination serum pools (1/2x LOD or 50, n=10). Closed and open symbols represent PD2 and PD3 timepoint values, respectively.

SEQUENCE IDENTIFIERS SEQ ID NO: 1 sets forth an amino acid sequence for a wild type type 1 fimbriae D-mannose specific adhesin [Escherichia coli FimH J96]. SEQ ID NO: 2 sets forth an amino acid sequence for a fragment of FimH, corresponding to aa residues 22-300 of SEQ ID NO: 1 (mature FimH protein).

SEQ ID NO: 3 sets forth an amino acid sequence for a FimH lectin domain. SEQ ID NO: 4 sets forth an amino acid sequence for a FimH pilin domain. SEQ ID NO: 5 sets forth an amino acid sequence for a polypeptide derived from E. coli FimH (pSB02198 - FimH mlgK signal pept / F22..Q300 J96 FimH N28S V48C L55C N91S N249Q / 7 AA linker / FimG A1..K14 / GGHis8 in pcDNA3.1(+)) SEQ ID NO: 6 sets forth an amino acid sequence for a polypeptide derived from E. coli FimH (pSB02307 - FimH mlgK signal pept / F22..Q300 J96 FimH N28S N91S N249Q / His8 in pcDNA3.1(+)) SEQ ID NO: 7 sets forth an amino acid sequence for a fragment of a polypeptide derived from E. coli FimH (pSB02083 FimH Lectin Domain Wild Type construct) SEQ ID NO: 8 sets forth an amino acid sequence for a fragment of a polypeptide derived from E. coli FimH (pSB02158 FimH Lectin Domain Lock Mutant) SEQ ID NO: 9 sets forth an amino acid sequence for a fragment of a polypeptide derived from E. coli FimG (FimG Al..K14) SEQ ID NO: 10 sets forth an amino acid sequence for a fragment of a polypeptide derived from E. coli FimC. SEQ ID NO: 11 sets forth an amino acid sequence for a 4 aa linker. SEQ ID NO: 12 sets forth an amino acid sequence for a 5 aa linker. SEQ ID NO: 13 sets forth an amino acid sequence for a 6 aa linker. SEQ ID NO: 14 sets forth an amino acid sequence for a 7 aa linker. SEQ ID NO: 15 sets forth an amino acid sequence for a 8 aa linker. SEQ ID NO: 16 sets forth an amino acid sequence for a 9 aa linker. SEQ ID NO: 17 sets forth an amino acid sequence for a 10 aa linker. SEQ ID NO: 18 sets forth an amino acid sequence for a FimH J96 signal sequence. SEQ ID NO: 19 sets forth an amino acid sequence for the signal peptide of SEQ ID NO: 5 (pSB02198 - FimH mlgK signal pept / F22..Q300 J96 FimH N28S V48C L55C N91S N249Q 7 AA linker/ FimG Al..K14 /GGHis8 in pcDNA3.1(+)). SEQ ID NO: 20 sets forth an amino acid sequence for a polypeptide derived from E. coli FimH according to SEQ ID NO: 5 (mature protein of pSB02198 - FimH mgK signal pept / F22..Q300 J96 FimH N28S V48C L55C N91S N249Q / 7 AA linker / FimG Al..K14 / GGHis8 in pcDNA3.1(+)). SEQ ID NO: 21 sets forth an amino acid sequence for a polypeptide derived from E. coli FimG. SEQ ID NO: 22 sets forth an amino acid sequence for the signal peptide of SEQ ID NO: 6 (pSB02307 - FimH mlgK signal pept / F22..Q300 J96 FimH N28S N91S N249Q / His8 in pcDNA3.1(+)).

SEQ ID NO: 23 sets forth an amino acid sequence for a polypeptide derived from E. coli FimH according to SEQ ID NO: 6 (mature protein of FimH mlgK signal pept / F22..Q300 J96 FimH N28S N91S N249Q / His8 in pcDNA3.1(+)). SEQ ID NO: 24 sets forth an amino acid sequence for a polypeptide derived from E. coli FimH according to SEQ ID NO: 7 (mature protein of pSB02083 FimH Lectin Domain Wild Type construct). SEQ ID NO: 25 sets forth an amino acid sequence for a His-tag. SEQ ID NO: 26 sets forth an amino acid sequence for a polypeptide derived from E. coli FimH according to SEQ ID NO: 8 (mature protein of pSB02158 FimH Lectin Domain Lock Mutant) SEQ ID NO: 27 sets forth an amino acid sequence for a polypeptide derived from E. coli FimH (pSBO1878). SEQ ID NO: 28 sets forth an amino acid sequence for a polypeptide derived from E. coli FimH (K12). SEQ ID NO: 29 sets forth an amino acid sequence for a polypeptide derived from E. coli FimH (UT189). SEQ ID NO: 30 sets forth a 025b 2401 WzzB amino acid sequence. SEQ ID NO: 31 sets forth a025a:K5:H1 WzzB amino acid sequence. SEQ ID NO: 32 sets forth a 025a ETEC ATCC WzzB amino acid sequence. SEQ ID NO: 33 sets forth a K12 W3110 WzzB amino acid sequence. SEQ ID NO: 34 sets forth a Salmonella LT2 WzzB amino acid sequence. SEQ ID NO: 35 sets forth a 025b 2401 FepE amino acid sequence. SEQ ID NO: 36 sets forth a 025a:K5:H1 FepE amino acid sequence. SEQ ID NO: 37 sets forth a 025a ETEC ATCC FepE amino acid sequence. SEQ ID NO: 38 sets forth a 0157 FepE amino acid sequence. SEQ ID NO: 39 sets forth a Salmonella LT2 FepE amino acid sequence. SEQ ID NO: 40 sets forth a primer sequence for LT2wzzBS. SEQ ID NO: 41 sets forth a primer sequence for LT2wzzBAS. SEQ ID NO: 42 sets forth a primer sequence for O25bFepES. SEQ ID NO: 43 sets forth a primer sequence for O25bFepEA. SEQ ID NO: 44 sets forth a primer sequence for wzzB P1_S. SEQ ID NO: 45 sets forth a primer sequence for wzzB P2_AS. SEQ ID NO: 46 sets forth a primer sequence for wzzB P3_S. SEQ ID NO: 47 sets forth a primer sequence for wzzB P4_AS. SEQ ID NO: 48 sets forth a primer sequence for 0157 FepES. SEQ ID NO: 49 sets forth a primer sequence for 0157 FepEAS. SEQ ID NO: 50 sets forth a primer sequence for pBAD33_adaptorS.

SEQ ID NO: 51 sets forth a primer sequence for pBAD33_adaptorAS. SEQ ID NO: 52 sets forth a primer sequence for JUMPSTARTr. SEQ ID NO: 53 sets forth a primer sequence for gndf. SEQ ID NO: 54 sets forth an amino acid sequence for a mouse IgK signal sequence. SEQ ID NO: 55 sets forth an amino acid sequence for a human IgG receptor FcRn large subunit p51 signal peptide. SEQ ID NO: 56 sets forth an amino acid sequence for a human IL10 protein signal peptide. SEQ ID NO: 57 sets forth an amino acid sequence for a human respiratory syncytial virus A (strain A2) fusion glycoprotein FO signal peptide. SEQ ID NO: 58 sets forth an amino acid sequence for an influenza A hemagglutinin signal peptide. SEQ ID NOs: 59-101 set forth amino acid and nucleic acid sequences for a nanostructure related polypeptide or fragment thereof. SEQ ID NOs: 102-109 set forth SignalP 4.1 (DTU Bioinformatics) sequences from various species used for signal peptide predictions. SEQ ID NO: 110 sets forth an amino acid sequence for a polypeptide derived from E. coli FimH (pSB02083 -- FimHLD (mgK signal pept, N28S, N91S)). SEQ ID NO: 111 sets forth an amino acid sequence for a polypeptide derived from E. coli FimH (pSB02158 -- FimHLD-LM (mgK signal pept, N28S N91S V48C L55C)). SEQ ID NO: 112 sets forth an amino acid sequence for a polypeptide derived from E. coli FimH (pSB02307 - FimH-DSG (mlgK signal pept, N28S N91S N249Q 7aa linker FimG A1..K14)). SEQ ID NO: 113 sets forth an amino acid sequence for a polypeptide derived from E. coli FimH (pSB02198 - FimH-DSG-LM (mgK signal pept, N28S N91S 249Q V48C L55C 7aa linker FimG A1..K14)).

DETAILED DESCRIPTION OF THE INVENTION The present disclosure relates to compositions comprising E. coli FimH polypeptides and O-antigen glycoconjugates, methods for producing and purifying the compositions, and methods of using said compositions. In one aspect, the invention includes a composition including the FimH polypeptide or fragment thereof described herein. The composition may include a polypeptide or fragment thereof that is suitable for in vivo administration. For example, the polypeptide or fragment thereof in such a composition may have a purity of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, by mass. In an embodimentthe polypeptide in such a composition may have a purity of at least 95% by mass.

In an embodimentthe polypeptide in such a composition may have a purity of at least 97% by mass. In an embodimentthe polypeptide in such a composition may have a purity of at least 98% by mass. In an embodimentthe polypeptide in such a composition may have a purity of at least 99% by mass. The composition may further comprise an adjuvant. In a further aspect, the invention includes a composition for use in inducing an immune response against E. coli or E.coli infection. Use of the composition described herein for inducing an immune response against E. color E.coliinfection and use of the composition described herein in the manufacture of a medicament for inducing an immune response against E. color E.coli infection, are also disclosed. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it was individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as"), provided herein is intended merely to further illustrate the disclosure and does not pose a limitation on the scope of the claims. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure. Several documents are cited throughout the text of this disclosure. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the present disclosure was not entitled to antedate such disclosure.

Definitions As used hereinhe term "about" means approximately or nearly, and in the context of a numerical value or range set forth herein in one embodiment means 20%, 10%, 5%, or 3% of the numerical value or range recited or claimed. The terms "a" and "an" and "the" and similar reference used in the context of describing the disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. "Fragment", with reference to an amino acid sequence (peptide or protein), relates to a part of an amino acid sequence, i.e. a sequence which represents the amino acid sequence shortened at the N-terminus and/or C-terminus. A fragment shortened at the C terminus (N-terminal fragment) is obtainable e.g. by translation of a truncated open reading frame that lacks the 3-end of the open reading frame. A fragment shortened at the N-terminus (C-terminal fragment) is obtainable e.g. by translation of a truncated open reading frame that lacks the 5-end of the open reading frame, as long as the truncated open reading frame comprises a start codon that serves to initiate translation. A fragment of an amino acid sequence comprises e.g. at least 50 %, at least 60 %, at least 70 %, at least 80%, at least 90% of the amino acid residues from an amino acid sequence. A fragment of an amino acid sequence preferably comprises at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from an amino acid sequence. As used herein, the term "wild type" or"VT" or "native" refers to an amino acid sequence that is found in nature, including allelic variations. A wild type amino acid sequence, peptide or protein has an amino acid sequence that has not been intentionally modified. As used herein, "variants" of an amino acid sequence (peptide, protein or polypeptide), or "mutants', or reference to a "mutated" polypeptide, comprise amino acid insertion variants/mutants, amino acid addition variants/mutants, amino acid deletion variants/mutants and/or amino acid substitution variants/mutants. The term "variant" or "mutant" includes all mutants, splice variants, posttranslationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, in particular those which are naturally occurring. The term "variant" or "mutant" includes, in particular, fragments of an amino acid sequence. Amino acid insertion variants comprise insertions of single or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are inserted into a particular site in an amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible. Amino acid addition variants comprise amino- and/or carboxy-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as by removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletions may be in any position of the protein. Amino acid deletion variants that comprise the deletion at the N-terminal and/or C-terminal end of the protein are also called N-terminal and/or C-terminal truncation variants. Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place. Preference is given to the modifications being in positions in the amino acid sequence which are not conserved between homologous proteins or peptides and/or to replacing amino acids with other ones having similar properties. Preferably, amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In one embodiment, conservative amino acid substitutions include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Preferably the degree of similarity, preferably identity between a given amino acid sequence and an amino acid sequence which is a variant of said given amino acid sequence will be at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is given preferably for an amino acid region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is given preferably for at least about 20, at least about 40, at least about60, atleastabout80, atleastabout100, atleast about120, atleastabout140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments continuous amino acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. The alignment for determining sequence similarity, preferably sequence identity can be done with art known tools, preferably using the best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS:needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

As used herein, "sequence similarity" indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences. The terms "% identical", "% identity" or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or "window of comparison", in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the United States National Center for Biotechnology Information (NCBI) website (e.g., at blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLASTSPEC=blast2seq&LINK_L OC=align2seq). In some embodiments, the algorithm parameters used for BLASTN algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to 0; (iv) Match/Mismatch Scores set to 1, -2; (v) Gap Costs set to Linear; and (vi) the filter for low complexity regions being used. In some embodiments, the algorithm parameters used for BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi) conditional compositional score matrix adjustment. Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100. In some embodiments, the degree of similarity or identity is given for a region which is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about

90% or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments continuous nucleotides. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence. Homologous amino acid sequences exhibit according to the disclosure at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, at least 98 or at least 99% identity of the amino acid residues. The amino acid sequence variants/mutants described herein may readily be prepared by the skilled person, for example, by recombinant DNA manipulation. The manipulation of DNA sequences for preparing peptides or proteins having substitutions, additions, insertions or deletions, is described in detail in Sambrook et al. (1989), for example. Furthermore, the peptides and amino acid variants described herein may be readily prepared with the aid of known peptide synthesis techniques such as, for example, by solid phase synthesis and similar methods. In one aspect, a fragment or variant/mutant of an amino acid sequence (peptide or protein) is preferably a "functional fragment" or "functional variant". The term "functional fragment" or "functional variant/mutant" of an amino acid sequence relates to any fragment or variant/mutant exhibiting one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., it is functionally equivalent. With respect to antigens or antigenic sequences, one particular function is one or more immunogenic activities displayed by the amino acid sequence from which the fragment or variant is derived. The term "functional fragment" or "functional variant/mutant", as used herein, in particular refers to a variant/mutant molecule or sequence that comprises an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence and that is still capable of fulfilling one or more of the functions of the parent molecule or sequence, e.g., inducing an immune response. In one aspect, the modifications in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced but still significantly present, e.g., immunogenicity of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the parent molecule or sequence. However, in other embodiments, immunogenicity of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

As used herein, "isolated" means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not "isolated", but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated". An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

1. Polypeptides Derived from E. coliand Fragments Thereof In one aspect, disclosed herein is a mammalian cell that includes a polynucleotide that encodes a polypeptide derived from E. coli or a fragment thereof. The term "derived from" as used herein refers to a polypeptide that comprises an amino acid sequence of a FimH polypeptide or FimCH polypeptide complex or a fragment thereof as described herein that has been altered by the introduction of an amino acid residue substitution, deletion or addition. Preferably, the polypeptide derived from E. color a fragment thereof includes a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to the sequence of the corresponding wild-type E. coli FimH polypeptide or fragment. In some embodiments, the polypeptide derived from E. color a fragment thereof has at least 85% identity to the sequence of the corresponding wild-type E. coli FimH polypeptide or fragment. In some embodiments, the polypeptide derived from E. color a fragment thereof has at least 90% identity to the sequence of the corresponding wild-type E. coli FimH polypeptide or fragment. In some embodiments, the polypeptide derived from E. color a fragment thereof has at least 95% identity to the sequence of the corresponding wild-type E. coli FimH polypeptide or fragment. In some embodiments, the polypeptide derived from E. color a fragment thereof has at least 98% identity to the sequence of the corresponding wild-type E. coli FimH polypeptide or fragment. In some embodiments, the polypeptide derived from E. color a fragment thereof has at least 99% identity to the sequence of the corresponding wild-type E. coli FimH polypeptide or fragment. In some embodiments, the polypeptide derived from E. color a fragment thereof has the identical total length of amino acids as the corresponding wild-type FimH polypeptide or FimCH polypeptide complex or a fragment thereof. In some embodiments, the polypeptide derived from E. coli has the identical total length of amino acids as the corresponding wild-type FimH polypeptide or FimCH polypeptide complex. The fragments should include at least n consecutive amino acids from the sequences and, depending on the particular sequence, n is 7 or more (eg. 8, 10, 12, 14, 16, 18, 20 or more). Preferably the fragments include an epitope from the sequence. In some embodiments, the fragment includes an amino acid sequence of at least 50 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues, or at least 250 consecutive amino acid residues of the amino acid sequence of a polypeptide derived from E. coli. In some embodiments, the fragment includes an amino acid sequence of at least 50 consecutive amino acid residues of the amino acid sequence of a polypeptide derived from E. coli. In some embodiments, the fragment includes an amino acid sequence of at least 100 consecutive amino acid residues of the amino acid sequence of a polypeptide derived from E. coli. In some embodiments, the fragment includes an amino acid sequence of at least 150 consecutive amino acid residues of the amino acid sequence of a polypeptide derived from E. coli. In some embodiments, the fragment includes an amino acid sequence of at least 200 consecutive amino acid residues of the amino acid sequence of a polypeptide derived from E. coli. In some embodiments, the fragment includes an amino acid sequence of at least 250 consecutive amino acid residues of the amino acid sequence of a polypeptide derived from E. coli. In some embodiments, the polypeptide derived from E. coli or a fragment thereof includes one or more non-classical amino acids, as compared to a corresponding wild type E. coli FimH polypeptide or fragment. As used herein, the term "FimH polypeptide" refers to any FimH polypeptide or fragment thereof as described herein that has been altered by the introduction of an amino acid substitution, deletion or addition, any FimH domain of the full-length wild type E. coli FimH polypeptide, any combination of domains of the full-length wild type E. coli FimH polypeptide, or the full-length E. coliFimH polypeptide. For example, in one embodiment the present disclosure provides a FimH polypeptide that is a FimHLD polypeptide, or a FimH-DSG polypeptide. In some embodiments, the polypeptide derived from E. coli or a fragment thereof possess a similar or identical function as a corresponding wild-type FimH polypeptide or a fragment thereof. In a preferred embodiment, polypeptides or polypeptide complexes or fragments thereof of the invention are isolated or purified. In some embodiments, the polynucleotide encoding the polypeptide derived from E. coli or a fragment thereof is integrated into the genomic DNA of the mammalian cell, and, when cultured in a suitable condition, said polypeptide derived from E. coli or a fragment thereof is expressed by the mammalian cell. In a preferred embodiment, the polypeptide derived from E. color a fragment thereof is soluble.

In some embodiments, the polypeptide derived from E. coli or a fragment thereof is secreted from the mammalian host cell. In some embodiments, the polypeptide derived from E. coli or a fragment thereof may include additional amino acid residues, such as N-terminal or C-terminal extensions. Such extensions may include one or more tags, which may facilitate detection (e.g. an epitope tag for detection by monoclonal antibodies) and/or purification (e.g. a polyhistidine-tag to allow purification on a nickel-chelating resin) of the polypeptide or fragment thereof. In some embodiments, the tag includes the amino acid sequence selected from any one of SEQ ID NO: 21 and SEQ ID NO: 25. Such affinity-purification tags are known in the art. Examples of affinity-purification tags include, e.g., His tag (hexahistidine, which may, for example, bind to metal ion), maltose-binding protein (MBP), which may, for example, bind to amylose), glutathione-S- transferase (GST), which may, for example, bind to glutathione, FLAG tag, which may, for example, bind to an anti-flag antibody), Strep tag, which may, for example, bind to streptavidin or a derivative thereof). In preferred embodiments, the polypeptide derived from E. coli or a fragment thereof does not include additional amino acid residues, such as N-terminal or C-terminal extensions. In some embodiments, the polypeptide derived from E. coli or a fragment thereof described herein does not include an exogenous tag sequence. While specific strains of E. colimay be referenced herein, it should be understood that the polypeptide derived from E. coli or a fragment thereof are not limited to specific strains unless specified. In some embodiments, the polypeptide derived from E. coli FimH or a fragment thereof includes a phenylalanine residue at the N-terminus of the polypeptide. In some embodiments, the polypeptide derived from FimH or fragment thereof includes a phenylalanine residue within the first 20 residue positions of the N-terminus. Preferably, the phenylalanine residue is located at position 1 of the polypeptide. For example, in some embodiments, the polypeptide derived from E. coli FimH or a fragment thereof does not include an additional glycine residue at the N terminus of the polypeptide derived from E. coli FimH or a fragment thereof. In some embodiments, the phenylalanine residue at position 1 of the wild-type mature E. coli FimH is replaced by an aliphatic hydrophobic amino acid, such as, for example, any one of Ile, Leu and Val residues. In some embodiments, a signal peptide may be used for expressing the polypeptide derived from E. coli or a fragment thereof. Signal sequences and expression cassettes for producing proteins are known in the art. In general, leader peptides are 5-30 amino acids long, and are typically present at the N-terminus of a newly synthesized polypeptide. The signal peptide generally contains a long stretch of hydrophobic amino acids that has a tendency to form a single alpha-helix. In addition, many signal peptides begin with a short positively charged stretch of amino acids, which may help to enforce proper topology of the polypeptide during translocation. At the end of the signal peptide there is typically a stretch of amino acids that is recognized and cleaved by signal peptidase. Signal peptidase may cleave either during or after completion of translocation to generate a free signal peptide and a mature protein. In some embodiments, the signal peptide includes the amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identity to any one of SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 22. In some embodiments, the signal peptide includes the amino acid sequence having at least 99% identity to any one of SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 22. In some embodiments, the signal peptide has the amino acid sequence selected from any one of SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 22.In some embodiments, the polypeptide derived from E. color a fragment thereof described herein may include a cleavable linker. Such linkers allow for the tag to be separated from the purified complex, for example by the addition of an agent capable of cleaving the linker. Cleavable linkers are known in the art. Such linkers may be cleaved for example, by irradiation of a photolabile bond or acid-catalyzed hydrolysis. Another example of a cleavable linker includes a polypeptide linker, which incorporates a protease recognition site and may be cleaved by the addition of a suitable protease enzyme. In some embodiments, the polypeptide derived from E. coli or a fragment thereof includes a modification as compared to the corresponding wild-type E. coli FimH polypeptide or fragment. The modification may include a covalent attachment of a molecule to the polypeptide. For example, such modifications may include glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. In some embodiments, the polypeptide derived from E. coli or a fragment thereof may include a modification, such as by chemical modifications using techniques known to those of skill in the art, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc., as compared to a corresponding wild-type E. coli FimH polypeptide or fragment. In another embodiment, the modification may include a covalent attachment of a lipid molecule to the polypeptide. In some embodiments, the polypeptide does not include a covalent attachment of a molecule to the polypeptide as compared to the corresponding wild-type E. coli FimH polypeptide or fragment thereof.

For example, proteins and polypeptides produced in cell culture may be glycoproteins that contain covalently linked carbohydrate structures including oligosaccharide chains. These oligosaccharide chains are linked to the protein via either N-linkages or 0-linkages. The oligosaccharide chains may comprise a significant portion of the mass of the glycoprotein. Generally, N-linked oligosaccharide is added to the amino group on the side chain of an asparagine residue within the target consensus sequence of Asn-X-Ser/Thr, where X may be any amino acid except proline. In some embodiments, the glycosylation site includes an amino acid sequence selected from any one of the following: asparagine-glycine-threonine (NGT), asparagine-isoleucine-threonine (NIT), asparagine-glycine-serine (NGS), asparagine-serine threonine (NST), and asparagine-threonine-serine (NTS). The polypeptide derived from E. coli or a fragment thereof produced in mammalian cells may by glycosylated. The glycosylation may occur at the N-linked glycosylation signal Asn-Xaa-Ser/Thr in the sequence of the polypeptide derived from E. coli or a fragment thereof. "N-linkedglycosylation" refers to the attachment of the carbohydrate moiety via GlcNAc to an asparagine residue in a polypeptide chain. TheN-linked carbohydrate contains a common Man 1-6(Man1-3)Manp1-4GcNAcp1 4GIcNAcp-R core structure, where R represents an asparagine residue of the produced polypeptide derived from E. coli or a fragment thereof. In some embodiments, a glycosylation site in the polypeptide derived from E. coli or a fragment thereof is removed by a mutation within the sequence of the polypeptide derived from E. coli or a fragment thereof. For example, in some embodiments, the Asn residue of a glycosylation motif (Asn-Xaa-Ser/Thr) may be mutated, preferably by a substitution. In some embodiments, the residue substitution is selected from any one of Ser, Asp, Thr, and GIn. In some embodiments, the Ser residue of a glycosylation motif may be mutated, preferably by a substitution. In some embodiments, the residue substitution is selected from any one of Asp, Thr, and GIn. In some embodiments, the Thr residue of a glycosylation motif may be mutated, preferably by a substitution. In some embodiments, the residue substitution is selected from any one of Ser, Asp, and GIn. In some embodiments, a glycosylation site (such as Asn-Xaa-Ser/Thr) in the polypeptide derived from E. coli or a fragment thereof is not removed or modified. In some embodiments, a compound to decrease or inhibit glycosylation may be added to the cell culture medium. In such embodiments, the polypeptide or protein includes at least one more unglycosylated (i.e., aglycosylated) site, that is, a completely unoccupied glycan site with no carbohydrate moiety attached thereto, or comprises at least one carbohydrate moiety less at the same potential glycosylation site than an otherwise identical polypeptide or protein which is produced by a cell under otherwise identical conditions but in the absence of a glycosylation inhibiting compound.

Such compounds are known in the art and may include, but are not limited to, tunicamycin, tunicaymycin homologs, streptovirudin, mycospocidin, amphomycin, tsushimycin, antibiotic 24010, antibiotic MM 19290, bacitracin, corynetoxin, showdomycin, duimycin, 1-deoxymannonojirimycin, deoxynojirimycin, N-methyl-1 dexoymannojirimycin, brefeldin A, glucose and mannose analogs, 2-deoxy- D-glucose, 2-deoxyglucose, D-(+)-mannose, D-(+) galactose, 2-deoxy-2-fluoro-D-glucose, 1 4 dideoxy- 1 ,4-imino-D-mannitol (DIM), fluoroglucose, fluoromannose, UDP-2 deoxyglucose, GDP-2-deoxyglucose, hydroxymethylglutaryl-CoA reductase inhibitors, 25-hydroxycholesterol, hydroxycholesterol, swainsonine, cycloheximide, puromycin, actinomycin D, monensin, m-Chlorocarbonyl-cyanide phenyhydrazone (CCCP), compactin, dolichyl-phosphoryl^-deoxyglucose, N-Acetyl-D-Glucosamine, hygoxanthine, thymidine, cholesterol, glucosamine, mannosamine, castanospermine, glutamine, bromoconduritol, conduritol epoxide and conduritol derivatives, glycosylmethyl-p nitrophenyltriazenes, p-Hydroxynorvaline, threo-p-fluoroasparagine, D-(+)-Gluconic acid 6-lactone, di(2-ethyl hexyl)phosphate, tributyl phosphate, dodecyl phosphate, 2 dimethylamino ethyl ester of (diphenyl methyl)-phosphoric acid, [2-(diphenyl phosphinyloxy)ethyl]trimethyl ammonium iodide, iodoacetate, and/or fluoroacetate One of ordinary skill in the art will readily recognize or will be able to determine glycosylation inhibiting substances that may be used in accordance with methods and compositions of the present invention without undue experimentation. In such embodiments, glycosylation of the polypeptide or fragment thereof may be controlled without the introduction of an amino acid mutation into the polypeptide or fragment thereof. In some embodiments, the level of glycosylation (e.g., number of glycan sites that are occupied on the polypeptide or fragment thereof, the size and/or complexity of glycoform at the site, and the like) of the polypeptide or fragment thereof produced by the mammalian cell are lower than levels of glycosylation of the polypeptide or fragment thereof produced under otherwise identical conditions in an otherwise identical medium that lacks such a glycolysis-inhibiting compound and/or mutation. In some embodiments, the sequence of a polypeptide derived from E. coli or a fragment thereof does not include a site ofN-linked protein glycosylation. In some embodiments, the sequence of a polypeptide derived from E. color a fragment thereof does not include at least one site of N-linked protein glycosylation. In some embodiments, the sequence of a polypeptide derived from E. color a fragment thereof does not include any sites ofN-linked protein glycosylation. In some embodiments, the sequence of a polypeptide derived from E. color a fragment thereof includes a site for N-linked protein glycosylation. In some embodiments, the sequence of a polypeptide derived from E. coli or a fragment thereof includes at most 1 site ofN-linked protein glycosylation. In some embodiments, the sequence of a polypeptide derived from E. coli or a fragment thereof includes at most 2 sites of N-linked protein glycosylation. A polypeptide derived from E. coli or a fragment thereof expressed by different cell lines or in transgenic animals may have different glycan site occupancies, glycoforms and/or glycosylation patterns compared with each other. In some embodiments, the invention encompasses a polypeptide derived from E. color a fragment thereof regardless of the the glycosylation, glycan occupancy or glycoform pattern of the polypeptide derived from E. coli or a fragment thereof produced in a mammalian cell. In some embodiments, the polypeptide derived from E. coli or a fragment thereof may be derived from an E. coli FimH polypeptide, wherein the amino acid residue at position 1 of the polypeptide is phenylalanine, not methionine, for example, a polypeptide having the amino acid sequence SEQ ID NO: 2. Preferably, the polypeptide derived from E. coli FimH comprises a phenylalanine at position 1 of the amino acid sequence of the polypeptide derived from E. coli. In another preferred embodiment, the polypeptide derived from E. coli FimH comprises the amino acid sequence SEQ ID NO: 3, preferably wherein the residue at position 1 of the amino acid sequence of the polypeptide derived from E. coli is phenylalanine. In some embodiments, the polypeptide derived from E. color a fragment thereof may include the amino acid sequence SEQ ID NO: 4, which may be derived from an E. coli FimH polypeptide. In some embodiments, the polypeptide derived from E. coli or a fragment thereof includes the amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%,80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, and SEQ ID NO: 113. In some embodiments, the polypeptide derived from E. coli or a fragment thereof includes the amino acid sequence having at least 95% identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, and SEQ ID NO: 113. In some embodiments, the polypeptide derived from E. color a fragment thereof includes the amino acid sequence having at least 99% identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ

ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, and SEQ ID NO: 113. In some embodiments, the polypeptide derived from E. coior a fragmentthereof may be derived from an E. coli FimG polypeptide, for example, having the amino acid sequence SEQ ID NO: 9. In some embodiments, the polypeptide derived from E. color a fragment thereof may be derived from an E. coli FimC polypeptide, for example, having the amino acid sequence SEQ ID NO: 10.

A. Polypeptides Derived from E. coli FimH and Fragments Thereof

In a preferred embodiment, the polypeptide or fragment thereof is derived from an E. coli FimH. In some embodiments, the polypeptide or fragment thereof includes full length E. coli FimH. Full length FimH includes two domains: an N-terminal lectin domain and a C-terminal pilin domain, which are connected by a short linker. In some embodiments, the full length of E. coli FimH includes 279 amino acids, which includes the full length of the mature protein of E. coli FimH. In some embodiments, the full length of E. coli FimH includes 300 amino acids, which includes the full length of the mature protein of E. coli FimH and a signal peptide sequence having 21 amino acids in length. The primary structure of the 300 amino acid-long wild type FimH is highly conserved across E. colistrains. An exemplary sequence for a full-length E. coli FimH is SEQ ID NO: 1. The full length FimH sequence includes a sequence for a lectin domain and a sequence for a pilin domain. The lectin domain of FimH contains the carbohydrate recognition domain, which is responsible for binding to the mannosylated uroplakin 1a on the urothelial cell surface. The pilin domain is anchored to the core of the pilus via a donor strand of the subsequent FimG subunit, which is a process termed donor strand complementation. Starting from the N-terminus, the names and in parenthesis the exemplary amino acid sequences of each domain of a full length FimH are as follows: FimH lectin (SEQ ID NO: 2) and FimH pilin (SEQ ID NO: 3). Other suitable polypeptides and fragments thereof derived from E. coli FimH include variants that have various degrees of identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112 and SEQ ID NO: 113, such as at least 70%, 71%, 72%, 73%, 74%,75%,76%,77%,78%,79%,80%,81%,82%,83%,84%,85%,86%,87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 20,

SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112 and SEQ ID NO: 113. Other suitable polypeptides have least 95% identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112 and SEQ ID NO: 113. Othersuitable polypeptides have least 99% identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112 and SEQ ID NO: 113. In certain embodiments, the FimH variant proteins: (i) form part of the FimH-FimC; (ii) comprise at least one epitope from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112 and SEQ ID NO: 113; and/or (iii) may elicit antibodies in vivo which immunologically cross react with an E. coli FimH. In some embodiments, the composition includes a polypeptide having at least n consecutive amino acids from any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112 and SEQ ID NO: 113, wherein n is 7 or more (eg. 8, 10, 12, 14, 16, 18, 20 or more). Preferably the fragments include an epitope from the sequence. In some embodiments, composition includes a polypeptide having at least 50 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues, or at least 250 consecutive amino acid residues of the amino acid sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112 and SEQ ID NO: 113. In some embodiments, composition includes a polypeptide having at least 50 consecutive amino acid residues of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112 and SEQ ID NO: 113. In some embodiments, composition includes a polypeptide having at least 100 consecutive amino acid residues of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112 and SEQ ID NO: 113. In some embodiments, composition includes a polypeptide having at least 200 consecutive amino acid residues of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID

NO: 28, SEQ ID NO: 29, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112 and SEQ ID NO: 113. In some embodiments, composition includes a polypeptide having at least 250 consecutive amino acid residues of the amino acid sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112 and SEQ ID NO: 113. In some embodiments, the composition includes a polypeptide having at least 70%,71%,72%,73%,74%,75%,76%,77%,78%,79%,80%,81%,82%,83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO: 1. In some embodiments, the composition includes a polypeptide having at least 90% identity to SEQ ID NO: 1. In some embodiments, the composition includes a polypeptide having at least 95% identity to SEQ ID NO: 1. In some embodiments, the composition includes a polypeptide having at least 99% identity to SEQ ID NO: 1. In some embodiments, the composition includes a polypeptide as set forth at SEQ ID NO: 1. In some embodiments, the composition includes a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO: 2. In some embodiments, the composition includes a polypeptide having at least 90% identity to SEQ ID NO: 2. In some embodiments, the composition includes a polypeptide having at least 95% identity to SEQ ID NO: 2. In some embodiments, the composition includes a polypeptide having at least 99% identity to SEQ ID NO: 2. In some embodiments, the composition includes a polypeptide as set forth at SEQ ID NO: 2. In some embodiments, the composition includes a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%,80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO: 3. In some embodiments, the composition includes a polypeptide having at least 90% identity to SEQ ID NO: 3. In some embodiments, the composition includes a polypeptide having at least 95% identity to SEQ ID NO: 3. In some embodiments, the composition includes a polypeptide having at least 99% identity to SEQ ID NO: 3. In some embodiments, the composition includes a polypeptide as set forth at SEQ ID NO: 3. In some embodiments, the composition includes a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%,76%,77%,78%,79%,80%,81%,82%,83%,84%,85%,86%,87%,88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO: 4. In some embodiments, the composition includes a polypeptide having at least 90% identity to SEQ ID NO: 4. In some embodiments, the composition includes a polypeptide having at least 95% identity to SEQ ID NO: 4. In some embodiments, the composition includes a polypeptide having at least 99% identity to SEQ ID NO: 4. In some embodiments, the composition includes a polypeptide as set forth at SEQ ID NO: 4. In some embodiments, the composition includes a polypeptide having at least 70%, 71%,72%,73%,74%,75%,76%,77%,78%,79%,80%,81%,82%,83%,84%,85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO: 5. In some embodiments, the composition includes a polypeptide having at least 90% identity to SEQ ID NO: 5. In some embodiments, the composition includes a polypeptide having at least 95% identity to SEQ ID NO: 5. In some embodiments, the composition includes a polypeptide having at least 99% identity to SEQ ID NO: 5. In some embodiments, the composition includes a polypeptide as set forth at SEQ ID NO: 5. In some embodiments, the composition includes a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%,79%,80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO: 6. In some embodiments, the composition includes a polypeptide having at least 90% identity to SEQ ID NO: 6. In some embodiments, the composition includes a polypeptide having at least 95% identity to SEQ ID NO: 6. In some embodiments, the composition includes a polypeptide having at least 99% identity to SEQ ID NO: 6. In some embodiments, the composition includes a polypeptide as set forth at SEQ ID NO: 6. In some embodiments, the composition includes a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO: 20. In some embodiments, the composition includes a polypeptide having at least 90% identity to SEQ ID NO: 20. In some embodiments, the composition includes a polypeptide having at least 95% identity to SEQ ID NO: 20. In some embodiments, the composition includes a polypeptide having at least 99% identity to SEQ ID NO: 20. In some embodiments, the composition includes a polypeptide as set forth at SEQ ID NO: 20. In some embodiments, the composition includes a polypeptide having at least 70%, 71%,72%,73%,74%,75%,76%,77%,78%,79%,80%,81%,82%,83%,84%,85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO: 23. In some embodiments, the composition includes a polypeptide having at least 90% identity to SEQ ID NO: 23. In some embodiments, the composition includes a polypeptide having at least 95% identity to SEQ ID NO: 23. In some embodiments, the composition includes a polypeptide having at least 99% identity to SEQ ID NO: 23. In some embodiments, the composition includes a polypeptide as set forth at SEQ ID NO: 23. In some embodiments, the composition includes a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%,79%,80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,

93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO: 24. In some embodiments, the composition includes a polypeptide having at least 90% identity to SEQ ID NO:24. In some embodiments, the composition includes a polypeptide having at least 95% identity to SEQ ID NO: 24. In some embodiments, the composition includes a polypeptide having at least 99% identity to SEQ ID NO: 24. In some embodiments, the composition includes a polypeptide as set forth at SEQ ID NO: 24. In some embodiments, the composition includes a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%,78%,79%,80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO: 26. In some embodiments, the composition includes a polypeptide having at least 90% identity to SEQ ID NO: 26. In some embodiments, the composition includes a polypeptide having at least 95% identity to SEQ ID NO: 26. In some embodiments, the composition includes a polypeptide having at least 99% identity to SEQ ID NO: 26. In some embodiments, the composition includes a polypeptide as set forth at SEQ ID NO: 26. In some embodiments, the composition includes a polypeptide having at least 70%, 71%,72%,73%,74%,75%,76%,77%,78%,79%,80%,81%,82%,83%,84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO: 27. In some embodiments, the composition includes a polypeptide having at least 90% identity to SEQ ID NO: 27. In some embodiments, the composition includes a polypeptide having at least 95% identity to SEQ ID NO: 27. In some embodiments, the composition includes a polypeptide having at least 99% identity to SEQ ID NO: 27. In some embodiments, the composition includes a polypeptide as set forth at SEQ ID NO: 27. In some embodiments, the composition includes a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO: 28. In some embodiments, the composition includes a polypeptide having at least 90% identity to SEQ ID NO: 28. In some embodiments, the composition includes a polypeptide having at least 95% identity to SEQ ID NO: 28. In some embodiments, the composition includes a polypeptide having at least 99% identity to SEQ ID NO: 28. In some embodiments, the composition includes a polypeptide as set forth at SEQ ID NO: 28. In some embodiments, the composition includes a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%,78%,79%,80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO: 29. In some embodiments, the composition includes a polypeptide having at least 90% identity to SEQ ID NO: 29. In some embodiments, the composition includes a polypeptide having at least 95% identity to SEQ ID NO: 29. In some embodiments, the composition includes a polypeptide having at least 99% identity to SEQ ID NO: 29. In some embodiments, the composition includes a polypeptide as set forth at SEQ ID NO: 29. In some embodiments, the composition includes a polypeptide having at least 70%, 71%,72%,73%,74%,75%,76%,77%,78%,79%,80%,81%,82%,83%,84%,85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO: 110. In some embodiments, the composition includes a polypeptide having at least 90%identitytoSEQIDNO:110. In some embodiments, the composition includes a polypeptide having at least 95% identity to SEQ ID NO: 110. In some embodiments, the composition includes a polypeptide having at least 99% identity to SEQ ID NO: 110. In some embodiments, the composition includes a polypeptide as set forth at SEQ ID NO: 110. In some embodiments, the composition includes a polypeptide having at least 70%, 71%, 72%, 73%, 74%,75%,76%,77%,78%,79%,80%,81%,82%,83%,84%,85%,86%,87%,88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO: 111. In some embodiments, the composition includes a polypeptide having at least 90% identity to SEQ ID NO: 111. In some embodiments, the composition includes a polypeptide having at least 95% identity to SEQ ID NO: 111. In some embodiments, the composition includes a polypeptide having at least 99% identity to SEQ ID NO: 111. In some embodiments, the composition includes a polypeptide as set forth at SEQ ID NO: 111. In some embodiments, the composition includes a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%,79%,80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO: 112. In some embodiments, the composition includes a polypeptide having at least 90% identity to SEQ ID NO: 112. In some embodiments, the composition includes a polypeptide having at least 95% identity to SEQ ID NO: 112. In some embodiments, the composition includes a polypeptide having at least 99% identity to SEQ ID NO: 112. In some embodiments, the composition includes a polypeptide as set forth at SEQ ID NO: 112. In some embodiments, the composition includes a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO: 113. In some embodiments, the composition includes a polypeptide having at least 90% identity to SEQ ID NO: 113. In some embodiments, the composition includes a polypeptide having at least 95% identity to SEQ ID NO: 113. In some embodiments, the composition includes a polypeptide having at least 99% identity to SEQ ID NO: 113. In some embodiments, the composition includes a polypeptide as set forth at SEQ ID NO: 113.

Another example of a suitable polypeptide and fragments thereof derived from E. coli FimH described herein is shown as SEQ ID NO: 2, which lacks the wild-type N terminal signal sequence, and corresponds to amino acid residues 22-300 of SEQ ID NO: 1. Another example of a FimH fragment includes the entire N- terminal signal sequence and the mature protein, such as set forth in SEQ ID NO: 1. In some embodiments, a glycosylation site in the polypeptide derived from E. coli or a fragment thereof is removed by a mutation within the sequence of the polypeptide derived from E. coli or a fragment thereof. For example, in some embodiments, the Asn residue at position 7 of a mature E. coli FimH polypeptide (e.g., according to the numbering of SEQ ID NO: 2) may be mutated, preferably by a substitution. In some embodiments, the Asn residue at position 7 of a lectin domain of an E. coli FimH polypeptide (e.g., according to the numbering of SEQ ID NO: 3) may be mutated, preferably by a substitution. In some embodiments, the residue substitution is selected from any one of Ser, Asp, Thr, and GIn. In some embodiments, the Thr residue at position 10 of a mature E. coli FimH polypeptide (e.g., according to the numbering of SEQ ID NO: 2) may be mutated, preferably by a substitution. In some embodiments, the Thr residue at position 7 of a lectin domain of an E. coli FimH polypeptide (e.g., according to the numbering of SEQ ID NO: 3) may be mutated, preferably by a substitution. In some embodiments, the residue substitution is selected from any one of Ser, Asp, and GIn. In some embodiments, the Asn residue at position N235 of a mature E. coli FimH polypeptide (e.g., according to the numbering of SEQ ID NO: 2) may be mutated, preferably by a substitution. In some embodiments, the Asn residue at position N228 of a mature E. coli FimH polypeptide (e.g., according to the numbering of SEQ ID NO: 2) may be mutated, preferably by a substitution. In some embodiments, the residue substitution is selected from any one of Ser, Asp, Thr, and GIn. In some embodiments, the Asn residue at position 70 of a mature E. coli FimH polypeptide (e.g., according to the numbering of SEQ ID NO: 2) may be mutated, preferably by a substitution. In some embodiments, the Asn residue at position 70 of a lectin domain of an E. coli FimH polypeptide (e.g., according to the numbering of SEQ ID NO: 3) may be mutated, preferably by a substitution. In some embodiments, the residue substitution is selected from any one of Ser, Asp, Thr, and GIn. In some embodiments, the Ser residue at position 72 of a mature E. coli FimH polypeptide (e.g., according to the numbering of SEQ ID NO: 2) may be mutated, preferably by a substitution. In some embodiments, the Ser residue at position 72 of a lectin domain of an E. coli FimH polypeptide (e.g., according to the numbering of SEQ

ID NO: 3) may be mutated, preferably by a substitution. In some embodiments, the residue substitution is selected from any one of Asp, Thr, and Gn. By the term "fragment" as used herein refers to a polypeptide and is defined as any discrete portion of a given polypeptide that is unique to or characteristic of that polypeptide. The term as used herein also refers to any discrete portion of a given polypeptide that retains at least a fraction of the activity of the full-length polypeptide. In certain embodiments, the fraction of activity retained is at least 10% of the activity of the full-length polypeptide. In certain embodiments, the fraction of activity retained is at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the activity of the full-length polypeptide. In certain embodiments, the fraction of activity retained is at least 95%, 96%, 97%, 98% or 99% of the activity of the full-length polypeptide. In certain embodiments, the fraction of activity retained is 100% or more of the activity of the full-length polypeptide. In some embodiments, a fragment includes at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more consecutive amino acids of the full-length polypeptide.

B. Complex of FimH, FimC, and fragments thereof In some embodiments, the polypeptide derived from E. coli FimH or fragment thereof is present in a complex with polypeptide derived from E. coli FimC or fragment thereof. In a preferred embodiment, the polypeptide derived from E. coli FimH or fragment thereof and the polypeptide derived from E. coli FimC or fragment thereof are present in a complex, preferably in a 1:1 ratio in the complex. Without being bound by theory or mechanism, the full length FimH may be stabilized in an active conformation by the periplasmic chaperone FimC, thereby making it possible to purify full-length FimH protein. Accordingly, in some embodiments, the polypeptide or fragment thereof includes full length FimH and full length FimC. In some embodiments, the polypeptide or fragment thereof includes a fragment of FimH and a fragment of FimC. In some embodiments, the polypeptide or fragment thereof includes full length FimH and a fragment of FimC. An exemplary sequence for E. coli FimC is set forth in SEQ ID NO: 10. In some embodiments, the polypeptide derived from E. coli or a fragment thereof includes complex-forming fragments of FimH. A complex-forming fragment of FimH may be any part or portion of the FimH protein that retain the ability to form a complex with FimC or a fragment thereof. A suitable complex forming fragment of FimH may also be obtained or determined by standard assays known in the art, such as co-immunoprecipitation assay, cross-linking, or co-localization by fluorescent staining, etc. SDS-PAGE or western blot mayalso be used (e.g., by showing that the FimH fragment and FimC or fragment thereof are in a complex as evidenced by gel electrophoresis). In certain embodiments, the complex-forming fragment of FimH (i) forms part of the FimH-FimC complex; (ii) comprises at least one epitope from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 110, and SEQ ID NO: 111; and/or (iii) may elicit antibodies in vivo which immunologically cross react with an E. coli FimH. In some embodiments, the polypeptide derived from E. coli or a fragment thereof includes full length FimH, wherein the FimH is not complexed with FimC. In further embodiments, the polypeptide or fragment thereof includes a fragment of FimH, wherein the fragment is not complexed with FimC. In some embodiments, the polypeptide derived from E. coli or a fragment thereof FimC includes SEQ ID NO: 10. In some embodiments, the the complex may be expressed from the same plasmid, preferably under the the control of separate promoters for each polypeptide or fragment thereof. In some embodiments, the polypeptide derived from E. coli FimH or a fragment thereof binds to a polypeptide derived from E. coli FimC or a fragment thereof, which may be engineered into the structure of the polypeptide derived from E. coli FimH or fragment thereof. The portion of the FimC molecule that binds to the FimH in the complex is called a "donor strand" and the mechanism of formation of the native FimH structure using the strand from FimC thatbinds to FimH in the FimCH complex is known as "donor strand complementation." In some embodiments, the polypeptide derived from E. coli FimH or a fragment thereof may be expressed by the appropriate donor strand complemented version of FimH, wherein the amino acid sequence of FimC that interacts with FimH in the FimCH complex is itself engineered at the C-terminal end of FimH to provide the native conformation without the need for the remainder of the FimC molecule to be present. In some embodiments, the polypeptide derived from E. coli FimH or a fragment thereof may be expressed in the form of a complex that includes isolated domains thereof, such as the lectin binding domain and the piling domain, and such domains may be linked together covalently or non-covalently. For example, in some embodiments, the linking segment may include amino acid sequences or other oligomeric structures, including simple polymer structures. The methods and compositions of the invention may include complexes described herein, in which said polypeptides or fragements thereof derived from E. coli are co-expressed or formed in a combined state.

C. Lectin domain, Pilin domain, and Variants thereof

Conformation and ligand-binding properties of the lectin domain of FimH may be under the allosteric control of the pilin domain of FimH. Under static conditions, the interaction of the two domains of full length FimH stabilizes the lectin domain in a low affinity to monomannose state (for example, K -300 pM), which is characterized by a shallow binding pocket. Binding to a mannoside ligand may induce a conformational change leading to a medium affinity state, in which the lectin and pilin domains remain in close contact. However, upon shear stress, the lectin and pilin domains may separate and induce the high affinity state (for example, K <1.2 pM). Because of the absence of negative allosteric regulation exerted by the pilin domain, isolated lectin domain of FimH is locked in the high-affinity state (for example, K <1.2 pM). The isolated, recombinant lectin domain, which is locked in the high-affinity state. Locking the adhesin in a low-affinity conformation (for example, K -300 pM), however, induces the production of adhesion-inhibiting antibodies. Accordingly, there is an interest in stabilizing the lectin domain in the low-affinity state. In some embodiments, the polypeptide derived from E. coli or a fragment thereof includes the lectin domain of an E. coli FimH. Exemplary sequences for a lectin domain include any one of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 110, and SEQ ID NO: 111. In some embodiments, the lectin domain of an E. coliFimH includes cysteine substitutions. In a preferred embodiment, the lectin domain of an E. coli FimH includes cysteine substitutions within the first 50 amino acid residues of the lectin domain. In some embodiments, the lectin domain may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cysteine substitutions. Preferably, the lectin domain includes 2 cysteine substitutions. See, for example, pSB02158 and pSB02198. Other suitable polypeptides and fragments thereof derived from E. coli FimH include FimH lectin domain variants that have various degrees of identity to SEQ ID NO: 3, such as at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to the sequence recited in SEQ ID NO: 3. In some embodiments, the composition includes a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO: 3. In some embodiments, the composition includes a polypeptide having at least 90% identity to SEQ ID NO: 3. In some embodiments, the composition includes a polypeptide having at least 95% identity to SEQ ID NO: 3. In some embodiments, the composition includes a polypeptide having at least 99% identity to SEQ ID NO: 3. In some embodiments, the composition includes a polypeptide as set forth at SEQ ID NO: 3. In some embodiments, the polypeptide derived from E. coli or a fragment thereof includes the pilin domain of an E. coliFimH. Other suitable polypeptides and fragments thereof derived from E. coli FimH include FimH pilin domain variants that have various degrees of identity to SEQ ID NO: 7, such as at least 70%, 71%, 72%, 73%, 74%,75%, 76%,77%,78%,79%,80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to the sequence recited in SEQ ID NO: 7. In some embodiments, the composition includes a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO: 7. In some embodiments, the composition includes a polypeptide having at least 90% identity to SEQ ID NO: 7. In some embodiments, the composition includes a polypeptide having at least 95% identity to SEQ ID NO: 7. In some embodiments, the composition includes a polypeptide having at least 99% identity to SEQ ID NO: 7. In some embodiments, the composition includes a polypeptide as set forth at SEQ ID NO: 7. Other suitable polypeptides and fragments thereof derived from E. coli FimH include FimH lectin domain variants that have various degrees of identity to SEQ ID NO: 8, such as at least 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%,78%,79%,80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to the sequence recited in SEQ ID NO: 8. In some embodiments, the composition includes a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO: 8. In some embodiments, the composition includes a polypeptide having at least 90% identity to SEQ ID NO: 8. In some embodiments, the composition includes a polypeptide having at least 95% identity to SEQ ID NO: 8. In some embodiments, the composition includes a polypeptide having at least 99% identity to SEQ ID NO: 8. In some embodiments, the composition includes a polypeptide as set forth at SEQ ID NO: 8. In some embodiments, the polypeptide derived from E. coli or a fragment thereof includes the pilin domain of an E. coli FimH. Other suitable polypeptides and fragments thereof derived from E. coli FimH include FimH pilin domain variants that have various degrees of identity to SEQ ID NO: 24, such as at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%, 98%, 99% or 99.9% identity to the sequence recited in SEQ ID NO: 24. In some embodiments, the composition includes a polypeptide having at least 70%, 71%, 72%, 73%,74%,75%,76%,77%,78%,79%,80%,81%,82%,83%,84%,85%,86%,87%,

88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO: 24. In some embodiments, the composition includes a polypeptide having at least 90% identity to SEQ ID NO: 24. In some embodiments, the composition includes a polypeptide having at least 95% identity to SEQ ID NO: 24. In some embodiments, the composition includes a polypeptide having at least 99% identity to SEQ ID NO: 24. In some embodiments, the composition includes a polypeptide as set forth at SEQ ID NO: 24. Other suitable polypeptides and fragments thereof derived from E. coli FimH include FimH lectin domain variants that have various degrees of identity to SEQ ID NO: 26, such as at least 70%, 71%, 72%, 73%, 74%, 75%,76%,77%,78%,79%,80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to the sequence recited in SEQ ID NO: 26. In some embodiments, the composition includes a polypeptide having at least 70%,71%,72%,73%,74%,75%,76%,77%,78%,79%,80%,81%,82%,83%,84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO: 26. In some embodiments, the composition includes a polypeptide having at least 90% identity to SEQ ID NO: 26. In some embodiments, the composition includes a polypeptide having at least 95% identity to SEQ ID NO: 26. In some embodiments, the composition includes a polypeptide having at least 99% identity to SEQ ID NO: 26. In some embodiments, the composition includes a polypeptide as set forth at SEQ ID NO: 26. Other suitable polypeptides and fragments thereof derived from E. coli FimH include FimH lectin domain variants that have various degrees of identity to SEQ ID NO: 110, such as at least 70%, 71%,72%,73%,74%,75%,76%,77%,78%,79%,80%,81%,82%,83%,84%,85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to the sequence recited in SEQ ID NO: 110. In some embodiments, the composition includes a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO: 110. In some embodiments, the composition includes a polypeptide having at least 90% identity to SEQ ID NO: 110. In some embodiments, the composition includes a polypeptide having at least 95% identity to SEQ ID NO: 110. In some embodiments, the composition includes a polypeptide having at least 99% identity to SEQ ID NO: 110. In some embodiments, the composition includes a polypeptide as set forth at SEQ ID NO: 110. Other suitable polypeptides and fragments thereof derived from E. coli FimH include FimH lectin domain variants that have various degrees of identity to SEQ ID NO: 111, such as at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%, 99% or 99.9% identity to the sequence recited in SEQ ID NO: 111. In some embodiments, the composition includes a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%,

77%,78%,79%,80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO: 111. In some embodiments, the composition includes a polypeptide having at least 90% identity to SEQ ID NO: 111. In some embodiments, the composition includes a polypeptide having at least 95% identity to SEQ ID NO: 111. In some embodiments, the composition includes a polypeptide having at least 99% identity to SEQ ID NO: 111. In some embodiments, the composition includes a polypeptide as set forth at SEQ ID NO: 111.

In some embodiments, the composition includes a polypeptide having at least n consecutive amino acids from any one of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 110, and SEQ ID NO: 111, wherein n is 7 or more (eg. 8, 10, 12, 14, 16, 18, 20 or more). Preferably the fragments include an epitope from the sequence. In some embodiments, the composition includes a polypeptide having at least 50 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues, or at least 250 consecutive amino acid residues of the amino acid sequence of any one of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 110, and SEQ ID NO: 111. In some embodiments, the composition includes a polypeptide having at least 50 consecutive amino acid residues of any one of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 110, and SEQ ID NO: 111. In some embodiments, the composition includes a polypeptide having at least 100 consecutive amino acid residues of any one of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 110, and SEQ ID NO: 111. In some embodiments, the composition includes a polypeptide having at least 150 consecutive amino acid residues of any one of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 110, and SEQ ID NO: 111. In some embodiments, the composition includes a polypeptide having at least 250 consecutive amino acid residues of the amino acid sequence of any one of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 110, and SEQ ID NO: 111. The location and length of a lectin domain of E. coli FimH or a homologue or a variant thereof may be predicted based on pairwise alignment of its sequence to any one of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 26,

SEQ ID NO: 110, and SEQ ID NO: 111, for example by aligning the amino acid sequence of a FimH to SEQ ID NO: 1, and identifying the sequence that aligns to residues 22-179 of SEQ ID NO: 1.

D. Wild-type N-terminal signal sequence In some embodiments, the N-terminal wild type signal sequence of full-length FimH is cleaved in a host cell to produce a mature FimH polypeptide. As such, the FimH expressed by the host cell may lack the N-terminal signal sequence. In preferred embodiments, the polypeptide derived from E. coli or a fragment thereof may be encoded by a nucleotide sequence that lacks the coding sequence for the wild type N-terminal signal sequence. In some embodiments, the polypeptide derived from E. coli or a fragment thereof includes the FimH-FimC complex forming fragments of FimH, the N-terminal signal sequence (such as, residues 1-21 of SEQ ID NO: 1), or a combination thereof. A complex-forming fragment of FimH may be any part or portion of the FimH protein that retains the ability to form a complex with FimC. In some embodiments, the polypeptide derived from E. coli or a fragment thereof may lack between 1 and 21 amino acid residues (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acid residues, or lack 1-21 residues, 1-20 residues, 1-15 residues, 1-10 residues, 2-20 residues, 2-15 residues, 2-10 residues, 5-20 residues, 5-15 residues, or 5-10 residues) at the N-terminus and/or C-terminus of the full-length FimH polypeptide, which may include the signal sequence, lectin domain, and pilin domain. In some embodiments, the polypeptide derived from E. color a fragment thereof lacks 1-21 residues at the N-terminus of the full-length FimH polypeptide. In some embodiments, the polypeptide derived from E. coli or a fragment thereof lacks 1- 10 residues at the N-terminus of the full length FimH polypeptide. In some embodiments, the polypeptide derived from E. color a fragment thereof lacks 5-15 residues at the N-terminus of the full-length FimH polypeptide. In some embodiments, the polypeptide derived from E. coli or a fragment thereof lacks 5-10 residues at the N-terminus of the full-length FimH polypeptide. In some embodiments, the polypeptide derived from E. coli or a fragment thereof lacks 1-21 residues at the C-terminus of the full-length FimH polypeptide. In some embodiments, the polypeptide derived from E. color a fragment thereof lacks 1- 10 residues at the C-terminus of the full-length FimH polypeptide. In some embodiments, the polypeptide derived from E. coli or a fragment thereof lacks 5-15 residues at the C-terminus of the full-length FimH polypeptide. In some embodiments, the polypeptide derived from E. coli or a fragment thereof lacks 5-10 residues at the C-terminus of the full-length FimH polypeptide.

II. Nucleic acids

In one aspect, nucleic acids encoding the polypeptide derived from E. coior a fragment thereof are disclosed. One or more nucleic acid constructs encoding the polypeptide derived from E. coli or a fragment thereof may be used for genomic integration and subsequent expression of the polypeptide derived from E. coiora fragment thereof. For example, a single nucleic acid construct encoding the polypeptide derived from E. coli or fragment thereof may be introduced to a host cell. Alternatively, the coding sequences for the polypeptide derived from E. coli or a fragment thereof may be carried by two or more nucleic acid constructs, which are then introduced into host cell simultaneously or sequentially. For example, in one exemplary embodiment, a single nucleic acid construct encodes the lectin domain and pilin domain of an E. coliFimH. In another exemplary embodiment, one nucleic acid construct encodes the lectin domain and a second nucleic acid construct encodes the pilin domain of an E. coli FimH. In some embodiments, genomic integration is achieved. The nucleic acid construct may comprise genomic DNA that comprises one or more introns, or cDNA. Some genes are expressed more efficiently when introns are present. In some embodiments, the nucleic acid sequence is suitable for the expression of exogenous polypeptides in said mammalian cell. In some embodiments, the nucleic acid encoding the polypeptide or fragment thereof is codon optimized to increase the level of expression in any particular cell. In some embodiments, the nucleic acid construct includes a signal sequence that encodes a peptide that directs secretion of the polypeptide derived from E. color a fragment thereof. In some embodiments, the nucleic acid includes the native signal sequence of the polypeptide derived from E. coli FimH. In some embodiments where the polypeptide derived from E. color a fragment thereof includes an endogenous signal sequence, the nucleic acid sequence encoding the signal sequence may be codon optimized to increase the level of expression of the protein in a host cell. In some embodiments, the signal sequence is any one of the following lengths: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 amino acids long. In some embodiments, the signal sequence is 20 amino acids long. In some embodiments, the signal sequence is 21 amino acids long. In some embodiments, where the polypeptide or fragment thereof includes a signal sequence, the endogenous signal sequence naturally associated with the polypeptide may be replaced with a signal sequence not associated with the wild type polypeptide to improve the level of expression of the polypeptide or fragment thereof in cultured cells. Accordingly, in some embodiments, the nucleic acid does not include the native signal sequence of the polypeptide derived from E. coli or a fragment thereof. In some embodiments, the nucleic acid does not include the native signal sequence of the polypeptide derived from E. coli FimH. In some embodiments, the polypeptide derived from E. color a fragment thereof may be expressed with a heterologous peptide, which is preferably a signal sequence or other peptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide derived from E. coli or a fragment thereof. For example, the polypeptide derived from E. coli FimH or a fragment thereof may be expressed with a heterologous peptide (e.g., IgK signal sequence), which is preferably a signal sequence or other peptide having a specific cleavage site at the N-terminus of the mature E. coli FimH protein. In preferred embodiments, the specific cleavage site at the N-terminus of the mature protein E. coli FimH occurs immediately before the initial phenylalanine residue of the mature E. coli FimH protein. The heterologous sequence selected is preferably one that is recognized and processed (i.e., cleaved by signal peptidase) by the host cell. In preferred embodiments, the signal sequence is an IgK signal sequence. In some embodiments, the nucleic acid encodes the amino acid sequence SEQ ID NO: 18. In some embodiments, the nucleic acid encodes the amino acid sequence SEQ ID NO: 19. In some embodiments, the nucleic acid encodes the amino acid sequence SEQ ID NO: 22. In preferred embodiments, the signal sequence is a mouse IgK signal sequence. Suitable mammalian expression vectors for producing the polypeptide derived from E. coli or fragments thereof are known in the art and may be commercially available, such as pSecTag2 expression vector from Invitrogen T M . An exemplary mouse Ig Kappa signal peptide sequence includes the sequence ETDTLLLWVLLLWVPGSTG (SEQ ID NO: 54). In some embodiments, the vector includes pBudCE4.1 mammalian expression vector from Thermo Fisher. Additional exemplary and suitable vectors include the pcDNA T3.1mammalian expression vector (Thermo Fisher). In some embodiments, the signal sequence does not include a hemagglutinin signal sequence. In some embodiments, the nucleic acid includes the native signal sequence of the polypeptide derived from E. coli or a fragment thereof. In some embodiments, the signal sequence is not an IgK signal sequence. In some embodiments, the signal sequence includes a hemagglutinin signal sequence. In one aspect, disclosed herein are vectors that include the coding sequences for the polypeptide derived from E. coli or a fragment thereof. Exemplary vectors include plasmids that are able to replicate autonomously or to be replicated in a mammalian cell. Typical expression vectors contain suitable promoters, enhancers, and terminators that are useful for regulation of the expression of the coding sequence(s) in the expression construct. The vectors may also include selection markers to provide a phenotypic trait for selection of transformed host cells (such as conferring resistance to antibiotics such as ampicillin or neomycin). Suitable promoters are known in the art. Exemplary promoters include, e.g., CMV promoter, adenovirus, EF1 a, GAPDH metallothionine promoter, SV-40 early promoter, SV-40 later promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, etc. Promoters may be constitutive or inducible. One or more vectors may be used (e.g., one vector encoding all subunits or domains or fragments thereof, or multiple vectors together encoding the subunits or domains or fragments thereof). Internal ribosome entry site (IRES) and 2A peptide sequences may also be used. IRES and 2A peptide provides alternative approaches for co-expression of multiple sequences. IRES is a nucleotide sequence that allows for translation initiation in the middle of a messenger RNA (mRNA) sequence as part of the greater process of protein synthesis. Usually, in eukaryotes, translation may be initiated only at the 5' end of the mRNA molecule. IRES elements allow expression of multiple genes in one transcript. IRES-based polycistronic vectors, which express multiple proteins from one transcript, mayreduce the escape of non-expressing clones from selection. The 2A peptide allows translation of multiple proteins in a single open reading frame into a polyprotein that is subsequently cleaved into individual proteins through a ribosome-skipping mechanism. 2A peptide mayprovide more balanced expression of multiple protein products. Exemplary IRES sequences include, e.g., EV71 IRES, EMCV IRES, HCV IRES. For genomic integration, the integration may be site-specific or random. Site-specific recombination may be achieved by introducing homologous sequence(s) into the nucleic acid constructs described herein. Such homologous sequence substantially matches the endogenous sequence at a specific target site in the host genome. Alternatively, random integration may be used. Sometimes, the expression level of a protein may vary depending upon the integration site. Therefore, it may be desirable to select a number of clones according to recombinant protein expression level to identify a clone that achieves the desired level of expression. Exemplary nucleic acid constructs are further described in the figures, such as any one of FIG. 2A-2T. In one aspect, the nucleic acid sequence encodes the amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%, 97%, 98%, 99%, 99.9% or 100% identity to any one of SEQ ID NO: 1, SEQ ID NO: 2,

SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, and SEQ ID NO: 113.

lil. Host Cells

In one aspect, the invention relates to cells in which the sequences encoding the polypeptide derived from E. coli or a fragment thereof are expressed in a mammalian host cell. In one embodiment, the polypeptide derived from E. color a fragment thereof is transiently expressed in the host cell. In another embodiment, the polypeptide derived from E. color a fragment thereof is stably integrated into the genome of the host cells, and, when cultured under a suitable condition, express the polypeptide derived from E. coli or a fragment thereof. In a preferred embodiment, the polynucleotide sequence is expressed with high efficiency and genomic stability. Suitable mammalian host cells are known in the art. Preferably, the host cell is suitable for producing protein at industrial manufacturing scale. Exemplary mammalian host cells include any one of the following and derivatives thereof: Chinese Hamster Ovary (CHO) cells, COS cells (a cell line derived from monkey kidney (African green monkey), Vero cells, Hela cells, baby hamster kidney (BHK) cells, Human Embryonic Kidney (HEK) cells, NSO cells (Murine myeloma cell line), and C127 cells (nontumorigenic mouse cell line). Further exemplary mammalian host cells include mouse Sertoli (TM4), buffalo rat liver (BRL 3A), mouse mammary tumor (MMT), rat hepatoma (HTC), mouse myeloma (NSO), murine hybridoma (Sp2/0), mouse thymoma (EL4), Chinese Hamster Ovary (CHO) and CHO cell derivatives, murine embryonic (NIH/3T3, 3T3 Li), rat myocardial (H9c2), mouse myoblast (C2C12), and mouse kidney (miMCD-3). Further examples of mammalian cell lines include NSO/1, Sp2/0, Hep G2, PER.C6, COS-7, TM4, CV1, VERO-76, MDCK, BRL 3A, W138, MMT 060562, TR1, MRC5, and FS4. Any cell susceptible to cell culture may be utilized in accordance with the present invention. In some embodiments, the cell is a mammalian cell. Non-limiting examples of mammalian cells that may be used in accordance with the present invention include BALB/c mouse myeloma line (NSO/l, ECACC No: 85110503); human retinoblasts (PER.C6, CruCell, Leiden, The Netherlands); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59,1977); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells +/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216, 1980); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO 76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68, 1982); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). In some preferred embodiment, the cells are CHO cells. In some preferred embodiments, the cells are GS-cells. Additionally, any number of commercially and non-commercially available hybridoma cell lines may be utilized in accordance with the present invention. The term "hybridoma" as used herein refers to a cell or progeny of a cell resulting from fusion of an immortalized cell and an antibody-producing cell. Such a resulting hybridoma is an immortalized cell that produces antibodies. Individual cells used to create the hybridoma can be from any mammalian source, including, but not limited to, rat, pig, rabbit, sheep, pig, goat, and human. In some embodiments, a hybridoma is a trioma cell line, which results when progeny of heterohybrid myeloma fusions, which are the product of a fusion between human cells and a murine myeloma cell line, are subsequently fused with a plasma cell. In some embodiments, a hybridoma is any immortalized hybrid cell line that produces antibodies such as, for example, quadromas (See, e.g., Milstein et al., Nature, 537:3053, 1983). One skilled in the art will appreciate that hybridoma cell lines might have different nutrition requirements and/or might require different culture conditions for optimal growth, and will be able to modify conditions as needed. In some embodiments, the cell comprises a first gene of interest, wherein the first gene of interest is chromosomally-integrated. In some embodiments, the first gene of interest comprises a reporter gene, a selection gene, a gene of interest (e.g., encoding a polypeptide derived from E. coli or a fragment thereof), an ancillary gene, or a combination thereof. In some embodiments, the gene of therapeutic interest comprises a gene encoding a difficult to express (DtE) protein. In some embodiments, the first gene of interest is located between two of the distinct recombination target sites (RTS) in a site-specific integration (SSI) mammalian cell, wherein two RTS are chromosomally-integrated within the NL1 locus or the NL2 locus. See, for example, United States Patent Application Publication No. 20200002727, for a description of the NL1 locus, the NL2 locus, the NL3 locus, the NL4 locus, the NL5 locus, and the NL6 locus. In some embodiments, the first gene of interest is located within the NL1 locus. In some embodiments, the cell comprises a second gene of interest, wherein the second gene of interest is chromosomally-integrated. In some embodiments, the second gene of interest comprises a reporter gene, a selection gene, a gene of therapeutic interest (such as a polypeptide derived from E. color a fragment thereof), an ancillary gene, or a combination thereof. In some embodiments, the gene of therapeutic interest comprises a gene encoding a DtE protein. In some embodiments, the second gene of interest is located between two of the RTS. In some embodiments, the second gene of interest is located within the NL1 locus or the NL2 locus. In some embodiments, the first gene of interest is located within the NL1 locus, and the second gene of interest is located within the NL2 locus. In some embodiments, the cell comprises a third gene of interest, wherein the third gene of interest is chromosomally-integrated. In some embodiments, the third gene of interest comprises a reporter gene, a selection gene, a gene of therapeutic interest (such as a polypeptide derived from E. coli or a fragment thereof), an ancillary gene, or a combination thereof. In some embodiments, the gene of therapeutic interest comprises a gene encoding a DtE protein. In some embodiments, the third gene of interest is located between two of the RTS. In some embodiments, the third gene of interest is located within the NL1 locus or the NL2 locus. In some embodiments, the third gene of interest is located within a locus distinct from the NL1 locus and the NL2 locus. In some embodiments, the first gene of interest, the second gene of interest, and the third gene of interest are within three separate loci. In some embodiments, at least one of the first genes of interest, the second gene of interest, and the third gene of interest is within the NL1 locus, and at least one of the first gene of interest, the second gene of interest, and the third gene of interest is within the NL2 locus. In some embodiments, the cell comprises a site-specific recombinase gene. In some embodiments, the site-specific recombinase gene is chromosomally-integrated. In some embodiments, the present disclosure provides a mammalian cell comprising at least four distinct RTS, wherein the cell comprises (a) at least two distinct RTS are chromosomally-integrated within the NL1 locus or NL2 locus; (b) a first gene of interest is integrated between the at least two RTS of (a), wherein the first gene of interest comprises a reporter gene, a gene encoding a DtE protein, an ancillary gene or a combination thereof; (c) and a second gene of interest is integrated within a second chromosomal locus distinct from the locus of (a), wherein the second gene of interest comprises a reporter gene, a gene encoding a DtE protein (such as a polypeptide derived from E. color a fragment thereof), an ancillary gene or a combination thereof.In some embodiments, the present disclosure provides a mammalian cell comprising at least four distinct RTS, wherein the cell comprises (a) at least two distinct RTS are chromosomally-integrated within the Fer1L4 locus; (b) at least two distinct RTS are chromosomally-integrated within the NL1 locus or the NL2 locus; (c) a first gene of interest is chromosomally-integrated within the Fer1L4 locus, wherein the first gene of interest comprises a reporter gene, a gene encoding a DtE protein, an ancillary gene or a combination thereof; and (d) a second gene of interest is chromosomally-integrated within the within the NL1 locus or NL2 locus of (b), wherein the second gene of interest comprises a reporter gene, a gene encoding a DtE protein (such as a polypeptide derived from E. color a fragment thereof), an ancillary gene or a combination thereof. In some embodiments, the present disclosure provides a mammalian cell comprising at least six distinct RTS, wherein the cell comprises (a) at least two distinct RTS and a first gene of interest are chromosomally-integrated within the Fer1L4 locus; (b) at least two distinct RTS and a second gene of interest are chromosomally integrated within the NL1 locus; and (c) at least two distinct RTS and a third gene of interest are chromosomally-integrated within the NL2 locus. As referred to herein, the terms "in operable combination," "in operable order," and "operably linked" refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced. In some embodiments, a gene of interest is operably linked to a promoter, wherein the gene of interest is chromosomally-integrated into the host cell. In some embodiments, the gene of interest is operably linked to a heterologous promoter; where in the gene of interest is chromosomally-integrated into the host cell. In some embodiments, an ancillary gene is operably linked to a promoter, wherein the ancillary gene is chromosomally-integrated into the host cell genome. In some embodiments, the ancillary gene is operably linked to a heterologous promoter; where in the ancillary gene is chromosomally-integrated into the host cell genome. In some embodiments, a gene encoding a DtE protein is operably linked to a promoter, wherein the gene encoding a DtE protein is chromosomally-integrated into the host cell genome. In some embodiments, the gene encoding a DtE protein is operably linked to a heterologous promoter, where in the gene encoding a DtE protein is chromosomally-integrated into the host cell genome. In some embodiments, a recombinase gene is operably linked to a promoter, wherein the recombinase gene is chromosomally-integrated into the host cell. In some embodiments, the recombinase gene is operably linked to a promoter, where in the recombinase gene is not integrated into the host cell genome. In some embodiments, a recombinase gene is operably linked to a heterologous promoter, wherein the recombinase gene is not chromosomally-integrated into the host cell genome. In some embodiments, the recombinase gene is operably linked to a heterologous promoter, wherein the recombinase gene is not chromosomally-integrated into the host cell genome. As referred to herein, the term "chromosomally-integrated" or "chromosomal integration" refers to the stable incorporation of a nucleic acid sequence into the chromosome of a host cell, e.g. a mammalian cell. i.e., a nucleic acid sequence that is chromosomally-integrated into the genomic DNA (gDNA) of a host cell, e.g. a mammalian cell. In some embodiments, a nucleic acid sequence that is chromosomally-integrated is stable. In some embodiments, a nucleic acid sequence that is chromosomally-integrated is not located on a plasmid or a vector. In some embodiments, a nucleic acid sequence that is chromosomally integrated is not excised. In some embodiments, chromosomal integration is mediated by the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated protein (Cas) gene editing system (CRISPR/CAS). In some embodiments, the host cells are suitable for growth in suspension cultures. Suspension competent host cells are generally monodisperse or grow in loose aggregates without substantial aggregation. Suspension competent host cells include cells that are suitable for suspension culture without adaptation or manipulation (e.g., hematopoietic cells, lymphoid cells) and cells that have been made suspension competent by modification or adaptation of attachment-dependent cells (e.g., epithelial cells, fibroblasts). In some embodiments, the expression level or activity of the polypeptide derived from E. coli or fragment thereof is increased by at least 2-fold, at least 3 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 90 fold, at least 100 fold, as compared to expression of the polypeptide derived from E. color a fragment thereof in a bacterial cell, such as, for example, an E. coli host cell. In some embodiments, the expression level of the polypeptide derived from E. color fragment thereof is increased by at least 2-fold as compared to expression of the polypeptide derived from E. color a fragment thereof in an E. colihost cell. In some embodiments, the expression level of the polypeptide derived from E. color fragment thereof is increased by at least 50-fold as compared to expression of the polypeptide derived from E. coli or a fragment thereof in an E. coli host cell. In some embodiments, the expression level of the polypeptide derived from E. coli or fragment thereof is increased by at least 100-fold as compared to expression of the polypeptide derived from E. color a fragment thereof in an E. coli host cell. The host cells described herein are suitable for large scale culture. For example, the cell cultures may be 10 L, 30 L, 50 L, 100 L, 150 L, 200 L, 300 L, 500 L, 1000 L, 2000 L, 3000 L, 4000 L, 5000 L, 10,000 L or larger. In some embodiments, the cell culture size may range from 10 L to 5000 L, from 10 L to 10,000 L, from 10 L, to 20,000 L, from 10 1, to 50,000 L, from

40 1, to 50,000 L, from 100 L to 50,000 L, from 500 L to 50,000 L, from 1000 L to 50,000 L, from 2000 L to 50,000 L, from 3000 1, to 50,000 L, from 4000 L to 50,000 L, from 4500 L to 50,000 L, from 1000 L to 10,000 L, from 1000 L to 20,000 L, from 1000 L to 25,000 L, from 1000 L to 30,000 L, from 15 L to 2000 L, from 40 L to 1000 L, from 100 L to 500 L, from 200 L to 400 L, or any integer there between. Media components for cell culture are known in the art, and may include, e.g., buffer, amino acid content, vitamin content, salt content, mineral content, serum content, carbon source content, lipid content, nucleic acid content, hormone content, trace element content, ammonia content, co factor content, indicator content, small molecule content, hydrolysate content and enzyme modulator content. The terms "medium", "cell culture medium" and "culture medium" as used herein refer to a solution containing nutrients which nourish growing mammalian cells. Typically, such solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by the cell for minimal growth and/or survival. Such a solution may also contain supplementary components that enhance growth and/or survival above the minimal rate, including, but not limited to, hormones and/or other growth factors, particular ions (such as sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds usually present at very low final concentrations), inorganic compounds present at high final concentrations (e.g., iron), amino acids, lipids, and/or glucose or other energy source. In some embodiments, a medium is advantageously formulated to a pH and salt concentration optimal for cell survival and proliferation. In some embodiments, a medium is a feed medium that is added after the beginning of the cell culture. In some embodiments, cells may be grown in one of a variety of chemically defined media, wherein the components of the media are both known and controlled. In some embodiments, cells may be grown in a complex medium, in which not all components of the medium are known and/or controlled. Chemically defined growth media for mammalian cell culture have been extensively developed and published over the last several decades. All components of defined media are well characterized, and so defined media do not contain complex additives such as serum or hydrolysates. Early media formulations were developed to permit cell growth and maintenance of viability with little or no concern for protein production. More recently, media formulations have been developed with the express purpose of supporting highly productive recombinant protein producing cell cultures. Such media are preferred for use in the method of the invention. Such media generally comprises high amounts of nutrients and in particular of amino acids to support the growth and/or the maintenance of cells at high density. If necessary, these media can be modified by the skilled person for use in the method of the invention. For example, the skilled person may decrease the amount of phenylalanine, tyrosine, tryptophan and/or methionine in these media for their use as base media or feed media in a method as disclosed herein. Not all components of complex media are well characterized, and so complex media may contain additives such as simple and/or complex carbon sources, simple and/or complex nitrogen sources, and serum, among other things. In some embodiments, complex media suitable for the present invention contains additives such as hydrolysates in addition to other components of defined medium as described herein. In some embodiments, defined media typically includes roughly fifty chemical entities at known concentrations in water. Most of them also contain one or more well-characterized proteins such as insulin, IGF-1, transferrin or BSA, but others require no protein components and so are referred to as protein-free defined media. Typical chemical components of the media fall into five broad categories: amino acids, vitamins, inorganic salts, trace elements, and a miscellaneous category that defies neat categorization. Cell culture medium may be optionally supplemented with supplementary components. The term "supplementary components" as used herein refers to components that enhance growth and/or survival above the minimal rate, including, but not limited to, hormones and/or other growth factors, particular ions (such as sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds usually present at very low final concentrations), amino acids, lipids, and/or glucose or other energy source. In some embodiments, supplementary components may be added to the initial cell culture. In some embodiments, supplementary components may be added after the beginning of the cell culture. Typically, trace elements refer to a variety of inorganic salts included at micromolar or lower levels. For example, commonly included trace elements are zinc, selenium, copper, and others. In some embodiments, iron (ferrous or ferric salts) can be included as a trace element in the initial cell culture medium at micromolar concentrations. Manganese is also frequently included among the trace elements as a divalent cation (MnCl2 or MnSO4) in a range of nanomolar to micromolar concentrations. Numerous less common trace elements are usually added at nanomolar concentrations. In some embodiments, the medium used in the method of the invention is a medium suitable for supporting high cell density, such as for example 1 x 10 6cells/mL, 5 x 106cells/mL, 1 x 107cells /mL, 5 x 107 cells/mL, 1X108 cells/mL or 5X108 cells/mL, in a cell culture. In some embodiments, the cell culture is a mammalian cell fed-batch culture, preferably a CHO cells fed batch culture.

In some embodiments, the cell culture medium comprises phenylalanine at a concentration below2mM, below 1mM, between 0.1 and 2mM, between 0.1 to 1mM, between 0.5 and 1.5mM or between 0.5 to 1mM. In some embodiments, the cell culture medium comprises tyrosine at a concentration below 2mM, below 1mM, between 0.1 and 2mM, between 0.1 to 1mM, between 0.5 and 1.5mM or between 0.5 to 1mM. In some embodiments, the cell culture medium comprises tryptophan at a concentration below 2mM, below 1mM, between 0.1 and 2mM, between 0.1 to 1mM, between 0.5 and 1.5mM or between 0.5 to 1mM. In some embodiments, the cell culture medium comprises methionine at a concentration below 2mM, below 1mM, between 0.1 and 2mM, between 0.1 to 1mM, between 0.5 and 1.5mM or between 0.5 to 1mM. In some embodiments, the cell culture medium comprises leucine at a concentration below 2mM, below 1mM, between 0.1 and 2mM, between 0.1 to 1mM, between 0.5 and 1.5mM or between 0.5 to 1mM. In some embodiments, the cell culture medium comprises serine at a concentration below 2mM, below 1mM, between 0.1 and 2mM, between 0.1 to 1mM, between 0.5 and 1.5mM or between 0.5 to 1mM. In some embodiments, the cell culture medium comprises threonine at a concentration below 2mM, below 1mM, between 0.1 and 2mM, between 0.1 to 1mM, between 0.5 and 1.5mM or between 0.5 to 1mM. In some embodiments, the cell culture medium comprises glycine at a concentration below 2mM, below 1mM, between 0.1 and 2mM, between 0.1 to 1mM, between 0.5 and 1.5mM or between 0.5 to 1mM. In some embodiments, the cell culture medium comprises two of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine and glycine at a concentration below 2mM, below 1mM, between 0.1 and 2mM, between 0.1 to 1mM, between 0.5 and 1.5mM or between 0.5 to 1mM. In some embodiments, the cell culture medium comprises phenylalanine and tyrosine at a concentration below 2mM, below 1mM, between 0.1 and 2mM, between 0.1 to 1mM, between 0.5 and 1.5mM or between 0.5 to 1mM. In some embodiments, the cell culture medium comprises phenylalanine and tryptophan at a concentration below 2mM, below 1mM, between 0.1 and 2mM, between 0.1 to 1mM, between 0.5 and 1.5mM or between 0.5 to 1mM. In some embodiments, the cell culture medium comprises phenylalanine and methionine at a concentration below 2mM, below 1mM, between 0.1 and 2mM, between 0.1 to 1mM, between 0.5 and 1.5mM or between 0.5 to 1mM. In some embodiments, the cell culture medium comprises tyrosine and tryptophan at a concentration below 2mM, below 1mM, between 0.1 and 2mM, between 0.1 to 1mM, between 0.5 and 1.5mM or between 0.5 to 1mM. In some embodiments, the cell culture medium comprises tyrosine and methionine at a concentration below 2mM, below 1mM, between 0.1 and 2mM, between 0.1 to 1mM, between 0.5 and 1.5mM or between 0.5 to 1mM. In some embodiments, the cell culture medium comprises tryptophan and methionine at a concentration below 2mM, below 1mM, between 0.1 and 2mM, between 0.1 to 1mM, between 0.5 and 1.5mM or between 0.5 to 1mM. In some embodiments, the cell culture medium comprises three of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine and glycine at a concentration below 2mM, below 1mM,between 0.1 and 2mM, between 0.1 to 1mM, between 0.5 and 1.5mM or between 0.5 to 1mM. In some embodiments, the cell culture medium comprises phenylalanine, tyrosine and tryptophan at a concentration below 2mM, below 1mM, between 0.1 and 2mM, between 0.1 to 1mM, between 0.5 and 1.5mM or between 0.5 to 1mM. In some embodiments, the cell culture medium comprises phenylalanine, tyrosine and methionine at a concentration below 2mM, below 1mM, between 0.1 and 2mM, between 0.1 to 1mM, between 0.5 and 1.5mM or between 0.5 to 1mM. In some embodiments, the cell culture medium comprises phenylalanine, tryptophan and methionine at a concentration below 2mM, below 1mM, between 0.1 and 2mM, between 0.1 to 1mM, between 0.5 and 1.5mM or between 0.5 to 1mM. In some embodiments, the cell culture medium comprises tyrosine, tryptophan and methionine at a concentration below 2mM, below 1mM, between 0.1 and 2mM, between 0.1 to 1mM, between 0.5 and 1.5mM or between 0.5 to 1mM. In some embodiments, the cell culture medium comprises four of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine and glycine at a concentration below 2mM, below 1mM, between 0.1 and 2mM, between 0.1 to 1mM, between 0.5 and 1.5mM or between 0.5 to 1mM. In some embodiments, the cell culture medium comprises phenylalanine, tyrosine, tryptophan and methionine at a concentration below 2mM, below 1mM, between 0.1 and 2mM, between 0.1 to 1mM, between 0.5 and 1.5mM or between 0.5 to 1mM. In some embodiments, the cell culture medium comprises five of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine and glycine at a concentration below 2mM, below 1mM, between 0.1 and 2mM, between 0.1 to 1mM, between 0.5 and 1.5mM or between 0.5 to 1mM. In some embodiments, the cell culture medium comprises six of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine and glycine at a concentration below 2mM, below 1mM, between 0.1 and 2mM, between 0.1 to 1mM, between 0.5 and 1.5mM or between 0.5 to 1mM. In some embodiments, the cell culture medium comprises seven of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine and glycine at a concentration below 2mM, below 1mM, between 0.1 and 2mM, between 0.1 to 1mM, between 0.5 and 1.5mM or between 0.5 to 1mM. Insome embodiments, the cell culture medium comprises phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine and glycine at a concentration below 2mM, below 1mM, between 0.1 and 2mM, between 0.1 to 1mM, between 0.5 and 1.5mM or between 0.5 to 1mM. In some embodiments, the cell culture medium further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 of glycine, valine, leucine, isoleucine, proline, serine, threonine, lysine, arginine, histidine, aspartate, glutamate and asparagine at a concentration above 2mM, 3mM, 4mM, 5mM, 10mM, 15mM, preferably 2mM. In some embodiments, the cell culture medium further comprises at least 5 of glycine, valine, leucine, isoleucine, proline, serine, threonine, lysine, arginine, histidine, aspartate, glutamate and asparagine at a concentration above 2mM, 3mM, 4mM, 5mM, 10mM, 15mM, preferably 2mM. In some embodiments, the cell culture medium further comprises glycine, valine, leucine, isoleucine, proline, serine, threonine, lysine, arginine, histidine, aspartate, glutamate and asparagine at a concentration above 2mM, 3mM, 4mM, 5mM, 10mM, 15mM, preferably 2mM. In some embodiments, the cell culture medium further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 of valine, isoleucine, proline, lysine, arginine, histidine, aspartate, glutamate and asparagine at a concentration above 2mM, 3mM, 4mM, 5mM, 10mM, 15mM, preferably 2mM. In some embodiments, the cell culture medium further comprises at least 5 of valine, isoleucine, proline, lysine, arginine, histidine, aspartate, glutamate and asparagine at a concentration above 2mM, 3mM, 4mM, 5mM, 10mM, 15mM, preferably 2mM. In some embodiments, the cell culture medium further comprises valine, isoleucine, proline, lysine, arginine, histidine, aspartate, glutamate and asparagine at a concentration above 2mM, 3mM, 4mM, 5mM, 10mM, 15mM, preferably 2mM. In some embodiments, the cell culture medium comprises serine at a concentration above 3mM, 5mM, 7mM, 10mM, 15mM or 20mM, preferably10mM. In some embodiments, the cell culture medium comprises valine at a concentration above 3mM, 5mM, 7mM, 10mM, 15mM or 20mM, preferably 10mM. In some embodiments, the cell culture medium comprises cysteine at a concentration above 3mM, 5mM, 7mM, 10mM, 15mM or20mM, preferably 10mM. Insome embodiments, the cell culture medium comprises isoleucine at a concentration above 3mM, 5mM, 7mM, 10mM, 15mM or 20mM, preferably 10mM. In some embodiments, the cell culture medium comprises leucine at a concentration above 3mM, 5mM, 7mM, 10mM, 15mMor20mM, preferably 10mM. In some embodiments, the above cell culture medium is for use in a method as disclosed herein. In some embodiments, the above cell culture medium is used in a method as disclosed herein as a base media. In some embodiments, the above cell culture medium is used a method as disclosed herein as a feed media.

IV. Method of Producing

In one aspect, the invention includes a method of producing a polypeptide derived from E. coli or a fragment thereof. The method includes culturing a mammalian cell under a suitable condition, thereby expressing the polypeptide derived from E. coli or a fragment thereof. The method may further include harvesting the polypeptide derived from E. coli or a fragment thereof from the culture. The process may further include purifying the polypeptide derived from E. coli or a fragment thereof. In some embodiments, the method produces the polypeptide or fragment thereof at a yield as 0.1 g/L to 0.5 g/L. In some embodiments, the cells may be grown in batch or fed-batch cultures, where the culture is terminated after sufficient expression of the polypeptide, after which the expressed polypeptide is harvested and optionally purified. In some embodiments, the cells may be grown in perfusion cultures, where the culture is not terminated and new nutrients and other components are periodically or continuously added to the culture, during which the expressed polypeptide is periodically or continuously harvested. In some embodiments, the cells may be grown in small scale reaction vessels ranging in volume from a few milliliters to several liters. In some embodiments, the cells may be grown in large scale commercial bioreactors ranging in volume from approximately least 1 liter to 10, 100, 250, 500, 1 ,000, 2,500, 5,000, 8,000, 10,000, 12,000 liters or more, or any volume in between. The temperature of the cell culture will be selected based primarily on the range of temperatures at which the cell culture remains viable, at which a high level of polypeptide is produced, the temperature at which production or accumulation of metabolic waste products is minimized, and/or any combination of these or other factors deemed important by the practitioner. As one non-limiting example, CHO cells grow well and produce high levels or protein or polypeptide at approximately 37°C. In general, most mammalian cells grow well and/or can produce high levels or protein or polypeptide within a range of about 25°C to 42°C, although methods taught by the present disclosure are not limited to these temperatures. Certain mammalian cells grow well and/or can produce high levels or protein or polypeptide within the range of about 35°C to 40°C. In certain embodiments, the cell culture is grown at a temperature of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31'C, 32°C,

33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C at one or more times during the cell culture process. The terms "culture" and "cell culture" as used herein refer to a cell population that is suspended in a medium under conditions suitable to survival and/or growth of the cell population. As will be clear to those of ordinary skill in the art, in some embodiments, these terms as used herein refer to the combination comprising the cell population and the medium in which the population is suspended. In some embodiments, the cells of the cell culture comprise mammalian cells.

The present invention may be used with any cell culture method that is amenable to the desired process (e.g., production of a recombinant protein (e.g., antibody)). As a non-limiting example, cells may be grown in batch or fed-batch cultures, where the culture is terminated after sufficient expression of the recombinant protein (e.g., antibody), after which the expressed protein (e.g., antibody) is harvested. Alternatively, as another non-limiting example, cells may be grown in batch-refeed, where the culture is not terminated and new nutrients and other components are periodically or continuously added to the culture, during which the expressed recombinant protein (e.g., antibody) is harvested periodically or continuously. Other suitable methods (e.g., spin tube cultures) are known in the art and can be used to practice the present invention. In some embodiments, a cell culture suitable for the present invention is a fed batch culture. The term "fed-batch culture" as used herein refers to a method of culturing cells in which additional components are provided to the culture at a time or times subsequent to the beginning of the culture process. Such provided components typically comprise nutritional components for the cells which have been depleted during the culturing process. A fed-batch culture is typically stopped at some point and the cells and/or components in the medium are harvested and optionally purified. In some embodiments, the fed-batch culture comprises a base medium supplemented with feed media. Cells may be grown in any convenient volume chosen by the practitioner. For example, cells may be grown in small scale reaction vessels ranging in volume from a few milliliters to several liters. Alternatively, cells may be grown in large scale commercial Bioreactors ranging in volume from approximately at least 1 liter to 10, 50, 100, 250, 500, 1000, 2500, 5000, 8000, 10,000, 12,000, 15000, 20000 or 25000 liters or more, or any volume in between. The temperature of a cell culture will be selected based primarily on the range of temperatures at which the cell culture remains viable and the range in which a high level of desired product (e.g., a recombinant protein) is produced. In general, most mammalian cells grow well and can produce desired products (e.g., recombinant proteins) within a range of about 25°C to 42°C, although methods taught by the present disclosure are not limited to these temperatures. Certain mammalian cells grow well and can produce desired products (e.g., recombinant proteins or antibodies) within the range of about 35°C to 40°C. In certain embodiments, a cell culture is grown at a temperature of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C,

32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C at

one or more times during the cell culture process. Those of ordinary skill in the art will be able to select appropriate temperature or temperatures in which to grow cells, depending on the particular needs of the cells and the particular production requirements of the practitioner. The cells may be grown for any amount of time, depending on the needs of the practitioner and the requirement of the cells themselves. In some embodiment, the cells are grown at 37°C. In some embodiments, the cells are grown at 36.5°C. In some embodiments, the cells may be grown during the initial growth phase (or growth phase) for a greater or lesser amount of time, depending on the needs of the practitioner and the requirement of the cells themselves. In some embodiments, the cells are grown for a period of time sufficient to achieve a predefined cell density. In some embodiments, the cells are grown for a period of time sufficient to achieve a cell density that is a given percentage of the maximal cell density that the cells would eventually reach if allowed to grow undisturbed. For example, the cells may be grown for a period of time sufficient to achieve a desired viable cell density of1,5,10,15,20,25,30,35,40,45,50, 55,60,65,70,75,80,85,90,95or99 percent of maximal cell density. In some embodiments, the cells are grown until the cell density does not increase by more than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% per day of culture. In some embodiments, the cells are grown until the cell density does not increase by more than 5% per day of culture. In some embodiment the cells are allowed to grow for a defined period of time. For example, depending on the starting concentration of the cell culture, the temperature at which the cells are grown, and the intrinsic growth rate of the cells, the cells may be grown for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days, preferably for 4 to 10 days. In some cases, the cells may be allowed to grow for a month or more. The practitioner of the present invention will be able to choose the duration of the initial growth phase depending on protein production requirements and the needs of the cells themselves. The cell culture may be agitated or shaken during the initial culture phase in order to increase oxygenation and dispersion of nutrients to the cells. In accordance with the present invention, one of ordinary skill in the art will understand that it can be beneficial to control or regulate certain internal conditions of the bioreactor during the initial growth phase, including but not limited to pH, temperature, oxygenation, etc. At the end of the initial growth phase, at least one of the culture conditions may be shifted so that a second set of culture conditions is applied and a metabolic shift occurs in the culture. A metabolic shift can be accomplished by, e.g., a change in the temperature, pH, osmolality or chemical inductant level of the cell culture. In one non-limiting embodiment, the culture conditions are shifted by shifting the temperature of the culture. However, as is known in the art, shifting temperature is not the only mechanism through which an appropriate metabolic shift can be achieved. For example, such a metabolic shift can also be achieved by shifting other culture conditions including, but not limited to, pH, osmolality, and sodium butyrate levels. The timing of the culture shift will be determined by the practitioner of the present invention, based on protein production requirements or the needs of the cells themselves. When shifting the temperature of the culture, the temperature shift may be relatively gradual. For example, it may take several hours or days to complete the temperature change. Alternatively, the temperature shift may be relatively abrupt. For example, the temperature change may be complete in less than several hours. Given the appropriate production and control equipment, such as is standard in the commercial large-scale production of polypeptides or proteins, the temperature change may even be complete within less than an hour. In some embodiments, once the conditions of the cell culture have been shifted as discussed above, the cell culture is maintained for a subsequent production phase under a second set of culture conditions conducive to the survival and viability of the cell culture and appropriate for expression of the desired polypeptide or protein at commercially adequate levels. As discussed above, the culture may be shifted by shifting one or more of a number of culture conditions including, but not limited to, temperature, pH, osmolality, and sodium butyrate levels. In some embodiments, the temperature of the culture is shifted. According to this embodiment, during the subsequent production phase, the culture is maintained at a temperature or temperature range that is lower than the temperature or temperature range of the initial growth phase. As discussed above, multiple discrete temperature shifts may be employed to increase cell density or viability or to increase expression of the recombinant protein. In some embodiments, the cells may be maintained in the subsequent production phase until a desired cell density or production titer is reached. In another embodiment of the present invention, the cells are allowed to grow for a defined period of time during the subsequent production phase. For example, depending on the concentration of the cell culture at the start of the subsequent growth phase, the temperature at which the cells are grown, and the intrinsic growth rate of the cells, the cells may be grown for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days. In some cases, the cells may be allowed to grow for a month or more. The practitioner of the present invention will be able to choose the duration of the subsequent production phase depending on polypeptide or protein production requirements and the needs of the cells themselves.

The cell culture may be agitated or shaken during the subsequent production phase in order to increase oxygenation and dispersion of nutrients to the cells. In accordance with the present invention, one of ordinary skill in the art will understand that it can be beneficial to control or regulate certain internal conditions of the bioreactor during the subsequent growth phase, including but not limited to pH, temperature, oxygenation, etc. In some embodiments, the cells express a recombinant protein and the cell culture method of the invention comprises a growth phase and a production phase. In some embodiments, step (ii) of any of the methods disclosed herein is applied during the totality of the cell culture method. In some embodiments, step (ii) of any of the methods disclosed herein is applied during a part of the cell culture method. In some embodiments, step (ii) is applied until a predetermined viable cell density is obtained. In some embodiments, the cell culture method of the invention comprises a growth phase and a production phase and step (ii) is applied during the growth phase. In some embodiments, the cell culture method of the invention comprises a growth phase and a production phase and step (ii) is applied during a part of the growth phase. In some embodiments, the cell culture method of the invention comprises a growth phase and a production phase and step (ii) is applied during the growth phase and the production phase. In step (ii) of any of the methods disclosed herein, the term "maintaining" can refer to maintaining the concentration of amino acid or metabolite below C1 or C2 for the entire culture process (until harvesting) or for a part of the culture process such as for example the growth phase, a part of the growth phase or until a predetermined cell density is obtained. In some embodiments of any of the above mentioned methods, cell growth and/or productivity are increased as compared to a control culture, said control culture being identical except that it does not comprise step (ii). In some embodiments of any of the above mentioned methods, the method of the invention is a method for improving cell growth. In some embodiment, the method of the invention is a method for improving cell growth in high density cell culture at high cell density. High cell density as used herein refers to cell density above 1 x 10 6cells/mL, 5 x 10 6cells/mL, 1 X 107cells /mL, 5 x 107 cells/mL, 1X108 cells/mL or 5X108 cells/mL, preferably above 1 X 107cells /mL, more preferably above 5 x 107 cells/mL. In some embodiments, the method of the invention is a method for improving cell growth in a cell culture where cell density is above 1 x 106cells/mL, 5 x 10 6cells/mL, 1 X 107cells /mL, 5 x 107 cells/mL, 1X108 cells/mL or 5X108 cells/mL . In some embodiments, the method of the invention is a method for improving cell growth in a cell culture where maximum cell density is above 1 x 10 6cells/mL, 5 x 10 6cells/mL, 1 X 107cells /mL, 5x 107 cells/mL, 1X10 8 cells/mL or 5X10 8 cells/mL.

In some embodiments, cell growth is determined by viable cell density (VCD), maximum viable cell density, or Integrated viable cell count (IVCC). In some embodiments, cell growth is determined by maximum viable cell density. The term "viable cell density" as used herein refers to the number of cells present in a given volume of medium. Viable cell density can be measured by any method known to the skilled person. Preferably, Viable cell density is measured using an automated cell counter such as Bioprofile Flex@. The term maximum cell density as used herein refers to the maximum cell density achieved during the cell culture. The term "cell viability" as used herein refers to the ability of cells in culture to survive under a given set of culture conditions or experimental variations. Those of ordinary skill in the art will appreciate that one of many methods for determining cell viability are encompassed in this invention. For example, one may use a dye (e.g., trypan blue) that does not pass through the membrane of a living cell, but can pass through the disrupted membrane of a dead or dying cell in order to determine cell viability. The term "Integrated viable cell count (IVCC)" as used herein refers to as the area under the viable cell density (VCD) curve. IVCC can be calculated using the following formula: IVCCt. 1= IVCCt+(VCD+VCDti)*(At)/2, where At is the time difference between t and t+1 time points. IVCCt=o can be assumed negligible. VCD and VCDi are viable cell densities at t and t+1 time points. The term "titer" as used herein refers, for example, to the total amount of recombinantly expressed protein produced by a cell culture in a given amount of medium volume. Titer is typically expressed in units of grams of protein per liter of medium. In some embodiments, cell growth is increased by at least 5%, 10%, 15%, 20% or 25% as compared to the control culture. In some embodiments, cell growth is increased by at least 10% as compared to the control culture. In some embodiments, cell growth is increased by at least 20% as compared to the control culture. In some embodiments, the productivity is determined by titer and/or volumetric productivity. The term "titer" as used herein refers, for example, to the total amount of recombinantly expressed protein produced by a cell culture in a given amount of medium volume. Titer is typically expressed in units of grams of protein per liter of medium. In some embodiments, the productivity is determined by titer. In some embodiments, the productivity is increased by at least 5%, 10%, 15%, 20% or 25% as compared to the control culture. In some embodiments, the productivity is increased by at least 10% as compared to a control culture. In some embodiments, the productivity is increased by at least 20% as compared to a control culture. In some embodiments, the maximum cell density of the cell culture is greater than 1 x 10 6cells/mL, 5 x 10 6cells/mL, 1 X 107cells /mL, 5 x 107 ocells/mL, 1X10 8 cells/mL or 5X10 8 cells/mL. In some embodiments, the maximum cell density of the cell culture is greater than 5 x 10 6cells/mL. In some embodiments, the maximum cell density of the cell culture is greater than 1X10 8 cells/mL.

V. Purification

In some embodiments, the method for producing a polypeptide derived from E. color a fragment thereof includes isolating and/or purifying the polypeptide derived from E. color a fragment thereof. In some embodiments, the expressed polypeptide derived from E. coli or a fragment thereof is secreted into the medium and thus cells and other solids may be removed by centrifugation and/or filtration. The polypeptide derived from E. color a fragment thereof produced in accordance with the methods described herein may be harvested from host cells and purified using any suitable method known to the skilled person. Suitable methods for purifying the polypeptide or fragment thereof include precipitation and various types of chromatography, such as hydrophobic interaction, ion exchange, affinity, chelation, and size exclusion, all of which are known in the art. Suitable purification schemes may include two or more of these or other suitable methods. In some embodiments, one or more of the polypeptide or fragments thereof derived from E. coli may include a "tag" that facilitates purification, such as an epitope tag or a HIS tag, Strep tag. Such tagged polypeptides may conveniently be purified, for example from conditioned media, by chelating chromatography or affinity chromatography. Optionally, the tag sequence may be cleaved post-purification. In some embodiments, the polypeptide derived from E. coli or a fragment thereof may include a tag for affinity purification. Affinity purification tags are known in the art. Examples include, e.g., His tag (binds to metal ion), an antibody, maltose-binding protein (MBP) (binds to amylose), glutathione-S- transferase (GST) (binds to glutathione), FLAG tag, Strep tag (binds to streptavidin or a derivative thereof). In a preferred embodiment, the polypeptide derived from E. color a fragment thereof does not include a purification tag. In some embodiments, the yield of the polypeptide derived from E. coli or a fragment thereof is at least about 1 mg/L, at least about 2 mg/L, at least about 3 mg/L, at least about 4 mg/L, at least about 5 mg/L, at least about 6 mg/L, at least about 7 mg/L, at least about 8 mg/L, at least about 9 mg/L, at least about 10 mg/L, at least about 11 mg/L, at least about 12 mg/L, at least about 13 mg/L, at least about 14 mg/L, at least about 15 mg/L, at least about 16 mg/L, at least about 17 mg/L, at least about 18 mg/L, at least about 19 mg/L, at least about 20 mg/L, at least about 25 mg/L, at least about 30 mg/L, at least about 35 mg/L, at least about 40 mg/L, at least about 45 mg/L, at least about 50 mg/L, at least about 55 mg/L, at least about 60 mg/L, at least about 65 mg/L, at least about 70 mg/L, at least about 75 mg/L, at least about 80 mg/L, at least about 85 mg/L, at least about 90 mg/L, at least about 95 mg/L, or at least about 100 mg/L. In some embodiments, the culture is at least about 10 liters in size, e.g., a volume of at least about 1OL, at least about 20L, at least about 30L, at least about 40L, atleastabout50L, at least about60 L, atleastabout70L, at least about80L, atleast about90L, at least about1OOL, atleastabout150L, atleastabout200L, atleastabout 250L, atleastabout300L, atleastabout400L, at least about500L, atleastabout600L, at least about700L, at least about 800L, at least about 900L, at least about 1000 L, at leastabout2000 L, atleastabout3000 L, atleastabout4000 L, at leastabout5000 L, at least about6000 L, at least about 10,000 L, at least about 15,000 L, at least about 20,000 L, at least about 25,000 L, at least about 30,000 L, at least about 35,000 L, at least about 40,000 L, at least about 45,000 L, at least about 50,000 L, at least about 55,000 L, at least about 60,000 L, at least about 65,000 L, at least about 70,000 L, at least about 75,000 L, at least about 80,000 L, at least about 85,000 L, at least about 90,000 L, at least about 95,000 L, at least about 100,000 L, etc.

VI. Compositions and Formulations

In one aspect, the invention includes a composition that includes a polypeptide derived from E. coli or a fragment thereof. In some embodiments, the composition elicits an immune response, including antibodies, that may confer immunity to pathogenic species of E. coli. In some embodiments, the composition includes the polypeptide derived from E. coli or fragment thereof as the only antigen. In some embodiments, the composition does not include a conjugate. In some embodiments, the composition includes the polypeptide derived from E. coli or fragment thereof and an additional antigen. In some embodiments, the composition includes the polypeptide derived from E. coli or fragment thereof and an additional E. coliantigen. In some embodiments, the composition includes the polypeptide derived from E. coli or fragment thereof and a glycoconjugate from E. coli. In some embodiments, the polypeptide or a fragment thereof is derived from E. coli FimH.

In some embodiments, the composition includes a polypeptide derived from E. coliFimC or a fragment thereof. In some embodiments, the composition includes a polypeptide derived from E. coli FimH or a fragment thereof; and a polypeptide derived from E. coli FimC or a fragment thereof. In one aspect, the invention includes a composition including a polypeptide derived from E. coli FimH or a fragment thereof; and a saccharide comprising a structure selected from any one of Formula 01 (e.g., Formula 01A, Formula 01B, and Formula 01C), Formula 02, Formula 03, Formula 04 (e.g., Formula 04:K52 and Formula 04:K6), Formula 05 (e.g., Formula 05ab and Formula 05ac (strain 180/C3)), Formula 06 (e.g., Formula 06:K2; K13; K15 and Formula 06:K54), Formula 07, Formula 08, Formula 09, Formula 010, Formula 011, Formula 012, Formula 013, Formula 014, Formula 015, Formula 016, Formula 017, Formula 018 (e.g., Formula 018A, Formula 018ac, Formula 018A1, Formula 018B, and Formula 018B1), Formula 019, Formula 020, Formula 021, Formula 022, Formula 023 (e.g., Formula 023A), Formula 024, Formula 025 (e.g., Formula 025a and Formula 025b), Formula 026, Formula 027, Formula 028, Formula 029, Formula 030, Formula 032, Formula 033, Formula 034, Formula 035, Formula 036, Formula 037, Formula 038, Formula 039, Formula 040, Formula 041, Formula 042, Formula 043, Formula 044, Formula 045 (e.g., Formula 045 and Formula 045rel), Formula 046, Formula 048, Formula 049, Formula 050, Formula 051, Formula 052, Formula 053, Formula 054, Formula 055, Formula 056, Formula 057, Formula 058, Formula 059, Formula 060, Formula 061, Formula 062, Formula 62D1, Formula 063, Formula 064, Formula 065, Formula 066, Formula 068, Formula 069, Formula 070, Formula 071, Formula 073 (e.g., Formula 073 (strain 73-1)), Formula 074, Formula 075, Formula 076, Formula 077, Formula 078, Formula 079, Formula 080, Formula 081, Formula 082, Formula 083, Formula 084, Formula 085, Formula 086, Formula 087, Formula 088, Formula 089, Formula 090, Formula 091, Formula 092, Formula 093, Formula 095, Formula 096, Formula 097, Formula 098, Formula 099, Formula 0100, Formula 0101, Formula 0102, Formula 0103, Formula 0104, Formula 0105, Formula 0106, Formula 0107, Formula 0108, Formula 0109, Formula 0110, Formula 0111, Formula 0112, Formula 0113, Formula 0114, Formula 0115, Formula 0116, Formula 0117, Formula 0118, Formula 0119, Formula 0120, Formula 0121, Formula 0123, Formula 0124, Formula 0125, Formula 0126, Formula 0127, Formula 0128, Formula 0129, Formula 0130, Formula 0131, Formula 0132, Formula 0133, Formula 0134, Formula 0135, Formula 0136, Formula 0137, Formula 0138, Formula 0139, Formula 0140, Formula 0141, Formula 0142, Formula 0143, Formula 0144, Formula 0145, Formula 0146, Formula 0147, Formula 0148, Formula 0149, Formula 0150, Formula 0151, Formula 0152, Formula 0153, Formula 0154, Formula 0155, Formula 0156, Formula 0157, Formula

0158, Formula 0159, Formula 0160, Formula 0161, Formula 0162, Formula 0163, Formula 0164, Formula 0165, Formula 0166, Formula 0167, Formula 0168, Formula 0169, Formula 0170, Formula 0171, Formula 0172, Formula 0173, Formula 0174, Formula 0175, Formula 0176, Formula 0177, Formula 0178, Formula 0179, Formula 0180, Formula 0181, Formula 0182, Formula 0183, Formula 0184, Formula 0185, Formula 0186, and Formula 0187, wherein n is an integer from 1 to 100. In some embodiments, the composition includes any one of the saccharides disclosed herein. In preferred embodiments, the composition includes any one of the conjugates disclosed herein. In some embodiments, the composition includes at least one glycoconjugate from E. coli serotype 025, preferably serotype 025b. In one embodiment, the composition includes at least one glycoconjugate from E. coli serotype 01, preferably serotype 01a. In one embodiment, the composition includes at least one glycoconjugate from E. coliserotype 02. In one embodiment, the composition includes at least one glycoconjugate from E. coliserotype 06. In one embodiment, the composition comprises at least one glycoconjugate selected from any one of the following E. coli serotypes 025, 01, 02, and 06, preferably 025b, 01a, 02, and 06. In one embodiment, the composition comprises at least two glycoconjugates selected from any one of the following E. coli serotypes 025, 01, 02, and 06, preferably 025b, 01a, 02, and 06. In one embodiment, the composition comprises at least three glycoconjugates selected from any one of the following E. coli serotypes 025, 01, 02, and 06, preferably 025b, 01a, 02, and 06. In one embodiment, the composition comprises a glycoconjugate from each of the following E. coli serotypes 025, 01, 02, and 06, preferably 025b, 01a, 02, and 06. In a preferred embodiment, the glycoconjugate of any of the above compositions is individually conjugated to CRM 19 7 . In another preferred embodiment, the glycoconjugate of any of the above compositions is individually conjugated to SCP. Accordingly, in some embodiments, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an 0-antigen from at least one E. coli serotype. In a preferred embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an 0-antigen from more than 1 E. coli serotype. For example, the composition may include an 0-antigen from two different E. coli serotypes (or "v", valences) to 12 different serotypes (12v). In one embodiment, the composition includes a polypeptide derived from E. color a fragment thereof; and an 0 antigen from 3 different serotypes. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an 0-antigen from 4 different

E. coli serotypes. In one embodiment, the composition includes an 0- antigen from 5 different E. coli serotypes. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an 0-antigen from 6 different E. coli serotypes. In one embodiment, the composition includes a polypeptide derived from E. color a fragment thereof; and an 0-antigen from 7 different E. coliserotypes. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an 0-antigen from 8 different E. coliserotypes. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an 0-antigen from 9 different E. coli serotypes. In one embodiment, the composition includes a polypeptide derived from E. color a fragment thereof; and an 0-antigen from 10 different E. coliserotypes. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an 0-antigen from 11 different E. coliserotypes. In one embodiment, the composition includes a polypeptide derived from E. color a fragment thereof; and an 0-antigen from 12 different serotypes. In one embodiment, the composition includes a polypeptide derived from E. color a fragment thereof; and an 0-antigen from 13 different serotypes. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an 0-antigen from 14 different serotypes. In one embodiment, the composition includes a polypeptide derived from E. color a fragment thereof; and an 0-antigen from 15 different serotypes. In one embodiment, the composition includes a polypeptide derived from E. color a fragment thereof; and an 0-antigen from 16 different serotypes. In one embodiment, the composition includes a polypeptide derived from E. color a fragment thereof; and an 0-antigen from 17 different serotypes. In one embodiment, the composition includes a polypeptide derived from E. color a fragment thereof; and an 0-antigen from 18 different serotypes. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an 0-antigen from 19 different serotypes. In one embodiment, the composition includes a polypeptide derived from E. color a fragment thereof; and an 0-antigen from 20 different serotypes. Preferably, the number of E. coli saccharides can range from 1 serotype (or "v", valences) to 26 different serotypes (26v). In one embodiment there is one serotype. In one embodiment there are 2 different serotypes. In one embodiment there are 3 different serotypes. In one embodiment there are 4 different serotypes. In one embodiment there are 5 different serotypes. In one embodiment there are 6 different serotypes. In one embodiment there are 7 different serotypes. In one embodiment there are 8 different serotypes. In one embodiment there are 9 different serotypes. In one embodiment there are 10 different serotypes. In one embodiment there are 11 different serotypes. In one embodiment there are 12 different serotypes. In one embodiment there are 13 different serotypes. In one embodiment there are 14 different serotypes. In one embodiment there are 15 different serotypes. In one embodiment there are 16 different serotypes. In one embodiment there are 17 different serotypes. In one embodiment there are 18 different serotypes. In one embodiment there are 19 different serotypes. In one embodiment there are 20 different serotypes. In one embodiment there are 21 different serotypes. In one embodiment there are 22 different serotypes. In one embodiment there are 23 different serotypes. In one embodiment there are 24 different serotypes. In an embodiment there are 25 different serotypes. In one embodiment there are 26 different serotypes. The saccharides are conjugated to a carrier protein to form glycoconjugates as described herein. In one aspect, the composition includes a polypeptide derived from E. color a fragment thereof; and a glycoconjugate that includes an 0-antigen from at least one E. coli serogroup, wherein the 0-antigen is conjugated to a carrier protein. In one embodiment, the composition includes a polypeptide derived from E. color a fragment thereof; and an 0-antigen from more than 1 E. coliserotype, wherein each 0-antigen is conjugated to a carrier protein. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an 0-antigen from 2 different E. coli serotypes, wherein each 0-antigen is conjugated to a carrier protein. In one embodiment, the composition includes a polypeptide derived from E. color a fragment thereof; and an 0-antigen from 3 different E. coliserotypes, wherein each 0-antigen is conjugated to a carrier protein. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an 0-antigen from 4 different E. coliserotypes, wherein each 0-antigen is conjugated to a carrier protein. In one embodiment, the composition includes a polypeptide derived from E. color a fragment thereof; and an 0-antigen from 5 different E. coliserotypes, wherein each 0-antigen is conjugated to a carrier protein. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an 0-antigen from 6 different E. coli serotypes, wherein each 0-antigen is conjugated to a carrier protein. In one embodiment, the composition includes a polypeptide derived from E. color a fragment thereof; and an 0-antigen from 7 different E. coliserotypes, wherein each 0-antigen is conjugated to a carrier protein. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an 0-antigen from 8 different E. coli serotypes, wherein each 0-antigen is conjugated to a carrier protein. In one embodiment, the composition includes a polypeptide derived from E. color a fragment thereof; and an 0-antigen from 9 different E. coliserotypes, wherein each 0-antigen is conjugated to a carrier protein. In one embodiment, the composition includes an 0 antigen from a polypeptide derived from E. color a fragment thereof; and 10 different E. coliserotypes, wherein each 0-antigen is conjugated to a carrier protein. In one embodiment, the composition includes an 0-antigen from a polypeptide derived from E. coli or a fragment thereof; and 11 different E. coli serotypes, wherein each 0-antigen is conjugated to a carrier protein. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an 0-antigen from 12 different serotypes, wherein each 0-antigen is conjugated to a carrier protein. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an0-antigen from 13 different serotypes, wherein each 0-antigen is conjugated to a carrier protein. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an 0-antigen from 14 different serotypes, wherein each 0-antigen is conjugated to a carrier protein. In one embodiment, the composition includes a polypeptide derived from E. color a fragment thereof; and an 0-antigen from 15 different serotypes, wherein each 0-antigen is conjugated to a carrier protein. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an 0-antigen from 16 different serotypes, wherein each 0-antigen is conjugated to a carrier protein. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an0-antigen from 17 different serotypes, wherein each 0-antigen is conjugated to a carrier protein. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an 0-antigen from 18 different serotypes, wherein each 0-antigen is conjugated to a carrier protein. In one embodiment, the composition includes a polypeptide derived from E. color a fragment thereof; and an 0-antigen from 19 different serotypes, wherein each 0-antigen is conjugated to a carrier protein. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an 0-antigen from 20 different serotypes, wherein each 0-antigen is conjugated to a carrier protein. In another aspect, the composition includes an 0-polysaccharide from at least one E. coli serotype. In a preferred embodiment, the composition includes an0-polysaccharide from more than 1 E. coliserotype. For example, the composition may include an0-polysaccharide from two different E. coli serotypes to 12 different E. coli serotypes. In one embodiment, the composition includes an 0-polysaccharide from 3 different E. coli serotypes. In one embodiment, the composition includes an 0-polysaccharide from 4 different E. coli serotypes. In one embodiment, the composition includes an O-polysaccharide from 5 different E. coli serotypes. In one embodiment, the composition includes an0-polysaccharide from 6 different E. coliserotypes. In one embodiment, the composition includes an 0-polysaccharide from 7 different E. coliserotypes. In one embodiment, the composition includes an 0-polysaccharide from 8 different E. coli serotypes. In one embodiment, the composition includes an 0 polysaccharide from 9 different E. coli serotypes. In one embodiment, the composition includes an 0-polysaccharide from 10 different E. coli serotypes. In one embodiment, the composition includes an O-polysaccharide from 11 different E. coli serotypes. In one embodiment, the composition includes an O-polysaccharide from 12 different serotypes. In one embodiment, the composition includes an O-polysaccharide from 13 different serotypes. In one embodiment, the composition includes an O-polysaccharide from 14 different serotypes. In one embodiment, the composition includes an O-polysaccharide from 15 different serotypes. In one embodiment, the composition includes an O-polysaccharide from 16 different serotypes. In one embodiment, the composition includes an 0 polysaccharide from 17 different serotypes. In one embodiment, the composition includes an 0-polysaccharide from 18 different serotypes. In one embodiment, the composition includes an 0-polysaccharide from 19 different serotypes. In one embodiment, the composition includes an 0-polysaccharide from 20 different serotypes. In a preferred embodiment, the composition includes an O-polysaccharide from at least one E. coli serotype, wherein the O-polysaccharide is conjugated to a carrier protein. In a preferred embodiment, the composition includes an0-polysaccharide from more than 1 E. coliserotype, wherein each 0-polysaccharide is conjugated to a carrier protein. For example, the composition may include an0-polysaccharide from two different E. coli serotypes to 12 different E. coli serotypes, wherein each 0 polysaccharide is conjugated to a carrier protein. In one embodiment, the composition includes an 0-polysaccharide from 3 different E. coli serotypes, wherein each 0 polysaccharide is conjugated to a carrier protein. In one embodiment, the composition includes an 0-polysaccharide from 4 different E. coli serotypes, wherein each 0 polysaccharide is conjugated to a carrier protein. In one embodiment, the composition includes an 0-polysaccharide from 5 different E. coli serotypes, wherein each 0 polysaccharide is conjugated to a carrier protein. In one embodiment, the composition includes an 0-polysaccharide from 6 different E. coli serotypes, wherein each 0 polysaccharide is conjugated to a carrier protein. In one embodiment, the composition includes an 0-polysaccharide from 7 different E. coli serotypes, wherein each 0 polysaccharide is conjugated to a carrier protein. In one embodiment, the composition includes an 0-polysaccharide from 8 different E. coli serotypes, wherein each 0 polysaccharide is conjugated to a carrier protein. In one embodiment, the composition includes an 0-polysaccharide from 9 different E. coli serotypes, wherein each 0 polysaccharide is conjugated to a carrier protein. In one embodiment, the composition includes an 0-polysaccharide from 10 different E. coli serotypes, wherein each 0 polysaccharide is conjugated to a carrier protein. In one embodiment, the composition includes an 0-polysaccharide from 11 different E. coli serotypes, wherein each 0 polysaccharide is conjugated to a carrier protein. In one embodiment, the composition includes an 0-polysaccharide from 12 different serotypes, wherein each 0-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition includes an 0 polysaccharide from 13 different serotypes, wherein each 0-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition includes an 0-polysaccharide from 14 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition includes an 0-polysaccharide from 15 different serotypes, wherein each 0-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition includes an 0-polysaccharide from 16 different serotypes, wherein each 0 polysaccharide is conjugated to a carrier protein. In one embodiment, the composition includes an 0-polysaccharide from 17 different serotypes, wherein each 0-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition includes an O-polysaccharide from 18 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition includes an 0-polysaccharide from 19 different serotypes, wherein each 0-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition includes an 0-polysaccharide from 20 different serotypes, wherein each 0 polysaccharide is conjugated to a carrier protein. In a most preferred embodiment, the composition includes an 0-polysaccharide from at least one E. coli serotype, wherein the 0-polysaccharide is conjugated to a carrier protein, and wherein the 0-polysaccharide includes the 0-antigen and core saccharide. In a preferred embodiment, the composition includes an 0-polysaccharide from more than 1 E. coli serotype, wherein each 0-polysaccharide is conjugated to a carrier protein, and wherein the 0 polysaccharide includes the 0-antigen and core saccharide. For example, the composition may include an O-polysaccharide from two different E. coli serotypes to 12 different E. coli serotypes, wherein each 0-polysaccharide is conjugated to a carrier protein, and wherein the 0-polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes an 0-polysaccharide from 3 different E. coliserotypes, wherein each 0 polysaccharide is conjugated to a carrier protein, and wherein the0-polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes an 0 polysaccharide from 4 different E. coli serotypes, wherein each 0-polysaccharide is conjugated to a carrier protein, and wherein the 0-polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes an O-polysaccharide from 5 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the 0-polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes an 0-polysaccharide from 6 different E. coli serotypes, wherein each 0-polysaccharide is conjugated to a carrier protein, and wherein the 0 polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes an 0-polysaccharide from 7 different E. coliserotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the 0 polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes an 0-polysaccharide from 8 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the 0 polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes an 0-polysaccharide from 9 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the 0 polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes an 0-polysaccharide from 10 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the 0 polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes an 0-polysaccharide from 11 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the 0 polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes an 0-polysaccharide from 12 different serotypes, wherein each 0 polysaccharide is conjugated to a carrier protein, and wherein the 0-polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes an 0-polysaccharide from 13 different serotypes, wherein each 0 polysaccharide is conjugated to a carrier protein, and wherein the 0-polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes an 0-polysaccharide from 14 different serotypes, wherein each 0 polysaccharide is conjugated to a carrier protein, and wherein the 0-polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes an 0-polysaccharide from 15 different serotypes, wherein each 0 polysaccharide is conjugated to a carrier protein, and wherein the0-polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes an 0-polysaccharide from 16 different serotypes, wherein each 0 polysaccharide is conjugated to a carrier protein, and wherein the0-polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes an 0-polysaccharide from 17 different serotypes, wherein each 0 polysaccharide is conjugated to a carrier protein, and wherein the 0-polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes an 0-polysaccharide from 18 different serotypes, wherein each 0 polysaccharide is conjugated to a carrier protein, and wherein the 0-polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes an 0-polysaccharide from 19 different serotypes, wherein each 0-polysaccharide is conjugated to a carrier protein, and wherein the 0-polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes an 0-polysaccharide from 20 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the 0-polysaccharide includes the 0-antigen and core saccharide. In a preferred embodiment, the carrier protein is CRM 1 97

. In another preferred embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an0-polysaccharide conjugated to CRM 197 , wherein the 0 polysaccharide includes Formula 025a, wherein n is at least 40, and the core saccharide. In a preferred embodiment, the composition further includes an 0-polysaccharide conjugated to CRM 197 , wherein the -polysaccharide includes Formula 025b, wherein n is at least 40, and the core saccharide. In another embodiment, the composition further includes an 0 polysaccharide conjugated to CRM 197 , wherein the 0-polysaccharide includes Formula 01a, wherein n is at least 40, and the core saccharide. In another embodiment, the composition further includes an 0-polysaccharide conjugated to CRM 197 , wherein the 0-polysaccharide includes Formula 02, wherein n is at least 40, and the core saccharide. In another embodiment, the composition further includes an0-polysaccharide conjugated to CRM 197

, wherein the 0-polysaccharide includes Formula 06, wherein n is at least 40, and the core saccharide. In another embodiment, the composition further includes an 0-polysaccharide conjugated to CRM 197 , wherein the 0-polysaccharide includes Formula 017, wherein n is at least 40, and the core saccharide. In another embodiment, the composition further includes an O-polysaccharide conjugated to CRM 1 97 , wherein the 0-polysaccharide includes Formula 015, wherein n is at least 40, and the core saccharide. In another embodiment, the composition further includes an 0-polysaccharide conjugated to CRM 197 , wherein the 0-polysaccharide includes Formula 018A, wherein n is at least 40, and the core saccharide. In another embodiment, the composition further includes an0-polysaccharide conjugated to CRM 197 ,

wherein the 0-polysaccharide includes Formula 075, wherein n is at least 40, and the core saccharide. In another embodiment, the composition further includes an 0-polysaccharide conjugated to CRM 197 , wherein the 0-polysaccharide includes Formula 04, wherein n is at least 40, and the core saccharide. In another embodiment, the composition further includes an 0 polysaccharide conjugated to CRM 197 , wherein the 0-polysaccharide includes Formula 016, wherein n is at least 40, and the core saccharide. In another embodiment, the composition further includes an 0-polysaccharide conjugated to CRM 197 , wherein the 0-polysaccharide includes Formula 013, wherein n is at least 40, and the core saccharide. In another embodiment, the composition further includes an0-polysaccharide conjugated to CRM 197 , wherein the 0-polysaccharide includes Formula 07, wherein n is at least 40, and the core saccharide. In another embodiment, the composition further includes an 0-polysaccharide conjugated to CRM 197 , wherein the 0-polysaccharide includes Formula 08, wherein n is at least 40, and the core saccharide. In another embodiment, the0-polysaccharide includes Formula 08, wherein n is 1-20, preferably 2-5, more preferably 3. Formula 08 is shown, e.g., in FIG. 1OB. In another embodiment, the composition further includes an O-polysaccharide conjugated to CRM 1 97 , wherein the 0-polysaccharide includes Formula 09, wherein n is at least 40, and the core saccharide. In another embodiment, the 0-polysaccharide includes Formula 09, wherein n is 1-20, preferably 4-8, more preferably 5. Formula 09 is shown, e.g., in FIG. 10B. In another embodiment, the 0 polysaccharide includes Formula 09a, wherein n is 1-20, preferably 4-8, more preferably 5. Formula 09a is shown, e.g., in FIG. 10B. In some embodiments, the 0-polysaccharide includes selected from any one of Formula 020ab, Formula 020ac, Formula 052, Formula 097, and Formula 0101, wherein n is 1-20, preferably 4-8, more preferably 5. See, e.g., FIG. 10B. As described above, the composition may include a polypeptide derived from E. coli or a fragment thereof; and any combination of conjugated0-polysaccharides (antigens). In one exemplary embodiment, the composition includes a polysaccharide that includes Formula 025b, a polysaccharide that includes Formula 01A, a polysaccharide that includes Formula 02, and a polysaccharide that includes Formula 06. More specifically, such as a composition that includes: (i) an O-polysaccharide conjugated to CRM 197 , wherein the 0-polysaccharide includes Formula 025b, wherein n is at least 40, and the core saccharide; (ii) an -polysaccharide conjugated to CRM 197 , wherein the 0-polysaccharide includes Formula 01a, wherein n is at least 40, and the core saccharide; (iii) an 0-polysaccharide conjugated to CRM 197 , wherein the 0 polysaccharide includes Formula 02, wherein n is at least 40, and the core saccharide; and (iv) an 0-polysaccharide conjugated to CRM 197 , wherein the 0-polysaccharide includes Formula 06, wherein n is at least 40, and the core saccharide. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and at least one O-polysaccharide derived from any E. coli serotype, wherein the serotype is not 025a. For example, in one embodiment, the composition does not include a saccharide that includes the Formula 025a. Such a composition may include, for example, an 0-polysaccharide that includes Formula 025b, an 0-polysaccharide that includes Formula 01A, an 0-polysaccharide that includes Formula 02, and an O-polysaccharide that includes Formula 06.

In one embodiment, the composition includes a polypeptide derived from E. color a fragment thereof; and an 0-polysaccharide from 2 different E. coli serotypes, wherein each 0 polysaccharide is conjugated to CRM 197, and wherein the 0-polysaccharide includes the 0 antigen and core saccharide. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an0-polysaccharide from 3 different E. coli serotypes, wherein each 0-polysaccharide is conjugated to CRM 197, and wherein the 0 polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes a polypeptide derived from E. color a fragment thereof; and an 0 polysaccharide from 4 different E. coli serotypes, wherein each 0-polysaccharide is conjugated to CRM 197 , and wherein the 0-polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an 0-polysaccharide from 5 different E. coli serotypes, wherein each 0 polysaccharide is conjugated to CRM 197, and wherein the 0-polysaccharide includes the 0 antigen and core saccharide. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an0-polysaccharide from 6 different E. coli serotypes, wherein each 0-polysaccharide is conjugated to CRM 197, and wherein the 0 polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes a polypeptide derived from E. color a fragment thereof; and an 0 polysaccharide from 7 different E. coli serotypes, wherein each 0-polysaccharide is conjugated to CRM 197 , and wherein the 0-polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an 0-polysaccharide from 8 different E. coli serotypes, wherein each 0 polysaccharide is conjugated to CRM 197, and wherein the 0-polysaccharide includes the 0 antigen and core saccharide. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an0-polysaccharide from 9 different E. coli serotypes, wherein each 0-polysaccharide is conjugated to CRM 197, and wherein the 0 polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes a polypeptide derived from E. color a fragment thereof; and an 0 polysaccharide from 10 different E. coliserotypes, wherein each 0-polysaccharide is conjugated to CRM 197 , and wherein the 0-polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes a polypeptide derived from E. color a fragment thereof; and an 0-polysaccharide from 11 different E. coli serotypes, wherein each O-polysaccharide is conjugated to CRM 197 , and wherein the O-polysaccharide includes the 0 antigen and core saccharide. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an0-polysaccharide from 12 different serotypes, wherein each 0-polysaccharide is conjugated to CRM 197, and wherein the 0-polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an 0 polysaccharide from 13 different serotypes, wherein each 0-polysaccharide is conjugated to CRM 197 and wherein the 0-polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an0-polysaccharide from 14 different serotypes, wherein each 0-polysaccharide is conjugated to CRM 197, and wherein the 0 polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes a polypeptide derived from E. color a fragment thereof; and an 0 polysaccharide from 15 different serotypes, wherein each 0-polysaccharide is conjugated to CRM 197 , and wherein the 0-polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an0-polysaccharide from 16 different serotypes, wherein each 0-polysaccharide is conjugated to CRM197, and wherein the 0 polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes a polypeptide derived from E. color a fragment thereof; and an 0 polysaccharide from 17 different serotypes, wherein each 0-polysaccharide is conjugated to CRM 197 , and wherein the 0-polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an0-polysaccharide from 18 different serotypes, wherein each 0-polysaccharide is conjugated to CRM 197, and wherein the 0 polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes a polypeptide derived from E. color a fragment thereof; and an 0 polysaccharide from 19 different serotypes, wherein each 0-polysaccharide is conjugated to CRM 197 , and wherein the 0-polysaccharide includes the 0-antigen and core saccharide. In one embodiment, the composition includes a polypeptide derived from E. coli or a fragment thereof; and an0-polysaccharide from 20 different serotypes, wherein each 0-polysaccharide is conjugated to CRM 197, and wherein the 0 polysaccharide includes the 0-antigen and core saccharide. In one aspect, the invention relates to a composition that includes a polypeptide derived from E. coli or a fragment thereof; and a conjugate including a saccharide covalently bound to a carrier protein, wherein the saccharide includes Formula 025b, wherein n is 15 ±2. In one aspect, the invention relates to a composition that includes a polypeptide derived from E. coli or a fragment thereof; and a conjugate including a saccharide covalently bound to a carrier protein, wherein the saccharide includes Formula 025b, wherein n is 17 ±2. In one aspect, the invention relates to a composition that includes a polypeptide derived from E. color a fragment thereof; and a conjugate including a saccharide covalently bound a carrier protein, wherein the saccharide includes Formula 025b, wherein n is 55± 2. In another aspect, the invention relates to a composition that includes a polypeptide derived from E. coli or a fragment thereof; and a conjugate including a saccharide covalently bound a carrier protein, wherein the saccharide includes Formula 025b, wherein n is 51± 2. In one embodiment, the saccharide further includes the E. coli R1 core saccharide moiety. In another embodiment, the saccharide further includes the E. coli K12 core saccharide moiety. In another embodiment, the saccharide further includes the KDO moiety. Preferably, the carrier protein is CRM 1 9 7 . In one embodiment, the conjugate is prepared by single end linked conjugation. In one embodiment, the conjugate is prepared by reductive amination chemistry, preferably in DMSO buffer. In one embodiment, the saccharide is conjugated to the carrier protein through a (2-((2-oxoethyl)thio)ethyl) carbamate (eTEC) spacer. Preferably, the composition further includes a pharmaceutically acceptable diluent. In one embodiment, the immunogenic composition elicits IgG antibodies in humans, said antibodies being capable of binding an E. coli serotype 025B polysaccharide at a concentration of at least 0.2 pg/ml, 0.3 pg/ml, 0.35 pg/ml, 0.4 pg/ml or 0.5 pg/ml as determined by ELISA assay. Therefore, comparison of OPA activity of pre- and post-immunization serum with the immunogenic composition of the invention can be conducted and compared for their response to serotype 025B to assess the potential increase of responders. In one embodiment, the immunogenic composition elicits IgG antibodies in humans, said antibodies being capable of killing E. coli serotype 025B as determined by in vitro opsonophagocytic assay. In one embodiment, the immunogenic composition elicits functional antibodies in humans, said antibodies being capable of killing E. coliserotype 025B as determined by in vitro opsonophagocytic assay. In one embodiment, the immunogenic composition of the invention increases the proportion of responders against E. coli serotype 025B (i.e., individual with a serum having a titer of at least 1:8 as determined by in vitro OPA) as compared to the pre immunized population. In one embodiment, the immunogenic composition elicits a titer of at least 1:8 against E. coli serotype 025B in at least 50% of the subjects as determined by in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention elicits a titer of at least 1:8 against E. coli serotype 025B in at least 60%, 70%, 80%, or at least 90% of the subjects as determined by in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention significantly increases the proportion of responders against E. coliserotypes 025B (i.e., individual with a serum having a titer of at least 1:8 as determined by in vitro OPA) as compared to the pre-immunized population. In one embodiment, the immunogenic composition of the invention significantly increases the OPA titers of human subjects against E. coli serotype 025B as compared to the pre-immunized population. In one aspect, the invention relates to a composition that includes a polypeptide derived from E. color a fragment thereof; and a conjugate including a saccharide covalently bound a carrier protein, wherein the saccharide includes Formula 01a, wherein n is 39 ±2. Inanother aspect, the invention relates to a composition that includes a polypeptide derived from E. coli or a fragment thereof; and a conjugate including a saccharide covalently bound a carrier protein, wherein the saccharide includes Formula 01a, wherein n is 13 ±2. In one embodiment, the saccharide further includes the E. coli R1 core saccharide moiety. In one embodiment, the saccharide further includes the KDO moiety. Preferably, the carrier protein is CRM 1 97 .In one embodiment, the conjugate is prepared by single end linked conjugation. In one embodiment, the conjugate is prepared by reductive amination chemistry, preferably in DMSO buffer. In one embodiment, the saccharide is conjugated to the carrier protein through a (2-((2-oxoethyl)thio)ethyl) carbamate (eTEC) spacer. Preferably, the composition further includes a pharmaceutically acceptable diluent. In one embodiment, the immunogenic composition elicits IgG antibodies in humans, said antibodies being capable of binding an E. coliserotype 01A polysaccharide at a concentration of at least 0.2 pg/ml, 0.3 pg/ml, 0.35 pg/ml, 0.4 pg/ml or 0.5 pg/ml as determined by ELISA assay. Therefore, comparison of OPA activity of pre- and post-immunization serum with the immunogenic composition of the invention can be conducted and compared for their response to serotype 01A to assess the potential increase of responders. In one embodiment, the immunogenic composition elicits IgG antibodies in humans, said antibodies being capable of killing E. coliserotype 01A as determined by in vitro opsonophagocytic assay. In one embodiment, the immunogenic composition elicits functional antibodies in humans, said antibodies being capable of killing E. coliserotype 01A as determined by in vitro opsonophagocytic assay. In one embodiment, the immunogenic composition of the invention increases the proportion of responders against E. coliserotype 01A (i.e., individual with a serum having a titer of at least 1:8 as determined by in vitro OPA) as compared to the pre immunized population. In one embodiment, the immunogenic composition elicits a titer of at least 1:8 against E. coli serotype 01A in at least 50% of the subjects as determined by in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention elicits a titer of at least 1:8 against E. coli serotype 01A in at least 60%, 70%, 80%, or at least 90% of the subjects as determined by in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention significantly increases the proportion of responders against E. coli serotypes 01A (i.e., individual with a serum having a titer of at least 1:8 as determined by in vitro OPA) as compared to the pre-immunized population. In one embodiment, the immunogenic composition of the invention significantly increases the OPA titers of human subjects against E. coli serotype 01A as compared to the pre-immunized population. In one aspect, the invention relates to a composition that includes a polypeptide derived from E. coli or a fragment thereof; and a conjugate including a saccharide covalently bound a carrier protein, wherein the saccharide includes Formula 02, wherein n is 43 ±2. In another aspect, the invention relates to a composition that includes a polypeptide derived from E. coli or a fragment thereof; and a conjugate including a saccharide covalently bound a carrier protein, wherein the saccharide includes Formula 02, wherein n is47 ±2. In another aspect, the invention relates to a composition that includes a conjugate including a saccharide covalently bound a carrier protein, wherein the saccharide includes Formula 02, wherein n is 17 ±2. In another aspect, the invention relates to a composition that includes a conjugate including a saccharide covalently bound a carrier protein, wherein the saccharide includes Formula 02, wherein n is 18 ±2. In one embodiment, the saccharide further includes the E. coli R1 core saccharide moiety. In another embodiment, the saccharide further includes the E. coli R4 core saccharide moiety. In another embodiment, the saccharide further includes the KDO moiety. Preferably, the carrier protein is CRM 1 9 7 . In one embodiment, the conjugate is prepared by single end linked conjugation. In one embodiment, the conjugate is prepared by reductive amination chemistry, preferably in DMSO buffer. In one embodiment, the saccharide is conjugated to the carrier protein through a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. Preferably, the composition further includes a pharmaceutically acceptable diluent. In one embodiment, the immunogenic composition elicits IgG antibodies in humans, said antibodies being capable of binding an E. coli serotype 02 polysaccharide at a concentration of at least 0.2 pg/ml, 0.3 pg/ml, 0.35 pg/ml, 0.4 pg/ml or 0.5 pg/ml as determined by ELISA assay. Therefore, comparison of OPA activity of pre- and post-immunization serum with the immunogenic composition of the invention can be conducted and compared for their response to serotype 02 to assess the potential increase of responders. In one embodiment, the immunogenic composition elicits IgG antibodies in humans, said antibodies being capable of killing E. coliserotype 02 as determined by in vitro opsonophagocytic assay. In one embodiment, the immunogenic composition elicits functional antibodies in humans, said antibodies being capable of killing E. coliserotype 02 as determined by in vitro opsonophagocytic assay. In one embodiment, the immunogenic composition of the invention increases the proportion of responders against E. coli serotype 02 (i.e., individual with a serum having a titer of at least 1:8 as determined by in vitro OPA) as compared to the pre-immunized population. In one embodiment, the immunogenic composition elicits a titer of at least 1:8 against E. coli serotype 02 in at least 50% of the subjects as determined by in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention elicits a titer of at least 1:8 against E. coiserotype 02 in at least 60%, 70%, 80%, or at least 90% of the subjects as determined by in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention significantly increases the proportion of responders against E. coliserotypes 02 (i.e., individual with a serum having a titer of at least 1:8 as determined by in vitro OPA) as compared to the pre immunized population. In one embodiment, the immunogenic composition of the invention significantly increases the OPA titers of human subjects against E. coli serotype 02 as compared to the pre-immunized population. In one aspect, the invention relates to a composition that includes a polypeptide derived from E. coli or a fragment thereof; and a conjugate including a saccharide covalently bound a carrier protein, wherein the saccharide includes Formula 06, wherein n is 42 ±2. In another aspect, the invention relates to a composition that includes a polypeptide derived from E. coli or a fragment thereof; and a conjugate including a saccharide covalently bound a carrier protein, wherein the saccharide includes Formula 06, wherein n is 50 ±2. In another aspect, the invention relates to a composition that includes a conjugate including a saccharide covalently bound a carrier protein, wherein the saccharide includes Formula 06, wherein n is 17 ±2. In another aspect, the invention relates to a composition that includes a conjugate including a saccharide covalently bound a carrier protein, wherein the saccharide includes Formula 06, wherein n is 18 ±2. In one embodiment, the saccharide further includes the E. coli R1 core saccharide moiety. In one embodiment, the saccharide further includes the KDO moiety. Preferably, the carrier protein is CRM1 97 . In one embodiment, the conjugate is prepared by single end linked conjugation. In one embodiment, the conjugate is prepared by reductive amination chemistry, preferably in DMSO buffer. In one embodiment, the saccharide is conjugated to the carrier protein through a (2-((2 oxoethyl)thio)ethyl) carbamate (eTEC) spacer. Preferably, the composition further includes a pharmaceutically acceptable diluent. In one embodiment, the immunogenic composition elicits IgG antibodies in humans, said antibodies being capable of binding an E. coliserotype 06 polysaccharide at a concentration of at least 0.2 pg/ml, 0.3 pg/ml, 0.35 pg/ml, 0.4 pg/ml or 0.5 pg/ml as determined by ELISA assay. Therefore, comparison of OPA activity of pre- and post immunization serum with the immunogenic composition of the invention can be conducted and compared for their response to serotype 06 to assess the potential increase of responders. In one embodiment, the immunogenic composition elicits IgG antibodies in humans, said antibodies being capable of killing E. coli serotype 06 as determined by in vitro opsonophagocytic assay. In one embodiment, the immunogenic composition elicits functional antibodies in humans, said antibodies being capable of killing E. coliserotype 06 as determined by in vitro opsonophagocytic assay. In one embodiment, the immunogenic composition of the invention increases the proportion of responders against E. coli serotype 06 (i.e., individual with a serum having a titer of at least 1:8 as determined by in vitro OPA) as compared to the pre-immunized population. In one embodiment, the immunogenic composition elicits a titer of at least 1:8 against E. coli serotype 06 in at least 50% of the subjects as determined by in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention elicits a titer of at least 1:8 against E. coli serotype 06 in at least 60%, 70%, 80%, or at least 90% of the subjects as determined by in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention significantly increases the proportion of responders against E. coli serotypes 06 (i.e., individual with a serum having a titer of at least 1:8 as determined by in vitro OPA) as compared to the pre-immunized population. In one embodiment, the immunogenic composition of the invention significantly increases the OPA titers of human subjects against E. coli serotype 06 as compared to the pre-immunized population. In one asoect, the composition includes a polypeptide derived from E. colior a fragment thereof; and a conjugate including a saccharide covalently bound to a carrier protein, wherein the saccharide includes a structure selected from any one of Formula 01 (e.g., Formula 01A, Formula 01B, and Formula 01C), Formula 02, Formula 03, Formula 04 (e.g., Formula 04:K52 and Formula 04:K6), Formula 05 (e.g., Formula 05ab and Formula 05ac (strain 180/C3)), Formula 06 (e.g., Formula 06:K2; K13; K15 and Formula 06:K54), Formula 07, Formula 08, Formula 09, Formula 010, Formula 011, Formula 012, Formula 013, Formula 014, Formula 015, Formula 016, Formula 017, Formula 018 (e.g., Formula 018A, Formula 018ac, Formula 018A1, Formula 018B, and Formula 018B1), Formula 019, Formula 020, Formula 021, Formula 022, Formula 023 (e.g., Formula 023A), Formula 024, Formula 025 (e.g., Formula 025a and Formula 025b), Formula 026, Formula 027, Formula 028, Formula 029, Formula 030, Formula 032, Formula 033, Formula 034, Formula 035, Formula 036, Formula 037, Formula 038, Formula 039, Formula 040, Formula 041, Formula 042, Formula 043, Formula 044, Formula 045 (e.g., Formula 045 and Formula 045rel), Formula 046, Formula 048, Formula 049, Formula 050, Formula 051, Formula 052, Formula 053, Formula 054, Formula 055, Formula 056, Formula 057, Formula 058, Formula 059, Formula 060, Formula 061, Formula 062, Formula 62D1, Formula 063, Formula 064, Formula 065, Formula 066, Formula 068, Formula 069, Formula 070, Formula 071, Formula 073 (e.g., Formula

073 (strain 73-1)), Formula 074, Formula 075, Formula 076, Formula 077, Formula 078, Formula 079, Formula 080, Formula 081, Formula 082, Formula 083, Formula 084, Formula 085, Formula 086, Formula 087, Formula 088, Formula 089, Formula 090, Formula 091, Formula 092, Formula 093, Formula 095, Formula 096, Formula 097, Formula 098, Formula 099, Formula 0100, Formula 0101, Formula 0102, Formula 0103, Formula 0104, Formula 0105, Formula 0106, Formula 0107, Formula 0108, Formula 0109, Formula 0110, Formula 0111, Formula 0112, Formula 0113, Formula 0114, Formula 0115, Formula 0116, Formula 0117, Formula 0118, Formula 0119, Formula 0120, Formula 0121, Formula 0123, Formula 0124, Formula 0125, Formula 0126, Formula 0127, Formula 0128, Formula 0129, Formula 0130, Formula 0131, Formula 0132, Formula 0133, Formula 0134, Formula 0135, Formula 0136, Formula 0137, Formula 0138, Formula 0139, Formula 0140, Formula 0141, Formula 0142, Formula 0143, Formula 0144, Formula 0145, Formula 0146, Formula 0147, Formula 0148, Formula 0149, Formula 0150, Formula 0151, Formula 0152, Formula 0153, Formula 0154, Formula 0155, Formula 0156, Formula 0157, Formula 0158, Formula 0159, Formula 0160, Formula 0161, Formula 0162, Formula 0163, Formula 0164, Formula 0165, Formula 0166, Formula 0167, Formula 0168, Formula 0169, Formula 0170, Formula 0171, Formula 0172, Formula 0173, Formula 0174, Formula 0175, Formula 0176, Formula 0177, Formula 0178, Formula 0179, Formula 0180, Formula 0181, Formula 0182, Formula 0183, Formula 0184, Formula 0185, Formula 0186, and Formula 0187, wherein n is an integer from 1 to 100, preferably from 31 to 90. In one embodiment, the saccharide further includes the E. coli R1 core saccharide moiety. In one embodiment, the saccharide further includes the E. coli R2 core saccharide moiety. In one embodiment, the saccharide further includes the E. coli R3 core saccharide moiety. In another embodiment, the saccharide further includes the E. coli R4 core saccharide moiety. In one embodiment, the saccharide further includes the E. coli K12 core saccharide moiety. In another embodiment, the saccharide further includes the KDO moiety. Preferably, the carrier protein is CRM19 7 . In one embodiment, the conjugate is prepared by single end linked conjugation. In one embodiment, the conjugate is prepared by reductive amination chemistry, preferably in DMSO buffer. In one embodiment, the saccharide is conjugated to the carrier protein through a (2-((2 oxoethyl)thio)ethyl)carbamate (eTEC) spacer. Preferably, the composition further includes a pharmaceutically acceptable diluent. In one embodiment, the composition furtherincludes atleast1,2, 3,4,5,6,7, 8,9,10,11,12,13,14,15,16,17,18,19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29 additional conjugates to at most 30 additional conjugates, each conjugate including a saccharide covalently bound to a carrier protein, wherein the saccharide includes a structure selected from any one of said Formulas.

A. Saccharide

In one embodiment, the saccharide is produced by expression (not necessarily overexpression) of different Wzz proteins (e.g., WzzB) to control of the size of the saccharide. As used herein, the term "saccharide" refers to a single sugar moiety or monosaccharide unit as well as combinations of two or more single sugar moieties or monosaccharide units covalently linked to form disaccharides, oligosaccharides, and polysaccharides. The saccharide may be linear or branched. In one embodiment, the saccharide is produced in a recombinant Gram-negative bacterium. In one embodiment, the saccharide is produced in a recombinant E. coli cell. In one embodiment, the saccharide is produced in a recombinant Salmonella cell. Exemplary bacteria include E. coli025K5H1, E. coli BD559, E. coli GAR2831, E. coli GAR865, E. coli GAR868, E. coliGAR869,E.coliGAR872,E.coliGAR878,E.coliGAR896,E.coliGAR1902,E.coli025a ETC NR-5, E. coli0157:H7:K-, Salmonella enterica serovar Typhimurium strain LT2, E. coli GAR2401, Salmonella enterica serotype Enteritidis CVD 1943, Salmonella enterica serotype Typhimurium CVD 1925, Salmonella enterica serotype Paratyphi A CVD 1902, and Shige//a flexneri CVD 1208S. In one embodiment, the bacterium is not E. coliGAR2401. This genetic approach towards saccharide production allows for efficient production of0-polysaccharides and O-antigen molecules as vaccine components. The term "wzz protein," as used herein, refers to a chain length determinant polypeptide, such as, for example, wzzB, wzz, wzzSF, wZZST, fepE, wzzfepE, wzzl and wzz2. The GenBank accession numbers for the exemplary wzz gene sequences are AF011910 for E4991/76, AF011911 for F186, AF011912 for M70/1-1, AF011913 for 79/311, AF011914 for Bi7509- 41, AF011915 for C664-1992, AF011916 for C258-94, AF011917 for C722-89, and AF011919 for EDL933. The GenBank accession numbers for the G7 and Bi316-41 wzz genes sequences are U39305 and U39306, respectively. Further GenBank accession numbers for exemplary wzz gene sequences are NP_459581 for Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 FepE; AIG66859 for E. coli0157:H7 Strain EDL933 FepE; NP_461024 for Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 WzzB. NP_416531 for E. coli K-12 substr. MG1655 WzzB, NP_415119 for E. coli K-12 substr. MG1655 FepE. In preferred embodiments, the wzz family protein is any one of wzzB, wzz, wzzSF, wZZST, fepE, wzzfepE, wZZ1 and wzz2, most preferably wzzB, more preferably fepE. Exemplary wzzB sequences include sequences set forth in SEQ ID Nos: 30-34. Exemplary FepE sequences include sequences set forth in SEQ ID Nos: 35-39.

In some embodiments, a modified saccharide (modified as compared to the corresponding wild-type saccharide) may be produced by expressing (not necessarily overexpressing) a wzz family protein (e.g., fepE) from a Gram-negative bacterium in a Gram negative bacterium and/or by switching off (i.e., repressing, deleting, removing) a second wzz gene (e.g., wzzB) to generate high molecular weight saccharides, such as lipopolysaccharides, containing intermediate or long O-antigen chains. For example, the modified saccharides may be produced by expressing (not necessarily overexpressing) wzz2 and switching off wzzl. Or, in the alternative, the modified saccharides may be produced by expressing (not necessarily overexpressing) wzzfepE and switching off wzzB. In another embodiment, the modified saccharides may be produced by expressing (not necessarily overexpressing) wzzB but switching off wzzfepE. In another embodiment, the modified saccharides may be produced by expressing fepE. Preferably, the wzz family protein is derived from a strain that is heterologous to the host cell. In some embodiments, the saccharide is produced by expressing a wzz family protein having an amino acid sequence that is at least 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39. In one embodiment, the wzz family protein includes a sequence selected from any one of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39. Preferably, the wzz family protein has at least 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34. In some embodiments, the saccharide is produced by expressing a protein having an amino acid sequence that is at least 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to an fepE protein. In one aspect, the invention relates to saccharides produced by expressing a wzz family protein, preferably fepE, in a Gram-negative bacterium to generate high molecular weight saccharides containing intermediate or long O-antigen chains, which have an increase of at least 1, 2, 3, 4, or 5 repeating units, as compared to the corresponding wild-type O-polysaccharide. In one aspect, the invention relates to saccharides produced by a Gram-negative bacterium in culture that expresses (not necessarily overexpresses) a wzz family protein (e.g., wzzB) from a Gram-negative bacterium to generate high molecular weight saccharides containing intermediate or long O-antigen chains, which have an increase of at least 1, 2, 3, 4, or 5 repeating units, as compared to the corresponding wild-type O-antigen. See description of0-polysaccharides and O-antigens below for additional exemplary saccharides having increased number of repeat units, as compared to the corresponding wild-type saccharides. A desired chain length is the one which produces improved or maximal immunogenicity in the context of a given vaccine construct. In another embodiment, the saccharide includes any one Formula selected from Table 1, wherein the number of repeat units n in the saccharide is greater than the number of repeat units in the corresponding wild-type -polysaccharide by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38, 39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63, 64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more repeat units. Preferably, the saccharide includes an increase of at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 repeat units, as compared to the corresponding wild-type O-polysaccharide. See, for example, Table 24. Methods of determining the length of saccharides are known in the art. Such methods include nuclear magnetic resonance, mass spectroscopy, and size exclusion chromatography, as described in Example 13. In a preferred embodiment, the invention relates to a saccharide produced in a recombinant E. coli host cell, wherein the gene for an endogenous wzz O-antigen length regulator (e.g., wzzB) is deleted and is replaced by a (second) wzz gene from a Gram-negative bacterium heterologous to the recombinant E. coli host cell (e.g., Salmonella fepE) to generate high molecular weight saccharides, such as lipopolysaccharides, containing intermediate or long O-antigen chains. In some embodiments, the recombinant E. coli host cell includes a wzz gene from Salmonella, preferably from Salmonella enterica. In one embodiment, the host cell includes the heterologous gene for a wzz family protein as a stably maintained plasmid vector. In another embodiment, the host cell includes the heterologous gene for a wzz family protein as an integrated gene in the chromosomal DNA of the host cell. Methods of stably expressing a plasmid vector in an E. coli host cell and methods of integrating a heterologous gene into the chromosome of an E. coli host cell are known in the art. In one embodiment, the host cell includes the heterologous genes for an O-antigen as a stably maintained plasmid vector. In another embodiment, the host cell includes the heterologous genes for an O-antigen as an integrated gene in the chromosomal DNA of the host cell. Methods of stably expressing a plasmid vector in an E. coli host cell and a Salmonella host cell are known in the art. Methods of integrating a heterologous gene into the chromosome of an E. colihost cell and a Salmonella host cell are known in the art.

In one aspect, the recombinant host cell is cultured in a medium that comprises a carbonsource. Carbon sources for culturing E. coliare known in the art. Exemplarycarbon sources include sugar alcohols, polyols, aldol sugars or keto sugars including but not limited to arabinose, cellobiose, fructose, glucose, glycerol, inositol, lactose, maltose, mannitol, mannose, rhamnose, raffinose, sorbitol, sorbose, sucrose, trehalose, pyruvate, succinate and methylamine. In a preferred embodiment, the medium includes glucose. In some embodiments, the medium includes a polyol or aldol sugar, for example, mannitol, inositol, sorbose, glycerol, sorbitol, lactose and arabinose as the carbon source. All of the carbon sources may be added to the medium before the start of culturing, or it may be added step by step or continuously during culturing. An exemplary culture medium for the recombinant host cell includes an element selected from any one of KH 2 PO 4 , K2 HPO 4 , (NH 4) 2 SO 4 , sodium citrate, Na 2 SO 4 , aspartic acid, glucose, MgSO 4 , FeSO 4-7H 20, Na 2MoO 4 -2H 2 0,H 3B0 3 , CoCl 2-6H 20, CuCl 2-2H 20, MnCl2-4H20, ZnCl2 and CaCl2-2H20. Preferably, the medium includes KH2PO4, K 2 HPO4 , (NH 4 ) 2 SO 4 , sodium citrate, Na 2 SO 4 , aspartic acid, glucose, MgSO 4 , FeSO 4

7H 20, Na 2MoO 4-2H 20, H 3B0 3, CoCl 2-6H 2 0, CuCl 2-2H 2 0, MnCl 2-4H 20, ZnCl2 and CaCl 2-2H 20. The medium used herein may be solid or liquid, synthetic (i.e. man-made) or natural, and may include sufficient nutrients for the cultivation of the recombinant host cell. Preferably, the medium is a liquid medium. In some embodiments, the medium may further include suitable inorganic salts. In some embodiments, the medium may further include trace nutrients. In some embodiments, the medium may further include growth factors. In some embodiments, the medium may further include an additional carbon source. In some embodiments, the medium may further include suitable inorganic salts, trace nutrients, growth factors, and a supplementary carbon source. Inorganic salts, trace nutrients, growth factors, and supplementary carbon sources suitable for culturing E. coli are known in the art. In some embodiments, the medium may include additional components as appropriate, such as peptone, N-Z Amine, enzymatic soy hydrosylate, additional yeast extract, malt extract, supplemental carbon sources and various vitamins. In some embodiments, the medium does not include such additional components, such as peptone, N-Z Amine, enzymatic soy hydrosylate, additional yeast extract, malt extract, supplemental carbon sources and various vitamins. Illustrative examples of suitable supplemental carbon sources include, but are not limited to other carbohydrates, such as glucose, fructose, mannitol, starch or starch hydrolysate, cellulose hydrolysate and molasses; organic acids, such as acetic acid, propionic acid, lactic acid, formic acid, malic acid, citric acid, and fumaric acid; and alcohols, such as glycerol, inositol, mannitol and sorbitol. In some embodiments, the medium further includes a nitrogen source. Nitrogen sources suitable for culturing E. coli are known in the art. Illustrative examples of suitable nitrogen sources include, but are not limited to ammonia, including ammonia gas and aqueous ammonia; ammonium salts of inorganic or organic acids, such as ammonium chloride, ammonium nitrate, ammonium phosphate, ammonium sulfate and ammonium acetate; urea; nitrate or nitrite salts, and other nitrogen-containing materials, including amino acids as either pure or crude preparations, meat extract, peptone, fish meal, fish hydrolysate, corn steep liquor, casein hydrolysate, soybean cake hydrolysate, yeast extract, dried yeast, ethanol-yeast distillate, soybean flour, cottonseed meal, and the like. In some embodiments, the medium includes an inorganic salt. Illustrative examples of suitable inorganic salts include, but are not limited to salts of potassium, calcium, sodium, magnesium, manganese, iron, cobalt, zinc, copper, molybdenum, tungsten and other trace elements, and phosphoric acid. In some embodiments, the medium includes appropriate growth factors. Illustrative examples of appropriate trace nutrients, growth factors, and the like include, but are not limited to coenzyme A, pantothenic acid, pyridoxine-HCI, biotin, thiamine, riboflavin, flavine mononucleotide, flavine adenine dinucleotide, DL-6,8-thioctic acid, folic acid, Vitamin B12 , other vitamins, amino acids such as cysteine and hydroxyproline, bases such as adenine, uracil, guanine, thymine and cytosine, sodium thiosulfate, p- or r-aminobenzoic acid, niacinamide, nitriloacetate, and the like, either as pure or partially purified chemical compounds or as present in natural materials. The amounts may be determined empirically by one skilled in the art according to methods and techniques known in the art. In another embodiment, the modified saccharide (as compared to the corresponding wild-type saccharide) described herein is synthetically produced, for example, in vitro. Synthetic production or synthesis of the saccharides may facilitate the avoidance of cost- and time-intensive production processes. In one embodiment, the saccharide is synthetically synthesized, such as, for example, by using sequential glycosylation strategy or a combination of sequential glycosylations and [3+2] block synthetic strategy from suitably protected monosaccharide intermediates. For example, thioglycosides and glycosyl trichloroacetimidate derivatives may be used as glycosyl donors in the glycosylations. In one embodiment, a saccharide that is synthetically synthesized in vitro has the identical structure to a saccharide produced by recombinant means, such as by manipulation of a wzz family protein described above.

The saccharide produced (by recombinant or synthetic means) includes a structure derived from any E. coli serotype including, for example, any one of the following E. coliserotypes: 01 (e.g., 01A, 01B, and 01C), 02, 03, 04 (e.g., 04:K52 and 04:K6), 05 (e.g., 05ab and 05ac (strain 180/C3)), 06 (e.g., 06:K2; K13; K15 and 06:K54), 07, 08, 09, 010, 011, 012, 013, 014, 015, 016, 017, 018 (e.g., 018A, 018ac, 018A1, 018B, and 018B1), 019, 020, 021, 022, 023 (e.g., 023A), 024, 025 (e.g., 025a and 025b), 026, 027, 028, 029, 030, 032, 033, 034, 035, 036, 037, 038, 039, 040, 041, 042, 043, 044, 045 (e.g., 045and 045rel), 046, 048, 049, 050, 051, 052, 053, 054, 055, 056, 057, 058, 059, 060, 061, 062, 62 D1, 063, 064, 065, 066, 068, 069, 070, 071, 073 (e.g., 073 (strain 73-1)), 074,075,076, 077, 078, 079, 080, 081, 082, 083, 084, 085, 086, 087, 088, 089, 090, 091, 092, 093, 095, 096, 097, 098, 099, 0100, 0101, 0102, 0103, 0104, 0105, 0106, 0107, 0108, 0109, 0110, 0111, 0112, 0113, 0114, 0115, 0116, 0117, 0118, 0119, 0120, 0121, 0123, 0124, 0125, 0126, 0127, 0128, 0129, 0130, 0131, 0132, 0133, 0134, 0135, 0136, 0137, 0138, 0139, 0140, 0141, 0142, 0143, 0144, 0145, 0146, 0147, 0148, 0149, 0150, 0151, 0152, 0153, 0154, 0155, 0156, 0157, 0158, 0159, 0160, 0161, 0162, 0163, 0164, 0165, 0166, 0167, 0168, 0169, 0170, 0171, 0172, 0173, 0174, 0175, 0176, 0177, 0178, 0179, 0180, 0181, 0182, 0183, 0184, 0185, 0186,and 0187. The individual polysaccharides are typically purified (enriched with respect to the amount of polysaccharide-protein conjugate) through methods known in the art, such as, for example, dialysis, concentration operations, diafiltration operations, tangential flow filtration, precipitation, elution, centrifugation, precipitation, ultra-filtration, depth filtration, and/or column chromatography (ion exchange chromatography, multimodal ion exchange chromatography, DEAE, and hydrophobic interaction chromatography). Preferably, the polysaccharides are purified through a method that includes tangential flow filtration. Purified polysaccharides may be activated (e.g., chemically activated) to make them capable of reacting (e.g., either directly to the carrier protein or via a linker such as an eTEC spacer) and then incorporated into glycoconjugates of the invention, as further described herein. In one preferred embodiment, the saccharide of the invention is derived from an E. coli serotype, wherein the serotype is 025a. In another preferred embodiment, the serotype is 025b. In another preferred embodiment, the serotype is 01A. In another preferred embodiment, the serotype is 02. In another preferred embodiment, the serotype is 06. In another preferred embodiment, the serotype is 017. In another preferred embodiment, the serotype is 015. In another preferred embodiment, the serotype is 018A. In another preferred embodiment, the serotype is 075. In another preferred embodiment, the serotype is 04. In another preferred embodiment, the serotype is 016. In another preferred embodiment, the serotype is 013. In another preferred embodiment, the serotype is 07. In another preferred embodiment, the serotype is 08. In another preferred embodiment, the serotype is 09. As used herein, reference to any of the serotypes listed above, refers to a serotype that encompasses a repeating unit structure (0-unit, as described below) known in the art and is unique to the corresponding serotype. For example, the term "025a" serotype (also known in the art as serotype "025")refers to a serotype that encompasses Formula 025 shown in Table 1. As another example, the term "025b"serotype refers to a serotype that encompasses Formula 025b shown in Table 1. As used herein, the serotypes are referred generically herein unless specified otherwise such that, for example, the term Formula "O18" refers generically to encompass Formula 018A, Formula 018ac, Formula 18A1, Formula 018B, and Formula 018B1. As used herein, the term "O1" refers generically to encompass the species of Formula that include the generic term "O1" in the Formula name according to Table 1, such as any one of Formula 01A, Formula OA, Formula 01B, and Formula 01C, each of which is shown in Table 1. Accordingly, an "O1 serotype" refers generically to a serotype that encompasses any one of Formula 01A, Formula 01Al, Formula 01B, and Formula 01 C. As used herein, the term "06"refers generically to species of Formula that include the generic term "06" in the Formula name according to Table 1, such as any one of Formula 06:K2; K13; K15; and 06:K54, each of which is shown in Table 1. Accordingly, an"06 serotype" refers generically to a serotype that encompasses any one of Formula 06:K2; K13; K15; and 06:K54. Other examples of terms that refer generically to species of a Formula that include the generic term in the Formula name according to Table 1 include: "04", "05","O18", and "045". As used herein, the term "02" refers to Formula 02 shown in Table 1. The term "020 antigen" refers to a saccharide that encompasses Formula 02 shown in Table 1. As used herein, reference to an O-antigen from a serotype listed above refers to a saccharide that encompasses the formula labeled with the corresponding serotype name. For example, the term "025B O-antigen" refers to a saccharide that encompasses Formula 025B shown in Table 1.

As another example, the term "010-antigen" generically refers to a saccharide that encompasses a Formula including the term "01," such as the Formula 01A, Formula 01A1, Formula 01B, and Formula 01C, each of which are shown in Table 1. As anotherexample, theterm "06 0-antigen" generically refersto a saccharidethat encompasses a Formula including the term "06,"such as Formula 06:K2; Formula 06:K13; Formula 06:K15 and Formula 06:K54, each of which are shown in Table 1.

B. O-Polysaccharide As used herein, the term "O-polysaccharide" refers to any structure that includes an O-antigen, provided that the structure does not include a whole cell or Lipid A. For example, in one embodiment, the 0-polysaccharide includes a lipopolysaccharide wherein the Lipid A is not bound. The step of removing Lipid A is known in the art and includes, as an example, heat treatment with addition of an acid. An exemplary process includes treatment with 1% acetic acid at 100°C for 90 minutes. This process is combined with a process of isolating Lipid A as removed. An exemplary process for isolating Lipid A includes ultracentrifugation. In one embodiment, the O-polysaccharide refers to a structure that consists of the O-antigen, in which case, the O-polysaccharide is synonymous with the term 0 antigen. In one preferred embodiment, the 0-polysaccharide refers to a structure that includes repeating units of the O-antigen, without the core saccharide. Accordingly, in one embodiment, the 0-polysaccharide does not include an E. coli R1 core moiety. In another embodiment, the 0-polysaccharide does not include an E. coli R2 core moiety. In another embodiment, the 0-polysaccharide does not include an E. coli R3 core moiety. In another embodiment, the 0-polysaccharide does not include an E. coli R4 core moiety. In another embodiment, the 0-polysaccharide does not include an E. coli K12 core moiety. In another preferred embodiment, the O-polysaccharide refers to a structure that includes an O-antigen and a core saccharide. In another embodiment, the O-polysaccharide refers to a structure that includes an O-antigen, a core saccharide, and a KDO moiety. Methods of purifying an O-polysaccharide, which includes the core oligosaccharide, from LPS are known in the art. For example, after purification of LPS, purified LPS may be hydrolyzed by heating in 1% (v/v) acetic acid for 90 minutes at 100 degrees Celsius, followed by ultracentrifugation at 142,000 x g for 5 hours at 4 degrees Celsius. The supernatant containing the 0-polysaccharide is freeze-dried and stored at 4 degrees Celsius. In certain embodiments, deletion of capsule synthesis genes to enable simple purification of 0-polysaccharide is described.

The O-polysaccharide can be isolated by methods including, but not limited to mild acid hydrolysis to remove lipid A from LPS. Other embodiments may include use of hydrazine as an agent for O-polysaccharide preparation. Preparation of LPS can be accomplished by known methods in the art. In certain embodiments, the O-polysaccharides purified from wild-type, modified, or attenuated Gram-negative bacterial strains that express (not necessarily overexpress) a Wzz protein (e.g., wzzB) are provided for use in conjugate vaccines. In preferred embodiments, the O-polysaccharide chain is purified from the Gram-negative bacterial strain expressing (not necessarily overexpressing) wzz protein for use as a vaccine antigen either as a conjugate or complexed vaccine. In one embodiment, the O-polysaccharide has a molecular weight that is increased by about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24 fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44-fold, 45-fold, 46-fold, 47 fold, 48-fold, 49-fold, 50-fold, 51-fold, 52-fold, 53-fold, 54-fold, 55-fold, 56-fold, 57-fold, 58-fold, 59-fold, 60-fold, 61-fold, 62-fold, 63-fold, 64-fold, 65-fold, 66-fold, 67-fold, 68-fold, 69-fold, 70 fold, 71-fold, 72-fold, 73-fold, 74-fold, 75-fold, 76-fold, 77-fold, 78-fold, 79-fold, 80-fold, 81-fold, 82-fold, 83-fold, 84-fold, 85-fold, 86-fold, 87-fold, 88-fold, 89-fold, 90-fold, 91-fold, 92-fold, 93 fold, 94-fold, 95-fold, 96-fold, 97-fold, 98-fold, 99-fold, 100-fold or more, as compared to the corresponding wild-type O-polysaccharide. In a preferred embodiment, the O-polysaccharide has a molecularweight that is increased by at least 1-fold and at most 5-fold, as compared to the corresponding wild-type O-polysaccharide. In another embodiment, the O-polysaccharide has a molecular weight that is increased by at least 2-fold and at most 4-fold, as compared to the corresponding wild-type O-polysaccharide. An increase in molecular weight of the 0 polysaccharide, as compared to the corresponding wild-type O-polysaccharide, is preferably associated with an increase in number of O-antigen repeat units. In one embodiment, the increase in molecular weight of the O-polysaccharide is due to the wzz family protein. In one embodiment, the O-polysaccharide has a molecular weight that is increased by about1,2,3,4,5,6,7,8,9,10,11,12,13,14, 15, 16,17,18,19,20,21,22,23,24,25,26, 27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51, 52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76, 77,78,79,80,81,82,83,84, 85,86,87,88,89,90,91,92,93,94,95,96,97, 98, 99,100 kDa or more, as compared to the corresponding wild-type O-polysaccharide. In one embodiment, the O-polysaccharide of the invention has a molecular weight that is increased by at least 1 and at most 200 kDa, as compared to the corresponding wild-type O-polysaccharide. In one embodiment, the molecular weight is increased by at least 5 and at most 200kDa. In one embodiment, the molecular weight is increased by at least 10 and at most 200kDa. In one embodiment, the molecular weight is increased by at least 12 and at most 200kDa. In one embodiment, the molecular weight is increased by at least 15 and at most 200kDa. In one embodiment, the molecular weight is increased by at least 18 and at most 200kDa. In one embodiment, the molecular weight is increased by at least 20 and at most 200kDa. In one embodiment, the molecular weight is increased by at least 21 and at most 200kDa. In one embodiment, the molecular weight is increased by at least 22 and at most 200kDa. In one embodiment, the molecular weight is increased by at least 30 and at most 200kDa. In one embodiment, the molecular weight is increased by at least 1 and at most 1OOkDa. In one embodiment, the molecular weight is increased by at least 5 and at most 1OOkDa. In one embodiment, the molecular weight is increased by at least 10 and at most 1OOkDa. In one embodiment, the molecular weight is increased by at least 12 and at most 1OOkDa. In one embodiment, the molecular weight is increased by at least 15 and at most 1OOkDa. In one embodiment, the molecular weight is increased by at least 20 and at most 1OOkDa. In one embodiment, the molecular weight is increased by at least 1 and at most 75kDa. In one embodiment, the molecular weight is increased by at least 5 and at most 75kDa. In one embodiment, the molecular weight is increased by at least 10 and at most 75kDa. In one embodiment, the molecular weight is increased by at least 12 and at most 75kDa. In one embodiment, the molecular weight is increased by at least 15 and at most 75kDa. In one embodiment, the molecular weight is increased by at least 18 and at most 75kDa. In one embodiment, the molecular weight is increased by at least 20 and at most 75kDa. In one embodiment, the molecular weight is increased by at least 30 and at most 75kDa. In one embodiment, the molecular weight is increased byat least 10 and at most90kDa. In one embodiment, the molecular weight is increased by at least 12 and at most 85kDa. In one embodiment, the molecular weight is increased by at least 10 and at most 75kDa. In one embodiment, the molecular weight is increased by at least 10 and at most 70kDa. In one embodiment, the molecular weight is increased by at least 10 and at most 60kDa. In one embodiment, the molecular weight is increased by at least 10 and at most 50kDa. In one embodiment, the molecular weight is increased by at least 10 and at most 49kDa. In one embodiment, the molecular weight is increased by at least 10 and at most 48kDa. In one embodiment, the molecular weight is increased by at least 10 and at most 47kDa. In one embodiment, the molecular weight is increased by at least 10 and at most 46kDa. In one embodiment, the molecular weight is increased by at least 20 and at most 45kDa. In one embodiment, the molecular weight is increased by at least 20 and at most 44kDa. In one embodiment, the molecular weight is increased by at least 20 and at most 43kDa. In one embodiment, the molecular weight is increased by at least 20 and at most 42kDa. In one embodiment, the molecular weight is increased by at least 20 and at most 41kDa. Such an increase in molecular weight of the 0 polysaccharide, as compared to the corresponding wild-type O-polysaccharide, is preferably associated with an increase in number of O-antigen repeat units. In one embodiment, the increase in molecular weight of the O-polysaccharide is due to the wzz family protein. See, for example, Table 21. In another embodiment, the 0-polysaccharide includes any one Formula selected from Table 1, wherein the number of repeat units n in the O-polysaccharide is greater than the number of repeat units in the corresponding wild-type -polysaccharide by 1, 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33, 34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58, 59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more repeat units. Preferably, the saccharide includes an increase of at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31,32,33, 34, 35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,or50 repeat units, as compared to the corresponding wild-type O-polysaccharide. See, for example, Table 21.

C. O-Antigen

The O-antigen is part of the lipopolysaccharide (LPS) in the outer membrane of Gram negative bacteria. The O-antigen is on the cell surface and is a variable cell constituent. The variability of the O-antigen provides a basis for serotyping of Gram-negative bacteria. The current E. coli serotyping scheme includes O-polysaccharides 1 to 181. The O-antigen includes oligosaccharide repeating units (0-units), the wild type structure of which usually contains two to eight residues from a broad range of sugars. The O-units of exemplary E. coliO-antigens are shown in Table 1, see also FIG. 9A-9C and FIG. 10A-1OB. In one embodiment, the saccharide of the invention may be one oligosaccharide unit. In one embodiment, the saccharide of the invention is one repeating oligosaccharide unit of the relevant serotype. In such embodiments, the saccharide may include a structure selected from any one of Formula 01a, Formula 02, Formula 06, Formula 08, Formula 09a, Formula 09, Formula O20ab, Formula O20ac, Formula 025b, Formula 052, Formula 097, and Formula 0101. In a further embodiment, the saccharide may include a structure selected from any one of Formula 01a, Formula 02, Formula 06, and Formula 025b. In one embodiment, the saccharide of the invention may be oligosaccharides. Oligosaccharides have a low number of repeat units (typically 5-15 repeat units) and are typically derived synthetically or by hydrolysis of polysaccharides. In such embodiments, the saccharide may include a structure selected from any one of Formula 01a, Formula 02, Formula 06, Formula 08, Formula 09a, Formula 09, FormulaO20ab, FormulaO20ac, Formula 025b, Formula 052, Formula 097, and Formula 0101. In a further embodiment, the saccharide may include a structure selected from any one of Formula 01a, Formula 02, Formula 06, and Formula 025b. Preferably, all of the saccharides of the present invention and in the immunogenic compositions of the present invention are polysaccharides. High molecular weight polysaccharides may induce certain antibody immune responses due to the epitopes present on the antigenic surface. The isolation and purification of high molecular weight polysaccharides are preferably contemplated for use in the conjugates, compositions and methods of the present invention. In some embodiments, the number of repeat 0 units in each individual O-antigen polymer (and therefore the length and molecular weight of the polymer chain) depends on the wzz chain length regulator, an inner membrane protein. Different wzz proteins confer different ranges of modal lengths (4 to >100 repeat units). The term "modal length" refers to the number of repeating O-units. Gram-negative bacteria often have two different Wzz proteins that confer two distinct OAg modal chain lengths, one longer and one shorter. The expression (not necessarily the overexpression) of wzz family proteins (e.g., wzzB) in Gram-negative bacteria may allow for the manipulation of 0 antigen length, to shift or to bias bacterial production of O-antigens of certain length ranges, and to enhance production of high-yield large molecular weight lipopolysaccharides. In one embodiment, a "short" modal length as used herein refers to a low number of repeat O-units, e.g., 1-20. In one embodiment, a "long" modal length as used herein refers to a number of repeat O-units greater than 20 and up to a maximum of 40. In one embodiment, a "very long" modal length as used herein refers to greater than 40 repeat O-units. In one embodiment, the saccharide produced has an increase of at least 10 repeating units, 15 repeating units, 20 repeating units, 25 repeating units, 30 repeating units, 35 repeating units, 40 repeating units, 45 repeating units, 50 repeating units, 55 repeating units, 60 repeating units, 65 repeating units, 70 repeating units, 75 repeating units, 80 repeating units, 85 repeating units, 90 repeating units, 95 repeating units, or 100 repeating units, as compared to the corresponding wild-type 0-polysaccharide. In another embodiment, the saccharide of the invention has an increase of 1, 2, 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27, 28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50, 51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,

74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98, 99, 100 or more repeat units, as compared to the corresponding wild-type0-polysaccharide. Preferably, the saccharide includes an increase of at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31,32,33, 34, 35,36,37,38,39,40,41,42,43,44,45,46,47,48,49, or50 repeat units, as compared to the corresponding wild-type 0-polysaccharide. See, for example, Table 21. Methods of determining the length of saccharides are known in the art. Such methods include nuclear magnetic resonance, mass spectroscopy, and size exclusion chromatography, as described in Example 13. Methods of determining the number of repeat units in the saccharide are also known in the art. For example, the number of repeat units (or "n" in the Formula) may be calculated by dividing the molecular weight of the polysaccharide (without the molecular weight of the core saccharide or KDO residue) by the molecular weight of the repeat unit (i.e., molecular weight of the structure in the corresponding Formula, shown for example in Table 1, which may be theoretically calculated as the sum of the molecular weight of each monosaccharide within the Formula). The molecular weight of each monosaccharide within the Formula is known in the art. The molecular weight of a repeat unit of Formula 025b, for example, is about 862 Da. The molecular weight of a repeat unit of Formula 01a, for example, is about 845 Da. The molecular weight of a repeat unit of Formula 02, for example, is about 829 Da. The molecular weight of a repeat unit of Formula 06, for example, is about 893 Da. When determining the number of repeat units in a conjugate, the carrier protein molecular weight and the protein:polysaccharide ratio is factored into the calculation. As defined herein, "n" refers to the number of repeating units (represented in brackets in Table 1) in a polysaccharide molecule. As is known in the art, in biological macromolecules, repeating structures may be interspersed with regions of imperfect repeats, such as, for example, missing branches. In addition, it is known in the art that polysaccharides isolated and purified from natural sources such as bacteria may be heterogenous in size and in branching. In such a case, n may represent an average or median value for n for the molecules in a population. In one embodiment, the O-polysaccharide has an increase of at least one repeat unit of an O-antigen, as compared to the corresponding wild-type 0-polysaccharide. The repeat units of O-antigens are shown in Table 1. In one embodiment, the 0-polysaccharide includes 1, 2, 3, 4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30, 31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55, 56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more total repeat units. Preferably, the saccharide has a total of at least 3 to at most 80 repeat units. In another embodiment, the -polysaccharide has an increase of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,

15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37, 38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60, 61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more repeat units, as compared to the corresponding wild-type O-polysaccharide. In one embodiment, the saccharide includes an O-antigen wherein n in any of the O-antigen formulas (such as, for example, the Formulas shown in Table 1 (see also FIG. 9A-9C and FIG. 10A-1OB)) is an integer of at least 1, 2, 3, 4, 5, 10, 20, 21, 22, 23, 24,25,26,27,28,29,30,31,32,33,34,35,36, 37, 38,39,40, and at most200,100, 99,98,97,96,95,94,93,92,91,90,89,88,87,86,81,80,79,78,77,76,75,74,73, 72,71,70,69,68,67,66,65,60,59,58,57,56,55,54,53,52,51, or50. Any minimum value and any maximum value may be combined to define a range. Exemplary ranges include, for example, at least 1 to at most 1000; at least 10 to at most 500; and at least 20 to at most 80, preferably at most 90. In one preferred embodiment, n is at least 31 to at most 90. In a preferred embodiment, n is 40 to 90, more preferably 60 to 85. In one embodiment, the saccharide includes an O-antigen wherein n in any one of the O-antigen Formulas is at least 1 and at most 200. In one embodiment, n in any one of the O-antigen Formulas is at least 5 and at most 200. In one embodiment, n in any one of the O-antigen Formulas is at least 10 and at most 200. In one embodiment, n in any one of the O-antigen Formulas is at least 25 and at most 200. In one embodiment, n in any one of the O-antigen Formulas is at least 50 and at most 200. In one embodiment, n in any one of the O-antigen Formulas is at least 75 and at most 200. In one embodiment, n in any one of the O-antigen Formulas is at least 100 and at most 200. In one embodiment, n in any one of the O-antigen Formulas is at least 125 and at most 200. In one embodiment, n in any one of the O-antigen Formulas is at least 150 and at most 200. In one embodiment, n in any one of the O-antigen Formulas is at least 175 and at most 200. In one embodiment, n in any one of the O-antigen Formulas is at least 1 and at most 100. In one embodiment, n in any one of the O-antigen Formulas is at least 5 and at most 100. In one embodiment, n in any one of the O-antigen Formulas is at least 10 and at most 100. In one embodiment, n in any one of the O-antigen Formulas is at least 25 and at most 100. In one embodiment, n in any one of the 0 antigen Formulas is at least 50 and at most 100. In one embodiment, n in any one of the O-antigen Formulas is at least 75 and at most 100. In one embodiment, n in any one of the O-antigen Formulas is at least 1 and at most 75. In one embodiment, n in any one of the O-antigen Formulas is at least 5 and at most 75. In one embodiment, n in any one of the 0-antigen Formulas is at least 10 and at most 75. In one embodiment, n in any one of the 0-antigen Formulas is at least 20 and at most 75. In one embodiment, n in any one of the 0 antigen Formulas is at least 25 and at most 75. In one embodiment, n in any one of the 0 antigen Formulas is at least 30 and at most 75. In one embodiment, n in any one of the 0 antigen Formulas is at least 40 and at most 75. In one embodiment, n in any one of the 0 antigen Formulas is at least 50 and at most 75. In one embodiment, n in any one of the 0 antigen Formulas is at least 30 and at most 90. In one embodiment, n in any one of the 0 antigen Formulas is at least 35 and at most 85. In one embodiment, n in any one of the 0 antigen Formulas is at least 35 and at most 75. In one embodiment, n in any one of the 0 antigen Formulas is at least 35 and at most 70. In one embodiment, n in any one of the 0 antigen Formulas is at least 35 and at most 60. In one embodiment, n in any one of the 0 antigen Formulas is at least 35 and at most 50. In one embodiment, n in any one of the 0 antigen Formulas is at least 35 and at most 49. In one embodiment, n in any one of the 0 antigen Formulas is at least 35 and at most 48. In one embodiment, n in any one of the 0 antigen Formulas is at least 35 and at most 47. In one embodiment, n in any one of the 0 antigen Formulas is at least 35 and at most 46. In one embodiment, n in any one of the 0 antigen Formulas is at least 36 and at most 45. In one embodiment, n in any one of the 0 antigen Formulas is at least 37 and at most 44. In one embodiment, n in any one of the 0 antigen Formulas is at least 38 and at most 43. In one embodiment, n in any one of the 0 antigen Formulas is at least 39 and at most 42. In one embodiment, n in any one of the 0 antigen Formulas is at least 39 and at most 41. For example, in one embodiment, n in the saccharide is 31, 32, 33, 34, 35, 36, 37, 38, 39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63, 64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88, 89, or 90, most preferably 40. In another embodiment, n is at least 35 to at most 60. For example, in one embodiment, n is any one of 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60, preferably 50. In another preferred embodiment, n is at least 55 to at most 75. For example, in one embodiment, n is 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, or 69, most preferably 60. The saccharide structure may be determined by methods and tools known art, such as, for example, NMR, including 1D, 1H, and/or 13C, 2D TOCSY, DQF-COSY, NOESY, and/or HMQC. In some embodiments, the purified polysaccharide before conjugation has a molecular weight of between 5 kDa and 400 kDa. In other such embodiments, the saccharide has a molecular weight of between 10 kDa and 400 kDa; between 5 kDa and 400 kDa; between 5 kDa and 300 kDa; between 5 kDa and 200 kDa; between 5 kDa and 150 kDa; between 10 kDa and 100 kDa; between 10 kDa and 75 kDa; between 10 kDa and 60 kDa; between 10 kDa and 40 kDa; between 10 kDa and 100 kDa; 10 kDa and 200 kDa; between 15 kDa and 150 kDa; between 12 kDa and 120 kDa; between 12 kDa and 75 kDa; between 12 kDa and 50 kDa; between 12 and 60 kDa; between 35 kDa and 75 kDa; between 40 kDa and 60 kDa; between 35 kDa and 60 kDa; between 20 kDa and 60 kDa; between 12 kDa and 20 kDa; or between 20 kDa and 50 kDa. In further embodiments, the polysaccharide has a molecular weight of between 7 kDa to 15 kDa; 8 kDa to 16 kDa; 9 kDato25 kDa;10kDato100;10kDato60kDa;10kDato70kDa;10kDato160kDa; 15 kDato600kDa;20kDato1000kDa;20kDato600kDa;20kDato400kDa;30kDa to 1,000 KDa; 30 kDa to 60 kDa; 30 kDa to 50 kDa or 5 kDa to 60 kDa. Any whole number integer within any of the above ranges is contemplated as an embodiment of the disclosure. As used herein, the term "molecular weight" of polysaccharide or of carrier protein- polysaccharide conjugate refers to molecular weight calculated by size exclusion chromatography (SEC) combined with multiangle laser light scattering detector (MALLS). A polysaccharide can become slightly reduced in size during normal purification procedures. Additionally, as described herein, polysaccharide can be subjected to sizing techniques before conjugation. Mechanical or chemical sizing maybe employed. Chemical hydrolysis may be conducted using acetic acid. Mechanical sizing may be conducted using High Pressure Homogenization Shearing. The molecular weight ranges mentioned above refer to purified polysaccharides before conjugation (e.g., before activation).

Table 1: E. coliserogroups/serotypes and O-unit moieties

Moiety structure Serogroupf Serogop/ Moiety Structure (0-unit) referred to eroype herein as:

OIAOIAI 01A,O01A1 [+3) -a-L -Rha-(1+3) -a-L -Rha-(1+3) -p-L- Rha-(1+4) -p-D -GcNAc-Foml Formula I 01A (1+ 1 P-D -ManNAc-(1+2) ].

01B 0113 ~[+3) -a- L- Rha-(1+2) -a-L -Rha-(1+2)-a-D -Gal-(1+3) -p-D -GlcNAc- Frua01 FormulaO01B (1+|3-D-M~anNAc-(1+2) ].

Moiety structure Serogroupf referred to Serotype Moiety Structure (0-unit) herein as:

01C [+3)-a-L-Rha-(1+2) -a-L-Rha-(1+3)-a-D-Gal-(1+3) -- D -GcNAc Formula O1C (1+|S-D-ManNAc-(1+2) ].

02 [+3)-a-L-Rha-(1+2)-a-L-Rha-(1+3)-0-L-Rha-(1+4)-0-D-GlcNAc Formula 02 (1+ 1 a-D -Fuc3NAc-(1+2) ],

03 ~ [0-L -RhaNAc(1+4)a-D -Glc-(1+4)|| +3) -p-D -GlcNAc-(1+3) -a-D - omla0 03 Gal-(13)--D-GlcNAc-(1]Formula3

04:K52 [+2)-a-L-Rha-(1+6)-a-D-Glc-(1+3)-a-L-FucNAc-(1+3)-0-D- Formula04:K52 GlcNAc(1+ ],

04:K6 [a-D-Glc-(1+3)|+2)-a-L-Rha-(1+6)-a-D-G c-(1+3)-a-L-FucNAc Formula04:K6 (1+3)-p-D -GlcNAc(1+ ],

05ab [+4)-0-D-Qui3NAc-(1+3)-0-D-Ribf-(1+4) -- D-Gal-(1+3)-a-D- Formula 05ab GalNAc(1+],

05ac (strain [+2)-0-D-Qui3NAc-(1+3)-0-D-Ribf-(1+4) -- D-Gal-(1+3)-a-D- Formula 05ac 180/C3) GalNAc(1+ ], (strain 180/C3)

06:K2; K13; [+4)-a-D-GalNAc-(1+3)-I-D-Man-(1+4)-0-D-Man-(1+3)-a-D- Formula 06:K2; K15 GlcNAc-(1+ | P-D -Gic-(1+2) ], K13; K15

06:K54 [+4)-a-D-GalNAc-(1+3)-I-D-Man-(1+4)-0-D-Man-(1+3)-a-D- Formula 06:K54 GlcNAc-(1+|P-D-GlcNAc-(1+2) ],

07 [a-L-Rha-(1+3)|+3)-p-D-Qui4NAc-(1+2)-a-D-Man-(1+4)-0-D Formula07 Gal-(1+3)-a-D-GlcNAc-(1+ ],

[+3)-a-L-Rha-(1+3) -a-L-Rha-(1+3)-a-D-Gal-(1+3) -p-D -GlcNAc 010 (1+| a-D-Fuc4NAcyl-(1+2) Acyl=acetyl (60%) or (R)-3- Formula 010 hydroxybutyryl (40%) ],

WO 2022/090893 PCT/1B2021/059820

Serogroupf Moiety structure Serotype Moiety Structure (0-unit) referred to herein as:

016 [2---af(6)aDGc(3)aLRaA(13-- Formula 016

017 [aDGc(4) 6---a-(4)aDMn(4)pDMn Formula 017

018A,018ac [-42)-a-L-Rha-(1-46)-a-D-Glc-(1-44)-a-D-Ga-(143)-a-D-GcNAc- Formula 018A, (1-4 1 -D-GlcNAc-(143) ],~ Formula 018ac

018AI [a-DGlc-(146)j 2) a-L Rha-(146) a-D Gic(144) a-D-Gal- Formula 018AI

018B [4)aLRa(4)aDGc(4)aDGi(4)aDGc~- Formula 018B

018BI [a-DGlc-(14)j 3) a-L Rha-(14) a-D Gic(1) aD-Gal- Formula 018BI

021 [-D-Gal-(144)j 43---a-14---i-13---aNc Formula 021

023A [--l(16j6)aDGc(4)-DGi(3)aDGNc Formula 023A

025/025a [P-D-Glc-(1-46)j -4)-a-D-Glc-(1-43)-a-L-FucNAc-(143)-3-D- Formula 025a

1-Glcp

025b Formula 025b 6

[ct-Rhap-(1--3)-ct-Glcp-(1 -- 3)-ct-Rhap2OAc-(1--3)-3 GlcpNAc-],

026 [+3)-a-L-Rha-(1+4)-a-L-FucNAc-(1+3)--D-GcNAc-(1+], Formula026

028 [2)-(R)-Gro-1-P+4)-0-D-GlcNAc-(1+3)-p-D-Galf2Ac-(1+3)-a Formula 028 D-GlcNAc-(1+ ],

[a-L-Rhap-(1+2)-a-L-Fucp 1 036 Formula 036 3 +4)-a-D-Manp-(1+3) -a-L-Fucp-(1+3)-0-D-GlcpNAc-(1+]n

044 [a-D-Glc-(1+4)|+6)-a-D-Man-(1+2)-a-D-Man-(1+2)-0-D-Man Formula044 (1+3)-a-D-GlcNAc(1+ ]n

045 [-+2)-0-D-Glc-(1+3)-a-L-6dTal2Ac-(1+3)-a-D-FucNAc-(1+]In Formula 045

045rel [-+2)-j-D-Glc-(1+3)-a-L-6dTal2Ac-(1+3)-p-D-GcNAc-(1+]In Formula 045rel

054 [->4)-a-d-GalpA-(1-> 2)-a-I-Rhap-(1 -> 2)-0-d-Ribf- Formula 054 (1 -> 4)-0-d-Galp-(1-> 3)-0-d-GIcpNAc-(1->]n

055 [+6)-0-D-GlcNAc-(1+3)-a-D-Gal-(1+3)-0-D-GalNAc-(1+Ia-Col Formula055 (1+2)-p-D -Gal-(1+3) ]n

056 [ +7)-a-Neu5Ac-(2+3)-0-D-Glc-(1+3)-0-D-GlcNAc-(1+| a-D Formula 056 Gal-(1+2) ]n

[+3) -a-D-Galp-(1+3) -a- L-FucpNAc-(1+3)-a-D-GlcpNAc-(1+]n 2 4 057 + ' Formula 057 1 1 a-D-GalpA2/3Ac S-D-Glcp

058 3-0-[(R)-1-carboxyethyl]-a-L-Rha -(1+3) |+4)-a-D-Man Formula 058 (1+4)-a-D-Man2Ac-(1+3)-0-D-GlcNAc-(1+In

064 [-D-Gal-(1+6)| +3)-a-D-ManNAc-(1+3)-0-D-GlcA-(1+3)-0-D- Formula064 Frua6 064 ~ Gal-(1+3)-p-D-GlcNAc(1+]n

[a-L-Rhap a-D-Glcp 1 1

068 Formula 068 3 3

Manp-(1+3)-a-D-GlcpNAC-(1+]n

069 [ +2)-a-L-Rha-(1+2)-a-L-Rha-(1+2)-a-D-Ga-(1+3)--D-GlcNAc- Formula069 (1+]In

073(Strain [a-D-Glc-(1+3)| +4)-a-D-Man-(1+2)-a-D-Man-(1+2)-0-D-Man- Formula073 73-1) (1+3)-a-D-GalNAc(1+]In (Strain 73-1)

-6)-a-D-GIcpNAc-(1-4)-p-D-GalpA-(1-3)-p-D-GIcpNAc-(1-]n 074 Formula 074

[p-D-Fucp3NAc-(1-3)

075 ~~~[0-D-Man-(1+4)1 +3)-a-D-Gal-(1+4)-a-L-Rha-(1+3)-0-D-Foml07 075 GcNAc-(1 nFormula075

076 [+4)-p-D-GlcpA-(1+4)-0-D-GalpNAc3Ac-(1+4)-a-D-GalpNAc Formula 076 (1+3)-p-D-GalpNAc-(1+]n

077 ~~[ +6)-a-D -Man-(1+2) -a-D -Man-(1+2)-0-D-Man-(1+3)-a-D- -oml 7 077 077 GcNAc(Formula

078 ~~[ +4)-0-D -GlcNAc-(1+4)-0-D -Man-(1+4) -a-D -Man-(1+3)-0-D- Foml07 078 GcNAc-(1 nFormula078

086 [ a-D-Gal-(1+3) |+4)-a-L-Fuc-(1+2)--D-Gal-(1+3)-a-D- Formula086 GalNAc-(1+3)--D-GalNAc-(1+ ]n

088 a-L-6dTal-(1+3) +4)-a-D-Man-(1+3)-a-D-Man-(1+3)-0-D Formula 088 GlcNAc-(1+ ].

090 [ +4)-a-L-Fuc2/3Ac-(1+2)-0-D-Gal-(1+3)-a-D-GalNAc-(1+3) - Formula 090 D-Ga[NAc-(1+ ],

098 [ +3)-a-L-QuiNAc-(1+4)-a-D-GalNAcA-(1+3)-a-L-QuiNAc Formula 098 (1+3)-0-D -GlcNAc-(1+ ],

0104 [ +4)-a-D-Gal-(1+4)-a-Neu5,7,9Ac3-(2+3)-p-D-Gal-(1+3)-p-D Formula 0104 GalNAc-(1+],

0111 [ a-Col-(1+6) +4)-a-D-Glc-(1+4)-a-D-Gal-(1+3)-0-D-GlcNAc Formula 0111 (1+ 1 a-Col-(1+3) ],

0113 [ +4)-a-D-GalNAc-(1+4)-a-D-GaA-(1+3)-a-D-Ga-(1+3)--D- Formula 0113 GlcNAc-(1+|I -D-Gal-(1+3) ],

0114 [ +4)-p-D-Qui3N(N-acetyl-L-seryl)-(1+3)-0-D-Ribf-(1+4)-P-D Formula 0114 Gal-(1+3)-a-D-GlcNAc(1+ ],

0119 [ S D-RhaNAc3NFo-(1+3) +2)-0-D-Man-(1+3)-a-D-Gal-(1+4) Formula 0119 a-L-Rha-(1+3)-a-D-GlcNAc-(1+ ]

0121 [ +3)-0-D-Qui4N(N-acetyl-glycyl)-(1+4)-a-D-GaNAc3AcA6N- Formula 0121 (1+4)-a-D-GalNAcA-(1+3) -a-D-GlcNAc-(1+ ],

0124 [ 4-0-[(R)-1-carboxyethyl]-3-D-Glc-(1+6)-a-D-Glc(1+4)1|3)-a Formula 0124 D-Gal-(1+6)-0-D-Galf-(1+3)--D-GalNAc-(1+ ]F

0125 [ a-D-Glc-(1+3) |+4)-0-D-GalNAc-(1+2)-a-D-Man-(1+3)-a-L Fuc-(1+3)-a-D-GalNAc-(1+| I-D-Gal-(1+3)] Formula0125

0126 [ +2)-0-D-Man-(1+3)-0-D-Gal-(1+3)-a-D-GlcNAc-(1+3)-p-D Formula 0126 GcNAc-(1+ | a-L -Fuc-(1+2) ],

0127 [2)-a-L-Fuc-(1+2)-p-D-Gal-(1+3)-a-D-GalNAc-(1+3)-a-D Formula 0127 Ga[NAc-(1+ ].

0128 [ a-L-Fuc-(1+2) |+6)-0-D-Gal-(1+3)-0-D-GalNAc-(1+4)-a-D Formula 0128 Gal-(1+3)-p-D-GalNAc-(1+]In

[ +4)-0-Pse5Ac7Ac-(2+4) -p-D-Gal-(1+4)-0-D -GlcNAc-(1+S 0136 Pse5Ac7Ac=5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-- Formula 0136 L-manno-nonulosonic acid]n

0138 0138 ~~[+42)-a-L -Rha-(1+3)-a-L-Rha-(1+4)-a-D-GalNAcA-(1+3)-0-D- nFormula Frua03 0138

[a-D-Galf-(1+2) -a-L-Rhap 1

0140 Formula 0140 4 +3) -0-a-D-Gcp-(1+4)--D-Gc-(1 (13) 0-D-GalpNAc-(1+]n

0141 [ a-L-Rha-(1+3)1|4)-a-D-Man-(1+3)-a-D-Man6Ac-(1+3)-0-D Formula 0141 GlcNAc-(1+|I -D -GlcA-(1+2) ]n

0142 [ +2)-a-L-Rha-(1+6)-a-D-GalNAc-(1+4)-a-D-GalNAc-(1+3)-a-D Formula 0142 Ga[NAc-(1+ I-D-GacNAc-(1+3) ]n

0143 [ +2)-0-D-GalA6R3,4Ac-(1+3)-a-D-GalNAc-(1+4)-p-D-GlcA Formula 0143 (1+3)-p-D-GlcNAc-(1+ R=1,3-dihydroxy-2-propylamino]n

0147 [ +2)-a-L-Rha-(1+2)-a-L-Rha-(1+4)-0-D-GalA-(1+3)-0-D Formula 0147 Ga[NAc-(1+ ]n

0149 [ +3)-0-D-GlcNAc-(S)-4,6Py-(1+3)-0-L-Rha-(1+4)-0-D-GlcNAc (1+ (S)-4,6Py=4,6-0-[(S)-1-carboxyethylidene]-] Formula0149

0152 [P-L-Rha-(1+4) |+3)-a-D-GlcNAc-(1-P+6)-a-D-Glc-(1+2)-p-D- Formula 0152 Glc-(1+3)-p-D-GlcNAc-(1+ ]n

0157 [ +2)-a-D-Rha4NAc-(1+3)-a-L-Fuc-(1+4)-p-D-Glc-(1+3)-a-D Formula 0157 Ga[NAc-(1+ ].

0158 [a-D-Glc-(1+6)|+4)-a-D-Glc-(1+3)-a-D-GalNAc-(1+3)-0-D Formula0158 Ga[NAc-(1+|I a-L-Rha-(1+3) ],

0159 [ a-L-Fuc-(1+4) +3)-0-D-GlcNAc-(1+4)-a-D-GalA-(1+3)-a-L Formula 0159 Fuc-(1+3)-p-D-GlcNAc-(1+ ],

0164 [ S D-Glc-(1+6)-a-D-Glc(1+4) +3)-p-D-Gal-(1+6)-p-D-Galf Formula 0164 (1+3)-p-D-GalNAc-(1+ ],

0173 [ a-L-Fuc-(1+4) |+3)-a-D-Glc-(1-P+6)-a-D-Glc-(1+2) -p-D -Glc Formula 0173 (1+3)-p-D-GlcNAc-(1+]u

62D 1 Suggested as [a-D-Gal(1+6) +2)-0-D-Qui3NAc-(1+3)-a-L-Rha-(1+3)-0-D Formula 62D1 Erwinia Gal-(1+3) -a-D -FucNAc-(1+ ]n herbicola

022 [-+6)-a-D-Glc-(1+4)--D-GlcA-(1+4)-p-D-GalNAc3Ac-(1+3)-a- Formula 022 D-Gal-(1+3)-p-D-GalNAc-(1+]n

035 [ +3)-a-L-Rha-(1+2)-a-L-Rha-(1+3)-a-L-Rha-(1+2)-a-L-Rha Formula 035 (1+3)-0-D-GlcNAc-(1+|I a-D -GalNAcA6N-(1+2) ]n

065 [ +2)-0-D-Qui3NAc-(1+4)-a-D-GalA6N-(1+4)-a-D-GalNAc Formula 065 (1+4)-0-D-GalA-(1+3)-a-D-GlcNAc-(1+ ]n

066 [ +2)-0-D-Man-(1+3)-a-D-GlcNAc-(1+2)-p-D-Glc3Ac-(1+3)-a-L- Formula066 6dTal-(1+3)-a-D-GlcNAc(1+ ]nF

083 +6)-a-D-Glc-(1+4)--D-GlcA-(1+6)-p-D-Gal-(1+4)-0-D-Gal- Formula083 oml 8 083 ~ ~(1+4)-p-D-GlcNAc-(1+ ],

[+44)-a-D-Qui3NAcyl-(1+4)-0-D-Gal-(1+4)-0-D-GlcNAc-(1+4) 091 0-D-GlcA6NGly-(1+3)-0-D-GlcNAc-(1+ Acyl=(R)-3- Formula091 hydroxybutyryl],

0105 [ S-D-Ribf-(1+3)-|4)-a-D-GlcA2Ac3Ac-(1+2)-a-L-Rha4Ac- Formula 105 (1+3)-0-L-Rha-(1+4)-0-L-Rha-(1+3)-0-D-GlcNAc6Ac-(1+ ] F

0116 0116 ~~[+42)-0-D-Qui4NAc-(1+6) -a-D-GlcNAc-(1+4)-a-D -GalNAc- (14)-a-D-GaA-(13)--D-GFormula Frua01 0116

0117 [ +4)-0-D-GalNAc-(1+3)-a-L-Rha-(1+4)-a-D-Glc-(1+4)-0-D-Gal Formula 0117 (1+3)-a-D-GalNAc-(1+],

0139 [ S D-Glc-(1+3) +3)-a-L-Rha-(1+4)-a-D-GalA-(1+2)-a-L-Rha Formula 0139 (1+3)-a-L-Rha-(1+2)-a-L-Rha-(1+3)-a-D -GlcNAc-(1+ ],

0153 [ +2)-0-D-Ribf-(1+4)-0-D-Gal-(1+4)-a-D-GlcNAc-(1+4)-p-D Formula 0153 Gal-(1+3)-a-D-GlcNAc-(1+ ],

0167 [a-D-Galf-(1+4)|+2)-0-D-GalA6N(L)Ala-(1+3)-a-D-GlcNAc Formula0167 (1+2)-0-D-Galf-(1+5)-0-D-Galf-(1+3)-0-D-GlcNAc-(1+ ],

0172 [ +3)-a-L-FucNAc-(1+4)-a-D-Glc6Ac-(1-P+4)-a-D-Glc-(1+3)-a- Formula 0172 L-FucNAc-(1+3)-a-D -GlcNAc-(1+ ],

08 [->2)-Ca-D-Man-(1-2)-Ca-D-Man-(1-3)-3-D-Man-(1->] Formula 08

[->2)-Ca-D-Man-(1-2)--D-Man-(1-3)-a-D-Man-(1-3)-a-D- Formula 09a 09a Man-(1- ],

[ -2)-[a-D -Man-(1-2)]2-CC-D -Man-(1-3)-CC-D -Man-(1->3)- Formula 09 09 C-D-Man-(1-> ]1

020ab [->2)-P-D-Ribf-(1->4)-a-D-Gal-(1- ]In Formula 020ab

020ac [CC-D-Gal-(1->3) I -2)--D-Ribf-(1->4)-a-D-Gal-(1- ], Formula 020ac

052 [ ->3)-p-D-Fucf-(1->3)--D-6dmanHep2Ac-(1- ]In Formula 052

[->3)-a-L-Rha-(1->3)-p-L-Rha-(1-> Ip-D-Xuf-(2->2)P-D- Formula 097 097 Xulf-(2->2) ]n

t 3-D-6dmanHep2Ac is 2-0-acetyl-6-deoxy-3-D-manno-heptopyranosyl. * 3-D-Xulfis 3-D-threo-pentofuranosyl.

D. Core Oligosaccharide

The core oligosaccharide is positioned between Lipid A and the O-antigen outer region in wild-type E. coli LPS. More specifically, the core oligosaccharide is the part of the polysaccharide that includes the bond between the O-antigen and the lipid A in wild type E. coli. This bond includes a ketosidic bond between the hemiketal function of the innermost 3-deoxy-d manno-oct-2-ulosonic acid (KDO)) residue and a hydroxyl-group of a GcNAc-residue of the lipid A. The core oligosaccharide region shows a high degree of similarity among wild-type E. coli strains. It usually includes a limited number of sugars. The core oligosaccharide includes an inner core region and an outer core region. More specifically, the inner core is composed primarily of L-glycero-D-manno-heptose (heptose) and KDO residues. The inner core is highly conserved. A KDO residue includes the following Formula KDO:

The outer region of the core oligosaccharide displays more variation than the inner core region, and differences in this region distinguish the five chemotypes in E. coli: R1, R2, R3, R4, and K-12. See FIG. 24, which illustrates generalized structures of the carbohydrate backbone of the outer core oligosaccharides of the five known chemotypes. HeplI is the last residue of the inner core oligosaccharide. While all of the outer core oligosaccharides share a structural theme, with a (hexose) 3 carbohydrate backbone and two side chain residues, the order of hexoses in the backbone and the nature, position, and linkage of the side chain residues can all vary. The structures for the R1 and R4 outer core oligosaccharides are highly similar, differing in only a single p-linked residue. The core oligosaccharides of wild-type E. coliare categorized in the art based on the structures of the distal oligosaccharide, into five different chemotypes: E. coli R1, E. coli R2, E. coli R3, E. coli R4, and E. coli K12. In a preferred embodiment, the compositions described herein include glycoconjugates in which the 0-polysaccharide includes a core oligosaccharide bound to the O-antigen. In one embodiment, the composition induces an immune response against at least any one of the core E. coli chemotypes E. coli R1, E. coli R2, E. coli R3, E. coli R4, and E. coli K12. In another embodiment, the composition induces an immune response against at least two core E. coli chemotypes. In another embodiment, the composition induces an immune response against at least three core E. coli chemotypes. In another embodiment, the composition induces an immune response against at least four core E. coli chemotypes. In another embodiment, the composition induces an immune response against all five core E. coli chemotypes. In another preferred embodiment, the compositions described herein include glycoconjugates in which the 0-polysaccharide does not include a core oligosaccharide bound to the O-antigen. In one embodiment, such a composition induces an immune response against at least any one of the core E. coli chemotypes E. coli R1, E. coli R2, E. coli R3, E. coli R4, and E. coli K12, despite the glycoconjugate having an 0 polysaccharide that does not include a core oligosaccharide. E. coliserotypes may be characterized according to one of the five chemotypes. Table 2 lists exemplary serotypes characterized according to chemotype. The serotypes in bold represent the serotypes that are most commonly associated with the indicated core chemotype. Accordingly, in a preferred embodiment, the composition induces an immune response against at least any one of the core E. coli chemotypes E. coli R1, E. coli R2, E. coli R3, E. coli R4, and E. coli K12, which includes an immune response against any one of the respective corresponding E. coliserotypes.

Table 2: Core Chemotype and associated E. coli Serotype

Core chemotype Serotype R1 025a, 06, 02, 01, 075, 04, 016, 08, 018, 09, 013, 020, 021, 091, and 0163.

R2 021, 044, 011, 089, 0162, 09 R3 025b, 015, 0153, 021, 017, 011, 0159, 022086,093 R4 02,01,086,07,0102,0160,0166 K-12 025b, 016

In some embodiments, the composition includes a saccharide that includes a structure derived from a serotype having an R1 chemotype, e.g., selected from a saccharide having Formula 025a, Formula 06, Formula 02, Formula 01, Formula 075, Formula 04, Formula 016, Formula 08, Formula 018, Formula 09, Formula 013, Formula 020, Formula 021, Formula 091, and Formula 0163, wherein n is 1 to 100. In some embodiments, the saccharide in said composition further includes an E. coli R1 core moiety, e.g., shown in FIG. 24. In some embodiments, the composition includes a saccharide that includes a structure derived from a serotype having an R1 chemotype, e.g., selected from a saccharide having Formula 025a, Formula 06, Formula 02, Formula 01, Formula 075, Formula 04, Formula 016, Formula 018, Formula 013, Formula 020, Formula 021, Formula 091, and Formula 0163, wherein n is 1 to 100, preferably 31 to 100, preferably from 31 to 90more preferably 35 to 90, most preferably 35 to 65. In some embodiments, the saccharide in said composition further includes an E. coli R1 core moiety in the saccharide. In some embodiments, the composition includes a saccharide that includes a structure derived from a serotype having an R2 chemotype, e.g., selected from a saccharide having Formula 021, Formula 044, Formula 011, Formula 089, Formula 0162, and Formula 09, wherein n is 1 to 100, preferably 31 to 100, preferably from 31 to 90, more preferably 35 to 90, most preferably 35 to 65. In some embodiments, the saccharide in said composition further includes an E. coli R2 core moiety, e.g., shown in FIG. 24. In some embodiments, the composition includes a saccharide that includes a structure derived from a serotype having an R3 chemotype, e.g., selected from a saccharide having Formula 025b, Formula 015, Formula 0153, Formula 021, Formula 017, Formula 011, Formula 0159, Formula 022, Formula 086, and Formula 093, wherein n is 1 to 100, preferably 31 to 100, preferably from 31 to 90, more preferably 35 to 90, most preferably 35 to 65. In some embodiments, the saccharide in said composition further includes an E. coli R3 core moiety, e.g., shown in FIG. 24. In some embodiments, the composition includes a saccharide that includes a structure derived from a serotype having an R4 chemotype, e.g., selected from a saccharide having Formula 02, Formula 01, Formula 086, Formula 07, Formula 0102, Formula 0160, and Formula 0166, wherein n is 1 to 100, preferably 31 to 100, preferably from 31 to 90, more preferably 35 to 90, most preferably 35 to 65. In some embodiments, the saccharide in said composition further includes an E. coli R4 core moiety, e.g., shown in FIG. 24. In some embodiments, the composition includes a saccharide that includes a structure derived from a serotype having an K-12 chemotype (e.g., selected from a saccharide having Formula 025b and a saccharide having Formula 016), wherein n is 1 to 1000, preferably 31 to 100, preferably from 31 to 90, more preferably 35 to 90, most preferably 35 to 65. In some embodiments, the saccharide in said composition further includes an E. coli K-12 core moiety, e.g., shown in FIG. 24. In some embodiments, the saccharide includes the core saccharide. Accordingly, in one embodiment, the O-polysaccharide further includes an E. coli R1 core moiety. In another embodiment, the O-polysaccharide further includes an E. coli R2 core moiety. In another embodiment, the 0-polysaccharide further includes an E. coli R3 core moiety. In another embodiment, the 0-polysaccharide further includes an E. coli R4 core moiety. In another embodiment, the 0-polysaccharide further includes an E. coli K12 core moiety. In some embodiments, the saccharide does not include the core saccharide. Accordingly, in one embodiment, the O-polysaccharide does not include an E. coli R1 core moiety. In another embodiment, the O-polysaccharide does not include an E. coli R2 core moiety. In another embodiment, the O-polysaccharide does not include an E. coli R3 core moiety. In another embodiment, the O-polysaccharide does not include an E. coli R4 core moiety. In another embodiment, the O-polysaccharide does not include an E. coli K12 core moiety.

E. Conjugated O-Antigens Chemical linkage of O-antigens or preferably O-polysaccharides to protein carriers may improve the immunogenicity of the O-antigens or O-polysaccharides. However, variability in polymer size represents a practical challenge for production. In commercial use, the size of the saccharide can influence the compatibility with different conjugation synthesis strategies, product uniformity, and conjugate immunogenicity. Controlling the expression of a Wzz family protein chain length regulator through manipulation of the 0- antigen synthesis pathway allows for production of a desired length of O-antigen chains in a variety of Gram-negative bacterial strains, including E. coli. In one embodiment, the purified saccharides are chemically activated to produce activated saccharides capable of reacting with the carrier protein. Once activated, each saccharide is separately conjugated to a carrier protein to form a conjugate, namely a glycoconjugate. As used herein, the term "glycoconjugate" refers to a saccharide covalently linked to a carrier protein. In one embodiment a saccharide is linked directly to a carrier protein. In another embodiment, a saccharide is linked to a protein through a spacer/linker. Conjugates may be prepared by schemes that bind the carrier to the 0-antigen at one or at multiple sites along the O-antigen, or by schemes that activate at least one residue of the core oligosaccharide. In one embodiment, each saccharide is conjugated to the same carrier protein. If the protein carrier is the same for 2 or more saccharides in the composition, the saccharides may be conjugated to the same molecule of the carrier protein (e.g., carrier molecules having 2 or more different saccharides conjugated to it). In a preferred embodiment, the saccharides are each individually conjugated to different molecules of the protein carrier (each molecule of protein carrier only having one type of saccharide conjugated to it). In said embodiment, the saccharides are said to be individually conjugated to the carrier protein. The chemical activation of the saccharides and subsequent conjugation to the carrier protein can be achieved by the activation and conjugation methods disclosed herein. After conjugation of the polysaccharide to the carrier protein, the glycoconjugates are purified (enriched with respect to the amount of polysaccharide- protein conjugate) by a variety of techniques. These techniques include concentration/diafiltration operations, precipitation/elution, column chromatography, and depth filtration. After the individual glycoconjugates are purified, they are compounded to formulate the immunogenic composition of the present invention. Activation. The present invention further relates to activated polysaccharides produced from any of the embodiments described herein wherein the polysaccharide is activated with a chemical reagent to produce reactive groups for conjugation to a linker or carrier protein. In some embodiments, the saccharide of the invention is activated prior to conjugation to the carrier protein. In some embodiments, the degree of activation does not significantly reduce the molecular weight of the polysaccharide. For example, in some embodiments, the degree of activation does not cleave the polysaccharide backbone. In some embodiments, the degree of activation does not significantly impact the degree of conjugation, as measured by the number of lysine residues modified in the carrier protein, such as, CRM1 97 (as determined by amino acid analysis). For example, in some embodiments, the degree of activation does not significantly increase the number of lysine residues modified (as determined by amino acid analysis) in the carrier protein by 3-fold, as compared to the number of lysine residues modified in the carrier protein of a conjugate with a reference polysaccharide at the same degree of activation. In some embodiments, the degree of activation does not increase the level of unconjugated free saccharide. In some embodiments, the degree of activation does not decrease the optimal saccharide/protein ratio. In some embodiments, the activated saccharide has a percentage of activation wherein moles of thiol per saccharide repeat unit of the activated saccharide is between 1-100%, such as, for example, between 2-80%, between 2-50%, between 3-30%, and between 4-25%. The degree of activation is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 2 20%, 2 30%, 2 40%, 2 50%, 2 60%, 2 70%, 2 80%, or 2 90%, or about 100%. Preferably, the degree of activation is at most 50%, more preferably at most 25%. In one embodiment, the degree of activation is at most 20%. Any minimum value and any maximum value may be combined to define a range. In one embodiment, the polysaccharide is activated with 1 -cyano-4 dimethylamino pyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated polysaccharide is then coupled directly or via a spacer (linker) group to an amino group on the carrier protein (preferably CRM 197 or tetanus toxoid). For example, the spacer may be cystamine or cysteamine to give a thiolated polysaccharide which could be coupled to the carrier via a thioether linkage obtained after reaction with a maleimide-activated carrier protein (for example using N-[Y maleimidobutyrloxy]succinimide ester (GMBS)) or a haloacetylated carrier protein (for example using iodoacetimide, N-succinimidyl bromoacetate (SBA; SIB), N succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), sulfosuccinimidyl(4 iodoacetyl)aminobenzoate (sulfo-SIAB), N-succinimidyl iodoacetate (SIA), or succinimidyl 3-[bromoacetamido]proprionate (SBAP)). In one embodiment, the cyanate ester (optionally made by CDAP chemistry) is coupled with hexane diamine or adipic acid dihydrazide (ADH) and the amino-derivatised saccharide is conjugated to the carrier protein (e.g., CRM 197) using carbodiimide (e.g., EDAC or EDC) chemistry via a carboxyl group on the protein carrier. Other suitable techniques for conjugation use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU. Conjugation may involve a carbonyl linker which may be formed by reaction of a free hydroxyl group of the saccharide with CDI followed by reaction with a protein to form a carbamate linkage. This may involve reduction of the anomeric terminus to a primary hydroxyl group, optional protection/deprotection of the primary hydroxyl group, reaction of the primary hydroxyl group with CDI to form a CDI carbamate intermediate and coupling the CDI carbamate intermediate with an amino group on a protein (CDI chemistry).

Molecular weight. In some embodiments, the glycoconjugate comprises a saccharide having a molecular weight of between 10 kDa and 2,000 kDa. In other embodiments, the saccharide has a molecular weight of between 50 kDa and 1,000 kDa. In other embodiments, the saccharide has a molecular weight of between 70 kDa and 900 kDa. In other embodiments, the saccharide has a molecular weight of between 100 kDa and 800 kDa. In other embodiments, the saccharide has a molecular weight of between 200 kDa and 600 kDa. In further embodiments, the saccharide has a molecular weight of 100 kDa to 1000 kDa; 100 kDa to900kDa;100kDato800kDa;100kDato700kDa;100kDato600kDa;100kDato500 kDa; 100 kDa to 400 kDa; 100 kDa to 300 kDa; 150 kDa to 1,000 kDa; 150 kDa to 900 kDa; 150kDato800kDa;150kDato700kDa;150kDato600kDa;150kDato500kDa;150kDa to400kDa;150kDato300kDa;200kDato1,000kDa;200kDato900kDa;200kDato800 kDa;200kDato700kDa;200kDato600kDa;200kDato500kDa;200kDato400kDa;200 kDato300;250kDato1,000kDa;250kDato900kDa;250kDato800kDa;250kDato700 kDa;250kDato600kDa;250kDato500kDa;250kDato400kDa;250kDato350kDa;300 kDato1,000kDa;300kDato900kDa;300kDato800kDa;300kDato700kDa;300kDato 600 kDa; 300 kDa to 500 kDa; 300 kDa to 400 kDa; 400 kDa to 1,000 kDa; 400 kDa to 900 kDa;400kDato800kDa;400kDato700kDa;400kDato600kDa;500kDato600kDa. In one embodiment, the glycoconjugate having such a molecular weight is produced by single-end conjugation. In another embodiment, the glycoconjugate having such a molecular weight is produced by reductive amination chemistry (RAC) prepared in aqueous buffer. Any whole number integer within any of the above ranges is contemplated as an embodiment of the disclosure. In some embodiments, the glycoconjugate of the invention has a molecular weight of between 400 kDa and 15,000 kDa; between 500 kDa and 10,000 kDa; between 2,000 kDa and 10,000 kDa; between 3,000 kDa and 8,000 kDa; or between 3,000 kDa and 5,000 kDa. In other embodiments, the glycoconjugate has a molecular weight of between 500 kDa and 10,000 kDa. In other embodiments, glycoconjugate has a molecular weight of between 1,000 kDa and 8,000 kDa. In still other embodiments, the glycoconjugate has a molecular weight of between 2,000 kDa and 8,000 kDa or between 3,000 kDa and 7,000 kDa. In further embodiments, the glycoconjugate of the invention has a molecular weight of between 200 kDa and 20,000 kDa; between 200 kDa and 15,000 kDa; between 200 kDa and 10,000 kDa; between 200 kDa and 7,500 kDa; between 200 kDa and 5,000 kDa; between 200 kDa and 3,000 kDa; between 200 kDa and 1,000 kDa; between 500 kDa and 20,000 kDa; between 500 kDa and 15,000 kDa; between 500 kDa and 12,500 kDa; between 500 kDa and 10,000 kDa; between 500 kDa and 7,500 kDa; between 500 kDa and 6,000 kDa; between 500 kDa and 5,000 kDa; between 500 kDa and 4,000 kDa; between 500 kDa and 3,000 kDa; between 500 kDa and 2,000 kDa; between 500 kDa and 1 ,500 kDa; between 500 kDa and 1,000 kDa; between 750 kDa and 20,000 kDa; between 750 kDa and 15,000 kDa; between 750kDa and 12,500 kDa; between 750kDa and 10,000 kDa; between 750kDa and 7,500 kDa; between 750 kDa and 6,000 kDa; between 750 kDa and 5,000 kDa; between 750 kDa and 4,000 kDa; between 750 kDa and 3,000 kDa; between 750 kDa and 2,000 kDa; between 750 kDa and 1,500 kDa; between 1,000 kDa and 15,000 kDa; between 1,000 kDa and 12,500 kDa; between 1,000 kDa and 10,000 kDa; between 1,000 kDa and 7,500 kDa; between 1,000 kDa and 6,000 kDa; between 1,000 kDa and 5,000 kDa; between 1,000 kDa and 4,000 kDa; between 1,000 kDa and 2,500 kDa; between 2,000 kDa and 15,000 kDa; between 2,000 kDa and 12,500 kDa; between 2,000 kDa and 10,000 kDa; between 2,000 kDa and 7,500 kDa; between 2,000 kDa and 6,000 kDa; between 2,000 kDa and 5,000 kDa; between 2,000 kDa and 4,000 kDa; or between 2,000 kDa and 3,000 kDa. In one embodiment, the glycoconjugate having such a molecular weight is produced by eTEC conjugation described herein. In another embodiment, the glycoconjugate having such a molecular weight is produced by reductive amination chemistry (RAC). In another embodiment, the glycoconjugate having such a molecular weight is produced by reductive amination chemistry (RAC) prepared in DMSO. In further embodiments, the glycoconjugate of the invention has a molecular weight of between 1,000 kDa and 20,000 kDa; between 1,000 kDa and 15,000 kDa; between 2,000 kDa and 10,000 kDa; between 2000 kDa and 7,500 kDa; between 2,000 kDa and 5,000 kDa; between 3,000 kDa and 20,000 kDa; between 3,000 kDa and 15,000 kDa; between 3,000 kDa and 12,500 kDa; between 4,000 kDa and 10,000 kDa; between 4,000 kDa and 7,500 kDa; between 4,000 kDa and 6,000 kDa; or between 5,000 kDa and 7,000 kDa. In one embodiment, the glycoconjugate having such a molecular weight is produced by reductive amination chemistry (RAC). In another embodiment, the glycoconjugate having such a molecular weight is produced by reductive amination chemistry (RAC) prepared in DMSO. In another embodiment, the glycoconjugate having such a molecular weight is produced by eTEC conjugation described herein. In further embodiments, the glycoconjugate of the invention has a molecular weight of between 5,000 kDa and 20,000 kDa; between 5,000 kDa and 15,000 kDa; between 5,000 kDa and 10,000 kDa; between 5,000 kDa and 7,500 kDa; between 6,000 kDa and 20,000 kDa; between 6,000 kDa and 15,000 kDa; between 6,000 kDa and 12,500 kDa; between 6,000 kDa and 10,000 kDa or between 6,000 kDa and 7,500 kDa. The molecular weight of the glycoconjugate may be measured by SEC-MALLS. Any whole number integer within any of the above ranges is contemplated as an embodiment of the disclosure. The glycoconjugates of the invention may also be characterized by the ratio (weight/weight) of saccharide to carrier protein. In some embodiments, the ratio of polysaccharide to carrier protein in the glycoconjugate (w/w) is between 0.5 and 3 (e.g., about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1 , about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, or about 3.0). In other embodiments, the saccharide to carrier protein ratio (w/w) is between 0.5 and 2.0, between 0.5 and 1.5, between 0.8 and 1.2, between 0.5 and 1.0, between 1.0 and 1.5 or between 1.0 and 2.0. In further embodiments, the saccharide to carrier protein ratio (w/w) is between 0.8 and 1.2. In a preferred embodiment, the ratio of polysaccharide to carrier protein in the conjugate is between 0.9 and 1.1. In some such embodiments, the carrier protein is CRM197 .

The glycoconjugates may also be characterized by their molecular size distribution (Kd).

Size exclusion chromatography media (CL-4B) can be used to determine the relative molecular size distribution of the conjugate. Size Exclusion Chromatography (SEC) is used in gravity fed columns to profile the molecular size distribution of conjugates. Large molecules excluded from the pores in the media elute more quickly than small molecules. Fraction collectors are used to collect the column eluate. The fractions are tested colorimetrically by saccharide assay. For the determination of K, columns are calibrated to establish the fraction at which molecules are fully excluded (Vo), (K=0), and the fraction representing the maximum retention (Vi), (Kd=1 ). The fraction at which a specified sample attribute is reached (Ve), is related to Kd by the expression, Kd = (Ve - Vo)/ (Vi - Vo). Free saccharide. The glycoconjugates and immunogenic compositions of the invention may include free saccharide that is not covalently conjugated to the carrier protein, but is nevertheless present in the glycoconjugate composition. The free saccharide may be non covalently associated with (i.e., non-covalently bound to, adsorbed to, or entrapped in or with) the glycoconjugate. In a preferred embodiment, the glycoconjugate comprises at most 50%, 45%, 40%, 35%, 30%, 25%, 20% or 15% of free polysaccharide compared to the total amount of polysaccharide. In a preferred embodiment the glycoconjugate comprises less than about 25% of free polysaccharide compared to the total amount of polysaccharide. In a preferred embodiment the glycoconjugate comprises at most about 20% of free polysaccharide compared to the total amount of polysaccharide. In a preferred embodiment the glycoconjugate comprises at most about 15% of free polysaccharide compared to the total amount of polysaccharide. In another preferred embodiment, the glycoconjugate comprises at most about 20%, 19%, 18%, 17%,16%,15%,14%,13%,12%,11%,10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of free polysaccharide compared to the total amount of polysaccharide. In a preferred embodiment the glycoconjugate comprises less than about 8% of free polysaccharide compared to the total amount of polysaccharide. In a preferred embodiment the glycoconjugate comprises at most about 6% of free polysaccharide compared to the total amount of polysaccharide. In a preferred embodiment the glycoconjugate comprises at most about 5% of free polysaccharide compared to the total amount of polysaccharide. See, for example, Table 19, Table 20, Table 21, Table 22, Table 23, Table 24, and Table 25. Covalent linkage. In other embodiments, the conjugate comprises at least one covalent linkage between the carrier protein and saccharide for every 5 to 10 saccharide repeat units; every 2 to 7 saccharide repeat units; every 3 to 8 saccharide repeat units; every 4 to 9 saccharide repeat units; every 6 to 1 1 saccharide repeat units; every 7 to 12 saccharide repeat units; every 8 to 13 saccharide repeat units; every 9 to 14 saccharide repeat units; every 10 to 15 saccharide repeat units; every 2 to 6 saccharide repeat units, every 3 to 7 saccharide repeat units; every 4 to 8 saccharide repeat units; every 6 to 10 saccharide repeat units; every 7 to 1 1 saccharide repeat units; every 8 to 12 saccharide repeat units; every 9 to 13 saccharide repeat units; every 10 to 14 saccharide repeat units; every 10 to 20 saccharide repeat units; every 4 to 25 saccharide repeat units or every 2 to 25 saccharide repeat units. In frequent embodiments, the carrier protein is CRM 197 . In another embodiment, at least one linkage between carrier protein and saccharide occurs for every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 saccharide repeat units of the polysaccharide. In one embodiment, the carrier protein is CRM 197. Any whole number integer within any of the above ranges is contemplated as an embodiment of the disclosure. Lysine residues. Another way to characterize the glycoconjugates of the invention is by the number of lysine residues in the carrier protein (e.g., CRM 197) that become conjugated to the saccharide which can be characterized as a range of conjugated lysines (degree of conjugation). The evidence for lysine modification of the carrier protein, due to covalent linkages to the polysaccharides, can be obtained by amino acid analysis using routine methods known to those of skill in the art. Conjugation results in a reduction in the number of lysine residues recovered, compared to the carrier protein starting material used to generate the conjugate materials. In a preferred embodiment, the degree of conjugation of the glycoconjugate of the invention is between 2 and 15, between 2 and 13, between 2 and 10, between 2 and 8, between 2 and 6, between 2 and 5, between 2 and 4, between 3 and 15, between 3 and 13, between 3 and 10, between 3 and 8, between 3 and 6, between 3 and 5, between 3 and 4, between 5 and 15, between 5 and 10, between 8 and 15, between 8 and 12, between

10 and 15 or between 10 and 12. In one embodiment, the degree of conjugation of the glycoconjugate of the invention is about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 1 1 , about 12, about 13, about 14 or about 15. In a preferred embodiment, the degree of conjugation of the glycoconjugate of the invention is between 4 and 7. In some such embodiments, the carrier protein is CRM19 7

. The frequency of attachment of the saccharide chain to a lysine on the carrier protein is another parameter for characterizing the glycoconjugates of the invention. For example, in some embodiments, at least one covalent linkage between the carrier protein and the polysaccharide for every 4 saccharide repeat units of the polysaccharide. In another embodiment, the covalent linkage between the carrier protein and the polysaccharide occurs at least once in every 10 saccharide repeat units of the polysaccharide. In another embodiment, the covalent linkage between the carrier protein and the polysaccharide occurs at least once in every 15 saccharide repeat units of the polysaccharide. In a further embodiment, the covalent linkage between the carrier protein and the polysaccharide occurs at least once in every 25 saccharide repeat units of the polysaccharide. 0-acetylation. In some embodiments, the saccharides of the invention are 0 acetylated. In some embodiments, the glycoconjugate comprises a saccharide which has a degree of 0-acetylation of between 10-100%, between 20-100%, between 30-100%, between 40-100%, between 50-100%, between 60-100%, between 70-100%, between 75-100%, 80 100%, 90-100%, 50- 90%, 60-90%, 70-90% or 80-90%. In other embodiments, the degree of 0-acetylation is 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, or 290%, or about 100%. By % of0-acetylation it is meant the percentage of a given saccharide relative to 100% (where each repeat unit is fully acetylated relative to its acetylated structure). In some embodiments, the glycoconjugate is prepared by reductive amination. In some embodiments, the glycoconjugate is a single-end-linked conjugated saccharide, wherein the saccharide is covalently bound to a carrier protein directly. In some embodiments, the glycoconjugate is covalently bound to a carrier protein through a (2-((2-oxoethyl)thio)ethyl) carbamate (eTEC) spacer. REDUCTIVE AMINATION. In one embodiment, the saccharide is conjugated to the carrier protein by reductive amination (such as described in U.S. Patent Appl. Pub. Nos. 2006/0228380, 2007/0231340, 2007/0184071 and 2007/0184072, WO 2006/110381, WO 2008/079653, and WO 2008/143709). Reductive amination includes (1) oxidation of the saccharide, (2) reduction of the activated saccharide and a carrier protein to form a conjugate. Before oxidation, the saccharide is optionally hydrolyzed. Mechanical or chemical hydrolysis may be employed. Chemical hydrolysis may be conducted using acetic acid.

The oxidation step may involve reaction with periodate. The term "periodate" as used herein refers to both periodate and periodic acid. The term also includes both metaperiodate (104-) and orthoperiodate (1035-) and the various salts of periodate (e.g., sodium periodate and potassium periodate). In one embodiment the polysaccharide is oxidized in the presence of metaperiodate, preferably in the presence of sodium periodate (NalO4). In another embodiment the polysaccharide is oxidized in the presence of orthoperiodate, preferably in the presence of periodic acid. In one embodiment, the oxidizing agent is a stable nitroxyl or nitroxide radical compound, such as piperidine-N-oxy or pyrrolidine-N-oxy compounds, in the presence of an oxidant to selectively oxidize primary hydroxyls. In said reaction, the actual oxidant is the N-oxoammonium salt, in a catalytic cycle. In an aspect, said stable nitroxyl or nitroxide radical compound are piperidine-N-oxy or pyrrolidine-N-oxy compounds. In an aspect, said stable nitroxyl or nitroxide radical compound bears a TEMPO (2,2,6,6 tetramethyl-1 -piperidinyloxy) or a PROXYL (2,2,5,5-tetramethyl-1 -pyrrolidinyloxy) moiety. In an aspect, said stable nitroxyl radical compound is TEMPO or a derivative thereof. In an aspect, said oxidant is a molecule bearing a N-halo moiety. In an aspect, said oxidant is selected from any one of N-ChloroSuccinimide, N-Bromosuccinimide, N lodosuccinimide, Dichloroisocyanuric acid, 1 ,3,5-trichloro-I ,3,5-triazinane-2,4,6-trione, Dibromoisocyanuric acid, 1 ,3,5-tribromo-I ,3,5-triazinane-2,4,6-trione, Diiodoisocyanuric acid and 1 ,3,5-triiodo-I ,3,5-triazinane-2,4,6-trione. Preferably said oxidant is N Chlorosuccinimide. Following the oxidation step of the saccharide, the saccharide is said to be activated and is referred to as "activated" herein below. The activated saccharide and the carrier protein may belyophilised (freeze-dried), either independently (discrete lyophilization) or together (co-lyophilized). In one embodiment the activated saccharide and the carrier protein are co-lyophilized. In another embodiment the activated polysaccharide and the carrier protein arelyophilized independently. In one embodiment the lyophilization takes place in the presence of a non reducing sugar, possible non-reducing sugars include sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol and palatinit. The next step of the conjugation process is the reduction of the activated saccharide and a carrier protein to form a conjugate (so-called reductive amination), using a reducing agent. Suitable reducing agents include the cyanoborohydrides, such as sodium cyanoborohydride, sodium triacetoxyborohydride or sodium or zinc borohydride in the presence of Bronsted or Lewis acids), amine boranes such as pyridine borane, 2-Picoline Borane, 2,6-diborane-methanol, dimethylamine-borane, t

BuMe'PrN-BH3, benzylamine-BH3 or 5-ethyl-2-methylpyridine borane (PEMB), borane-pyridine, or borohydride exchange resin. In one embodiment the reducing agent is sodium cyanoborohydride. In an embodiment, the reduction reaction is carried out in aqueous solvent (e.g.

, selected from PBS, MES, HEPES, Bis-tris, ADA, PIPES, MOPSO, BES, MOPS, DIPSO, MOBS, HEPPSO, POPSO, TEA, EPPS, Bicine or HEPB, at a pH between 6.0 and 8.5, 7.0 and 8.0, or 7.0 and 7.5), in another embodiment the reaction is carried out in aprotic solvent. In an embodiment, the reduction reaction is carried out in DMSO (dimethylsulfoxide) or in DMF (dimethylformamide) solvent. The DMSO or DMF solvent may be used to reconstitute the activated polysaccharide and carrier protein which has beenlyophilized. At the end of the reduction reaction, there may be unreacted aldehyde groups remaining in the conjugates, these may be capped using a suitable capping agent. In one embodiment this capping agent is sodium borohydride (NaBH 4). Following the conjugation (the reduction reaction and optionally the capping), the glycoconjugates may be purified (enriched with respect to the amount of polysaccharide-protein conjugate) by a variety of techniques known to the skilled person. These techniques include dialysis, concentration/diafiltration operations, tangential flow filtration precipitation/elution, column chromatography (DEAE or hydrophobic interaction chromatography), and depth filtration. The glycoconjugates maybe purified by diafiltration and/or ion exchange chromatography and/or size exclusion chromatography. In an embodiment, the glycoconjugates are purified by diafiltration or ion exchange chromatography or size exclusion chromatography. In one embodiment the glycoconjugates are sterile filtered. In a preferred embodiment, a glycoconjugate from an E. coiserotype is selected from any one of 025B, 01, 02, and 06 is prepared by reductive amination. In a preferred embodiment, the glycoconjugates from E. coli serotypes 025B, 01, 02, and 06 are prepared by reductive amination. In one aspect, the invention relates to a conjugate that includes a carrier protein, e.g., CRM , 19 7 linked to a saccharide of Formula 025B, presented by D-Glc

D-G1c---L-Rha2Ac- D-GlcNAc t f

L-Rha , wherein n is any integer greater than or equal to 1. In a preferred embodiment, n is an integer of at least 31, 32, 33, 34, 35, 36, 37, 38, 39,40, and at most200,100,99,98,97,96,95,94,93,92,91,90, 89, 88,87,86,81,80,79, 78,77,76,75,74,73,72,71,70,69,68,67,66,65,60,59,58,57, 56,55,54,53,52,51, or 50. Any minimum value and any maximum value may be combined to define a range. Exemplary ranges include, for example, at least 1 to at most 1000; at least 10 to at most 500; and at least 20 to at most 80. In one preferred embodiment, n is at least 31 to at most 90, more preferably 40 to 90, most preferably 60 to 85. In anotheraspect, the invention relates to a conjugate that includes a carrierprotein, e.g., CRM 1 97, linked to a saccharide having any one of the following structures shown in Table 1 (see also FIG. 9A-9C and FIG. 10A-1OB), wherein n is an integer greater than or equal to 1. Without being bound by theory or mechanism, in some embodiments, a stable conjugate is believed to require a level of saccharide antigen modification that is balanced against preserving the structural integrity of the critical immunogenic epitopes of the antigen. Activation and formation of an Aldehyde. In some embodiments, the saccharide of the invention is activated and results in the formation of an aldehyde. In such embodiments wherein the saccharide is activated, the percentage (%) of activation (or degree of oxidation (DO)) (see, e.g., Example 31) refers to moles of a saccharide repeat unit per moles of aldehyde of the activated polysaccharide. For example, in some embodiments, the saccharide is activated by periodate oxidation of vicinal diols on a repeat unit of the polysaccharide, resulting in the formation of an aldehyde. Varying the molar equivalents (meq) of sodium periodate relative to the saccharide repeat unit and temperature during oxidation results in varying levels of degree of oxidation (DO). The saccharide and aldehyde concentrations are typically determined by colorimetric assays. An alternative reagent is TEMPO (2,2,6,6-tetramethylpiperidine 1 oxyl radical)-N-chlorosuccinimide (NCS) combination, which results in the formation of aldehydes from primary alcohol groups. In some embodiments, the activated saccharide has a degree of oxidation wherein the moles of a saccharide repeat unit per moles of aldehyde of the activated saccharide is between 1-100, such as, for example, between 2-80, between 2-50, between 3-30, and between 4-25. The degree of activation is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 2 20, 2 30, 2 40, 2 50, 2 60, 2 70, 2 80, or> 90, or about 100. Preferably, the degree of oxidation (DO) is at least 5 and at most 50, more preferably at least 10 and at most 25. In one embodiment, the degree of activation is at least 10 and at most 25. Any minimum value and any maximum value may be combined to define a range. A degree of oxidation value may be represented as percentage (%) of activation. For example, in one embodiment, a DO value of 10 refers to one activated saccharide repeat unit out of a total of 10 saccharide repeat units in the activated saccharide, in which case the DO value of 10 may be represented as 10% activation.

In some embodiments, the conjugate prepared by reductive amination chemistry includes a carrier protein and a saccharide, wherein the saccharide includes a structure selected from any one of Formula 01 (e.g., Formula 01A, Formula 01B, and Formula 01C), Formula 02, Formula 03, Formula 04 (e.g., Formula 04:K52 and Formula 04:K6), Formula 05 (e.g., Formula 05ab and Formula 05ac (strain 180/C3)), Formula 06 (e.g., Formula 06:K2; K13; K15 and Formula 06:K54), Formula 07, Formula 08, Formula 09, Formula 010, Formula 011, Formula 012, Formula 013, Formula 014, Formula 015, Formula 016, Formula 017, Formula 018 (e.g., Formula 018A, Formula 018ac, Formula 018A1, Formula 018B, and Formula 018B1), Formula 019, Formula 020, Formula 021, Formula 022, Formula 023 (e.g., Formula 023A), Formula 024, Formula 025 (e.g., Formula 025a and Formula 025b), Formula 026, Formula 027, Formula 028, Formula 029, Formula 030, Formula 032, Formula 033, Formula 034, Formula 035, Formula 036, Formula 037, Formula 038, Formula 039, Formula 040, Formula 041, Formula 042, Formula 043, Formula 044, Formula 045 (e.g., Formula 045 and Formula 045rel), Formula 046, Formula 048, Formula 049, Formula 050, Formula 051, Formula 052, Formula 053, Formula 054, Formula 055, Formula 056, Formula 057, Formula 058, Formula 059, Formula 060, Formula 061, Formula 062, Formula 62D1, Formula 063, Formula 064, Formula 065, Formula 066, Formula 068, Formula 069, Formula 070, Formula 071, Formula 073 (e.g., Formula 073 (strain 73-1)), Formula 074, Formula 075, Formula 076, Formula 077, Formula 078, Formula 079, Formula 080, Formula 081, Formula 082, Formula 083, Formula 084, Formula 085, Formula 086, Formula 087, Formula 088, Formula 089, Formula 090, Formula 091, Formula 092, Formula 093, Formula 095, Formula 096, Formula 097, Formula 098, Formula 099, Formula 0100, Formula 0101, Formula 0102, Formula 0103, Formula 0104, Formula 0105, Formula 0106, Formula 0107, Formula 0108, Formula 0109, Formula 0110, Formula 0111, Formula 0112, Formula 0113, Formula 0114, Formula 0115, Formula 0116, Formula 0117, Formula 0118, Formula 0119, Formula 0120, Formula 0121, Formula 0123, Formula 0124, Formula 0125, Formula 0126, Formula 0127, Formula 0128, Formula 0129, Formula 0130, Formula 0131, Formula 0132, Formula 0133, Formula 0134, Formula 0135, Formula 0136, Formula 0137, Formula 0138, Formula 0139, Formula 0140, Formula 0141, Formula 0142, Formula 0143, Formula 0144, Formula 0145, Formula 0146, Formula 0147, Formula 0148, Formula 0149, Formula 0150, Formula 0151, Formula 0152, Formula 0153, Formula 0154, Formula 0155, Formula 0156, Formula 0157, Formula 0158, Formula 0159, Formula 0160, Formula 0161, Formula 0162, Formula 0163, Formula 0164, Formula 0165, Formula 0166, Formula 0167, Formula 0168, Formula 0169, Formula 0170, Formula 0171, Formula 0172, Formula 0173, Formula 0174, Formula 0175, Formula 0176, Formula 0177, Formula 0178, Formula 0179, Formula 0180, Formula 0181, Formula 0182, Formula 0183, Formula 0184, Formula 0185,

Formula 0186, and Formula 0187. In some embodiments, the saccharide in the conjugate includes a Formula, wherein n is an integer from 1 to 1000, from 5 to 1000, preferably 31 to 100, more preferably 35 to 90, most preferably 35 to 65. SINGLE-END LINKED CONJUGATES. In some embodiments, the conjugate is single-end-linked conjugated saccharide, wherein the saccharide is covalently bound at one end of the saccharide to a carrier protein. In some embodiments, the single-end linked conjugated polysaccharide has a terminal saccharide. For example, a conjugate is single-end linked if one of the ends (a terminal saccharide residue) of the polysaccharide is covalently bound to a carrier protein. In some embodiments, the conjugate is single-end linked if a terminal saccharide residue of the polysaccharide is covalently bound to a carrier protein through a linker. Such linkers may include, for example, a cystamine linker (Al), a 3,3'-dithio bis(propanoic dihydrazide) linker (A4), and a 2,2'-dithio-N,N'-bis(ethane-2,1-diyl)bis(2-(aminooxy)acetamide) linker (A6). In some embodiments, the saccharide is conjugated to the carrier protein through a 3-deoxy-d-manno-oct-2-ulosonic acid (KDO) residue to form a single-end linked conjugate. See, for example, Example 26, Example 27, Example 28, and FIG. 17. In some embodiments, the conjugate is preferably not a bioconjugate. The term "bioconjugate" refers to a conjugate between a protein (e.g., a carrier protein) and an antigen, e.g., an 0 antigen (e.g.,025B) prepared in a host cell background, wherein host cell machinery links the antigen to the protein (e.g., N-links). Glycoconjugates include bioconjugates, as well as sugar antigen (e.g., oligo- and polysaccharides) protein conjugates prepared by means that do not require preparation of the conjugate in a host cell, e.g., conjugation by chemical linkage of the protein and saccharide. Thiol Activated Saccharides. In some embodiments, the saccharide of the invention is thiol activated. In such embodiments wherein the saccharide is thiol activated, the percentage (%) of activation refers to moles of thiol per saccharide repeat unit of the activated polysaccharide. The saccharide and thiol concentrations are typically determined by Ellman's assay for quantitation of sulfhydryls. For example, in some embodiments, the saccharide includes activation of 2-Keto-3-deoxyoctanoic acid (KDO) with a disulfide amine linker. See, for example, Example 10 and FIG. 31. In some embodiments, the saccharide is covalently bound to a carrier protein through a bivalent, heterobifunctional linker (also referred to herein as a "spacer"). The linker preferably provides a thioether bond between the saccharide and the carrier protein, resulting in a glycoconjugate referred to herein as a "thioether glycoconjugate." In some embodiments, the linker further provides carbamate and amide bonds, such as, for example, (2 ((2-oxoethyl)thio)ethyl) carbamate (eTEC). See, for example, Example 21. In some embodiments, the single-end linked conjugate includes a carrier protein and a saccharide, wherein the saccharide includes a structure selected from any one of Formula 01 (e.g., Formula 01A, Formula 01B, and Formula 01C), Formula 02, Formula 03, Formula 04 (e.g., Formula 04:K52 and Formula 04:K6), Formula 05 (e.g., Formula 05ab and Formula 05ac (strain 180/C3)), Formula 06 (e.g., Formula 06:K2; K13; K15 and Formula 06:K54), Formula 07, Formula 08, Formula 09, Formula 010, Formula 011, Formula 012, Formula 013, Formula 014, Formula 015, Formula 016, Formula 017, Formula 018 (e.g., Formula 018A, Formula 018ac, Formula 018A1, Formula 018B, and Formula 018B1), Formula 019, Formula 020, Formula 021, Formula 022, Formula 023 (e.g., Formula 023A), Formula 024, Formula 025 (e.g., Formula 025a and Formula 025b), Formula 026, Formula 027, Formula 028, Formula 029, Formula 030, Formula 032, Formula 033, Formula 034, Formula 035, Formula 036, Formula 037, Formula 038, Formula 039, Formula 040, Formula 041, Formula 042, Formula 043, Formula 044, Formula 045 (e.g., Formula 045 and Formula 045rel), Formula 046, Formula 048, Formula 049, Formula 050, Formula 051, Formula 052, Formula 053, Formula 054, Formula 055, Formula 056, Formula 057, Formula 058, Formula 059, Formula 060, Formula 061, Formula 062, Formula 62 D1, Formula 063, Formula 064, Formula 065, Formula 066, Formula 068, Formula 069, Formula 070, Formula 071, Formula 073 (e.g., Formula 073 (strain 73-1)), Formula 074, Formula 075, Formula 076, Formula 077, Formula 078, Formula 079, Formula 080, Formula 081, Formula 082, Formula 083, Formula 084, Formula 085, Formula 086, Formula 087, Formula 088, Formula 089, Formula 090, Formula 091, Formula 092, Formula 093, Formula 095, Formula 096, Formula 097, Formula 098, Formula 099, Formula 0100, Formula 0101, Formula 0102, Formula 0103, Formula 0104, Formula 0105, Formula 0106, Formula 0107, Formula 0108, Formula 0109, Formula 0110, Formula 0111, Formula 0112, Formula 0113, Formula 0114, Formula 0115, Formula 0116, Formula 0117, Formula 0118, Formula 0119, Formula 0120, Formula 0121, Formula 0123, Formula 0124, Formula 0125, Formula 0126, Formula 0127, Formula 0128, Formula 0129, Formula 0130, Formula 0131, Formula 0132, Formula 0133, Formula 0134, Formula 0135, Formula 0136, Formula 0137, Formula 0138, Formula 0139, Formula 0140, Formula 0141, Formula 0142, Formula 0143, Formula 0144, Formula 0145, Formula 0146, Formula 0147, Formula 0148, Formula 0149, Formula 0150, Formula 0151, Formula 0152, Formula 0153, Formula 0154, Formula 0155, Formula 0156, Formula 0157, Formula 0158, Formula 0159, Formula 0160, Formula 0161, Formula 0162, Formula 0163, Formula 0164, Formula 0165, Formula 0166, Formula 0167, Formula 0168, Formula 0169, Formula 0170, Formula 0171, Formula 0172, Formula 0173, Formula 0174, Formula 0175, Formula

0176, Formula 0177, Formula 0178, Formula 0179, Formula 0180, Formula 0181, Formula 0182, Formula 0183, Formula 0184, Formula 0185, Formula 0186, and Formula 0187. In some embodiments, the saccharide in the conjugate includes a Formula, wherein n is an integer from 1 to 1000, from 5 to 1000, preferably 31 to 100, more preferably 35 to 90, most preferably 35 to 65. For example, in one embodiment, the single-end linked conjugate includes a carrier protein and a saccharide having a structure selected from Formula 08, Formula 09a, Formula 09, Formula O20ab, Formula O20ac, Formula 052, Formula 097, and Formula 0101, wherein n is an integer from 1 to 10.

F. eTECCONJUGATES In one aspect, the invention relates generally to glycoconjugates comprising a saccharide derived from E. coli described above covalently conjugated to a carrier protein through a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer (as described, for example, in US Patent 9517274 and International Patent Application Publication W02014027302, incorporated by reference herein in their entireties), including immunogenic compositions comprising such glycoconjugates, and methods for the preparation and use of such glycoconjugates and immunogenic compositions. Said glycoconjugates comprise a saccharide covalently conjugated to a carrier protein through one or more eTEC spacers, wherein the saccharide is covalently conjugated to the eTEC spacer through a carbamate linkage, and wherein the carrier protein is covalently conjugated to the eTEC spacer through an amide linkage. The eTEC spacer includes seven linear atoms (i.e., -C(O)NH(CH 2) 2SCH 2C(O)- ) and provides stable thioether and amide bonds between the saccharide and carrier protein. The eTEC linked glycoconjugates of the invention may be represented by the general formula (I):

where the atoms that comprise the eTEC spacer are contained in the central box. In said glycoconjugates of the invention, the saccharide may be a polysaccharide or an oligosaccharide. The carrier proteins incorporated into the glycoconjugates of the invention are selected from the group of carrier proteins generally suitable for such purposes, as further described herein or known to those of skill in the art. In particular embodiments, the carrier protein is CRM1m.

In another aspect, the invention provides a method of making a glycoconjugate comprising a saccharide described herein conjugated to a carrier protein through an eTEC spacer, comprising the steps of a) reacting a saccharide with a carbonic acid derivative in an organic solvent to produce an activated saccharide; b) reacting the activated saccharide with cystamine or cysteamine or a salt thereof, to produce a thiolated saccharide; c) reacting the thiolated saccharide with a reducing agent to produce an activated thiolated saccharide comprising one or more free sulfhydryl residues; d) reacting the activated thiolated saccharide with an activated carrier protein comprising one or more a-haloacetamide groups, to produce a thiolated saccharide-carrier protein conjugate; and e) reacting the thiolated saccharide-carrier protein conjugate with (i) a first capping reagent capable of capping unconjugated a-haloacetamide groups of the activated carrier protein; and/or (ii) a second capping reagent capable of capping unconjugated free sulfhydryl residues of the activated thiolated saccharide; whereby an eTEC linked glycoconjugate is produced. In frequent embodiments, the carbonic acid derivative is 1,1'-carbonyl-di-(1,2,4-triazole) (CDT) or 1,1'-carbonyldiimidazole (CDI). Preferably, the carbonic acid derivative is CDT and the organic solvent is a polar aprotic solvent, such as dimethylsulfoxide (DMSO). In preferred embodiments, the thiolated saccharide is produced by reaction of the activated saccharide with the bifunctional symmetric thioalkylamine reagent, cystamine or a salt thereof. Alternatively, the thiolated saccharide may be formed by reaction of the activated saccharide with cysteamine or a salt thereof. The eTEC linked glycoconjugates produced by the methods of the invention may be represented by general Formula (I). In frequent embodiments, the first capping reagent is N-acetyl-L-cysteine, which reacts with unconjugated a-haloacetamide groups on lysine residues of the carrier protein to form an S-carboxymethylcysteine (CMC) residue covalently linked to the activated lysine residue through a thioether linkage. In other embodiments, the second capping reagent is iodoacetamide (IAA), which reacts with unconjugated free sulfhydryl groups of the activated thiolated saccharide to provide a capped thioacetamide. Frequently, step e) comprises capping with both a first capping reagent and a second capping reagent. In certain embodiments, step e) comprises capping with N acetyl-L-cysteine as the first capping reagent and IAA as the second capping reagent. In some embodiments, the capping step e) further comprises reaction with a reducing agent, for example, DTT, TCEP, or mercaptoethanol, after reaction with the first and/or second capping reagent. The eTEC linked glycoconjugates and immunogenic compositions of the invention may include free sulfhydryl residues. In some instances, the activated thiolated saccharides formed by the methods provided herein will include multiple free sulfhydryl residues, some of which may not undergo covalent conjugation to the carrier protein during the conjugation step. Such residual free sulfhydryl residues are capped by reaction with a athiol-reactive capping reagent, for example, iodoacetamide (IAA), to cap the potentially reactive functionality. Other thiol-reactive capping reagents, e.g., maleimide containing reagents and the like are also contemplated. In addition, the eTEC linked glycoconjugates and immunogenic compositions of the invention may include residual unconjugated carrier protein, which may include activated carrier protein which has undergone modification during the capping process steps. In some embodiments, step d) further comprises providing an activated carrier protein comprising one or more a-haloacetamide groups prior to reacting the activated thiolated saccharide with the activated carrier protein. In frequent embodiments, the activated carrier protein comprises one or more a-bromoacetamide groups. In another aspect, the invention provides an eTEC linked glycoconjugate comprising a saccharide described herein conjugated to a carrier protein through an eTEC spacer produced according to any of the methods disclosed herein. In some embodiments, the carrier protein is CRM 197 and the covalent linkage via an eTEC spacer between the CRM 197 and the polysaccharide occurs at least once in every 4, 10, 15 or 25 saccharide repeat units of the polysaccharide. For each of the aspects of the invention, in particular embodiments of the methods and compositions described herein, the eTEC linked glycoconjugate comprises a saccharide described herein, such as, a saccharide derived from E. coli. In another aspect, the invention provides a method of preventing, treating or ameliorating a bacterial infection, disease or condition in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the invention, wherein said immunogenic composition comprises an eTEC linked glycoconjugate comprising a saccharide described herein. In some embodiments, the saccharide is derived from E. coli. In some embodiments, the eTEC linked glycoconjugate comprises a carrier protein and a saccharide, in which said saccharide comprises a structure selected from any one of Formula 01 (e.g., Formula 01A, Formula 01B, and Formula 01C), Formula 02, Formula 03, Formula 04 (e.g., Formula 04:K52 and Formula 04:K6), Formula 05 (e.g., Formula 05ab and Formula 05ac (strain 180/C3)), Formula 06 (e.g., Formula 06:K2; K13; K15 and Formula 06:K54), Formula 07, Formula 08, Formula 09, Formula 010, Formula 011, Formula 012, Formula 013, Formula 014, Formula 015, Formula 016, Formula 017, Formula 018 (e.g., Formula 018A, Formula 018ac, Formula

018A1, Formula 018B, and Formula 018B1), Formula 019, Formula 020, Formula 021, Formula 022, Formula 023 (e.g., Formula 023A), Formula 024, Formula 025 (e.g., Formula 025a and Formula 025b), Formula 026, Formula 027, Formula 028, Formula 029, Formula 030, Formula 032, Formula 033, Formula 034, Formula 035, Formula 036, Formula 037, Formula 038, Formula 039, Formula 040, Formula 041, Formula 042, Formula 043, Formula 044, Formula 045 (e.g., Formula 045 and Formula 045rel), Formula 046, Formula 048, Formula 049, Formula 050, Formula 051, Formula 052, Formula 053, Formula 054, Formula 055, Formula 056, Formula 057, Formula 058, Formula 059, Formula 060, Formula 061, Formula 062, Formula 62D1, Formula 063, Formula 064, Formula 065, Formula 066, Formula 068, Formula 069, Formula 070, Formula 071, Formula 073 (e.g., Formula 073 (strain 73-1)), Formula 074, Formula 075, Formula 076, Formula 077, Formula 078, Formula 079, Formula 080, Formula 081, Formula 082, Formula 083, Formula 084, Formula 085, Formula 086, Formula 087, Formula 088, Formula 089, Formula 090, Formula 091, Formula 092, Formula 093, Formula 095, Formula 096, Formula 097, Formula 098, Formula 099, Formula 0100, Formula 0101, Formula 0102, Formula 0103, Formula 0104, Formula 0105, Formula 0106, Formula 0107, Formula 0108, Formula 0109, Formula 0110, Formula 0111, Formula 0112, Formula 0113, Formula 0114, Formula 0115, Formula 0116, Formula 0117, Formula 0118, Formula 0119, Formula 0120, Formula 0121, Formula 0123, Formula 0124, Formula 0125, Formula 0126, Formula 0127, Formula 0128, Formula 0129, Formula 0130, Formula 0131, Formula 0132, Formula 0133, Formula 0134, Formula 0135, Formula 0136, Formula 0137, Formula 0138, Formula 0139, Formula 0140, Formula 0141, Formula 0142, Formula 0143, Formula 0144, Formula 0145, Formula 0146, Formula 0147, Formula 0148, Formula 0149, Formula 0150, Formula 0151, Formula 0152, Formula 0153, Formula 0154, Formula 0155, Formula 0156, Formula 0157, Formula 0158, Formula 0159, Formula 0160, Formula 0161, Formula 0162, Formula 0163, Formula 0164, Formula 0165, Formula 0166, Formula 0167, Formula 0168, Formula 0169, Formula 0170, Formula 0171, Formula 0172, Formula 0173, Formula 0174, Formula 0175, Formula 0176, Formula 0177, Formula 0178, Formula 0179, Formula 0180, Formula 0181, Formula 0182, Formula 0183, Formula 0184, Formula 0185, Formula 0186, and Formula 0187. In some embodiments, the saccharide in the conjugate includes a Formula, wherein n is an integer from 1 to 1000, from 5 to 1000, preferably 31 to 100, more preferably 35 to 90, most preferably 35 to 65. The number of lysine residues in the carrier protein that become conjugated to the saccharide can be characterized as a range of conjugated lysines. For example, in some embodiments of the immunogenic compositions, the CRM 197 may comprise 4 to 16 lysine residues out of 39 covalently linked to the saccharide. Another way to express this parameter is that about 10% to about 41% of CRM 197 lysines are covalently linked to the saccharide. In other embodiments, the CRM 197 may comprise 2 to 20 lysine residues out of 39 covalently linked to the saccharide. Another way to express this parameter is that about 5% to about 50% of CRM 197 lysines are covalently linked to the saccharide. In frequent embodiments, the carrier protein is CRM 197 and the covalent linkage via an eTEC spacer between the CRM 197 and the polysaccharide occurs at least once in every 4, 10, 15 or 25 saccharide repeat units of the polysaccharide. In other embodiments, the conjugate comprises at least one covalent linkage between the carrier protein and saccharide for every 5 to 10 saccharide repeat units; every 2 to 7 saccharide repeat units; every 3 to 8 saccharide repeat units; every 4 to 9 saccharide repeat units; every 6 to 11 saccharide repeat units; every 7 to 12 saccharide repeat units; every 8 to 13 saccharide repeat units; every 9 to 14 saccharide repeat units; every 10 to 15 saccharide repeat units; every 2 to 6 saccharide repeat units, every 3 to 7 saccharide repeat units; every 4 to 8 saccharide repeat units; every 6 to 10 saccharide repeat units; every 7 to 11 saccharide repeat units; every 8 to 12 saccharide repeat units; every 9 to 13 saccharide repeat units; every 10 to 14 saccharide repeat units; every 10 to 20 saccharide repeat units; or every 4 to 25 saccharide repeat units. In another embodiment, at least one linkage between carrier protein and saccharide occurs for every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 saccharide repeat units of the polysaccharide.

G. CARRIER PROTEINS

A component of the glycoconjugate of the invention is a carrier protein to which the saccharide is conjugated. The terms "protein carrier" or "carrier protein" or "carrier" may be used interchangeably herein. Carrier proteins should be amendable to standard conjugation procedures. One component of the conjugate is a carrier protein to which the 0 polysaccharide is conjugated. In one embodiment, the conjugate includes a carrier protein conjugated to the core oligosaccharide of the O-polysaccharide (see FIG. 24). In one embodiment, the conjugate includes a carrier protein conjugated to the O-antigen of the O-polysaccharide. The terms "protein carrier" or "carrier protein" or "carrier" may be used interchangeably herein. Carrier proteins should be amendable to standard conjugation procedures. In a preferred embodiment, the carrier protein of the conjugates is independently selected from any one of TT, DT, DT mutants (such as CRM 197), H. influenzae protein D, PhtX, PhtD, PhtDE fusions (particularly those described in WO 01/98334 and WO

03/54007), detoxified pneumolysin, PorB, N19 protein, PspA, OMPC, toxin A or B of C. Difficile and PsaA. In an embodiment, the carrier protein of the conjugates of the invention is DT (Diphtheria toxoid). In another embodiment, the carrier protein of the conjugates of the invention is TT (tetanus toxoid). In another embodiment, the carrier protein of the conjugates of the invention is PD (Haemophilus influenzae protein D - see, e.g., EP 0 594 610 B). In some embodiments, the carrier protein includes poly(L-lysine) (PLL). In a preferred embodiment, the saccharides are conjugated to CRM 197 protein. The CRM 197 protein is a nontoxic form of diphtheria toxin but is immunologically indistinguishable from the diphtheria toxin. CRM 197 is produced by C. diphtheriae infected by the nontoxigenic phage p197tox- created by nitrosoguanidine mutagenesis of the toxigenic corynephage beta. The CRM 197 protein has the same molecular weight as the diphtheria toxin but differs therefrom by a single base change (guanine to adenine) in the structural gene. This single base change causes an aminoacid substitution glutamic acid for glycine) in the mature protein and eliminatesthe toxic properties of diphtheria toxin. The CRM197 protein is a safe and effective T-cell dependent carrier for saccharides. Accordingly, in some embodiments, the conjugates of the invention include CRM 197 as the carrier protein, wherein the saccharide is covalently linked to CRM 197

. In a preferred embodiment, the carrier protein of the glycoconjugates is selected in the group consisting of DT (Diphtheria toxin), TT (tetanus toxoid) or fragment C of TT, CRM197 (a nontoxic but antigenically identical variant of diphtheria toxin), other DT mutants (such as CRM176, CRM228, CRM45 (Uchida et al J. Biol. Chem. 218; 3838-3844, 1973), CRM9, CRM45, CRM102, CRM103 or CRM107; and other mutations described by Nicholls and Youle in Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992; deletion or mutation of Glu-148 to Asp, GIn or Ser and/or Ala 158 to Gly and other mutations disclosed in US 4709017 or US 4950740; mutation of at least one or more residues Lys 516, Lys 526, Phe 530 and/or Lys 534 and other mutations disclosed in US 5917017 or US 6455673; or fragment disclosed in US 5843711), pneumococcal pneumolysin (Kuo et al (1995) Infect Immun 63; 2706-13) including ply detoxified in some fashion for example dPLY-GMBS (WO 04081515, PCT/EP2005/010258) or dPLY-formol, PhtX, including PhtA, PhtB, PhtD, PhtE (sequences of PhtA, PhtB, PhtD or PhtE are disclosed in WO 00/37105 or WO 00/39299) and fusions of Pht proteins for example PhtDE fusions, PhtBE fusions, Pht A-E (WO 01/98334, WO 03/54007, W02009/000826), OMPC (meningococcal outer membrane protein - usually extracted from N. meningitidis serogroup B - EP0372501 ), PorB (from N. meningitidis), PD (Haemophilus influenzae protein D - see, e.g., EP 0 594 610 B), or immunologically functional equivalents thereof, synthetic peptides (EP0378881 , EP0427347), heat shock proteins (WO 93/17712, WO 94/03208), pertussis proteins (WO 98/58668, EP0471 177), cytokines, lymphokines, growth factors or hormones (WO 91/01146), artificial proteins comprising multiple human CD4+ T cell epitopes from various pathogen derived antigens (Falugi et al (2001 ) Eur J Immunol 31 ; 3816-3824) such as N19 protein (Baraldoi et al (2004) Infect Immun 72; 4884-7) pneumococcal surface protein PspA (WO 02/091998), iron uptake proteins (WO 01/72337), toxin A or B of C. difficile (WO 00/61761), transferrin binding proteins, pneumococcal adhesion protein (PsaA), recombinant Pseudomonas aeruginosa exotoxin A (in particular non-toxic mutants thereof (such as exotoxin A bearing a substitution at glutamic acid 553 (Uchida Cameron DM, RJ Collier. 1987. J. Bacteriol. 169:4967-4971)). Other proteins, such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD) also can be used as carrier proteins. Other suitable carrier proteins include inactivated bacterial toxins such as cholera toxoid (e.g., as described in Int'l Patent Application No. WO 2004/083251), E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa. In some embodiments, the carrier protein is selected from any one of, for example, CRM 197, diphtheria toxin fragment B (DTFB), DTFB C8, Diphtheria toxoid (DT), tetanus toxoid (TT), fragment C of TT, pertussis toxoid, cholera toxoid, or exotoxin A from Pseudomonas aeruginosa; detoxified Exotoxin A of P. aeruginosa (EPA), maltose binding protein (MBP), flagellin, detoxified hemolysin A of S. aureus, clumping factor A, clumping factor B, Cholera toxin B subunit (CTB), Streptococcus pneumoniae Pneumolysin and detoxified variants thereof, C. jejuni AcrA, C. jejuni natural glycoproteins and Streptococcal C5a peptidase (SCP). In one embodiment, the carrier protein is detoxified Pseudomonas exotoxin (EPA). In another embodiment, the carrier protein is not detoxified Pseudomonas exotoxin (EPA). In one embodiment, the carrier protein is flagellin. In another embodiment, the carrier protein is not flagellin. In a preferred embodiment, the carrier protein of the glycoconjugates is independently selected from the group consisting of TT, DT, DT mutants (such as CRM 197), H. influenzae protein D, PhtX, PhtD, PhtDE fusions (particularly those described in WO 01/98334 and WO 03/54007), detoxified pneumolysin, PorB, N19 protein, PspA, OMPC, toxin A or B of C. Difficile and PsaA. In an embodiment, the carrier protein of the glycoconjugates of the invention is DT (Diphtheria toxoid). In another embodiment, the carrier protein of the glycoconjugates of the invention is TT (tetanus toxoid). In another embodiment, the carrier protein of the glycoconjugates of the invention is PD (Haemophilus influenzae protein D - see, e.g., EP 0 594 610 B). In a preferred embodiment, the capsular saccharides of the invention are conjugated to CRM 197 protein. The CRM 197 protein is a nontoxic form of diphtheria toxin but is immunologically indistinguishable from the diphtheria toxin. CRM 197 is produced by

C. diphtheriae infected by the nontoxigenic phage P197tox- created by nitrosoguanidine mutagenesis of the toxigenic corynephage beta (Uchida, T. et al. 1971, Nature New Biology 233:8-11). The CRM 197 protein has the same molecular weight as the diphtheria toxin but differs therefrom by a single base change (guanine to adenine) in the structural gene. This single base change causes an amino acid substitution glutamic acid for glycine) in the mature protein and eliminates the toxic properties of diphtheria toxin. The CRM 197 protein is a safe and effective T-cell dependent carrier for saccharides. Further details about CRM 197 and production thereof can be found e.g. in US 5,614,382 Accordingly, in frequent embodiments, the glycoconjugates of the invention comprise CRM 197 as the carrier protein, wherein the capsular polysaccharide is covalently linked to CRM 197 .

In a further embodiment, the carrier protein of the glycoconjugates is SCP (Streptococcal C5a Peptidase). All human isolates of p-hemolytic streptococci produce a highly conserved cell-wall protein SCP (Streptococcal C5a Peptidase) that specifically inactivates C5a. The scp genes encode a polypeptide containing between 1,134 and 1,181 amino acids (Brown et al., PNAS, 2005, vol. 102, no. 51 pages 18391-18396). The first 31 residues are the export signal presequence and are removed upon passing through the cytoplasmic membrane. The next 68 residues serve as a pro-sequence and must be removed to produce active SCP. The next 10 residues can be removed without loss of protease activity. At the other end, starting with Lys-1034, are four consecutive 17-residue motifs followed by a cell sorting and cell-wall attachment signal. This combined signal is composed of a 20-residue hydrophilic sequence containing an LPTTND sequence, a 17-residue hydrophobic sequence, and a short basic carboxyl terminus. SCP can be divided in domains (see figure 1B of Brown et al., PNAS, 2005, vol. 102, no. 51 pages 18391-18396). These domains are the Pre/Pro domain (which comprises the export signal presequence (commonly the first 31 residues) and the pro-sequence (commonly the next 68 residues)), the protease domain (which is splitted in two part (protease part 1 commonly residues 89-333/334 and protease domain part 2 and commonly residues 467/468-583/584), the protease-associated domain (PA domain) (commonly residues 333/334-467/468), three fibronectin type III (Fn) domains (Fn1, commonly residues 583/584-712/713; Fn2, commonly residues 712/713-928/929/930; commonly Fn3, residues 929/930-1029/1030/1031) and a cell wall anchor domain (commonly redisues 1029/1030/1031 to the C-terminus). In an embodiment, the carrier protein of the glycoconjugates of the invention is an SCP from GBS (SCPB). An example of SCPB is provided at SEQ. ID.NO: 3 of W097/26008. See also SEQ ID NO: 3 of WOOO/34487.

In another embodiment, the carrier protein of the glycoconjugate of the invention is an SCP from GAS (SCPA). Examples of SCPA can be found at SEQ.ID.NO:1 and SEQ.ID.NO:2 of W097/26008. See also SEQ ID NOs: 1, 2 and 23 of WOOO/34487. In a further embodiment, the carrierprotein of the glyconjugate ofthe invention isan SCP as set forth in SEQ ID NO: 150 or 151 of WO2014/136064.

H. DOSAGES OF THE COMPOSITIONS Dosage regimens may be adjusted to provide the optimum desired response. For example, a single dose of the polypeptide derived from E. coli or fragment thereof may be administered, several divided doses may be administered overtime, or the dose may be proportionally reduced or increased as indicated by the exigencies of the situation. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated, and may include single or multiple doses. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. Determining appropriate dosages and regimens for administration of the therapeutic protein are well-known in the relevant art and would be understood to be encompassed by the skilled artisan once provided the teachings disclosed herein. In some embodiments, the amount of the polypeptide derived from E. colior fragment thereof in the composition, may range from about 10 pg to about 300 pg of each protein antigen. In some embodiments, the amount of the polypeptide derived from E. coli or fragment thereof in the composition may range from about 20 pg to about 200 pg of each protein antigen. The amount of glycoconjugate(s) in each dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccines. Such amount will vary depending upon which specific immunogen is employed and how it is presented. The amount of a particular glycoconjugate in an immunogenic composition can be calculated based on total polysaccharide for that conjugate (conjugated and non conjugated). For example, a glycoconjugate with 20% free polysaccharide will have about 80 pg of conjugated polysaccharide and about 20 pg of non-conjugated polysaccharide in a 100 pg polysaccharide dose. The amount of glycoconjugate can vary depending upon the E. coliserotype. The saccharide concentration can be determined by the uronic acid assay. The "immunogenic amount" of the different polysaccharide components in the immunogenic composition, may diverge and each may comprise about 1.0 pg, about 2.0 pg, about 3.0 pg, about 4.0 pg, about 5.0 pg, about 6.0 pg, about 7.0 pg, about 8.0 pg, about 9.0 pg, about 10.0 pg, about 15.0 pg, about 20.0 pg, about 30.0 pg, about 40.0 pg, about 50.0 pg, about 60.0 pg, about 70.0 pg, about 80.0 pg, about 90.0 pg, or about 100.0 pg of any particular polysaccharide antigen. Generally, each dose will comprise 0.1 pg to 100 pg of polysaccharide for a given serotype, particularly 0.5 pg to 20 pg, more particularly 1 pg to 10 pg, and even more particularly 2 pg to 5 pg. Any whole number integer within any of the above ranges is contemplated as an embodiment of the disclosure. In one embodiment, each dose will comprise 1 pg, 2 pg, 3 pg, 4 pg, 5 pg, 6 pg, 7 pg, 8 pg, 9 pg, 10 pg, 15 pg or 20 pg of polysaccharide for a given serotype. Carrier protein amount. Generally, each dose will comprise 5 pg to 150 pg of carrier protein, particularly 10 pg to 100 pg of carrier protein, more particularly 15 pg to 100 pg of carrier protein, more particularly 25 pg to 75 pg of carrier protein, more particularly 30 pg to 70 pg of carrier protein, more particularly 30 pg to 60 pg of carrier protein, more particularly 30 pg to 50 pg of carrier protein and even more particularly 40 pg to 60 pg of carrier protein. In one embodiment, said carrier protein is CRM 197 .In one embodiment, each dose will comprise about 25 pg, about26 pg, about27 pg, about28 pg, about29 pg, about30 pg, about31 pg, about32 pg, about33 pg, about34 pg, about35 pg, about36 pg, about37 pg, about38 pg, about39 pg, about40 pg, about41 pg, about42 pg, about43 pg, about44 pg, about45 pg, about46 pg, about47 pg, about48 pg, about49 pg, about50 pg, about51 pg, about52 pg, about53 pg, about54 pg, about55 pg, about56 pg, about57 pg, about58 pg, about59 pg, about60 pg, about61 pg, about62 pg, about63 pg, about64 pg, about65 pg, about66 pg, about67 pg,68 pg, about69 pg, about70 pg, about71 pg, about72 pg, about73 pg, about74 pg or about 75 pg of carrier protein. In one embodiment, said carrier protein is CRM 197. In another embodiment, said carrier protein is SCP.

1. ADJUVANT

In some embodiments, the immunogenic compositions disclosed herein may further comprise at least one, two or three adjuvants. In some embodiments, the immunogenic compositions disclosed herein may further comprise at least one adjuvant. In some embodiments, the immunogenic compositions disclosed herein may further comprise one adjuvant. In some embodiments, the immunogenic compositions disclosed herein may further comprise two adjuvants. The term "adjuvant" refers to a compound or mixture that enhances the immune response to an antigen. Antigens may act primarily as a delivery system, primarily as an immune modulator or have strong features of both. Suitable adjuvants include those suitable for use in mammals, including humans. Examples of known suitable delivery-system type adjuvants that can be used in humans include, but are not limited to, alum (e.g., aluminum phosphate, aluminum sulfate or aluminum hydroxide), calcium phosphate, liposomes, oil-in-water emulsions such as MF59 (4.3% w/v squalene, 0.5% w/v polysorbate 80 (Tween 80), 0.5% w/v sorbitan trioleate (Span 85)), water-in-oil emulsions such as Montanide, and poly(D,L-lactide-co glycolide) (PLG) microparticles or nanoparticles. In an embodiment, the immunogenic compositions disclosed herein comprise aluminum salts (alum) as adjuvant (e.g., aluminum phosphate, aluminum sulfate or aluminum hydroxide). In a preferred embodiment, the immunogenic compositions disclosed herein comprise aluminum phosphate or aluminum hydroxide as adjuvant. In an embodiment, the immunogenic compositions disclosed herein comprise from 0.1 mg/mL to 1 mg/mL or from 0.2 mg/mL to 0.3 mg/mL of elemental aluminum in the form of aluminum phosphate. In an embodiment, the immunogenic compositions disclosed herein comprise about 0.25 mg/mL of elemental aluminum in the form of aluminum phosphate. Examples of known suitable immune modulatory type adjuvants that can be used in humans include, but are not limited to, saponin extracts from the bark of the Aquilla tree (QS21, Quil A), TLR4 agonists such as MPLA (Monophosphoryl Lipid A), 3DMPL (3-0 deacylated MPL) or GLA-AQ, LT/CT mutants, cytokines such as the various interleukins (e.g., IL-2, IL-12) or GM-CSF, ASO1, and the like. Examples of known suitable immune modulatory type adjuvants with both delivery and immune modulatory features that can be used in humans include, but are not limited to, ISCOMS (see, e.g., Sjdlander et al. (1998) J. Leukocyte Biol. 64:713; WO 90/03184, WO 96/11711, WO 00/48630, WO 98/36772, WO 00/41720, WO 2006/134423 and WO 2007/026190) or GLA-EM which is a combination of a TLR4 agonist and an oil-in-water emulsion. For veterinary applications including but not limited to animal experimentation, one can use Complete Freund's Adjuvant (CFA), Freund's Incomplete Adjuvant (IFA), Emulsigen, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3 hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. Further exemplary adjuvants to enhance effectiveness of the immunogenic compositions disclosed herein include, but are not limited to (1) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (b) RIBI TM adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components such as monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), TM TM preferably MPL+CWS (DETOX ); (2) saponin adjuvants, such as QS21, STIMULON (Cambridge Bioscience, Worcester, Mass.), ABISCO@ (Isconova, Sweden), or ISCOMATRIX@ (Commonwealth Serum Laboratories, Australia), may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes), which ISCOMS may be devoid of additional detergent (e.g., WO 00/07621); (3) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (4) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (e.g., WO 99/44636)), interferons (e.g., gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (5) monophosphoryl lipid A (MPL) or 3-0 deacylated MPL (3dMPL) (see, e.g., GB2220211, EP0689454) (see, e.g., WO 00/56358); (6) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions (see, e.g., EP0835318, EP0735898, EP0761231); (7) a polyoxyethylene ether or a polyoxyethylene ester (see, e.g., WO 99/52549); (8) a polyoxyethylene sorbitan ester surfactant in combination with an octoxynol (e.g., WO 01/21207) or a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional non-ionic surfactant such as an octoxynol (e.g., WO 01/21152); (9) a saponin and an immunostimulatory oligonucleotide (e.g., a CpG oligonucleotide) (e.g., WO 00/62800); (10) an immunostimulant and a particle of metal salt (see, e.g., WO 00/23105); (11) a saponin and an oil-in-water emulsion (e.g., WO 99/11241); (12) a saponin (e.g., QS21)+3dMPL+1M2 (optionally+a sterol) (e.g., WO 98/57659); (13) other substances that act as immunostimulating agents to enhance the efficacy of the composition. Muramyl peptides include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25 acetyl normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutarninyl-L alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE), etc. In another embodiment, the adjuvant is a liposomal QS21 formulation as set forth in Example 35. In a further embodiment, the adjuvant is a liposomal MPLA formulation as set forth in Example 35. In a still further embodiment, the adjuvant is a liposomal MPLA/QS21 formulation as set forth in Example 35. In an embodiment ofthe present invention, the immunogenic compositions as disclosed herein comprise a CpG Oligonucleotide as adjuvant. A CpG oligonucleotide as used herein refers to an immunostimulatory CpG oligodeoxynucleotide (CpG ODN), and accordingly these terms are used interchangeably unless otherwise indicated. Immunostimulatory CpG oligodeoxynucleotides contain one or more immunostimulatory CpG motifs that are unmethylated cytosine-guanine dinucleotides, optionally within certain preferred base contexts. The methylation status of the CpG immunostimulatory motif generally refers to the cytosine residue in the dinucleotide. An immunostimulatory oligonucleotide containing at least one unmethylated CpG dinucleotide is an oligonucleotide which contains a 5' unmethylated cytosine linked by a phosphate bond to a 3'guanine, and which activates the immune system through binding to Toll-like receptor 9 (TLR-9). In another embodiment the immunostimulatory oligonucleotide may contain one or more methylated CpG dinucleotides, which will activate the immune system through TLR9 but not as strongly as if the CpG motif(s) was/were unmethylated. CpG immunostimulatory oligonucleotides may comprise one or more palindromes that in turn may encompass the CpG dinucleotide. CpG oligonucleotides have been described in a number of issued patents, published patent applications, and other publications, including U.S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; and 6,339,068. In an embodiment of the present invention, the immunogenic compositions as disclosed herein comprise any of the CpG Oligonucleotide described at page 3, line 22, to page 12, line 36, of WO 2010/125480. Different classes of CpG immunostimulatory oligonucleotides have been identified. These are referred to as A, B, C and P class, and are described in greater detail at page 3, line 22, to page 12, line 36, of WO 2010/125480. Methods of the invention embrace the use of these different classes of CpG immunostimulatory oligonucleotides.

VII. Nanoparticles

In another aspect, disclosed herein is an immunogenic complex that includes 1) a nanostructure; and 2) at least one fimbrial polypeptide antigen or fragment thereof. Preferably, the fimbrial polypeptide or fragment thereof is derived from E. coli fimbrial H (fimH). In a preferred embodiment, the fimbrial polypeptide is selected from any one of the fimbrial polypeptides described above. For example, the fimbrial polypeptide may comprise any one amino acid sequence selected from SEQ ID NOs:1-10, 18, 20, 21, 23, 24, 26-29 and 110-113.

In some embodiments, the antigen is fused or conjugated to the nanostructure exterior to stimulate development of adaptive immune responses to the displayed epitopes. In some embodiments, the immunogenic complex further includes an adjuvant or other immunomodulatory compounds attached to the exterior and/or encapsulated in the cage interior to help tailor the type of immune response generated for each pathogen. In some embodiments, the nanostructure includes a single assembly including a plurality of identical first nanostructure-related polypeptides. In alternative embodiments, the the nanostructure includes a plurality assembly, including a plurality of identical first nanostructure-related polypeptides and a plurality of second assemblies, each second assembly comprising a plurality of identical second nanostructure related polypeptides. Various nanostructure platforms can be employed in generating the immunogenic compositions described herein. In some embodiments, the nanostructures employed are formed by multiple copies of a single subunit. In some embodiments, the nanostructures employed are formed by multiple copies of multiple different subunits. The nanostructures are typically ball-like shaped, and/or have rotational symmetry (e.g., with 3-fold and 5-fold axis), e.g., with an icosahedral structure exemplified herein. In some embodiments, the antigen is presented on self-assembling nanoparticles such as self-assembling nanostructures derived from ferritin (FR), E2p, Qp, and 13-01. E2p is a redesigned variant of dihydrolipoyl acyltransferase from Bacillus stearothermophilus. 13-01 is an engineered protein that may self-assemble into hyperstable nanoparticles. Sequences of the subunits of these proteins are known in the art. In a first apsect, disclosed herein is a nanostructure-related polypeptide comprising an amino acid sequence that is at least 75% identical over its length, and identical at least at one identified interface position, to the amino acid sequence of a nanostructure-related polypeptide selected from the group consisting of SEQ ID NOS: 59-92. The nanostructure-related polypeptides can be used, for example, to prepare the nanostructures. The nanostructure-related polypeptides were designed for their ability to self-assemble in pairs to form nanostructures, such as icosahedral nanostructures. In some embodiments, the nanostructure includes (a) a plurality of first assemblies, each first assembly comprising a plurality of identical first nanostructure-related polypeptides, wherein the first nanostructure-related polypeptides comprise the amino acid sequence of a nanostructure-related polypeptide selected from the group consisting of SEQ ID NOS: 59-92; and (b) a plurality of second assemblies, each second assembly comprising a plurality of identical second nanostructure-related polypeptides, wherein the second nanostructure-related polypeptides comprise the amino acid sequence of a nanostructure-related polypeptide selected from the group consisting of SEQ ID NOS: 59-92, and wherein the second nanostructure-related polypeptide differs from the first nanostructure-related polypeptide; wherein the plurality of first assemblies non-covalently interact with the plurality of second assemblies to form a nanostructure. The nanostructures include symmetrically repeated, non-natural, non-covalent polypeptide-polypeptide interfaces that orient a first assembly and a second assembly into a nanostructure, such as one with an icosahedral symmetry. SEQ ID NOS: 59-92 provide the amino acid sequence of exemplary nanostructure-related polypeptides. The number of interface residues for the exemplary nanostructure-related polypeptides of SEQ ID NO:59-92 range from 4-13 residues. In various embodiments, the nanostructure-related polypeptides comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its length, and identical at least at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 identified interface positions (depending on the number of interface residues for a given nanostructure-related polypeptide), to the amino acid sequence of a nanostructure-related polypeptide selected from the group consisting of SEQ ID NOS: 59-92. In other embodiments, the nanostructure-related polypeptides comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its length, and identical at least at 20%, 25%, 33%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 100% of the identified interface positions, to the amino acid sequence of a nanostructure-related polypeptide selected from the group consisting of SEQ ID NOS: 59-92. In further embodiments, the nanostructure-related polypeptides include a nanostructure-related polypeptide having the amino acid sequence of a nanostructure-related polypeptide selected from the group consisting of SEQ ID NOS: 59-98. In one non-limiting embodiment, the nanostructure-related polypeptides can be modified to facilitate covalent linkage to a "cargo" of interest. In one non-limiting example, the nanostructure-related polypeptides can be modified, such as by introduction of various cysteine residues at defined positions to facilitate linkage to one or more antigens of interest, such that a nanostructure of the nanostructure-related polypeptides would provide a scaffold to provide a large number of antigens for delivery as a vaccine to generate an improved immune response. In some embodiments, some or all native cysteine residues that are present in the nanostructure-related polypeptides but not intended to be used for conjugation may be mutated to other amino acids to facilitate conjugation at defined positions. In another non-limiting embodiment, the nanostructure-related polypeptides may be modified by linkage (covalent or non-covalent) with a moiety to help facilitate "endosomal escape."

For applications that involve delivering molecules of interest to a target cell, such as targeted delivery, a critical step can be escape from the endosome-a membrane-bound organelle that is the entry point of the delivery vehicle into the cell. Endosomes mature into lysosomes, which degrade their contents. Thus, if the delivery vehicle does not somehow "escape" from the endosome before it becomes a lysosome, it will be degraded and will not perform its function. There are a variety of lipids or organic polymers that disrupt the endosome and allow escape into the cytosol. Thus, in this embodiment, the nanostructure-related polypeptides can be modified, for example, by introducing cysteine residues that will allow chemical conjugation of such a lipid or organic polymer to the monomer or resulting assemly surface. In another non limiting example, the nanostructure-related polypeptides can be modified, for example, by introducing cysteine residues that will allow chemical conjugation of fluorophores or other imaging agents that allow visualization of the nanostructures in vitro or in vivo. Surface amino acid residues on the nanostructure-related polypeptides can be mutated in order to improve the stability or solubility of the protein subunits or the assembled nanostructures. As will be known to one of skill in the art, if the nanostructure-related polypeptide has significant sequence homology to an existing protein family, a multiple sequence alignment of other proteins from that family can be used to guide the selection of amino acid mutations at non-conserved positions that can increase protein stability and/or solubility, a process referred to as consensus protein design (9). Surface amino acid residues on the nanostructure-related polypeptides can be mutated to positively charged (Arg, Lys) or negatively charged (Asp, Glu) amino acids in order to endow the protein surface with an overall positive or overall negative charge. In one non-limiting embodiment, surface amino acid residues on the nanostructure-related polypeptides can be mutated to endow the interior surface of the self-assembling nanostructure with a high net charge. Such a nanostructure can then be used to package or encapsulate a cargo molecule with the opposite net charge due to the electrostatic interaction between the nanostructure interior surface and the cargo molecule. In one non-limiting embodiment, surface amino acid residues on the nanostructure-related polypeptides can be mutated primarily to Arginine or Lysine residues in order to endow the interior surface of the self-assembling nanostructure with a net positive charge. Solutions containing the nanostructure-related polypeptides can then be mixed in the presence of a nucleic acid cargo molecule such as a dsDNA, ssDNA, dsRNA, ssRNA, cDNA, miRNA., siRNA, shRNA, piRNA, or other nucleic acid in order to encapsulate the nucleic acid inside the self-assembling nanostructure. Such a nanostructure could be used, for example, to protect, deliver, or concentrate nucleic acids. In one embodiment, the nanostructure has icosahedral symmetry. In this embodiment, the nanostructure may comprise 60 copies of the first nanostructure-related polypeptide and 60 copies of the second nanostructure-related polypeptide. In one such embodiment, the number of identical first nanostructure-related polypeptides in each first assembly is different than the number of identical second nanostructure-related polypeptides in each second assembly. For example, in one embodiment, the nanostructure comprises twelve first assemblies and twenty second assemblies; in this embodiment, each first assembly may; for example, comprise five copies of the identical first nanostructure related polypeptide, and each second assembly may, for example, comprise three copies of the identical second nanostructure-related polypeptide. In another embodiment, the nanostructure comprises twelve first assemblies and thirty second assemblies; in this embodiment, each first assembly may, for example, comprise five copies of the identical first nanostructure-related polypeptide, and each second assembly may, for example, comprise two copies of the identical second nanostructure related polypeptide. In a further embodiment, the nanostructure comprises twenty first assemblies and thirty second assemblies; in this embodiment, each first assembly may, for example, comprise three copies of the identical first nanostructure-related polypeptide, and each second assembly may, for example, comprise two copies of the identical second nanostructure-related polypeptide. All of these embodiments are capable of forming synthetic nanomaterials with regular icosahedral symmetry.

Vill. COMBINATION WITH A SACCHARIDE AND/OR POLYPEPTIDE OR FRAGMENT THEREOF DERIVED FROM KLEBSIELLA PNEUMONIAE Klebsiella pneumoniae (K. pneumoniae) is a Gram-negative pathogen, known to cause urinary tract infections, bacteremia, and sepsis. Multidrug-resistant Klebsiella pneumoniae infections are an increasing cause of mortality in vulnerable populations at risk. The 0-antigen serotypes are highly prevalent among strains causing invasive disease globally and derived 0-antigen glycoconjugates are attractive as vaccine antigens. In one aspect, any of the compositions disclosed herein may further comprise at least one saccharide that is, or is derived from, at least one K. pneumoniae serotype selected from 01 (and d-Gal-Ill variants), 02 (and d-Gal-Ill variants), 2ac, 03, 04, 05, 07, 08, and 012. In a preferred embodiment, any of the compositions disclosed herein may further comprise a polypeptide derived from K. pneumoniae selected from a polypeptide derived from K. pneumoniae Type I fimbrial protein or an immunogenic fragment thereof; or a polypeptide derived from K. pneumoniae Type III fimbrial protein or an immunogenic fragment thereof; or a combination thereof.

As is known in the art, K. pneumoniae 01 and 020-antigens and their corresponding v1 and v2 subtypes are polymeric galactans that differ in the structures of their repeat units. K. pneumoniae 01 and 02 antigens contain homopolymer galactose units (or galactans). K. pneumoniae 01 and 02 antigens each contain D-galactan I units (sometimes referred to as the 02a repeat unit), but 01 antigens differ in that 01 antigens have a D-galactan II cap structure. D-galactan III (d-Gal-III) is a variant of D-galactan I. Structures of the base galactans I and III that define the two distinct serotype 02 subtypes, 02v1 and 02v2; and structures of the derived chimeras resulting from capping by galactan IIwhich yields subtypes 01v1 and 01v2, are shown in Kelly SD, et al. J Biol Chem 2019; 294:10863-76; and Clarke BR, et al. J Biol Chem 2018; 293:4666-79. In some embodiments, the saccharide derived from K. pneumoniae 01 includes a repeat unit of [->3)-p-D-Galf-(1->3)-a-D-Galp-(1->]. In some embodiments, the saccharide derived from K. pneumoniae 01 includes a repeat unit of [->3)-a-D- Galp-(1->3)- p-D-Galp (1->]. In some embodiments, the saccharide derived from K. pneumoniae 01 includes a repeat unit of [->3)-p-D-Galf -(1->3)-a-D-Gap-(1->], and a repeat unit of [->3)-a-D- Galp-(1->3)- P-D Galp-(1->]. In some embodiments, the saccharide derived from K. pneumoniae 01 includes a repeat unit of ->3)-p-D-Galf -(1->3)-[a-D-Galp-(1->4)]-a-D-Galp-(1->] (referred to as the D-Gal III repeat unit). (Kol 0., et al. (1992) Carbohydr. Res. 236, 339-344; Whitfield C., et al. (1991) J. Bacteriol. 173, 1420-1431). In some embodiments, the saccharide derived from K. pneumoniae 02 includes a repeat unit of [->3)-a-D-Galp-(1->3)-p-D-Galf-(1->] (which may be an element of K. pneumoniae serotype 02a antigen). In some embodiments, the saccharide derived from K. pneumoniae 02 includes a repeat unit of [->3)-p-D-GcpNAc-(1->5)-p-D-Galf-(1->] (which may be an element of K. pneumoniae serotype 02c antigen). In some embodiments, the saccharide derived from K. pneumoniae 02 includes a modification of the 02a repeat unit by side chain addition of (1-4) linked Galp residues (which may be an element of the K. pneumoniae O2afg antigen). In some embodiments, the saccharide derived from K. pneumoniae 02 includes a modification of the 02a repeat unit by side chain addition of (1->2)-linked Galp residues (which may be an element of the K. pneumoniae O2aeh antigen). (Whitfield C., et al. (1992) J. Bacteriol. 174, 4913-4919). Without being bound by mechanism or theory, 0-antigen polysaccharide structure of K. pneumoniae serotypes 03 and 05 are disclosed in the art to be identical to those of E. coli serotypes 09a (Formula 09a) and 08 (Formula 08), respectively. In some embodiments, the saccharide derived from K. pneumoniae 04 includes a repeat unit of [->4)-a-D-Galp-(1->2)-P-D-Ribf-(1->)]. In some embodiments, the saccharide derived from K. pneumoniae 07 includes a repeat unit of [->2-a-L-Rhap-(1->2)-p-D-Ribf (1->3)-a-L-Rhap-(1->3)-a-L-Rhap-(1->]. In some embodiments, the saccharide derived from K.

pneumoniae 08 serotype includes the same repeat-unit structure as K. pneumoniae 02a, but is nonstoichiometrically O-acetylated. In some embodiments, the saccharide derived from K. pneumoniae 012 serotype includes a repeat unit of [a-Rhap-(1 ->3)-p GlcpNAc] disaccharide repeat unit. In one aspect, the invention includes a composition including a polypeptide derived from E. coli FimH or a fragment thereof; and at least one saccharide that is, or derived from, at least one K. pneumoniae serotype selected from 01 (and d-Gal-ll variants), 02 (and d-Gal-Illvariants), 2ac, 03, 04, 05, 07, 08, and 012. In some embodiments, the composition includes saccharides from or derived from one or more of serotypes 01, 02, 03, and 05, or a combination thereof. In some embodiments, the composition includes saccharides from or derived from each of serotypes 01, 02, 03, and 05. In another aspect, the invention includes a composition including at least one saccharide that is, or is derived from, at least one K. pneumoniae serotype selected from 01 (and d-Gal-Ill variants), 02 (and d-Gal-Illvariants), O2ac, 03, 04, 05, 07, 08, and 012; and a saccharide derived from an E.ColiO-antigen having a structure selected from any one of Formula 01 (e.g., Formula 01A, Formula 01B, and Formula 01C), Formula 02, Formula 03, Formula 04 (e.g., Formula 04:K52 and Formula 04:K6), Formula 05 (e.g., Formula 05ab and Formula 05ac (strain 180/C3)), Formula 06 (e.g., Formula 06:K2; K13; K15 and Formula 06:K54), Formula 07, Formula 08, Formula 09, Formula 010, Formula 011, Formula 012, Formula 013, Formula 014, Formula 015, Formula 016, Formula 017, Formula 018 (e.g., Formula 018A, Formula 018ac, Formula 018A1, Formula 018B, and Formula 018B1), Formula 019, Formula 020, Formula 021, Formula 022, Formula 023 (e.g., Formula 023A), Formula 024, Formula 025 (e.g., Formula 025a and Formula 025b), Formula 026, Formula 027, Formula 028, Formula 029, Formula 030, Formula 032, Formula 033, Formula 034, Formula 035, Formula 036, Formula 037, Formula 038, Formula 039, Formula 040, Formula 041, Formula 042, Formula 043, Formula 044, Formula 045 (e.g., Formula 045 and Formula 045rel), Formula 046, Formula 048, Formula 049, Formula 050, Formula 051, Formula 052, Formula 053, Formula 054, Formula 055, Formula 056, Formula 057, Formula 058, Formula 059, Formula 060, Formula 061, Formula 062, Formula 62D1, Formula 063, Formula 064, Formula 065, Formula 066, Formula 068, Formula 069, Formula 070, Formula 071, Formula 073 (e.g., Formula 073 (strain 73-1)), Formula 074, Formula 075, Formula 076, Formula 077, Formula 078, Formula 079, Formula 080, Formula 081, Formula 082, Formula 083, Formula 084, Formula 085, Formula 086, Formula 087, Formula 088, Formula 089, Formula 090, Formula 091,

Formula 092, Formula 093, Formula 095, Formula 096, Formula 097, Formula 098, Formula 099, Formula 0100, Formula 0101, Formula 0102, Formula 0103, Formula 0104, Formula 0105, Formula 0106, Formula 0107, Formula 0108, Formula 0109, Formula 0110, Formula 0111, Formula 0112, Formula 0113, Formula 0114, Formula 0115, Formula 0116, Formula 0117, Formula 0118, Formula 0119, Formula 0120, Formula 0121, Formula 0123, Formula 0124, Formula 0125, Formula 0126, Formula 0127, Formula 0128, Formula 0129, Formula 0130, Formula 0131, Formula 0132, Formula 0133, Formula 0134, Formula 0135, Formula 0136, Formula 0137, Formula 0138, Formula 0139, Formula 0140, Formula 0141, Formula 0142, Formula 0143, Formula 0144, Formula 0145, Formula 0146, Formula 0147, Formula 0148, Formula 0149, Formula 0150, Formula 0151, Formula 0152, Formula 0153, Formula 0154, Formula 0155, Formula 0156, Formula 0157, Formula 0158, Formula 0159, Formula 0160, Formula 0161, Formula 0162, Formula 0163, Formula 0164, Formula 0165, Formula 0166, Formula 0167, Formula 0168, Formula 0169, Formula 0170, Formula 0171, Formula 0172, Formula 0173, Formula 0174, Formula 0175, Formula 0176, Formula 0177, Formula 0178, Formula 0179, Formula 0180, Formula 0181, Formula 0182, Formula 0183, Formula 0184, Formula 0185, Formula 0186, and Formula 0187, wherein n is an integer from 1 to 100. In some embodiments, the composition includes a saccharide from or derived from one or more of K. pneumoniae serotypes 01, 02, 03 and 05, or a combination thereof. In some embodiments, the composition includes a saccharide from or derived from each of K. pneumoniae serotypes 01, 02, 03 and 05. In some embodiments, the composition includes a saccharide derived from an E.Coli0-antigen having Formula 09 and does not include a saccharide derived from K. pneumoniae serotype 03. In some embodiments, the composition includes a saccharide derived from an E.Coli0-antigen having Formula 08 and does not include a saccharide derived from K. pneumoniae serotype 05. In another aspect, the invention relates to a composition including a polypeptide derived from E. coli FimH or a fragment thereof; at least one saccharide that is, or derived from, at least one K. pneumoniae serotype selected from 01 (and d-Gal-Ill variants), 02 (and d-Gal-ll variants), O2ac, 03, 04, 05, 07, 08, and 012; and a saccharide having a structure selected from any one of Formula 01 (e.g., Formula 01A, Formula 01B, and Formula 01C), Formula 02, Formula 03, Formula 04 (e.g., Formula 04:K52 and Formula 04:K6), Formula 05 (e.g., Formula 05ab and Formula 05ac (strain 180/C3)), Formula 06 (e.g., Formula 06:K2; K13; K15 and Formula 06:K54), Formula 07, Formula 08, Formula 09, Formula 010, Formula 011, Formula 012, Formula 013, Formula 014, Formula 015, Formula 016, Formula 017, Formula 018 (e.g., Formula 018A, Formula 018ac, Formula 018A1, Formula 018B, and Formula 018B1), Formula 019, Formula 020, Formula 021, Formula 022, Formula 023 (e.g., Formula 023A), Formula 024, Formula 025 (e.g., Formula 025a and Formula 025b), Formula 026,

Formula 027, Formula 028, Formula 029, Formula 030, Formula 032, Formula 033, Formula 034, Formula 035, Formula 036, Formula 037, Formula 038, Formula 039, Formula 040, Formula 041, Formula 042, Formula 043, Formula 044, Formula 045 (e.g., Formula 045 and Formula 045rel), Formula 046, Formula 048, Formula 049, Formula 050, Formula 051, Formula 052, Formula 053, Formula 054, Formula 055, Formula 056, Formula 057, Formula 058, Formula 059, Formula 060, Formula 061, Formula 062, Formula 62D1, Formula 063, Formula 064, Formula 065, Formula 066, Formula 068, Formula 069, Formula 070, Formula 071, Formula 073 (e.g., Formula 073 (strain 73-1)), Formula 074, Formula 075, Formula 076, Formula 077, Formula 078, Formula 079, Formula 080, Formula 081, Formula 082, Formula 083, Formula 084, Formula 085, Formula 086, Formula 087, Formula 088, Formula 089, Formula 090, Formula 091, Formula 092, Formula 093, Formula 095, Formula 096, Formula 097, Formula 098, Formula 099, Formula 0100, Formula 0101, Formula 0102, Formula 0103, Formula 0104, Formula 0105, Formula 0106, Formula 0107, Formula 0108, Formula 0109, Formula 0110, Formula 0111, Formula 0112, Formula 0113, Formula 0114, Formula 0115, Formula 0116, Formula 0117, Formula 0118, Formula 0119, Formula 0120, Formula 0121, Formula 0123, Formula 0124, Formula 0125, Formula 0126, Formula 0127, Formula 0128, Formula 0129, Formula 0130, Formula 0131, Formula 0132, Formula 0133, Formula 0134, Formula 0135, Formula 0136, Formula 0137, Formula 0138, Formula 0139, Formula 0140, Formula 0141, Formula 0142, Formula 0143, Formula 0144, Formula 0145, Formula 0146, Formula 0147, Formula 0148, Formula 0149, Formula 0150, Formula 0151, Formula 0152, Formula 0153, Formula 0154, Formula 0155, Formula 0156, Formula 0157, Formula 0158, Formula 0159, Formula 0160, Formula 0161, Formula 0162, Formula 0163, Formula 0164, Formula 0165, Formula 0166, Formula 0167, Formula 0168, Formula 0169, Formula 0170, Formula 0171, Formula 0172, Formula 0173, Formula 0174, Formula 0175, Formula 0176, Formula 0177, Formula 0178, Formula 0179, Formula 0180, Formula 0181, Formula 0182, Formula 0183, Formula 0184, Formula 0185, Formula 0186, and Formula 0187, wherein n is an integer from 1 to 100, preferably from 31 to 90. In some embodiments, the composition includes a saccharide derived from an E.Coli 0-antigen having Formula 09 and does not include a saccharide derived from K. pneumoniae serotype 03. In some embodiments, the composition includes a saccharide derived from an E.Coli0-antigen having Formula 08 and does not include a saccharide derived from K. pneumoniae serotype 05.

In some embodiments, the composition includes at least one saccharide derived from any one K. pneumoniae type selected from the group consisting of 01, 02, 03, and 05. In some embodiments, the composition includes at least one saccharide derived from K. pneumoniae type 01. In one aspect of this embodiment, the K.pneumoniae 0 antigen is selected from subtype v1 (01v1) or subtype v2 (01v2). In one aspect of this embodiment, the K.pneumoniae 0-antigen is selected from subtype v1 (Olvi) and subtype v2 (01v2). In some embodiments, the composition includes at least one saccharide derived from K. pneumoniae type 02. In one aspect of this embodiment, the K.pneumoniae 0-antigen is selected from subtype v1 (02v1) or subtype v2 (O2v2). In one aspect of this embodiment, the K.pneumoniae 0-antigen is selected from subtype v1 (02v1) and subtype v2 (O2v2). In another aspect, the K.pneumoniae 0-antigen is selected from the group consisting of: a) serotype 01 subtype v1 (01v1), b) serotype 01 subtype v2 (01v2), c) serotype 02 subtype v1 (2v1), and d) serotype 02 subtype v2 (O2v2). In one aspect of this embodiment, the K.pneumoniae 0 antigen is subtype v1 (01v). In one aspect of this embodiment, the K.pneumoniae0-antigen is subtype v2 (01v2). In one aspect of this embodiment, the K.pneumoniae 0-antigen is subtype v (02v1). In one aspect of this embodiment, the K.pneumoniae 0-antigen is subtype v2 (O2v2). In another aspect of this embodiment, the composition comprises one, two, three or four K.pneumoniae 0-antigen selected from the group consisting of: a) serotype 01 subtype v (01v), b) serotype 01 subtype v2 (01v2), c) serotype 02 subtype v (O2v),and d) serotype 02 subtype v2 (02v2).In some embodiments, the composition includes a combination of saccharides derived from K. pneumoniae, wherein a first saccharide is derived from any one of K. pneumoniae types selected from the group consisting of 01, 02, 03, and 05; and a second saccharide is derived from a saccharide is derived from any one of K. pneumoniae types selected from the group consisting of 01 (and d-Gal-Ill variants), 02 (and d-Gal-Ill variants), 02ac, 03, 04, 05, 07, 08, and 012. For example, in some embodiments, the composition includes at least one saccharide derived from K. pneumoniae type 01 and at least one saccharide derived from K. pneumoniae type 02. In a preferred embodiment, the saccharide derived from K. pneumoniae is conjugated to a carrier protein; and the saccharide derived from E. coli is conjugated to a carrier protein. In another aspect, the invention includes a composition including a polypeptide derived from E. coli FimH or a fragment thereof; and at least one saccharide derived from any one K. pneumoniae type selected from the group consisting of 01, 02, 03, and 05. In another aspect, the invention includes at least one saccharide derived from any one K. pneumoniae type selected from the group consisting of 01, 02, 03, and 05; and at least one saccharide derived from E. coli having a structure selected from any one of Formula 01 (e.g., Formula 01A, Formula 01B, and Formula 01C), Formula 02, Formula 03, Formula 04

(e.g., Formula 04:K52 and Formula 04:K6), Formula 05 (e.g., Formula 05ab and Formula 05ac (strain 180/C3)), Formula 06 (e.g., Formula 06:K2; K13; K15 and Formula 06:K54), Formula 07, Formula 08, Formula 09, Formula 010, Formula 011, Formula 012, Formula 013, Formula 014, Formula 015, Formula 016, Formula 017, Formula 018 (e.g., Formula 018A, Formula 018ac, Formula 018A1, Formula 018B, and Formula 018B1), Formula 019, Formula 020, Formula 021, Formula 022, Formula 023 (e.g., Formula 023A), Formula 024, Formula 025 (e.g., Formula 025a and Formula 025b), Formula 026, Formula 027, Formula 028, Formula 029, Formula 030, Formula 032, Formula 033, Formula 034, Formula 035, Formula 036, Formula 037, Formula 038, Formula 039, Formula 040, Formula 041, Formula 042, Formula 043, Formula 044, Formula 045 (e.g., Formula 045 and Formula 045rel), Formula 046, Formula 048, Formula 049, Formula 050, Formula 051, Formula 052, Formula 053, Formula 054, Formula 055, Formula 056, Formula 057, Formula 058, Formula 059, Formula 060, Formula 061, Formula 062, Formula 62 D1, Formula 063, Formula 064, Formula 065, Formula 066, Formula 068, Formula 069, Formula 070, Formula 071, Formula 073 (e.g., Formula 073 (strain 73-1)), Formula 074, Formula 075, Formula 076, Formula 077, Formula 078, Formula 079, Formula 080, Formula 081, Formula 082, Formula 083, Formula 084, Formula 085, Formula 086, Formula 087, Formula 088, Formula 089, Formula 090, Formula 091, Formula 092, Formula 093, Formula 095, Formula 096, Formula 097, Formula 098, Formula 099, Formula 0100, Formula 0101, Formula 0102, Formula 0103, Formula 0104, Formula 0105, Formula 0106, Formula 0107, Formula 0108, Formula 0109, Formula 0110, Formula 0111, Formula 0112, Formula 0113, Formula 0114, Formula 0115, Formula 0116, Formula 0117, Formula 0118, Formula 0119, Formula 0120, Formula 0121, Formula 0123, Formula 0124, Formula 0125, Formula 0126, Formula 0127, Formula 0128, Formula 0129, Formula 0130, Formula 0131, Formula 0132, Formula 0133, Formula 0134, Formula 0135, Formula 0136, Formula 0137, Formula 0138, Formula 0139, Formula 0140, Formula 0141, Formula 0142, Formula 0143, Formula 0144, Formula 0145, Formula 0146, Formula 0147, Formula 0148, Formula 0149, Formula 0150, Formula 0151, Formula 0152, Formula 0153, Formula 0154, Formula 0155, Formula 0156, Formula 0157, Formula 0158, Formula 0159, Formula 0160, Formula 0161, Formula 0162, Formula 0163, Formula 0164, Formula 0165, Formula 0166, Formula 0167, Formula 0168, Formula 0169, Formula 0170, Formula 0171, Formula 0172, Formula 0173, Formula 0174, Formula 0175, Formula 0176, Formula 0177, Formula 0178, Formula 0179, Formula 0180, Formula 0181, Formula 0182, Formula 0183, Formula 0184, Formula 0185, Formula 0186, and Formula 0187. In some embodiments, the composition includes a saccharide derived from an E.Coli0-antigen having Formula 09 and does not include a saccharide derived from K. pneumoniae serotype 03. In some embodiments, the composition includes a saccharide derived from an E.Coli0-antigen having Formula 08 and does not include a saccharide derived from K. pneumoniae serotype 05. In some embodiments, the composition includes at least one saccharide derived from K. pneumoniae type 01; and at least one saccharide derived from E. coli having a structure selected from the group consisting of Formula 08 and Formula 09. In another embodiment, the composition includes at least one saccharide derived from K. pneumoniae type 02; and at least one saccharide derived from E. coli having a structure selected from the group consisting of Formula 08 and Formula 09. In another embodiment, the composition includes at least one saccharide derived from K. pneumoniae type 01; at least one saccharide derived from K. pneumoniae type 02; and at least one saccharide derived from E. coli having a structure selected from the group consisting of Formula 08 and Formula 09. In one embodiment, the invention provides a method of inducing an immune response to K. pneumoniae in a subject comprising administering to the subject an immunologically effective amount of an immunogenic composition comprising at least one glycoconjugate from E. coli serotype 08 or 09, wherein said immunogenic composition does not comprise glycoconjugates from K. pneumoniae serotype 05 or 03. In one aspect, the composition includes a saccharide derived from an E. coli0-antigen having Formula 08 and does not include a saccharide derived from K. pneumoniae serotype 05. In another aspect, the composition includes a saccharide derived from an E. coli0-antigen having Formula 09 and does not include a saccharide derived from K. pneumoniae serotype 03. In another embodiment, the invention provides a method of inducing an immune response to E. coli in a subject comprising administering to the subject an immunologically effective amount of an immunogenic composition comprising at least one glycoconjugate from K. pneumoniae serotype 05 or 03, or a variant thereof, wherein said immunogenic composition does not comprise glycoconjugates from E. coli serotype 08 or 09. In one aspect, the composition includes a saccharide derived from K. pneumoniae serotype 05 and does not include a saccharide derived from an E. coli 0-antigen having Formula 08. In another aspect, the composition includes a saccharide derived from K. pneumoniae serotype 03 and does not include a saccharide derived from an E. coli0-antigen having Formula 09. In some embodiments, the composition includes at least one saccharide that is, or is derived from, at least one K. pneumoniae serotype selected from 01 (and d-Gal-Ill variants), 02 (and d-Gal-Ill variants), O2ac, 03, 04, 05, 07, 08, and 012; at least one saccharide derived from E. coli having a structure selected from the group consisting of Formula 08 and

Formula 09. In some embodiments, the composition includes at least one saccharide that is, or derived from, at least one K. pneumoniae serotype selected from 01 (and d Gal-III variants), 02 (and d-Gal-Illvariants), 2ac, 03, 04, 05, 07, 08, and 012; at least one saccharide derived from E. coli having a structure selected from the group consisting of Formula 01A, Formula 01B, Formula 02, Formula 06, and Formula 025B. In some embodiments, the composition further includes a polypeptide derived from K. pneumoniae selected from a polypeptide derived from K. pneumoniae Type I fimbrial protein or an immunogenic fragment thereof; or a polypeptide derived from K. pneumoniae Type III fimbrial protein or an immunogenic fragment thereof, or a combination thereof. The sequences of said polypeptides are known in the art.

IX. Uses of the Compositions In one aspect, the disclosure provides use of the compositions described herein, nucleic acids encoding E.coli FimH polypeptides or vectors for expressing them, or compositions comprising a polypeptide or nucleic acids as a medicament, or in the manufacture of a medicament, for eliciting an immune response against E.coli or K. pneumoniae or for preventing E.coli or K. pneumoniae infection in a subject. In other aspects, the present disclosure provides a method of eliciting an immune response against E.colior K. pneumoniae in a subject, such as a human, comprising administering to the subject an effective amount of a composition described herein or a nucleic acid molecule encoding an E.coli or K. pneumoniae polypeptide. The present disclosure also provides a method of preventing E.coli or K. pneumoniae infection in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition, such as a vaccine, comprising a E.coli or K. pneumoniae composition described herein. As used herein, "subject" means a mammal, preferably a human. In some particular embodiments, the human is a child, such as an infant. In some other particular embodiments, the human is a woman, particularly a pregnant woman. The compositions of the invention may be administered to the subject with or without administration of an adjuvant. The effective amount administered to the subject is an amount that is sufficient to elicit an immune response against an E. color K. pneumoniae antigen in the subject. Subjects that can be selected for treatment include those that are at risk for developing an E. coli or K. pneumoniae infection because of exposure or the possibility of exposure to E. coli or K. pneumoniae. Because humans may be infected with E. coli or K. pneumoniae by the age of 2, the entire birth cohort is included as a relevant population for immunization. This could be done, for example, by beginning an immunization regimen anytime from birth to 6 months of age, from 6 months of age to 5 years of age, in pregnant women (or women of child-bearing age) to protect their infants by passive transfer of antibody, infants still in utero, and subjects greater than 50 years of age. Administration of the compositions provided by the present disclosure, such as pharmaceutical compositions, can be carried out using standard routes of administration. Non limiting embodiments include parenteral administration, such as intradermal, intramuscular, subcutaneous, transcutaneous, mucosal, or oral administration. The total dose of the composition provided to a subject during one administration can be varied as is known to the skilled practitioner. It is also possible to provide one or more booster administrations of one or more of the vaccine compositions. If a boosting vaccination is performed, typically, such a boosting vaccination will be administered to the same subject at a moment between one week and 10 years, preferably between two weeks and six months, after administering the composition to the subject for the first time (which is in such cases referred to as "priming vaccination"). In alternative boosting regimens, it is also possible to administer different vectors, e.g., one or more adenovirus, or other vectors such as modified vaccinia virus of Ankara (MVA), or DNA, or protein, to the subject after the priming vaccination. It is, for instance, possible to administer to the subject a recombinant viral vector hereof as a prime, and boosting with a composition described herein. In certain embodiments, the administration comprises a priming administration and at least one booster administration. In certain other embodiments, the administration is provided annually. In still other embodiments, the administration is provided annually together with an influenza vaccine. The vaccines provided by the present disclosure may be used together with one or more other vaccines. For example, in adults they may be used together with an influenza vaccine, Prevnar, tetanus vaccine, diphtheria vaccine, and pertussis vaccine. For pediatric use, vaccines provided by the present disclosure may be used with any other vaccine indicated for pediatric patients.

EXAMPLES

In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner. The following Examples illustrate some embodiments of the invention.

EXAMPLE Bacterial fimbrial adhesins FimH and FmIH allow Escherichiacolito exploitdistinct urinary tract microenvironments through recognition of specific host cell glycoproteins. FimH binds to manosylated uroplakin receptors in the uroepithelium whereas FmH binds to galactose or N-acetylgalactosamine 0-glycans on epithelial surface proteins in the kidney and inflamed bladder. FimH fimbriae also play a role in colonization of enterotoxigenic E.coli (ETEC) and multidrug-resistant invasive E.coliin the gut through binding to highly mannosylated proteins on the intestinal epithelia. Full length FimH is composed of two domains: the N-terminal lectin domain and the C-terminal pilin domain, which are connected by a short linker. The lectin domain of FimH contains the carbohydrate recognition domain, which is responsible for binding to the mannosylated uroplakin 1a on the urothelial cell surface. The pilin domain is anchored to the core of the pilus via a donor strand of the subsequent FimG subunit, which is a process termed donor strand complementation. Conformation and ligand-binding properties of the lectin domain of FimH are under the allosteric control of the pilin domain of FimH. Under static conditions, the interaction of the two domains of full length FimH stabilizes the lectin domain in the low affinity to monomannose (for example, Kd -300 pM) state, which is characterized by a shallow binding pocket. Binding to a mannoside ligand induces a conformational change leading to a medium affinity state, where the lectin and pilin domains remain in close contact. However, upon shear stress, the lectin and pilin domains separate, thereby inducing the high-affinity state (for example, K <1.2 pM). Because of the absence of negative allosteric regulation exerted by the pilin domain, the isolated lectin domain of FimH is locked in the high-affinity state. The isolated, recombinant lectin domain, which is locked in the high-affinity state, exhibits high stability. Locking the adhesin in a low-binding conformation, however, induces the production of adhesion-inhibiting antibodies. Accordingly, there is an interest in stabilizing the lectin domain in the low-affinity state. Table 3 sets forth the FimH constructs used to make various constructs to address these needs.

Table 3: Summary of FimH constructs

Construct Plasmid Signal Protein Linker Additional Backbone Mass sequence Description protein variant FimH pSB01877 FimH FimH J96 none none pcDNA3.1(+) lectin signal F22..G181 or domain sequence pCAG vector

FimH pSB01878 mIgK FimH J96 none none pcDNA3.1(+) Fully lectin signal F22..G181 or reduced domain sequence pCAG vector mass, with His tag: 18117.48 Observed non reduced mass, with His tag: 18117.90

Mass without tag: 17022.08

FimH/C pSB01879 FimH FimH J96 none none pBudCE4.1 signal F22..Q300 Dual sequence promoter vector (CMV &EFl)

FimH/C pSBO1880 mIgK FimH J96 none none pBudCE4.1 signal F22..Q300 Dual sequence promoter vector (CMV &EFla) FimH/C pSBO1881 mIgK FimC none none pBudCE4.1 signal G37..E241 Dual sequence (according promoter to SEQ ID vector (CMV NO: 18) & EFla)

FimH- pSBO1882 FimH FimH J96 DNKQ FimG dscG signal F22..Q300 A1..K14 sequence (SEQ ID NO: 17)

FimH- pSBO1883 FimH FimH J96 GGSGG FimG dscG signal F22..Q300 A1..K14 sequence (SEQ ID NO: 17) FimH- pSBO1884 FimH FimH J96 GGSSGG FimG dscG signal F22..Q300 A1..K14 sequence (SEQ ID NO: 17)

FimH- pSBO1885 FimH FimH J96 GGSSGGG FimG N-terminus dscG signal F22..Q300 A1..K14 residue at sequence (SEQID W20, and NO: 17) therefore does not appearto have been processed at preferred position; a small amount of protein present exhibiting preferred processing as indicated by the small amount of the FACK peptide being detected FimH- pSBO1886 FimH FimH J96 GGGSSGGG FimG dscG signal F22..Q300 A1..K14 sequence (SEQ ID NO: 17)

FimH- pSBO1887 FimH FimH J96 GGGSGSGGG FimG dscG signal F22..Q300 A1..K14 sequence (SEQ ID NO: 17)

FimH- pSBO1888 FimH FimH J96 GGGSGGSGGG FimG dscG signal F22..Q300 A1..K14 sequence (SEQ ID NO: 17)

FimH- pSBO1889 mIgK FimH J96 DNKQ FimG dscG signal F22..Q300 A1..K14 sequence (SEQ ID NO: 17)

FimH- pSBO1890 mIgK FimH J96 GGSGG FimG dscG signal F22..Q300 A1..K14 sequence (SEQID NO: 17)

FimH- pSBO1891 mIgK FimH J96 GGSSGG FimG dscG signal F22..Q300 A1..K14 sequence (SEQ ID NO: 17)

FimH- pSBO1892 mIgK FimH J96 GGSSGGG FimG appears to dscG signal F22..Q300 A1..K14 have had sequence (SEQID the signal NO: 17) peptide processed with the F22 being the preferred N terminal residue; identity of the peptide was confirmed by MS/MS FimH- pSBO1893 mIgK FimH J96 GGGSSGGG FimG dscG signal F22..Q300 A1..K14 sequence (SEQ ID NO: 17)

FimH- pSBO1894 mIgK FimH J96 GGGSGSGGG FimG dscG signal F22..Q300 A1..K14 sequence (SEQ ID NO: 17)

FimH- pSBO1895 mIgK FimH J96 GGGSGGSGGG FimG dscG signal F22..Q300 A1..K14 sequence (SEQ ID NO: 17)

FimH pSB02081 mIgK F22..G181 lectin signal J96 FimH domain sequence N28Q N91S / His8 in pcDNA3.1(+) FimH pSB02082 mIgK F22..G181 lectin signal J96 FimH domain sequence N28Q N91S / His8 in pcDNA3.1(+) FimH pSB02083 mIgK F22..G181 lectin signal J96 FimH domain sequence N28S N91S/ His8 in pcDNA3.1(+) FimH pSB02088 mIgK F22..G181 lectin signal J96 FimH domain sequence V48C L55C/ His8 in pcDNA3.1(+) FimH pSB02089 mIgK F22..G181 lectin signal J96 FimH domain sequence N28QV48C L55C N91S/ His8 in pcDNA3.1(+) FimH pSB02158 mIgK F22..G181 lectin signal J96 FimH domain sequence N28S V48C L55C N91S/ His8 in pcDNA3.1(+) FimH- pSB02159 dscG FimH- mIgK FimH mIgK dscG signal signal pept/ pSB02198 sequence F22..Q300

J96 FimH N28S V48C L55C N91S N249Q/7 AA linker/ FimG A1..K14/ GGHis8 in pcDNA3.1(+) FimH- FimH mIgK dscG signal pept/ F22..Q300 J96 FimH N28S V48C L55C N91S N256Q/7 AA linker/ FimG mIgK A1..K14/ signal GGHis8 in pSB02199 sequence pcDNA3.1(+) FimH- FimH mIgK dscG signal pept/ F22..Q300 J96 FimH N28S V48C L55C N91S N249Q N256Q/7 AA linker/ FimG mIgK A1..K14/ signal GGHis8 in pSB02200 sequence pcDNA3.1(+) FimH- mIgK FimH mIgK dscG signal signal pept/ pSB02304 sequence F22..Q300

J96 FimH N28S V48C L55C N91S T251A / 7 AA linker/ FimG A1..K14/ GGHis8 in pcDNA3.1(+) FimH- FimH mIgK dscG signal pept/ F22..Q300 J96 FimH N28S V48C L55C N91S T258A / 7 AA linker/ FimG mIgK A1..K14/ signal GGHis8 in pSB02305 sequence pcDNA3.1(+) FimH- FimH mIgK dscG signal pept/ F22..Q300 J96 FimH N28S V48C L55C N91S T251A T258A / 7 AA linker/ FimG mIgK A1..K14/ signal GGHis8 in pSB02306 sequence pcDNA3.1(+) FimH- mIgK FimH mIgK dscG signal signal pept/ pSB02307 sequence F22..Q300

J96 FimH N28S N91S N249Q/7 AA linker/ FimG A1..K14/ GGHis8 in pcDNA3.1(+) FimH- FimH mIgK dscG signal pept/ F22..Q300 J96 FimH N28S N91S N256Q/7 AA linker/ FimG mIgK A1..K14/ signal GGHis8 in pSB02308 sequence pcDNA3.1(+)

Allof the FimH constructs studied were monomeric proteins of expected molecular weight.

Table 4

Protein Sedimentation Mw,app M.,expected Homogeneity Coefficient, S E.coli expression Cytosolic FimH-LD 1.9 S 18 kDa 18 kDa 98% Periplasmic FimH-LD 1.9 S 18 kDa 18 kDa 98% FimH-LD lock mutant 2.0S 19 kDa 18 kDa 97%

Mammalian expression FimH-LD 1.9 S 18 kDa 18 kDa >99% FimH-LD lock mutant 1.9 S 18 kDa 18 kDa 98% FimH wild type 2.7S 36 kDa 34 kDa 96% FimH lock mutant 2.7S 34 kDa 34 kDa 94%

Expected molecular weight of FimC-FimH complex is 53.1 kDa; Expected molecular weight of FimC is 24 kDa.

EXAMPLE 2: Mammalian expression of FimH lectin binding domain The present non-limiting example relates to producing a polypeptide derived from E. coli or a fragment thereof in a HEK cell line. The yields were relatively high, as compared to expression of the polypeptide derived from E. color a fragment thereof in an E. coli host cell. To accomplish the production of FimH variants from mammalian cells, a SignalP prediction algorithm was used to analyze different heterologous signal sequences for secretion of proteins and fragments. The wild type FimH leader sequence was also analyzed. The predictions indicated that the wild type FimH leader sequence may work for secretion of the FimH variants in mammalian cells, however, the secreted variant was predicted to be cleaved at the W20 residue of the full-length wild type FimH (see SEQ ID NO: 1), rather than the F22 residue of the full-length wild type FimH (see SEQ ID NO: 1). A hemagglutinin signal sequence was predicted not to work. The murine IgK signal sequence was predicted to produce an N terminus of F22 of SEQ ID NO: 1, or F1 residue of the mature protein. Based on these analyses, DNA was synthesized and recombinantly produced constructs to express the FimH lectin binding domain with the wild-type FimH leader. Constructs were also prepared to express the FimH lectin binding domain with the mgK signal sequence. Affinity purification tags, such as His tag, were introduced to the C-terminus of the polypeptide derived from E. coli or a fragment thereof to facilitate purification. The expression plasmid was transfected into HEK host cells, namely EXP1293 mammalian cells. The polypeptides or fragments thereof derived from E. coliwere successfully expressed. For example, the preferred N-terminal processing using the mlgK signal sequence fused to the mature start of FimH at F22 was demonstrated for the pSB01892 FimHdscG construct by MS. The processing is believed correct for the lectin domain construct pSB01878 and the mass spec data supports this. The preferred N-terminal processing (i.e., processing at F22 of SEQ ID NO: 1) was not shown with the native FimH leader peptide. pSB01877 and pSB01878 constructs are in pcDNA3.1(+) mammalian expression vectors. The cells were diluted and subsequently used in 20 ml transfections. 1 ug/ml DNA for each construct was used and transfected cells in 125ml flasks using Expifectamine protocol. After 72 hours, the cell viability was still good so the expression was allowed to continue until 96 hours. Samples were taken at 72 hours and ran 10 ul of each on SDS PAGE gels to check for expression.

After 96 hours, conditioned media was harvested and 0.25ml of Nickel Excel resin was added with batch binding O/N at 40C with rotation. Eluted in TrisC pH8.0, NaCI, imidazole. See FIG. 4. pSBO1878 has expected mass consistent with N-terminal F22. Glycosylation present on 1 or 2 sites (+1 mass from each deamidation of N-D). Glycosylation mutants were constructed. See, for example, pSB02081, pSB02082, pSB02083, pSB02088, and pSB02089. The glycosylation mutants expressed the polypeptides of interest. See FIG. 5 for results. A FimH lectin domain lock mutant was also constructed. See, for example, pSB02158. Results of the expression of the pSB02158 construct is shown in FIG. 6B. Fluorescence polarization assay using 0.5 pmoles fluorescein-conjugated aminophenyl-mannopyranoside (APMP). The assay was performed at room temperature, 300 RPM for 64 hrs. Results shown in FIG. 6C.

EXAMPLE 3: Mammalian expression of FimH/C complex, pSB01879 and pSBO1880 For production of the FimH/C complex, dual expression constructs of the FimC under the EF1alpha promoter and the FimH with either the wild type or mgK signal peptide were prepared. These were cloned into a pBudCE4.1 mammalian expression vector (ThermoFisher) and a C-term His tag was added to the FimC. The FimC variant was designed for secretion using the mlgK signal peptide as it resulted in a postive prediction to yield the G37 FimC as the first residue of the mature protein based on SignalP analysis. More specifically, these constructs were designed to have the FimC fragment under the EF1alpha promoter in the vector pBudCE4.1 and the FimH fragment inserts under the CMV promoter in the same vector. The vector pBudCE4.1 is an expression vector from Thermo Fisher that has 2 promoters for expression in mammalian cells. The FimC fragment insert (pSBO1881 insert) was subcloned by digesting with Notl and Xhol and subcloning into the pBudCE4.1 vector at the same sites. These were plated onto 2xYT zeocin 50 ug/ml plates. Colonies were inoculated into 2xYT with zeocin 50ug/ml, grew overnight at 37 0C and plasmid prepped. These were digested with Notl and Xhol to check for insert and all colonies had insert size of -722 bp. pSBO1881 was digested with HindlIl and BamHl and the pSBO1879 insert and pSBO1880 insert DNA was digested with HindlIl and BamHl. These fragments were gel isolated and subcloned into the pSBO1881 vector and plated onto 2xYTzeo5O ug/ml plates. Colonies from each were inoculated into 2xYT zeo50ug/ml, grown overnight at 37 0C, plasmid prepped and digested with Notl and Xhol to test for FimC insert and

HindIl and BamHl to test for FimH inserts. All clones had expected sized inserts at both cloning sites. The pSB01879-1 and pSB01880-1 clones were subsequently used for expression. The FimH/FimC complex has been demonstrated to express in EXP1293 cells as well. Expression may be optimized by switching promoters, such as EF1a, CAG, Ub, Tub, or other promoters. The preferred N-terminal processing (i.e., processing at F22 of SEQ ID NO: 1) was not shown with the native FimH leader peptide. Exemplary results from SignalP 4.1 (DTU Bioinformatics) used for signal peptide predictions are shown below. Additional signal peptides are predicted to produce the preferred N-terminus of Phe at position 1 of the mature FimH polypeptide or fragment thereof. The following is only a representative sample set of 4 common signal sequences. The following signal peptide sequences were predicted to yield the preferred N-terminus of Phe at position 1 of the mature FimH polypeptide or fragment thereof:

Table 5

signal peptide sequence SEQ ID NO:

>sp IP558991FCGRNHUMAN IgG receptor MGVPRPQPWALGLLLFLLPGSLG SEQ

FcRn large subunit p51 OS=Homo sapiens ID NO: OX=9606 GN=FCGRT PE=1 SV=1 55 >trIQ6FGW4|Q6FGW4_HUMAN ILi0 protein MHSSALLCCLVLLTGVRA SEQ ID OS=Homo sapiens OX=9606 GN=L1O PE=2 SV=1 NO: 56

The following signal peptide sequences were NOT predicted to yield the preferred N terminus of Phe at position 1 of the mature FimH polypeptide or fragment thereof:

Table 6

signal peptide sequence SEQ ID NO:

>sp|P034201FUSHRSVA Fusion glycoprotein FO SEQ MELLILKANAITTILTAVTFCFASG ID OS=Human respiratory syncytial virus A (strain A2) NO:

OX=11259 GN=F PE=1 SV=1 57

>spIP03451|HEMA_157A0 Hemagglutinin OS=Influenza A MAIIYLILLFTAVRG SEQ ID virus (strain A/Japan/305/1957 H2N2) OX=387161 GN=HA NO:

PE=1SV=1 58

Table 7

SignalIP 4.1 used for predictions

Fusion sequence

>sp I P558991FCGRNHUMAN IgG receptor The signal peptide from the protein listed in the FcRn large subunit p51 OS=Homo sapiens respective left column is shown below in CAPITAL OX=9606 GN=FCGRT PE=1 SV=1 LETTERS. The N-terminus of FimH is depicted in lower case. MGVPRPQPWALGLLLFLLPGSLGAESHLSLLY HLTAVSSPAPGTPAFWVSGWLGPQQYLS MGVPRPQPWALGLLLFLLPGSLGfacktangtaipigggs YNSLRGEAEPCGAWVWENQVSWYWEKETT anvyvnlapvvnvgqnlvvdls (SEQ ID NO: 103) DLRIKEKLFLEAFKALGGKGPYTLQGLLGCE LGPDNTSVPTAKFALNGEEFMNFDLKQGTWG GDWPEALAISQRWQQQDKAANKELTFLLF SCPHRLREHLERGRGNLEWKEPPSMRLKARPS SPGFSVLTCSAFSFYPPELQLRFLRNGL AAGTGQGDFGPNSDGSFHASSSLTVKSGDEH HYCCIVQHAGLAQPLRVELESPAKSSVLV VGIVIGVLLLTAAAVGGALLWRRMRSGLPAP WISLRGDDTGVLLPTPGEAQDADLKDVNV IPATA (SEQ ID NO: 102)

# Measure Position Value Cutoff signal peptide? max. C 24 0.664 max. Y 24 0.788 max. S 9 0.966 mean S 1-23 0.935 D 1-23 0.867 0.450 YES Name=Sequence SP='YES' Cleavage site between pos. 23 and 24: SLG-FA D=0.867 D-cutoff=0.450 Networks=SignalP-noTM

>spIP03420|FUSHRSVA Fusion glycoprotein The signal peptide from the protein listed in the FO OS=Human respiratory syncytial virus A respective left column is shown below in CAPITAL LETTERS. The N-terminus of FimH is depicted in (strain A2) OX=11259 GN=F PE=1 SV=1 lower case.

MELLILKANAITTILTAVTFCFASGfacktangtaipigggsanvy vnlapvvnvgqnlvvdls (SEQ ID NO: 105) MELLILKANAITTILTAVTFCFASGQNITEEFYQ STCSAVSKGYLSALRTGWYTSVITIE LSNIKENKCNGTDAKVKLIKQELDKYKNAVTE #Measure Position Value Cutoff signal peptide? LQLLMQSTPPTNNRARRELPRFMNYTLN NAKKTNVTLSKKRKRRFLGFLLGVGSAIASG max. C 28 0.188 VAVSKVLHLEGEVNKIKSALLSTNKAVVS LSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSC max. Y 28 0.263 SISNIETVIEFQQKNNRLLEITREFSVN AGVTTPVSTYMLTNSELLSLINDMPITNDQKK max. S 11 0.478 LMSNNVQIVRQQSYSIMSIIKEEVLAYV VQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNI mean S 1-27 0.387 CLTRTDRGWYCDNAGSVSFFPQAETCKV QSNRVFCDTMNSLTLPSEINLCNVDIFNPKYD D 1-27 0.312 0.500 NO CKIMTSKTDVSSSVITSLGAIVSCYGKT KCTASNKNRGIIKTFSNGCDYVSNKGMDTVS Name=Sequence SP='NO'D=0.312 D-cutoff=0.500 VGNTLYYVNKQEGKSLYVKGEPIINFYDP Networks=SignalP-TM LVFPSDEFDASISQVNEKINQSLAFIRKSDELL HNVNAGKSTTNIMITTIIIVIIVILLS LIAVGLLLYCKARSTPVTLSKDQLSGINNIAFS N (SEQ ID NO: 104)

>trIQ6FGW4|Q6FGW4_HUMAN IL10 protein The signal peptide from the protein listed in the OS=Homo sapiens OX=9606 GN=lL10 PE=2 respective left column is shown below in CAPITAL LETTERS. The N-terminus of FimH is depicted in SV=1 lower case.

MHSSALLCCLVLLTGVRAfacktangtaipigggsanvyvnlapvv nvgqnlvvdls (SEQ ID NO: 107) MHSSALLCCLVLLTGVRASPGQGTQSENSC THFPGNLPNMLRDLRDAFSRVKTFFQMKDQ # Measure Position Value Cutoff signal peptide? max. C 19 0.726

LDNLLLKESLLEDFKGYLGCQALSEMIQFYLE EVMPQAENQDPDIKAHVNSLGENLKTLR LRLRRCHRFLPCENKSKAVEQVKNAFNKLQE KGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 106) max. Y 19 0.829 max. S 4 0.973 mean S 1-18 0.947 D 1-18 0.893 0.450 YES Name=Sequence SP='YES' Cleavage site between pos. 18 and 19: VRA-FA D=0.893 D-cutoff=0.450 Networks=SignalP-noTM

>sp|P034511HEMA_157A0 Hemagglutinin OS=Influenza A virus (strain A/Japan/305/1957 The signal peptide from the protein listed in the H2N2) OX=387161 GN=HA PE=1 SV=1 respective left column is shown below in CAPITAL LETTERS. The N-terminus of FimH is depicted in lower case.

MAIIYLILLFTAVRGfacktangtaipigggsanvyvnlapvvnvgq nlvvdls (SEQ ID NO: 109) MAIIYLILLFTAVRGDQICIGYHANNSTEKVDT NLERNVTVTHAKDILEKTHNGKLCKLN GIPPLELGDCSIAGWLLGNPECDRLLSVPEW #Measure Position Value Cutoff signal peptide? SYIMEKENPRDGLCYPGSFNDYEELKHLL SSVKHFEKVKILPKDRWTQHTTTGGSRACAV max. C 18 0.524 SGNPSFFRNMVWLTKEGSDYPVAKGSYNN TSGEQMLIIWGVHHPIDETEQRTLYQNVGTY max. Y 18 0.690 VSVGTSTLNKRSTPEIATRPKVNGQGGRM EFSWTLLDMWDTINFESTGNLIAPEYGFKISK max. S 1 0.951 RGSSGIMKTEGTLENCETKCQTPLGAIN TTLPFHNVHPLTIGECPKYVKSEKLVLATGLR mean S 1-17 0.895 NVPQIESRGLFGAIAGFIEGGWQGMVDG WYGYHHSNDQGSGYAADKESTQKAFDGITN D 1-17 0.800 0.450 YES KVNSVIEKMNTQFEAVGKEFGNLERRLENL NKRMEDGFLDVWTYNAELLVLMENERTLDF Name=Sequence SP='YES' Cleavage site between HDSNVKNLYDKVRMQLRDNVKELGNGCFEF pos. 17 and 18: GFA-CK D=0.800 D-cutoff=0.450 Networks=SignalP-noTM YHKCDDECMNSVKNGTYDYPKYEEESKLNR NEIKGVKLSSMGVYQILAIYATVAGSLSLA

IMMAGISFWMCSNGSLQCRICI (SEQ ID NO: 108)

EXAMPLE 4: Mammalian Expression of Donor Strand Complement Fusion of FimH with the FimG peptide Several linker lengths were tested. Recombinant expression with these linkers fusing the FimH to the N-terminal FimG peptide in both the wild type FimH and the mgK signal peptide fused to F22 of FimH were prepared. The FimH donor strand complement FimG constructs have also been shown to have robust expression in EXP1293 cells. The preferred N-terminal processing (i.e., processing at F22 of SEQ ID NO: 1) was not shown with the native FimH leader peptide. For the donor strand complement constructs, oligonucleotides were designed to produce base constructs in pcDNA3.1(+) that contained the various linkers and FimG peptide. A unique BstEll site was incorporated at G294 V295 T296 residues, according to the numbering of SEQ ID NO: 1 of FimH. The same BstEll site was incorporated in the linkers to produce base constructs. The base constructs for pSB01882-01895 were constructed. Primers were used to PCR amplify pcDNA3.1(+) with ACCUPRIME PFX DNA Polymerase (Thermo Fisher), digest the PCR products with Ndel (in CMV promoter) and BamHl and cloned into pcDNA3.1(+) that was digested with Ndel and BamHl and gel isolated to remove the fragment. Another transient transfection was performed with pSB01877, 01878, 01879, 01880, 01885, and 01892 alongside EXP1293 cells as control. Constructs pSB01882 through pSB01895 were used in transient transfection expression tests in EXP1293 cells from Thermo Fisher as per the manufacturer's protocol. See FIG. 3, which shows the results following expression in 20 mL EXP1293 cells, 72 hours, 10 ul of conditioned media loaded; high levels of expression observed; the FimH/FimC complex present following expression from pSB01879 & pSB01880 constructs; 20 ml conditioned media batch bound to Nickel Excel, 40 CV wash, elution in lmdidazole. Additional FimH-donor strand complement constructs were prepared. See, for example, pSB02198, pSB02199, pSB02200, pSB02304, pSB02305, pSB02306, pSB02307, pSB02308 constructs. The expression of pSB2198 FimH dscG lock mutant construct is shown in FIG. 7. The pSB2198 FimH dscG Lock Mutant yielded 12 mg/L from transient expression.

According to Vi-CELL XR 2.04 (Beckman Coulter, Inc.), the following were observed (actual cell type used for expression was HEK cells):

Table 8

Sample Cell type Viability (%) Total cells/ml Viable cells/ml Avg. diam. 6 6 parameter (x10 ) (x10 ) (microns) entered EXPI P13 CHO 97.8 3.56 3.48 19.33 pSB01882 CHO 90.9 4.98 4.53 17.39 pSB01889 CHO 89.2 5.23 4.67 17.14 cells CHO 88.9 6.66 5.92 16.91 Expi Start CHO 93.7 3.35 3.14 18.72

Samples at harvest ~ 85-86 hours after transfection: 1877 SF-9 57.3 4.32 2.48 16.00 pSB01878 SF-9 57.6 3.88 2.24 15.49 pSB01879 SF-9 59.1 5.24 3.10 15.32

pSB01880 SF-9 56.8 5.97 3.39 15.10

pSB01885 SF-9 63.1 6.95 4.39 16.08 pSB01892 SF-9 56.2 4.89 2.75 15.91 187772 SF-9 79.5 5.14 4.09 18.36 187872 SF-9 72.6 5.26 3.81 17.35 expicont SF-9 75.5 4.95 3.74 18.62

EXAMPLE 5: Molecular weight fragments are with processed signal peptide

Table 9

pSB01877 FimH J96 ELL41155.1 [E. coli J96] Analysis Analysis Fragment 15-189 Entire Protein Length 175 aa 189 aa Molecular Weight 18948.34 20522.36 m.w. 1 microgram= 52.775 pMoles 48.727 pMoles Molecular Extinction 35800 35800 Coefficient 1 A(280) corr. to: 0.53 mg/ml 0.57 mg/ml A[280] of 1 mg/ml 1.89 AU 1.74 AU

Isoelectric Point 6.81 8 Charge at pH7 -0.48 1.52

pSB01878 FimH J96 ELL41155.1 [E. coli J96] Analysis Analysis Fragment 21-188 Entire Protein Length 168 aa 188 aa Molecular Weight 18117.48 20344.08 m.w. 1 microgram= 55.195 pMoles 49.154 pMoles Molecular Extinction 24420 35800 Coefficient 1 A(280) corr. to: 0.74 mg/ml 0.57 mg/ml A[280] of 1 mg/ml 1.35 AU 1.76 AU Isoelectric Point 6.81 6.29 Charge at pH7 -0.48 -2.47

pSB01885 FimH J96 ELL41155.1 [E. coli J96] Analysis Analysis Fragment 20-331 Entire Protein Length 312 aa 331 aa Molecular Weight 32406.19 34537.79 m.w. 1 microgram= 30.858 pMoles 28.954 pMoles Molecular Extinction 38030 43720 Coefficient 1 A(280) corr. to: 0.85 mg/ml 0.79 mg/ml A[280] of 1 mg/ml 1.17 AU 1.27 AU Isoelectric Point 7.25 8.32 Charge at pH7 0.5 2.5

pSB01892 FimH J96 ELL41155.1 [E. coli J96] Analysis Analysis Fragment 21-330 Entire Protein Length 310 aa 330 aa Molecular Weight 32132.91 34359.51 m.w. 1 microgram= 31.121 pMoles 29.104 pMoles Molecular Extinction 32340 43720 Coefficient 1 A(280) corr. to: 0.99 mg/ml 0.79 mg/ml

A[280] of 1 mg/ml 1.01 AU 1.27 AU Isoelectric Point 7.25 6.51 Charge at pH7 0.5 -1.49

pSB01893 FimH J96 ELL41155.1 [E. coli J96] Analysis Analysis Entire Protein Length 331 aa Molecular Weight 34416.56 1 microgram= 29.056 pMoles Molecular Extinction 43720 Coefficient 1 A(280) corr. to: 0.79 mg/ml A[280] of 1 mg/ml 1.27 AU Isoelectric Point 6.51 Charge at pH7 -1.49

pSB01894 FimH J96 ELL41155.1 [E. coli J96] Analysis Analysis Fragment 21-332 Entire Protein Length 312 aa 332 aa Molecular Weight 32247.01 34473.61 m.w. 1 microgram= 31.011 pMoles 29.008 pMoles Molecular Extinction 32340 43720 Coefficient 1 A(280) corr. to: 1.00 mg/ml 0.79 mg/ml A[280] of 1 mg/ml 1.00 AU 1.27 AU Isoelectric Point 7.25 6.51 Charge at pH7 0.5 -1.49

pSB02083 Analysis Fragment 21-188 Entire Protein Length 168 aa 188 aa Molecular Weight 18063.42 20290.02 m.w. 1 microgram = 55.361 pMoles 49.285 pMoles Molar Extinction 24420 35800 coefficient

1 A(280) corr. to: 0.74 mg/ml 0.57 mg/ml A[280] of 1 mg/ml 1.35 AU 1.76 AU Isoelectric Point 6.81 6.29 Charge at pH 7 -0.48 -2.47

pSB02198 FimH PSB 2198 1.45 mg/ml Sample 5 ml 20190918SS Volume (mIs) 25 Conc. (mg/ml) 1.45 Total Amount (mgs) 36.25 Aliquots 5ml x5 Yield 12mg/L Buffer: 50 mM TrisCi pH8.0, 300 mM NaCI

pSB02307 Fim H 2307 0.48mglml Sample Name 5ml 20190918SS Volume (mIs) 22.5 mis Conc. (mg/ml) 0.48 mg/ml Total Amount (mgs) 10.8mg Yield 3.6mg/L Buffer: 50 mM TrisCi pH8.0, 300 mM NaCI

EXAMPLE 6: The N-terminal a-amino group of Phel (according to the numbering of SEQ ID NO: 2) in the FimH mature protein provides critical polar recognition for D-mannose

Without being bound by theory or mechanism, it is suggested that the correct signal peptide cleavage just ahead of Phel (according to the numbering of SEQ ID NO: 2) of the FimH mature protein is important to express functional FimH protein. Changes at the N-terminal a amino group, such as by adding an amino acid at the N-terminus ahead of Phel of the FimH protein can abolish the hydrogen bond interactions with 02-, 05- and 06-atoms of the D-mannose and introduce steric repulsion with D-mannose, thereby blocking mannose binding. This is confirmed with our experimental observation that adding an extra Gly residue ahead of the Phel of SEQ ID NO: 2 leads to no detection of mannose binding. Following an analysis of the crystal structure of FimH bound to D-mannose, the following were observed: The N-Terminal a-amino group of Phel along with sidechains of Asp54 of the FimH according to the numbering of SEQ ID NO: 2 and Gln133 of the FimH according to the numbering of SEQ ID NO: 2 provide critical polar recognition motifs for D-mannose, and mutations and changes of these polar interactions lead to no mannose binding.

EXAMPLE 7: The sidechain of Phel in FimH does not interact directly with D-mannose but is rather buried inside of FimH, suggesting that Phel can be replaced by other residues, e.g. aliphatic hydrophobic residues (lie, Leu, or Val) Analysis of crystal structures of FimH in complex with D-mannose and its analogs (e.g. PDB ID: 1QUN) shows that the sidechain of Phel (according to the numbering of SEQ ID NO: 2) does not interact directly with D-mannose but rather stabilizes the binding pocket by stacking its aromatic rings with the sidechains of Va56, Tyr95, Gln133 and Phel44 (according to the numbering of SEQ ID NO: 2). Alternative N-terminal residue instead of Phe may stabilize the FimH protein, accommodate mannose binding, and allow correct signal peptide cleavage. Such residues may be identified by suitable method known in the art, such as by visual inspection of a crystal structure of FimH, or more quantitative selection using computational protein design software, such as BioLuminateTM [BioLuminate, Schrodinger LLC, New York, 2017], Discovery StudioTM [Discovery Studio Modeling Environment, Dassault Systemes, San Diego, 2017], MOE TM [Molecular Operating Environment, Chemical Computing Group Inc., Montreal, 2017], and RosettaTM[Rosetta, University of Washington, Seattle, 2017]. An illustrative example is shown FIG. 9A-9C. The replacement amino acids can be aliphatic hydrophobic amino acids (e.g. Ile, Leu and Val). FIG. 11 depicts computational mutagenesis scanning of Phel with other amino acids having aliphatic hydrophobic sidechains, e.g. Ile, Leu and Val, which may stabilize the FimH protein and accommodate mannose binding.

EXAMPLE 8: Mutations of Asn7 according to the numbering of SEQ ID NO: 2 in a FimH protein can remove the putative N-glycosylation site and prevent deamidation, without impacting mannose, mAb2l, or mAb475 binding. Over-expression of secreted E. coliFimH from mammalian cell lines may lead to N-linked glycosylation at residue Asn7, according to the numbering of SEQ ID NO: 2. In addition, residue Asn7 is solvent exposed and followed with a Gly residue, making it very prone to deamidation. Analysis of crystal structures of FimH in complex with D-mannose and its analogs (e.g. PDB ID: 1QUN) indicates that Asn7 is more than 20 A away from the mannose binding site and a mutation at the site should not impact mannose binding. Thus, mutations of Asn7 to other amino acids (e.g. Ser, Asp and GIn) can effectively remove the putative N-glycosylation site and prevent deamidation.

EXAMPLE 9: E. coliand S. enterica strains Clinical strains and derivatives are listed in Table 10. Additional reference strains included: 025K5H1, a clinical 025a serotype strain; and S. enterica serovar Typhimurium strain LT2. Gene knockouts in E. colistrains removing the targeted open-reading frame but leaving a short scar sequence were constructed. The hydrolyzed O-antigen chain and core sugars are indicated subsequently as 0 Polysaccharide (OPS) for simplicity.

Table 10: E. coli Strains Strain Strain Genotype Serotype Alias GAR2401 PFEEC0100 wt (blood isolate) 025b

'2401AwzzB - AwzzB 025b '2401AAraAA(OPS) - AAraA A(rflB-wzzB) OPS 025K5H1 PFEEC0101 wt 025a O25K5H1AwzzB AwzzB 025a BD559 - W3110 AAraA AfhuA ArecA ops BD559AwzzB - W3110AAmA AfhuA Ogg_ ArecAAwzzB BD559A(OPS) - BD559 A(rflB-wzzB) ops

GAR2831 PFEECO102 wt (blood isolate) 025b GAR865 PFEECO103 wt (blood isolate) 02 GAR868 PFEEC0104 wt (blood isolate) 02 GAR869 PFEECO105 wt (blood isolate) 015

Strain Strain Genotype Serotype Alias GAR872 PFEECO106 wt (blood isolate) 01 GAR878 PFEECO107 wt (blood isolate) 075 GAR896 PFEECO108 wt (blood isolate) 015 GAR1902 PFEECO109 wt (blood isolate) 06 Atlas187913 PFEECO068 wt (blood isolate) 025b Salmonella entericaserovar Typhimurium -- wt N/A strain LT2

EXAMPLE 10: Oligonucleotide primers for wzzB, fepEand 0-antigen gene cluster cloning

Table 11: Oligonucleotide Primers

Name Primer Sequence Comments LT2wzzBS GAAGCAAACCGTACGCGTAAAG (SEQ ID NO: based on Genbank 40) GCA_000006945.2 Salmonella enterica LT2wzzB_AS CGACCAGCTCTTACACGGCG (SEQ ID NO: 41) serovar Typhimurium strain LT2 O25bFepE_S GAAATAGGACCACTAATAAATACACAAATTAAT Based on Genbank AAC (SEQ ID NO: 42) GCA_000285655.3 025b EC958 strain O25bFepE_A ATAATTGACGATCCGGTTGCC (SEQ ID NO: 43) ST131 assembly and O25b GAR2401 WGS data wzzB P1_S GCTATTTACGCCCTGATTGTCTTTTGT (SEQ ID based on E. coli K-12 NO: 44) strain sequence, Genbank MG1655 wzzB P2_AS ATTGAGAACCTGCGTAAACGGC (SEQ ID NO: NC_000913.3 or W3110 45) assembly wzzB P3_S TGAAGAGCGGTTCAGATAACTTCC (SEQ ID NO: GCA_000010245.1 46) (UDP-glucose-6-dehydrogenase)

wzzB P4_AS CGATCCGGAAACCTCCTACAC (SEQ ID NO:47) (Phosphoribosyl-AMP cyclohydrolase/ Phosphoribosyl-ATP pyrophosphohydrolase)

0157 FepE_S GATTATTCGCGCAACGCTAAACAGAT (SEQ ID E. coli0157 fepE NO: 48) (based on Genbank EDL933 strain GCA_000732965.1) 0157 TGATCATTGACGATCCGGTAGCC (SEQ ID NO: FepE AS 49) pBAD33_ada CGGTAGCTGTAAAGCCAGGGGCGGTAGCGTG Adaptor has central ptorS GTTTAAACCCAAGCAACAGATCGGCGTCGTCG Pmel site and homology GTATGGA (SEQ ID NO: 50) to conserved 5' OAg operon promoter and 3' pBAD33_ada AGCTTCCATACCGACGACGCCGATCTGTTGCT gnd gene sequences ptor_AS TGGGTTTAAACCACGCTACCGCCCCTGGCTTT ACAGCTACCGAGCT (SEQ ID NO: 51)

JUMPSTART_ GGTAGCTGTAAAGCCAGGGGCGGTAGCGTG Universal Jumpstart r (SEQ ID NO: 52) (OAg operon promoter)

gnd-f CCATACCGACGACGCCGATCTGTTGCTTGG Universal 3'OAg (gnd) (SEQ ID NO: 53) operon antisense primer

EXAMPLE 11: Plasmids Plasmid vectors and subclones are listed in Table 12. PCR fragments harboring various E. coli and Salmonella wzzB and fepE genes were amplified from purified genomic DNA and subcloned into the high copy number plasmid provided in the Invitrogen PCR@Blunt cloning kit FIG. 12A-12B. This plasmid is based on the pUC replicon. Primers P3 and P4 were used to amplify E. coli wzzB genes with their native promoter, and are designed to bind to regions in proximal and distal genes encoding UDP-glucose-6-dehydrogenase and phosphoribosyladenine nucleotide hydrolase respectively (annotated in Genbank MG1655 NC_000913.3). A PCR fragment containing Salmonella fepE gene and promoter were amplified using primers previously described. Analogous E. coli fepE primers were designed based on available Genbank genome sequences or whole genome data generated internally (in case of GAR2401 and 025K5H1). Low copy number plasmid pBAD33 was used to express 0-antigen biosynthetic genes under control of the arabinose promoter. The plasmid was first modified to facilitate cloning (via Gibson method ) of long PCR fragments amplified using universal primers homologous to the 5' promoter and 3'6-phosphogluconate dehydrogenase (gnd) gene Table 12. The pBAD33 subclone containing the 025b biosynthetic operon is illustrated in FIG. 12A 12B.

Table 12

Plasmids

Name Replicon Resistance Comments marker PCR@Blunt || TOPO pUC KanR Invitrogen PCR cloning vector pBAD33 P15a CamR Arabinose inducible vector pBAD33-OAg P15a CamR OAg operon Gibson cloning vector pBAD33-025b P15a CamR 025b OAg expression plasmid pBAD33-021 P15a CamR 021 OAg expression plasmid pBAD33-016 P15a CamR 016 OAg expression plasmid pBAD33-075 P15a CamR 075 OAg expression plasmid pBAD33-01 P15a CamR 01 OAg expression plasmid pBAD33-02 P15a CamR 02 OAg expression plasmid pTOPO-025b 2401 wzzB pUC KanR GAR 2401 gDNA template pTOPO-025b 2401 fepE pUC KanR pTOPO-K12 wzzB pUC KanR E. coli K-12 strain gDNA template pTOPO-025a wzzB pUC KanR E. coli 025a strain 025K5H1 pTOPO-025a fepE pUC KanR gDNA template pTOPO-Salmonella LT2 pUC KanR Salmonella enterica serovar wzzB Typhimurium strain LT2 gDNA pTOPO-Salmonella LT2 pUC KanR template fepE pTOPO-025a ETEC wzzB pUC KanR 025a ETEC strain gDNA pTOPO-025a ETEC fepE pUC KanR purchased from ATCC ("NR-5" E2539-C1) pTOPO-0157fepE pUC KanR 0157:H7:K- Shigella toxin strain gDNA purchased from ATCC (EDL933 #43895D-5)

EXAMPLE 12: 0-antigen Purification

The fermentation broth was treated with acetic acid to a final concentration of 1 - 2% (final pH of 4.1). The extraction of OAg and delipidation were achieved by heating the acid treated broth to 100°C for 2 hours. At the end of the acid hydrolysis, the batch was cooled to ambient temperature and 14% NH 40H was added to a final pH of 6.1. The neutralized broth was centrifuged and the centrate was collected. To the centrate was added CaCl2 in sodium phosphate and the resulting slurry was incubated for 30 mins at room temperature. The solids were removed by centrifugation and the centrate was concentrated 12-fold using a 1OkDa membrane, followed by two diafiltrations against water. The retentate which contained OAg was then purified using a carbon filter. The carbon filtrate was diluted 1:1 (v/v) with 4.M ammonium sulfate. The final ammonium sulfate concentration was 2M. The ammonium sulfate treated carbon filtrate was further purified using a membrane with 2M ammonium sulfate as the running buffer. The OAg was collected in the flow through. For the long OAg the HIC filtrate was concentrated and then buffer exchanged against water (20 diavolumes) using a 5kDa membrane. For the short (native) OAg polysaccharide, the MWCO was further reduced to enhance yield.

EXAMPLE 13: Conjugation of 025b long0-antigen to CRM1 97 The first set of long chain025b polysaccharide-CRM 197 conjugates were produced using periodate oxidation followed by conjugation using reductive amination chemistry (RAC) (Table 14). Conjugate variants with three activation levels (low, medium and high) by varying the oxidation levels. Conjugates were produced by reacting thelyophilized activated polysaccharides with lyophilized CRM 197, reconstituted in DMSO medium, using sodium cyanoborohydride as the reducing agent. Conjugation reactions were carried out at 23 °C for 24 hrs, followed by capping using sodium borohydride for 3 hrs. Following the conjugation quenching step, conjugates were purified by ultrafiltration/diafiltration with 100K MWCO regenerated cellulose membrane, using 5mM Succinate/0.9% NaCI, pH 6.0. Final filtration of the conjugates were performed using a 0.22 pm membrane. Unless expressly stated otherwise, the conjugates disclosed throughout the following Examples include a core saccharide moiety.

Long 0-antigen expression conferred by heterologous polymerase chain length regulators. Initial E. coli strain construction focused on the 025 serotype. Goal was to overexpress heterologous wzzB or fepE genes to see if they confer longer chain length in 025wzzB knockout strains. First, blood isolates were screened by PCR to identify strains of the 025a and

025b subtype. Next, strains were screened for sensitivity to ampicillin. A single ampicillin sensitive 025b isolate GAR2401 was identified into which a wzzB deletion was introduced. Similarly, a wzzB deletion was made in 025a strain 025K5H1. For genetic complementation of these mutations, wzzB genes from GAR 2401 and 025K5H1 were subcloned into the high copy PCR-Blunt II cloning vector and introduced into both strains by electroporation. Additional wzzB genes from E. coli K-12 and S. enterica serovar Typhimurium LT2 were similarly cloned and transferred; likewise fepE genes from E. coli 025K5H1, GAR 2401, 025a ETEC NR-5, 0157:H7:K- and S. enterica serovarTyphimurium LT2. Bacteria were grown overnight in LB medium and LPS was extracted with phenol, resolved by SDS PAGE (4-12% acrylamide) and stained. Each well of the gel was loaded with LPS extracted from the same number of bacterial cells (approximately 2 ODoo units). Size of LPS was estimated from an internal native E. coliLPS standard and by counting the ladder discernable from a subset of samples showing a broad distribution of chain lengths (differing by one repeat unit). On the left side of FIG. 13A, LPS profiles of plasmid transformants of 025a 025K5HAwzzB are shown; and on the right, analogous profiles of 025b GAR 2401AwzzB transformants. An immunoblot of a replicate gel probed with 025-specific sera is shown in FIG. 13B. Results from this experiment show that introduction of the homologous wzzB gene into the E. coliO25aAwzzB host restores expression of short 025 LPS (10-20x), as does the Salmonella LT2 wzzB. Introduction of the 025b wzzB gene from GAR2401 does not, suggesting the WzzB enzyme from this strain is defective. A comparison of E. coli WzzB amino acid sequences suggests that A21OE and P253S substitutions may be responsible. Significantly, Salmonella LT2 fepE and E. coli fepE from 025a 025K5H1 conferred the ability to express very long (VL) OAg LPS, with the Salmonella LT2 fepE resulting in OAg exceeding in size that conferred by E. coli fepE. A similar pattern of expression was observed with GAR2401AwzzB transformants: E. coli025a or K12 strain wzzB restored ability to produce short LPS. The Salmonella LT2 fepE generated the longest LPS, the E. coli fepE a slightly shorter LPS, while the Salmonella LT2 wzzB yielded an intermediate sized long LPS (L). The ability of other E. coli fepE genes to produce very long LPS was assessed in a separate experiment with transformants of E. coli O25aAwzzB. The fepE genes from GAR2401, an 025a ETEC strain and an 0157 Shigela toxin producing strain also conferred the ability to produce very long LPS, but not as long as the LPS generated with the Salmonella LT2 fepE (FIG. 14). Having established in serotype 025a and 025b strains that Salmonella LT2 fepE generates the longest LPS of the polymerase regulators evaluated, we next sought to determine whether it would also produce very long LPS in other E. coliserotypes. Wild-type bacteremia isolates of serotype 01, 02, 06, 015 and 075 were transformed with the Salmonella fepE plasmid and LPS extracted. The results shown in FIG. 15 confirm that Salmonella fepE can confer the ability to make very long LPS in other prevalent serotypes associated with blood-infections. Results also show that plasmid-based expression of Salmonella fepE appears to override the control of chain length normally exerted by endogenous wzzB in these strains.

Plasmid-based expression of O-antigens in a common E. coli host strain.

From the perspective of bioprocess development, the ability to produce O-antigens of different serotypes in a common E. coli host instead of multiple strains would greatly simplify the manufacturing of individual antigens. To this end, O-antigen gene clusters from different serotypes were amplified by PCR and cloned into a low-copy number plasmid (pBAD33) under control of an arabinose regulated promoter. This plasmid is compatible (can coexist) with the Salmonella LT2 fepE plasmid in E. colias it harbors a different (p15a) replicon and different selectable marker (chloramphenicol vs kanamycin). In a first experiment, a pBAD33 025b operon plasmid subclone was cotransfected with the Salmonella LT2 fepE plasmid into GAR2401AwzzB and transformants grown in the presence or absence of 0.2% arabinose. Results shown in FIG. 16A-16B demonstrated that very long O-antigen LPS was produced in an arabinose-dependent manner. O-antigen gene clusters cloned from other serotypes were similarly evaluated and the results shown in FIG. 17. Co-expression of Salmonella LT2 fepE and pBAD33-OAg plasmids resulted in detectable long chain LPS corresponding to 01, 02 (for two out of four clones), 016, 021 and 075 serotypes. For unknown reasons, the pBAD33-06 plasmid failed to yield detectable LPS in all four isolates tested. Although expression level was variable, results show that expression of long chain O-antigens in a common host is feasible. However, in some cases further optimization to improve expression may be required, for example by modification of plasmid promoter sequences. The profiles of LPS from different serotype 025 E. colistrains with or without the Salmonella LT2 fepE plasmid are shown in FIG. 18. Two strains were studied for fermentation, extraction and purification of O-antigens: GAR2831, for the production of native short 025b OAg; and GAR2401AwzzB/fepE, for the production of long 025b OAg. The corresponding short and long form LPSs shown in the FIG. 18 SDS-PAGE gel are highlighted in red. Polysaccharides were extracted directly from fermented bacteria with acetic acid and purified. Size exclusion chromatography profiles of purified short and long or very long 025b polysaccharides are shown in FIG. 19A-19B. The properties of two lots of short polysaccharide (from GAR2831) are compared with a single very long polysaccharide preparation (from strain

GAR2401wzzB/fepE). The molecular mass of the long O-antigen is 3.3-fold greater than that of the short O-antigen, and the number of repeat units was estimated to be -65 (very long) vs -20. See Table 13.

Table 13

Poly Lot # Native Native Modified (long chain) Poly Lot # 709766-24A 709722-24B 709766-25A Poly MW (kDa) 17.3 16.3 55.3 # Repeat Units 20 19 64

The very long 025b O-antigen polysaccharide was conjugated to diphtheria toxoid CRM 19 7using a conventional reductive amination process. Three different lots of glycoconjugate were prepared with varying degree of periodate activation: medium (5.5%), low (4.4%) and high (8.3%). The resulting preparations and unconjugated polysaccharide were shown to be free of endotoxin contamination) (Table 14). Groups of four rabbits (New Zealand White females) were each vaccinated with 10 mcg of glycoconjugate and 20 mcg of QS21 adjuvant and serum sampled (VAC 2017-PRL-EC-0723) according to the schedule shown in FIG. 20A. It is worth noting that a 10 mcg dose is at the low end of the range customarily given to rabbits in the evaluation of bacterial glycoconjugates (20-50 mcg is more typical). A group of rabbits was also vaccinated in a separate study (VAC-2017-PRL-GB-0698) with unconjugated polysaccharide using the same dose (10 mcg polysaccharide + 20mcg QS21 adjuvant) and identical administration schedule. Rabbit antibody responses to the three025b glycoconjugate preparations were evaluated in a LUMINEX assay in which carboxy beads were coated with methylated human serum albumin prebound with unconjugated 025b long polysaccharide. The presence of 025b specific IgG antibodies in serum samples was detected with a phycoerythrin(PE)-labelled anti IgG secondary antibody. The profiles of immune responses observed in sera sampled at week 0 (pre-immune), week 6 (post-dose 2, PD2), week 8 (post-dose 3, PD3) and week 12 (post dose 4, PD4) in best-responding rabbits (one from each group of four) are shown in FIG. 21A 21C. No significant pre-immune serum IgG titers were detected in any of the 12 rabbits. In contrast, 025b antigen-specific antibody responses were detected in post-vaccination sera from rabbits in all three groups, with the low-activation glycoconjugate group responses trending slightly higher than the medium or high activation glycoconjugate groups. Maximal responses were observed by the post-dose 3 timepoint. One rabbit in the low activation group and one rabbit from the high activation group failed to respond to vaccination (non-responders).

To assess the impact of CRM 197 carrier protein conjugation on immunogenicity of the long 025b OAg polysaccharide, the presence of antibodies in sera from rabbits vaccinated with unconjugated polysaccharide was compared with sera from rabbits vaccinated with the low activation CRM 197 glycoconjugate FIG. 22A-22F. Remarkably, the free polysaccharide was not immunogenic, eliciting virtually no IgG responses in immune vs preimmune sera (FIG. 22A). In contrast, 025b OAg-specific IgG mean fluorescence intensity values (MFIs) of approximately ten-fold above pre-immune serum levels were observed in PD4 sera from three out of four rabbits vaccinated with 025b OAg- CRM 197, across a range of serum dilutions (from 1:100 to 1:6400). These results demonstrate the necessity of carrier protein conjugation to generate IgG antibodies to the 025b OAg polysaccharide at the 10 mcg dose level. Bacteria grown on TSA plates were suspended in PBS, adjusted to OD00 of 2.0 and fixed in 4% paraformaldehyde in PBS. After blocking in 4% BSA/PBS for 1h, bacteria were incubated with serial dilutions of pre-immune and PD3 immune sera in 2% BSA/PBS, and bound IgG detected with PE-labeled secondary F(ab) antibody. Specificity of the 025b antibodies elicited by the025b OAg-CRM 197 was demonstrated in flow cytometry experiments with intact bacteria. Binding of IgG to whole cells was detected with PE-conjugated F(ab') 2 fragment goat anti-rabbit IgG in an Accuri flow cytometer. As shown in FIG. 23A-23C, pre-immune rabbit antibodies failed to bind to wild-type serotype 025b isolates GAR2831and GAR2401 or to a K-12 E. coli strain, whereas matched PD3 antibodies stained the 025b bacteria in a concentration dependent manner. Negative control K-12 strain which lacks the ability to express OAg showed only very weak binding of PD3 antibodies, most likely due to the presence of exposed inner core oligosaccharide epitopes on its surface. Introduction of the Salmonella fepE plasmid into the wild-type 025b isolates resulted in significantly enhanced staining, consistent with the higher density of immunogenic epitopes provided by the longer OAg polysaccharide. Conclusion: The results described show that not only is Salmonella fepE the determinant of very long O-antigen polysaccharides in Salmonella species, but that it also can confer on E. coli strains of different O-antigen serotypes the ability to make very long OAgs. This property can be exploited to produce O-antigen vaccine polysaccharides with improved properties for bioprocess development, by facilitating purification and chemical conjugation to appropriate carrier proteins, and by potentially enhancing immunogenicity through the formation of higher molecular weight complexes.

EXAMPLE 14: Initial rabbit studies generated first polyclonal antibody reagents and IgG responses to RAC025b OAg-CRM 197

Long chain 025b polysaccharide-CRM 197 conjugates were produced using periodate oxidation followed by conjugation using reductive amination chemistry (RAC) (Table 14). See also Table 24.

Table 14

CRM197 132242-28 132242-27 132242-29 709766-29 conjugate Medium 5.5% Low 4.5% High 8.3% Free 025b activation activation activation polysaccharide Polysaccharide 0.7 0.6 0.67 1 concentration (mg/mL) Endotoxin 0.02 0.02 0.02 <0.6 EU (EU/ug) Matrix 5 um Succinate buffer/saline, pH 6.0

In Rabbit Study 1 (VAC-2017-PRL-EC-0723) (also described above in Example 13) five (5) rabbits/group, with 10ug L-, M- or H-activation RAC (+QS21) received a composition according to the schedule shown in FIG. 20A. Unconjugated free 025b polysaccharide was observed not to be immunogenic in a follow-up rabbit Study (VAC-2017-PRL-GB-0698) (see FIG. 25). In Rabbit Study 2 (VAC-2018-PRL-EC-077) - 2 rabbits/group, with L-RAC (AOH 3 , QS21, or no adjuvant) received a composition according to the schedule shown in FIG. 20B. Rabbits 4-1, 4-2, 5-1, 5-2, 6-1, and 6-2 received the very long unconjugated 025b polysaccharide described in Example 13, and week 18 sera were tested. More specifically, a composition including 50 ug unconjugated 025b, 100 ug AIOH3 adjuvant was administered to Rabbit 4-1. A composition including 50 ug unconjugated 025b, 100 ug AIOH 3 adjuvant was administered to Rabbit 4-2. A composition including 50 ug unconjugated 025b, 50 ug QS-21 adjuvant was administered to Rabbit 5-1. A composition including 50 ug unconjugated 025b, 50 ug QS-21 adjuvant was administered to Rabbit 5-2. A composition including 50 ug unconjugated 025b, no adjuvant was administered to Rabbit 6-1. A composition including 50 ug unconjugated 025b, no adjuvant was administered to Rabbit 6-2.

EXAMPLE 15: Rabbit studies with 025b RAC conjugate: dLIA serum dilution titers

Rabbit Study 2 (VAC-2018-PRL-EC-077) 025b dLIA serum dilution titers vs best responding rabbit from study 1 (VAC-2017-PRL-EC-0723). For these experiments a modified direct binding Luminex assay was implemented in which a polylysine conjugate of 025b long 0 antigen was passively adsorbed onto the Luminex carboxy beads instead of the methylated serum albumin long O-antigen mixture described previously. The use of the polylysine-025b conjugate improved the sensitivity of the assay and the quality of IgG concentration dependent responses, permitting determination of serum dilution titers through use of curve-fitting (four parameter non-linear equation). 025b IgG titers in sera from highest titer rabbit from first study is compared with sera from second study rabbits in Table 15.

Table 15

025b-CRM Low 025b-CRM Low 025b-CRM Low Activation Activation Activation Conjugate Conjugate Conjugate with with Alum Adjuvant without QS21IAdjuvant m (so as serum Adjuvant (ECso (ECso as serum dilution) as serum dilution)dilution)

Rabbit1- Rabbit2- Rabbit2- Rabbi Rabbit Rabbit 1-1 2 1 2 t 3-1 3-2 Week 3Antisera (3 ~1:20 -1:200 -1:200 <1:100 <1:100 -1:200 wks after primary) 0 Week 7Antisera (1 1:1600 1:4000 1:250 1:500 1:250 1:1500 wk after boost 1) Week10Antisera(1 1:1100 1:1900 1:250 1:500 1:800 1:1200 wk after boost 2) Week 18 Antisera (1 1:140 1:1600 1:4000 1:1300 1:1200 1:1600 wk after boost 4) 0 Average of 6 replicates of best antisera from rabbit 2-3 (assay standard from first study) ECso = 1:1700

Higher doses in second rabbit study (50/20ug vs 1Oug) did not improve IgG titers. Two month rest boosts IgG responses (not observed with shorter intervals). Alum appears to enhance IgG response in rabbits compared with QS21 or no adjuvant.

An opsonophagocytic assay (OPA) with baby rabbit complement (BRC) and HL60 cells as source of neutrophils was established to measure the functional immunogenicity of 0 antigen glycoconjugates. Pre-frozen bacterial stocks of E. coliGAR2831 were grown in Luria broth (LB) media at 37°C. Cells were pelleted and suspended to a concentration of 1 ODoo unit per ml in PBS supplemented with 20% glycerol and frozen. Pre-titered thawed bacteria were diluted to 0.5X 105 CFU/ml in HBSS (Hank's Balanced Salt Solution) with 1% Gelatin ) and 10 tL (103 CFU) combined with 20 tLof serially diluted sera in a U-bottomed tissue culture

microplate and the mixture shaken at 700 rpm BELLCO Shaker) for 30 min at 370C in a 5% C02 incubator.10 I of 2.5% complement (Baby Rabbit Serum, PEL-FREEZ 31061-3, prediluted in HBG) and 20 L of HL-60 cells (0.75X 107 /ml) and 40 tL of HBG added to the U-bottomed tissue culture microplate and the mixture shaken at 700 rpm BELLCO Shaker) for 45min at 370C in a 5% C02 incubator. Subsequently, 10 tL of each 100tL reaction was transferred into the corresponding wells of a pre-wetted MILLIPORE MULTISCREENHTS HV filter plate prepared by applying 100 tL water, filter vacuumed, and applying 150 tL of 50% LB. The filter plate was vacuum filtered and incubated overnight at 370C in a 5% C02 incubator. The next day the colonies were enumerated after fixing, staining, and destaining with COOMASSIE dye and Destain solutions, using an IMMUNOSPOT@ analyzer and IMMUNOCAPTURE software. To establish the specificity of OPA activity, immune sera were preincubated with 100 tg/mL purified long 025b O-antigen prior to combining with the other assay components in the OPA reaction. The OPA assay includes control reactions without HL60 cells or complement, to demonstrate dependence of any observed killing on these components. Matched pre-immune and post-vaccination serum samples from representative rabbits from both rabbit studies were evaluated in the assay and serum dilution titers determined (Table 16, FIG. 26A-26B). Preincubation with unconjugated 025b long O-antigen polysaccharide blocked bactericidal activity demonstrating specificity of the OPA (FIG. 19C). Table 16 OPA titers Rabbit 2-3 was dosed as follows: Rabbit 2-3 dosing: 10/10/10/1Oug RAC conjugate +

QS21, post-dose (PD) 4 bleed. Rabbit 1-2 was dosed as follows: 50/20/20/20ug RAC conjugate + AI(OH)3, PD4 bleed.

Table 16

Sample Titer Rabbit 2-3 Pre-immune serum 537 Rabbit 2-3 wk13 serum (terminal bleed) 13686 Rabbit 1-2 Pre-immune serum <200

Rabbit 1-2 wk19 serum (terminal bleed) 22768

EXAMPLE 16: O-antigen 025b IgG levels elicited by unconjugated 025b long O-antigen polysaccharide and derived 025b RAC/DMSO long O-antigen glycoconjugate. Groups of ten CD-1 mice were dosed by sub-cutaneous injection with 0.2 or 2.0 pg/animal of 025b RAC/DMSO long O-antigen glycoconjugate at weeks 0, 5 and 13, with bleeds taken at week 3 (post-dose 1, PD1), week 6 (post-dose 2, PD2) and week 13 (post-dose 3, PD3) timepoints for immunogenicity testing. Levels of antigen-specific IgG were determined by quantitative Luminex assay (see details in Example 15) with 025b-specific mouse mAb as internal standard. Baseline IgG levels (dotted line) were determined in serum pooled from 20x randomly selected unvaccinated mice. The free unconjugated 025b long O-antigen polysaccharide immunogen did not induce IgG above baseline levels at any timepoint. In contrast, IgG responses were observed after two doses of 25b-CRM197 RAC long conjugate glycoconjugate: robust uniform IgG responses were observed by PD3, with intermediate and more variable IgG levels at PD2. GMT IgG values (ng/ml) are indicated with 95% Cl error bars. See FIG. 27A-27C.

EXAMPLE 17: Specificity of the 025b baby rabbit complement (BRC) OPA.

A-B) 025b RAC/DMSO long O-antigen post-immune serum from rabbits 2-3 and 1-2 (but not matched pre-immune control serum) shows bactericidal OPA activity. C) OPA activity of immune serum from rabbit 1-2 was blocked by pre-incubation with 100pg/mL long O-antigen 025b polysaccharide. Strain GAR2831 bacteria were incubated with HL60s, 2.5% BRC and serial dilutions of serum for 1h at 370C and surviving bacteria enumerated by counting microcolonies (CFUs) on filter plates. See FIG. 26A-26C.

EXAMPLE 18: RAC and eTEC 025b long glycoconjugates are more immunogenic than single end glycoconjugates.

BRC OPA assay with carbapenem-resistant fluoroquinlone-resistant MDR strain Atlas187913. Groups of 20 CD-1 mice were vaccinated with 2pg of glycoconjugate according to the same schedule as shown in FIG. 28A-28B and OPA responses determined at post-dose 2 (PD2) (FIG. 28A) and post-dose 3 (PD3) (FIG. 28B) timepoints. Bars indicate GMTs with 95% Cl. Responder rates above unvaccinated baseline are indicated. Log transformed data from different groups were evaluated to assess if differences were statistically significant using unpaired t-test with Welch's correction (Graphpad Prism). Results are summarized in the Table 17. SeeFIG.28A-28B. In mice that were vaccinated with 2 pg of eTEC 01a long glycoconjugates, OPA titers against 01a, PD2 and PD3 (data not shown), were observed to be greater than the OPA titers against 025b, PD2 and PD3, respectively, shown in Table 17.

Table 17

DESCRIPTION % Responders (n/N)* GEOMEAN % Responders GEOMEAN TITER PD2 (n/N)* TITER PD3 Single end short, 2 pg 45(9/20) 1,552 85(17/20) 17,070 Single end long, 2pg 30(6/20) 763 85(17/20) 10,838 RAC/DMSO long, 2pg 65(13/20) 8,297 95(19/20) 163,210 eTEC (10%) long, 2pg 90(18/20) 27,368 100(19/19) 161,526

EXAMPLE 19: OPA immunogenicity of eTEC chemistry may be improved by modifying levels of polysaccharide activation.

BRC OPA assay with carbapenem-resistant fluoroquinlone-resistant MDR strain Atlas187913. Groups of 20 CD-1 mice were vaccinated with 0.2pg or 2pg of the indicated long 025b eTEC glycoconjugate and OPA responses determined at PD2 timepoint. Aggregated log transformed data from 4% activation vs 17% activation groups were evaluated to confirm that differences in OPA responses were statistically significant using unpaired t-test with Welch's correction (Graphpad Prism). GMTs and responder rates for individual groups are summarized in Table 18. See FIG. 29.

Table 18

Description % Responders (n/N) GeoMean Titer eTEC long 4% activation (0.2 pg) 35(7/20) 628 eTEC long 4% activation (0.2 pg) 65(13/20) 8,185 eTEC long 10% activation (0.2 pg) 45(9/20) 1,085 eTEC long 10% activation (0.2 pg) 90(18/20) 27,368 eTEC long 17% activation (0.2 pg) 70(14/20) 3,734 eTEC long 17% activation (0.2 pg) 80(16/20) 25,461

EXAMPLE 20: Challenge study indicates long E. coli 025b eTEC conjugates elicit protection after three doses. Groups of 20x CD-1 mice immunized with a 2pg dose according to the indicated schedule were challenged IP with 1 x 109 bacteria of strain GAR2831. Subsequent survival was monitored for six days. Groups of mice vaccinated with eTEC glycoconjugates activated at 4%,

10% or 17% levels were protected from lethal infection, whereas unvaccinated control mice or mice vaccinated with 2pg unconjugated 025b long polysaccharide were not. See FIG. 30A 30B.

EXAMPLE 21: Process for Preparation of eTEC Linked Glycoconjugates Activation of Saccharide and Thiolation with Cystamine dihydrochloride. The saccharide is reconstituted in anhydrous dimethylsulfoxide (DMSO). Moisture content of the solution is determined by Karl Fischer (KF) analysis and adjusted to reach a moisture content of 0.1 and 1.0%, typically 0.5%. To initiate the activation, a solution of 1,1'-carbonyl-di-1,2,4-triazole (CDT) or 1,1' carbonyldiimidazole (CDI) is freshly prepared at a concentration of 100 mg/mL in DMSO. The saccharide is activated with various amounts of CDT/CDI (1 - 10 molar equivalents) and the reaction is allowed to proceed for 1 - 5 hours at rt or 350C. Water was added to quench any residual CDI/CDT in the activation reaction solution. Calculations are performed to determine the added amount of water and to allow the final moisture content to be 2 - 3% of total aqueous. The reaction was allowed to proceed for 0.5 hour at rt. Cystamine dihydrochloride is freshly prepared in anhydrous DMSO at a concentration of 50 mg/mL. The activated saccharide is reacted with 1 - 2 mol. eq. of cystamine dihydrochloride. Alternatively, the activated saccharide is reacted with 1 - 2 mol. eq. of cysteamine hydrochloride. The thiolation reaction is allowed to proceed for 5 20 hours at rt, to produce a thiolated saccharide. The thiolation level is determined by the added amount of CDT/CDI. Reduction and Purification of Activated Thiolated Saccharide. To the thiolated saccharide reaction mixture a solution of tris(2-carboxyethyl)phosphine (TCEP), 3 - 6 mol. eq., is added and allowed to proceed for 3 - 5 hours at rt. The reaction mixture is then diluted 5 - 10 fold by addition to pre-chilled 10 mM sodium phosphate monobasic, and filtered through a 5pm filter. Dialfiltration of thiolated saccharide is performed against 30 - 40-fold diavolume of pre chilled 10 mM sodium phosphate monobasic. An aliquot of activated thiolated saccharide retentate is pulled to determine the saccharide concentration and thiol content (Ellman) assays. Activation and Purification of Bromoacetylated Carrier Protein. Free amino groups of the carrier protein are bromoacteylated by reaction with a bromoacetylating agent, such as bromoacetic acid N-hydroxysuccinimide ester (BAANS), bromoacetylbromide, or another suitable reagent. The carrier protein (in 0.1M Sodium Phosphate, pH 8.0 ±0.2) is first kept at 8 ±3C, at about pH 7 prior to activation. To the protein solution, the N-hydroxysuccinimide ester of bromoacetic acid (BAANS) as a stock dimethylsulfoxide (DMSO) solution (20 mg/mL) is added in a ratio of 0.25 - 0.5 BAANS: protein (w/w). The reaction is gently mixed at 5 + 3 °C for 30

60 minutes. The resulting bromoacetylated (activated) protein is purified, e.g., by ultrafiltration/diafiltration using 10 kDa MWCO membrane using 10 mM phosphate (pH 7.0) buffer. Following purification, the protein concentration of the bromoacetylated carrier protein is estimated by Lowry protein assay. The extent of activation is determined by total bromide assay by ion-exchange liquid chromatography coupled with suppressed conductivity detection (ion chromatography). The bound bromide on the activated bromoacetylated protein is cleaved from the protein in the assay sample preparation and quantitated along with any free bromide that may be present. Any remaining covalently bound bromine on the protein is released by conversion to ionic bromide by heating the sample in alkaline 2-mercaptoethanol. Activation and Purification of Bromoacetylated CRM 197. CRM 197 was diluted to 5 mg/mL with 10 mM phosphate buffered 0.9% NaCI pH 7 (PBS) and then made 0.1 M NaHC0O 3 pH 7.0 using 1 M stock solution. BAANS was added at a CRM 197 : BAANS ratio 1 :0.35 (w:w) using a BAANS stock solution of 20 mg/mL DMSO. The reaction mixture was incubated at between 3 °C and 11 °C for 30 mins-1 hour then purified by ultrafiltration/diafiltration using a 10K MWCO membrane and 10mM Sodium Phosphate/0.9% NaCI, pH 7.0. The purified activated CRM 197 was assayed by the Lowry assay to determine the protein concentration and then diluted with PBS to 5 mg/mL. Sucrose was added to 5% wt/vol as a cryoprotectant and the activated protein was frozen and stored at -25 °C until needed for conjugation. Bromoacetylation of lysine residues of CRM 197 was very consistent, resulting in the activation of 15 to 25 lysines from 39 lysines available. The reaction produced high yields of activated protein. Conjugation of Activated Thiolated Saccharide to Bromoacetylated Carrier Protein. Bromoacetylated carrier protein and activated thiolated saccharide are subsequently added. The saccharide/protein input ratio is 0.8 ±0.2. The reaction pH is adjusted to 9.0 ±0.1 with 1 M NaOH solution. The conjugation reaction is allowed to proceed at 5 °C for 20± 4 hours. Capping of Residual Reactive Functional Groups. The unreacted bromoacetylated residues on the carrier protein are quenched by reacting with 2 mol. eq. of N-acetyl-L-cysteine as a capping reagent for 3 - 5 hours at 5 °C. Residual free sulfhydryl groups are capped with 4 mol. eq. of iodoacetamide (IAA) for 20 - 24 hours at 5 °C. Purification of eTEC-linked Glycoconjugate. The conjugation reaction (post-IAA capped) mixture is filtered through 0.45 pm filter. Ultrafiltration/dialfiltration of the glycoconjugate is performed against 5 mM succinate-0.9% saline, pH 6.0. The glycoconjugate retentate is then filtered through 0.2 pm filter. An aliquot of glycoconjugate is pulled for assays.

The remaining glycoconjugate is stored at 5 0C. See Table 21, Table 22, Table 23, Tale 24, and Table 25.

EXAMPLE 22: PREPARATION OF E. COLI-025B ETEC CONJUGATES

Activation Process - Activation of E. coli-025b Lipopolysaccharide. The lyophilized E. coli-025b polysaccharide was reconstituted in anhydrous dimethylsulfoxide (DMSO). Moisture content of the lyophilized 025b/DMSO solution was determined by Karl Fischer (KF) analysis. The moisture content was adjusted by adding WFI to the 025b/DMSO solution to reach a moisture content of 0.5%. To initiate the activation, 1,1'-carbonyldiimidazole (CDI) was freshly prepared as 100 mg/mL in DMSO solution. E. coli-025b polysaccharide was activated with various amounts of CDI prior to the thiolation step. The CDI activation was carried out at rt or 35°C for 1 - 3 hours. Water was added to quench any residual CD in the activation reaction solution. Calculations are performed to determine the added amount of water and to allow the final moisture content to be 2 - 3% of total aqueous. The reaction was allowed to proceed for 0.5 hour at rt. Thiolation of Activated E. coli-025b Polysaccharide. Cystamine-dihydrochloride was freshly prepared in anhydrous DMSO and 1 - 2 mol. eq. of cystamine dihydrochloride was added to the activated polysaccharide reaction solution. The reaction was allowed to proceed for 20± 4 hours at rt. Reduction and Purification of Activated Thiolated E. coli-025b Polysaccharide. To the thiolated saccharide reaction mixture a solution of tris(2-carboxyethyl)phosphine (TCEP), 3 6 mol. eq., was added and allowed to proceed for 3 - 5 hours at rt. The reaction mixture was then diluted 5 - 10-fold by addition to pre-chilled 10 mM sodium phosphate monobasic and filtered through a 5pm filter. Dialfiltration of thiolated saccharide was performed against 40-fold diavolume of pre-chilled 10 mM sodium phosphate monobasic with 5K MWCO ultrafilter membrane cassettes. The thiolated 025b polysaccharide retentate was pulled for both saccharide concentration and thiol (Ellman) assays. A flow diagram of the activation process is provided in FIG. 32A). Conjugation Process - Conjugation of Thiolated E. coli-025b Polysaccharide to Bromoacetylated CRM 1 97. The CRM 197 carrier protein was activated separately by bromoacetylation, as described in Example 21, and then reacted with the activated E. coli 025b polysaccharide for the conjugation reaction. Bromoacetylated CRM 197 and thiolated 025b polysaccharide were mixed together in a reaction vessel. The saccharide/protein input ratio was 0.8 ±0.2. The reaction pH was adjusted to 8.0 - 10.0. The conjugation reaction was allowed to proceed at 5 °C for 20± 4 hours.

Capping of Reactive Groups on Bromoacetylated CRM 19 7 and Thiolated E. col 025b Polysaccharide. The unreacted bromoacetylated residues on CRM 197 proteins were capped by reacting with 2 mol. eq. of N-acetyl-L-cysteine for 3 - 5 hours at 5 °C, followed by capping any residual free sulfhydryl groups of the thiolated 025b-polysaccharide with 4 mol. eq. of iodoacetamide (IAA) for 20 - 24 hours at 5 °C. Purification of eTEC-linked E. coli-025b Glycoconjugate. The conjugation solution was filtered through a 0.45 pm or 5pm filter. Dialfiltration of the 025b glycoconjugate was carried out with 100K MWCO ultrafilter membrane cassettes. Diafiltration was performed against 5 mM succinate-0.9% saline, pH 6.0. The E. coli-025b glycoconjugate 1OOK retentate was then filtered through a 0.22 pm filter and stored at 5 °C. A flow diagram of the conjugation process is provided in FIG. 32B.

Results The reaction parameters and characterization data for several batches of E. coli-025b eTEC glycoconjugates are shown in Table 19. The CDI activation-thiolation with cystamine dihydrochloride generated glycoconjugates having from 41 to 92% saccharide yields and <5 to 14% free saccharides. See also Table 21, Table 22, Table 23, Table 24 and Table 25.

Table 19. Experimental Parameters and Characterization Data of E. coli-025b eTEC Conjugates

Conjugate Batch 025b-1A 025b-2B 025b-3C 025b-4D 025b-5E 025b-6F Activation level (mol of thiol/mol of polysaccharide), % 10 20 22 17 25 24 Input Sacc/Prot Ratio 0.8 0.8 0.8 0.8 0.8 0.8 Saccharide yield (%) 56 57 79 92 41 59 Output Sacc/Prot Ratio 0.88 1 1.18 1.32 2.9 1.4 Free Saccharide, % 8 <5 6 5 14 5 Free Protein, % <1 <1 <1 <1 <1 <1 Conjugate Mw, kDa 1057 4124 2259 2306 1825 1537

Total CMCA 3 na na 7.2 na na

EXAMPLE 23: Procedure for the preparation of E. coliO-antigen polysaccharide-CRM197 eTEC conjugates (applied to O-antigens from E. coliserotypes 025b, O1a, 02, and 06 Activation of polysaccharide. The E. coliO-antigen polysaccharide is reconstituted in anhydrous dimethylsulfoxide (DMSO). To initiate the activation, various amounts of 1,1' carbonyldiimidazole (CDI) (1 - 10 molar equivalents) is added to the polysaccharide solution and the reaction is allowed to proceed for 1 - 5 hours at rt or 350C. Then, water (2 - 3%, v/v) was added to quench any residual CDI in the activation reaction solution. After the reaction was allowed to proceed for 0.5 hour at rt, 1 - 2 mol. eq. of cystamine dihydrochloride is added. The reaction is allowed to proceed for 5 - 20 hours at rt, and then treated with 3 - 6 mol. eq of tris(2 carboxyethyl)phosphine (TCEP) to produce a thiolated saccharide. The thiolation level is determined by the added amount of CDI. The reaction mixture is then diluted 5 - 10-fold by addition to pre-chilled 10 mM sodium phosphate monobasic, and filtered through a 5pm filter. Dialfiltration of thiolated saccharide is performed against 30 - 40-fold diavolume of pre-chilled 10 mM sodium phosphate monobasic. An aliquot of activated thiolated saccharide retentate is pulled to determine the saccharide concentration and thiol content (Ellman) assays. Activation of Carrier Protein (CRM1 97). The CRM 197 (in 0.1M Sodium Phosphate, pH 8.0 ±0.2) is first kept at 8 3 °C, at about pH 8 prior to activation. To the protein solution, the N-hydroxysuccinimide ester of bromoacetic acid (BAANS) as a stock dimethylsulfoxide (DMSO) solution (20 mg/mL) is added in a ratio of 0.25 - 0.5 BAANS: protein (w/w). The reaction is gently mixed at 5 + 3 °C for 30 - 60 minutes. The resulting bromoacetylated (activated) protein is purified, e.g., by ultrafiltration/diafiltration using 10 kDa MWCO membrane using 10 mM phosphate (pH 7.0) buffer. Following purification, the protein concentration of the bromoacetylated carrier protein is estimated by Lowry protein assay. Conjugation. Activated CRM 197 and activated E. coliO-antigen polysaccharide are subsequently added to a reactor and mixed. The saccharide/protein input ratio is 1 ±0.2. The reaction pH is adjusted to 9.0 ±0.1 with 1 M NaOH solution. The conjugation reaction is allowed to proceed at 5 °C for 20± 4 hours. The unreacted bromoacetylated residues on the carrier protein are quenched by reacting with 2 mol. eq. of N-acetyl-L-cysteine as a capping reagent for 3 - 5 hours at 5 °C. Residual free sulfhydryl groups are capped with 4 mol. eq. of iodoacetamide (IAA) for 20 - 24 hours at 5 °C. Then, the reaction mixture is purified using ultrafiltration/dialfiltration performed against 5 mM succinate-0.9% saline, pH 6.0. The purified conjugate is then filtered through 0.2 pm filter. See Table 21, Table 22, Table 23, Table 24 and Table 25.

EXAMPLE 24: General procedure- Conjugation of 0-antigen (from E. coliserotypes 01, 02, 06, 25b) Polysaccharide by Reductive mination Chemistry (RAC) Conjugation in dimethylsulfoxide(RACIDMSO)

Activating Polysaccharide. Polysaccharide oxidation was carried out in 100 mM sodium phosphate buffer (pH 6.0 ±0.2) by sequential addition of calculated amount of 500 mM sodium phosphate buffer (pH 6.0) and water for injection (WFI) to give final polysaccharide concentration of 2.0 g/L. If required, the reaction pH was adjusted to pH 6.0, approximately. After pH adjustment, the reaction temperature was cooled to 4 0C. Oxidation was initiated by the addition of approximately 0.09 - 0.13 molar equivalents of sodium periodate. The oxidation reaction was performed at 5 ±3 C for 20± 4 hrs, approximately. Concentration and diafiltration of the activated polysaccharide was carried out using 5K MWCO ultrafiltration cassettes. Diafiltration was performed against 20-fold diavolumes of WF. The purified activated polysaccharide was then stored at 5 ±3C. The purified activated saccharide is characterized, inter alia, by (i) saccharide concentration by colorimetric assay; (ii) aldehyde concentration by colorimetric assay; (iii) degree of oxidation; and (iv) molecular weight by SEC-MALLS.

Compounding Activated Polysaccharide with Sucrose Excipient, and Lyophilizing. The activated polysaccharide was compounded with sucrose to a ratio of 25 grams of sucrose per gram of activated polysaccharide. The bottle of compounded mixture was thenlyophilized. Following lyophilization, bottles containing lyophilized activated polysaccharide were stored at 20± 5C. Calculated amount of CRM 197 protein was shell-frozen and lyophilized separately. Lyophilized CRM 197 was stored at -20± 5C.

Reconstituting Lyophilized Activated Polysaccharide and Carrier Protein. Lyophilized activated polysaccharide was reconstituted in anhydrous dimethyl sulfoxide (DMSO). Upon complete dissolution of polysaccharide, an equal amount of anhydrous DMSO was added tolyophilized CRM 197 for reconstitution.

Conjugating and Capping. Reconstituted activated polysaccharide was combined with reconstituted CRM 197 in the reaction vessel, followed by mixing thoroughly to obtain a clear solution before initiating the conjugation with sodium cyanoborohydride. The final polysaccharide concentration in reaction solution was approximately 1 g/L. Conjugation was initiated by adding 0.5 - 2.0 MEq of sodium cyanoborohydride to the reaction mixture and incubating at 23± 2 C for 20-48 hrs. The conjugation reaction was terminated by adding 2 MEq of sodium borohydride (NaBH 4) to cap unreacted aldehydes. This capping reaction continued at 23± 2C for 3 ±1 hrs. Purifying the Conjugate. The conjugate solution was diluted 1:10 with chilled 5 mM succinate-0.9% saline (pH 6.0) in preparation for purification by tangential flow filtration using 100-300K MWCO membranes. The diluted conjugate solution was passed through a 5 pm filter, and diafiltration was performed using 5 mM succinate / 0.9% saline (pH 6.0) as the medium. After the diafiltration was completed, the conjugate retentate was transferred through a 0.22pm filter. The conjugate was diluted further with 5 mM succinate / 0.9% saline (pH 6), to a target saccharide concentration of approximately 0.5 mg/mL. Alternatively, the conjugate is purified using 20 mM Histidine-0.9% saline (pH 6.5) by tangential flow filtration using 100-300K MWCO membranes. Final 0.22pm filtration step was completed to obtain the immunogenic conjugate. See Table 21, Table 22, Table 23, Table 24 and Table 25.

EXAMPLE 25: Conjugation in aqueous buffer (RAC/Aqueous), as applied to from E. col serotypes 025B, O1A, 02, and 06

Polysaccharides activation and diafiltration was performed in the same manner as the one for DMSO based conjugation. The filtered activated saccharide was compounded with CRM1 97 at a polysaccharide to protein mass ratio ranging from 0.4 to 2 w/w depending on the serotype. This input ratio was selected to control the polysaccharide to CRM 197 ratio in the resulting conjugate. The compounded mixture was thenlyophilized. Upon conjugation, the polysaccharide and protein mixture was dissolved in 0.1M sodium phosphate buffer at the polysaccharide concentration ranging from 5 to 25 g/L depending on the serotype, pH was adjusted between 6.0 to 8.0 depending on the serotype. Conjugation was initiated by adding 0.5 - 2.0 MEq of sodium cyanoborohydride to the reaction mixture and incubating at 23± 2 C for 20-48 hrs. The conjugation reaction was terminated by adding 1-2 MEq of sodium borohydride (NaBH 4) to cap unreacted aldehydes. Alternatively, the filtered activated saccharide and calculated amount of CRM 197 protein was shell-frozen and lyophilized separately, and then combined upon dissolving in 0.1M sodium phosphate buffer, subsequent conjugation can then be proceeded as described above.

Table 20. Summary of results from both conjugations prepared in DMSO and aqueous buffer

RAC/DMSO RAC/Aqueous Poly MW (kDa) 48K 46K Degree of Oxidation (DO) 12 12 Saccharide/Protein Ratio 0.8 1.0 % Free Saccharide <5% 32% Conjugate MW by SEC-MALLS, kDa 7950 260

EXAMPLE 26: Procedure for the preparation of E. coli0-antigen polysaccharide-CRM 197 single-ended conjugates Lipopolysaccharides (LPS), which are common components of the outer membrane of Gram-negative bacteria, comprise lipid A, the core region, and the 0 antigen (also refer to as the0-specific polysaccharide or0-polysaccharide). Different serotype of 0-antigen repeating units differ in their composition, structure and serological features. The 0-antigen used in this invention is attached to the core domain which contains a sugar unit called 2-Keto-3-deoxyoctanoic acid (KDO) at its chain terminus. Unlike some conjugation methods based on random activation of the polysaccharide chain (e.g. activation with sodium periodate, or carbodiimide). This invention discloses a conjugation process involving selective activation of KDO with a disulfide amine linker, upon unmasking of thiol functional group, it is then conjugated to bromo activated CRM 197 protein as depicted in FIG. 31 (Preparation of Single-Ended Conjugates).

Conjugation based on cystamine linker (Al). 0-antigen polysaccharide and cystamine (50-250mol. eq of KDO) were mixed in phosphate buffer, adjust pH to 6.0-7.0. To the mixture, sodium cyanoborohydride (NaCNBH 3) (5-30 mol. eq of KDO) was added and the mixture was stirred at 370C for 48-72hrs. Upon cooling to room temperature and diluted with equal volume of phosphate buffer, the mixture was treated with tris(2-carboxyethyl)phosphine (TCEP) (1.2 mol, eq of cystamine added). The mixture was then purified through diafiltration using 5 KDa MWCO membrane against 10 mM sodium phosphate monobasic solution, to furnish thiol containing 0 antigen polysaccharide. The thiol content can be determined by Ellman assays. The conjugation was then proceeded by mixing above thiol activated 0-antigen polysaccharide with bromo activated CRM 197 protein at a ratio of 0.5-2.0. The pH of the reaction mixture is adjusted to 8.0 -10.0 with 1 M NaOH solution. The conjugation reaction was proceeded at 5 °C for 24± 4 hours. The unreacted bromo residues on the carrier protein were quenched by reacting with 2 mol. eq. of N-acetyl-L-cysteine for 3 - 5 hours at 5 °C. The addition of 3 mol. eq. of iodoacetamide (related to N-acetyl-L-Cysteine added) wad then followed to cap the residual free sulfhydryl groups. This capping reaction was proceeded for another 3-5 hours at 5 °C, and pH of both capping steps was maintained at 8.0-10.0 by addition of 1M NaOH. The resulting conjugate was obtained after ultrafiltration/dialfiltration using 30 KDa MWCO membrane against 5 mM succinate-0.9% saline, pH 6.0. See Table 21, Table 22, Table 23, Table 24 and Table 25.

EXAMPLE 27: Conjugation based on 3,3'-dithio bis(propanoic dihydrazide) linker (A4)

0-antigen polysaccharide and 3,3'-dithio bis(propanoic dihydrazide) (5-50mol. eq of KDO) were mixed in acetate buffer, adjust pH to 4.5-5.5. To the mixture, sodium cyanoborohydride (NaCNBH 3) (5-30 mol. eq of KDO) was added and the mixture was stirred at 23-37°C for 24-72 hrs. The mixture was then treated with tris(2-carboxyethyl)phosphine (TCEP) (1.2 mol, eq of 3,3'-dithio bis(propanoicdihydrazide) linker added). The mixture was then purified through diafiltration using 5 KDa MWCO membrane against 10 mM sodium phosphate monobasic solution, to furnish thiol containing 0-antigen polysaccharide. The thiol content can be determined by Ellman assays. The conjugation was then proceeded by mixing above thiol activated 0-antigen polysaccharide with bromo activated CRM 19 7protein at a ratio of 0.5-2.0. The pH of the reaction mixture is adjusted to 8.0 -10.0 with 1 M NaOH solution. The conjugation reaction was proceeded at 5 C for 24± 4 hours. The unreacted bromo residues on the carrier protein were quenched by reacting with 2 mol. eq. of N-acetyl-L-cysteine for 3 - 5 hours at 5 °C. The addition of 3 mol. eq. of iodoacetamide (related to N-acetyl-L-Cysteine added) wad then followed to cap the residual free sulfhydryl groups. This capping reaction was proceeded for another 3-5 hours at 5 °C, and pH of both capping steps was maintained at 8.0-10.0 by addition of 1M NaOH. The resulting conjugate was obtained after ultrafiltration/dialfiltration using 30 KDa MWCO membrane against 5 mM succinate-0.9% saline, pH 6.0.

EXAMPLE 28: Conjugation based on 2,2'-dithio-N,N'-bis(ethane-2,1-diyl)bis(2 (aminooxy)acetamide) linker (A6) 0-antigen polysaccharide and 2,2'-dithio-N,N'-bis(ethane-2,1-diyl)bis(2 (aminooxy)acetamide) (5-50mol. eq of KDO) were mixed in acetate buffer, adjust pH to 4.5-5.5. The mixture was then stirred at 23-37°C for 24-72 hrs, followed by the addition of sodium cyanoborohydride (NaCNBH 3) (5-30 mol. eq of KDO) and the mixture was stirred for another 3 24 hrs. The mixture was then treated with tris(2-carboxyethyl)phosphine (TCEP) (1.2 mol, eq of linker added). The mixture was then purified through diafiltration using 5 KDa MWCO membrane against 10 mM sodium phosphate monobasic solution, to furnish thiol containing 0-antigen polysaccharide. The thiol content can be determined by Ellman assays. The conjugation was then proceeded by mixing above thiol activated 0-antigen polysaccharide with bromo activated CRM 197 protein at a ratio of 0.5-2.0. The pH of the reaction mixture is adjusted to 8.0 -10.0 with 1 M NaOH solution. The conjugation reaction was proceeded at 5 °C for 24± 4 hours. The unreacted bromo residues on the carrier protein were quenched by reacting with 2 mol. eq. of N-acetyl-L-cysteine for 3 - 5 hours at 5 °C. The addition of 3 mol. eq. of iodoacetamide (related to N-acetyl-L Cysteine added) was then followed to cap the residual free sulfhydryl groups. This capping reaction was proceeded for another 3-5 hours at 5 °C, and pH of both capping steps was maintained at 8.0-10.0 by addition of 1M NaOH. The resulting conjugate was obtained after ultrafiltration/dialfiltration using 30 KDa MWCO membrane against 5 mM succinate-0.9% saline, pH 6.0.

EXAMPLE 29A: Preparation of Bromo activated CRM 197 The CRM 197 was prepared in 0.1M Sodium Phosphate, pH 8.0 ±0.2 solution, and was cooled to 5 ±3 C. To the protein solution, the N-hydroxysuccinimide ester of bromoacetic acid (BAANS) as a stock dimethylsulfoxide (DMSO) solution (20 mg/mL) is added in a ratio of 0.25 - 0.5 BAANS: protein (w/w). The reaction is gently mixed at 5

+ 3 °C for 30 - 60 minutes. The resulting bromoacetylated (activated) protein is purified, e.g., by ultrafiltration/diafiltration using 10 kDa MWCO membrane using 10 mM phosphate (pH 7.0) buffer. Following purification, the protein concentration of the bromoacetylated carrier protein is estimated by Lowry protein assay. Table 21: 01a Conjugates Conjugate Lot# 132240-112-2132242-106 132242-124 132242-127132242-130 Poly Lot# 709756-160 709756-160[709756-160710958-116710958-116 Poly Type Long Chain Short Chain Poly MW (kDa) 33 33 33 11 11 Variant eTEC Single-End RAC/DMSOSingle-End RAC/DMSC

Activation 8% SH 2.1% SH DO: 13 6.4% SH DO: 16

Conjugate Data Yield (%) 30 26 77 45 35 SPRatio 0.6 0.5 1.0 0.7 0.6 Free Sacc (%) 9 9 20 5 6 MW (kDa) 1035 331 1284 280 2266

Sacc Conc (mg.mL)0.31 0.37 0.58 0.59 0.37 Endotoxin (EU/ug) 0.03 0.02 0.01 0.01 0.01 Buffer 5 mM Succ/Saline, pH 6.0

Table 22: 02 Conjugates

Conjugate Lot# 00709749-0003-1132242-161 132242-152 132242-159132242-157

Poly Lot# 709766-33 709766-65 710958-141-2

Poly Type Long Chain Short Chain

Poly MW (kDa) 36 39 14 Variant eTEC Single-End RAC/DMSO Single-End RAC/DMSO Activation 6.8% SH 1.6% SH DO: 17 6.3% SH DO: 19 Conjugate Data Yield (%) 26 33 50 38 36 SPRatio 1.5 0.8 0.8 1.0 0.6 Free Sacc (%) 11 24% <5 <5 6 MW (kDa) 1161 422 3082 234 1120 Endotoxin (EU/ug)0.025 0.02 0.01 0.01 0.01 Buffer 5 mM Succ/Saline, pH 6.0

Table 23: 06 Conjugates

Conjugate Lot# 132240-117-1[132242-1341132242-137 132242-146132242-145

Poly Lot# 710958-121-1 710958-143-3 Poly Type Long Chain Short Chain Poly MW (kDa) 44 15 Variant eTEC Single-End RAC/DMSOSingle-End RAC/DMSO Activation 18% SH 2.2% SH DO: 16.5 6.1% SH DO: 22 Conjugate Data Yield (%) 27 23 58 48 30 SPRatio 0.78 0.6 0.82 0.7 0.6 Free Sacc (%) 9 4 4 <5 8 MW (kDa) 1050 340 1910 256 2058 Sacc Conc (mg.mL)0.39 0.45 0.59 0.88 0.41

Endotoxin (EU/ug) 0.03 0.02 0.01 0.004 0.005 Buffer 5 mM Succ/Saline, pH 6.0

Table 24: 025b Conjugates

Conjugate 132242-132240-132240-132240-132242- 132242-27 132242-29 132242-28 132242-121 Lot# 98 73-1-1 62-1 81 116 709766-709766-709766-709766-710958-710958- 709766-28 709766-28 Poly Lot# 709766-28 29 30 30 30 117/118 117/118

Poly type Long Chain Short Chain Long Long Chain Chain

Poly MW 51 48 48 48 48 14 14 51 51 (kDa)

Variant RAC/DMSOSingle- eTEC eTEC eTEC Single- RAC/DMSORAC/DMSORAC/DMSO End End 2.4% 6.6% 21 12 Activation DO: 18 10% SH4% SH 17% SH DO:17 SH SH Conjugate Data Yield (%) 82 26 56 32 92 28 18 71 80 SPRatio 0.9 0.82 0.88 0.64 1.32 0.7 0.36 0.81 0.84 Free Sacc 8.3 <5 7.2 5 <5 11 <5 <5 <5 (%) Conjugate 4415 840 1057 1029 2306 380 9114 3303 7953 MW (kDa) Sacc 0.6 0.67 Conc 0.7 0.4 0.43 0.36 0.9 0.45 0.19 (mg.mL) Endotoxin 0.02 0.22 0.01 0.02 0.08 0.08 0.01 0.01 0.01 (EU/ug) Conjugate (DS) 5 mM Succ/Saline, pH 6.0 Buffer matrix

Table 25: 025b K-12 Conjugates

Conjugate Lot# 709749-015-2709744-0016 Poly Lot# 710958-137 Poly Type Long Chain(K12) Poly MW (kDa) 44 Variant eTEC RAC/DMSO Activation SH: 24% DO: 19 Conjugate Data Yield (%) 59% 33% SPRatio 1.4 0.83 Free Sacc (%) 5% 5.2% MW (kDa) 1537 4775 Sacc Conc (mg.mL)0.91 0.29 Endotoxin (EU/ug) 0.08 0.01

Buffer 5 mM Succ/Saline, pH 6.0

EXAMPLE 29B: Preparation of E. colO-Ag-TT conjugates

E. coli serotype 025b long polysaccharide, Lot# 709766-30 (about 6.92 mg/mL, MW: about 39kDa), 50 mg, lyophilized was used for Tetanus Toxoid (TT) conjugation. E. coli serotype 01a long polysaccharide 710958-142-3 (about 6.3 mg/mL, MW: about 44.3 kDa) (50mg, 7.94 mL) waslyophilized. E. coli serotype 06 long polysaccharide, 710758-121-1 (about 16.8 mg/mL, MW: about 44 kDa) (50mg, 2.98 mL) waslyophilized. Each of the lyophilized polysaccharides listed above was dissolved in WFI to make at approx 5-10 mg/mL to it, 0.5 mL (100 mg (1-cyano-4-dimethylaminopyridinum tetrafluoroborate (CDAP) solution in 1 mL acetonitrile) was added and stirred at RT. Triethylamine (TEA) 0.2M (2mL) was added and stirred at RT. Preparation of Tetanus toxoid (TT): TT (100 mg, 47 ml) was concentrated to approximately 20 mL and washed twice with saline (2x5OmL) using filteration tubes. After that it was diluted with HEPES and saline to make final HEPES conc as about 0.25M. TT was prepared as described above and pH of the reaction was adjusted to about 9.1-9.2. The reaction mixture was stirred at RT.

After 20-24 hrs the reaction was quenched with Glycine (0.5 mL). After that it was concentrated to using MWCO regenerated cellulose membranes and diafiltration was performed against saline. Filtered and analyzed. See Table 26.

Table 26

Exemplary embodiments:

E. coli serotype 025b-TT conjugate E. coilserotype 06-TT conjugate Volume: 41 mL Volume: 42 mL Sacc Conc (Anthrone): 1.122 mg/mL (92% yield) Sacc Conc (Anthrone): Protein Conc (Lowry): 1.133 mg/mL 0.790 mg/mL (66% yield) SPRatio: 0.99 Protein Conc (Lowry): Free Sacc (DOC): 74.7% 1.895 mg/mL The product obtained was concentrated to 15 mL using MWCO SPRatio: 0.42 regenerated cellulose membranes and diafiltration was Free Sacc (DOC): <5% performed against saline (40X diavolumes). Filtered through MW (kDa): 1192 0.22 um filter and analyzed. Endotoxin (EU/ug:) 0.022 Volume: 27 mL Sacc Conc (Anthrone): 1.041 mg/mL (56% yield) Protein Conc (Lowry): 1.012 mg/mL SPRatio: 1.03 Free Sacc (DOC): 60.6% (poly recovery 100%)

EXAMPLE 30: Additional results from O-antigen Fermentation, Purification, and Conjugation

The exemplary processes described below is generally applicable to all E. coli serotypes. The production of each polysaccharide included a batch production fermentation followed by chemical inactivation prior to downstream purification. Strains and storage. Strains employed for biosynthesis of short chain 0 antigen were clinical wild type strains of E. coli. Long chain 0-antigen was produced with derivatives of the short chain-producers that had been engineered by the Wanner Datsenko method to possess a deletion of the native wzzb gene and were complemented by the "long-chain" extender function fepE from Salmonella. The fepE function was expressed from its native promoter on either a high copy colEl-based

"topo" vector or a low copy derivative of the colEl-based vector pET30a, from which the T7 promoter region had been deleted. Cell banks were prepared by growing cells in either animal free LB or minimal medium to an OD00 of at least 3.0. The broth was then diluted in fresh medium and combined with 80% glycerol to obtain a 20% glycerol final concentration with 2.0 OD6 0oo/mL. Media used for seed culture and fermentation. The seed and fermentation medium employed share the following formulation: KH 2 PO 4 , K2 HPO 4 , (NH 4) 2 SO 4 , sodium citrate, Na 2 SO 4 , aspartic acid, glucose, MgS 4 , FeS 4-7H 20, Na 2M0 4-2H 2 0, H3B03, CoCl 2 -6H 20, CuCl 2-2H 2 0, MnCl 2-4H 20, ZnCl2 and CaCl 2-2H 20. Seed and fermentation conditions. Seeds were inoculated at 0.1% from a single seed vial. The seed flask was incubated at 370C for 16-18 hours and typically achieved 10-20 OD6oo/mL. Fermentation was performed in a 1OL stainless steel, steam in place fermentor. Inoculation of the fermentor was typically 1:1000 from a 10 ODoo seed. The batch phase, which is the period during which growth proceeds on the 10 g/L batched glucose, typically lasts 8 hours. Upon glucose exhaustion, there was a sudden rise in dissolved oxygen, at which point glucose was fed to the fermentation. The fermentation typically then proceeds for 16-18 hours with harvest giving > 120 ODeoo/mL. Initial evaluation of short/long chain O-antigen production for serotypes O1a, 02, 06 and 025b. Wild type strains for 01a, 02, 06 and025b were fermented in a supplemented minimal medium in batch mode to an ODeoo = 15-20. Upon glucose exhaustion, which results in a sudden decrease in oxygen consumption, a growth limiting glucose feed was applied from a glucose solution for 16-18 hours. Cell densities of 124-145 ODoo units/mL were reached. The pH of the harvest broths was subsequently adjusted to about 3.8 and heated to 950C for 2 hours. The hydrolyzed broth was then cooled to 250C, brought to pH 6.0 and centrifuged to remove solids. The resulting supernatant was then applied to a SEC-HPLC column for quantitation of the O-antigen. Productivities in the range of 2240-4180 mg/L were obtained. The molecular weight of purified short-chain O-antigen from these batches was found to range from 10-15 kDa. It was also noted that SEC chromatography of the 02 and 06 hydrolysates revealed a distinct and separable contaminating polysaccharide that was not evident in the 01a and 025b hydrolysates. Long chain versions of the 01a, 02, 06 and 025b O-antigens where accessed through fermentation of a wzzb deletion version of each strain which carried a heterologous, complementing fepE gene on a high-copy, kanamycin-selectable topo plasmid. Fermentation was performed as for the short chain, albeit with kanamycin selection. The final cell densities observed at 124-177 ODeoo/mL were associated with O-antigen productivities of 3500 9850 mg/L. The complementation-based synthesis of long chain O-antigen was at least as productive as in the parental short chain strain and in some cases more so. The molecular weights of purified O-antigen polysaccharide were 33-49 kDa or about 3 times the size of the corresponding short chain. It was noted that the long chain hydrolysates for 02 and 06 showed evidence of a contaminating polysaccharide peak that, in the case of long chain antigen, was observed as a shoulder on the main O-antigen peak; 01 and 025b showed no evidence of production of a contaminating polysaccharide, as was seen earlier with the short chain parent. Growth rate suppression was found to be associated with the presence of the topo replicon absent the fepE. Additionally, the Awzzb mutation itself had not adverse effect on growth rate, indicating that the disturbed growth rates were conveyed by the plasmid vector. Evaluation of strains for production of 011, 013, 016, 021 and 075 0 antigen. Multiple wild-type strains of serotypes 011, 013, 016, 021 and 075 were evaluated for their propensity to produce unwanted polysaccharide in fermentation by SEC-HPLC. Strains for 011, 013, 016, 021 and 075 were selected as absent contaminating polysaccharide, as well as for their ability to produce > 1000 mg/L 0 antigen and for the display of an antibiotic sensitivity profile that allowed Wanner Datsenko recombineering for introduction of the Awzzb trait. Chloramphenicol-selectable versions of topo-fepE and pET-fepE were constructed that allowed for the introduction of fepE into the 011, 013, 016, 021 and 075 Awzzb strains that in general were found to be kanamycin-resistant. The resulting topo-fepE and pET-fepE bearing strains were fermented with chloramphenicol selection and the supernatant from acid-hydrolyzed broth was evaluated by SEC-HPLC. Both the high (topo) and low copy (pET) fepE constructs directed the synthesis of O-antigen with productivities for each that were equivalent to the parental wild-type. Expression of potentially interfering polysaccharides was not observed. An evaluation of growth rates for wzzb plasmid-bearing strains showed that the 011, 013 and021 were retarded by the presence of topo-fepE but not by pET-fepE; strains 016 and 075 strains showed acceptable growth rates irrespective of replicon choice.

Table 27

short

-(SC) fep E final Oag MW or final cell SEC antigen IHMA type plasmid marker productivity long density0D 0 oductivrity type chain type (mg/L) kDa

(LC) Ola wt SC None None 125 2550 11 N Ola Awzzb/fepE LC topo Kana 130 5530 33 N Ola Awzzb/fepE LC pET Kana Not done (ND) ND ND ND 02 wt SC None None 127 2240 13 Y 02 Awzzb/fepE LC topo Kana 177 3750 49 Y 02 x LC pET x NA NA NA NA 06 wt SC None None 145 4180 16 Y 06 Awzzb/fepE LC topo Kana 124 9850 44 Y 06 Awzzb/fepE LC pET Kana ND ND ND ND 011 wt SC None None 194 4720 x N 011 Awzzb/fepE LC topo Kana 142 7220 x N 011 x LC pET x NA NA NA NA 013 wt SC None x 113 4770 x N 013 Awzzb/fepE LC topo cam 101 4680 x N 013 Awzzb/fepE LC pET cam 108 4600 x N 016 wt SC None x 154 1870 x N 016 Awzzb/fepE LC topo cam 129 1180 x N 016 Awzzb/fepE LC pET cam 137 1280 x N 021 wt SC None x 140 1180 x N 021 Awzzb/fepE LC topo cam ND ND x N 021 Awzzb/fepE LC pET cam 131 820 x N 025b 2831 SC None None 126 3550 10 N 025b Awzzb/fepE LC topo Kana 152 3500 49 N 025b x LC pET x NA NA NA NA 075 wt SC None x 149 1690 x N 075 Awzzb/fepE LC topo cam 132 1500 x N 075 Awzzb/fepE LC pET cam 138 1520 x N

The purification process for the polysaccharides included acid hydrolysis to release the O-antigens. A crude suspension of serotype specific E. coli culture in fermentation reactor was directly treated with acetic acid to the final pH of 3.5±0.5 and the acidified broth was heated to the temperature of 95±50C for at least one hour. This treatment cleaves the labile linkage between KDO, at the proximal end of the oligosaccharide and the lipid A, thus releasing the O-Ag chain. The acidified broth that contains the released O-Ag was cooled to 20 ±10C before being neutralized to pH 7 ±1.0 using NH 40H. The process further included severalcentrifugation, filtration, and concentration/ diafiltration operations steps.

Table 28

Increase Purified Purified Number in M.W. Conjugate Serotype ExpectedTiter Poly of NMRM.W. Conjugation (core) Polysize(g/L) M.W. Repeat kDa Lot

# over (~) Lt (kDa) Units short

5365 132242-28 (RAC/DMSO) AwzzB + 1423 132242-98 LT2FepE Long 5.3 47 55 34 (Single-end) 025b 1258 132240-73-1 (R1) 1 (eTEC) 380 132242-116 AwzzB + Short 2.3 13/14 15 NA V (Single-end) 025a wzzB 9114 132242-121 (RAC/DMSO) 1537 709749-015 AwzzB+ 2(eTEC) 025b T2FepE Long 3.5 44 51 27 V 4 707401 LT2FepE 4775 709744-0016 (K12) (RAC/DMSO

wt Short 3.5 17 17 NA /

1035 132240-112 2(eTEC) AwzzB + 331 132242-106 01a (R1) LT2FepE Long 5.5 33 39 22 (Single-end) 1284 132242-124 (RAC/DMSO)

280 132242-127

wt Short 2.5 11 13 NA V 2266 132242-130 (RAC/DMSO) 1161 00707947 36 43 22 / 0003-1 (eTEC) AwzzB+ Long 4.9 422 132242-161 LT2FepE Ln . (single-end) 39 47 25 02 (Ri) 3082 132242-152 (RAC/DMSO) 234 132242-159

wt Short 2.8 14 17 NA V 1120 1322421-157 (RAC/DMSO) AwzzB+ NA NA NA NA LT2FepE Long 5.1 02 (R4) wt Short 2.1 14.7 18 NA V

AwzzB+ 37.2 42 22.2 V LT2FepE Long 6.9

256 132242-146 06 (R) 17 NA (Single-end_ wt Short 3.5 15 V 2058 123342-145 (RAC/DMSO) 1050 132240-117 1(eTEC)

340 132242-134 AwzzB + Long 8.4 44.4 50 28.2 V (Single-end) 06 (Ri) LT2FepE 124-3 132242-137

1910 (RAC/DMSO)

wt Short 3.6 16.2 18 NA V

EXAMPLE 31: Conjugation towards O-antigen (04, 011, 021, 075) studied (RAC/DMSO)

Table 29: 04 conjugates

Conjugate Lot# 709744-70 709744-73 709744-72

Poly Lot# 709740-168

Poly MW (kDa) 52

DO 26 19 15

Act poly Mw (kDa) 51

Conjugation

Input SP 1.0 1.0 1.0

SPRatio 0.85 1.0 1.0

Free Sacc (%) <5% <5% <5%

MW (kDa) 4764 4758 3423

Yield (%) 72 80 82

Endotoxin (EU/ug)0.003 0.001 0.005

Table 30: 011 conjugates

Conjugate Lot# 709744-64709744-66709744-65709744-67

Poly Lot# 709740-162

Poly MW (kDa) 39

DO 21 14

Act poly Mw (kDa) 40

Conjugation

Input SP 1.0 1.3 1.0 1.3

SPRatio 0.5 0.64 0.65 0.75

Free Sacc (%) <5% <5% <5% <5%

MW (kDa) 10520 7580 4814 4338

Yield (%) 30 30 44 38

Endotoxin (EU/ug)0.005 0.005 0.005 0.005

Table 31: 021 conjugates

Conjugate Lot# 709749-113709749-111709749-112709749-115709749-116

Poly Lot# 709740-165

Poly MW (kDa) 40

DO 25 18 15

Act poly Mw (KDa)40 41 40

Conjugation

Input SP 1.0 1.0 0.8 1.0 1.25

SPRatio 0.6 0.6 0.5 0.9 1.1

Free Sacc (%) 6% 5% <5% 12% 7%

MW (kDa) 6920 5961 9729 2403 1960

Yield (%) 31 36 37 52 54

Endotoxin (EU/ug) 0.02 0.02 0.03 0.01 0.009

Table 32: 075 conjugates

Conjugate Lot# 709749-101 709749-102 709749-103

Poly Lot# 709766-080B

Poly MW (kDa) 48

DO 18 25

Act poly Mw (kDa) 43 44

Conjugation

Input SP 1.0 0.8 1.0

SPRatio 0.94 0.76 0.78

Free Sacc (%) <5% 6% 6%

MW (kDa) 2304 2427 5229

Yield (%) 62 65 45

Endotoxin (EU/ug) 0.02 0.01 0.01

EXAMPLE 32: PLL conjugates prepared

Table 33

Serotype 011 075 021 04

Conjugate Lot# 00707779-0413 00707779-0414 00707779-0415 00707779-0416

Poly Lot# 709740-162 709766-080B 709740-165 709740-168

Poly MW (kDa) 39 48 40 52

Conjugate Data

SPRatio 13.5 16.8 18.1 21.2

Free Sacc (%) 9.8% <5% <5% 6.9%

Sacc Conc 789 pg/mL 676 pg/mL 978 pg/mL 837 pg/mL

PLL Conc 58.3 pg/mL 40.3 pg/mL 54.0 pg/mL 39.4 pg/mL

Endotoxin (EU/ug) 0.002 0.002 0.005 0.004

Conjugate (DS) Matrix 1X PBS, 1M NaCI

EXAMPLE 33: Stable mammalian cell expression of E. coli polypeptides Stable CHO clones expressing FimH GSD or FimH LD were generated using a SSI (Site Specific Integration) stable expression system. The host CHO cell is an engineered cell line from a CHOK1SV GS-KO background (see, for example, United States Patent Application 20200002727, for a description of the CHOK1SV GS-KO host cell line). Briefly a landing pad with green fluorescent protein (GFP) gene surrounded by two FRT sites were targeted into a transcription hot spot in the genome of the host cell. The GFP gene can be exchanged with GS gene and the gene of interest which are also surrounded by FRT sites from the LVEC vector co expressed with flippase recombinase (FLPe). This system not only has growth and productivity profiles that compare favorably with random integration but also displays genotypic and phenotypic stability to at least 100 generations. As referred to herein, the term "FRT site" refers to a nucleotide sequence at which the product of the flippase (FLP) gene of the yeast 2 pm plasmid, FLP recombinase, can catalyze a site-specific recombination. A variety of non-identical FRT sites are known to the art. The sequences of the various FRT sites are similar in that they all contain identical 13-base pair inverted repeats flanking an 8-base pair asymmetric core region in which the recombination occurs. It is the asymmetric core region that is responsible for the directionality of the site and for the variation among the different FRT sites. Illustrative (non-limiting) examples of these include the naturally occurring FRT (F), and several mutant or variant FRT sites such as FRT F1and FRTF2. As referred to herein, the term "landing pad" refers to a nucleic acid sequence comprising a first recombination target site chromosomally-integrated into a host cell. In some embodiments, a landing site comprises two or more recombination target sites chromosomally integrated into a host cell. In some embodiments, the cell comprises 1, 2, 3, 4, 5, 6, 7, or 8 landing pads. In some embodiments, the cell comprises 1, 2, or 3 landing pads. In some embodiments, the cell comprises 4 landing pads. In some embodiments, landing pads are integrated at up to 1, 2, 3, 4, 5, 6, 7, or 8 distinct chromosomal loci. In some embodiments, landing pads are integrated at up to 1, 2, or 3 distinct chromosomal loci. In some embodiments, landing pads are integrated at 4 distinct chromosomal loci. The LVEC expression vector for FimH GSD or FimH LD and the FLPe expression vector were co-transfected into a SSI host cell by electroporation either with BioRad Gene Pulser Xcell or Amaxa 4D-Nucleofector. Then cells were cultured in media without glutamine to select cells that has GS gene integrated at the landing pad site. Usually cells recover in 2-3 weeks. Then single cell cloning were carried out in 96 well plates either by FACS or limiting dilution. Titers from wells with cells were ranked to narrow down to top 48 clones. A second round of fed batch screening in 24 deep-well plates was conducted to narrow down the clones to top 12. A third round of fed batch screening in Ambr15 was executed to narrow down the clones to top 3. Ambr250 experiments were used to identify the best clone. Master cell bank and working cell bank were generated for the top clone after its identification.

EXAMPLE 34: Cell line development and production reactor expression of FimH-DSG WT and FimHLD WT proteins The example described herein, describes an exemplary production of both FimH-DSG WT and FimHLD VVT proteins from stable CHO cell lines, where the coding sequences foreach protein has been stably intergraded into the CHO genome. In a production bioreactor setting, the stable CHO cell lines selected were able to produce the target protein at around 1gram per liter of culture for FimH-DSG WT, and 250 miligrams per liter of culture for FimHLD WT. The seed train for the production reactor was continuously scaled up from vial thaw of a working cell bank and expanded in shake flasks using an inoculation viable cell density of 0.3x1OA6 cells/ml through three passage cycles in shake flasks to provide enough cells for the production reactor. The cells were grown at 36.5 deg C, at 5% C02 for three-four days. The production reactor was seeded from the final shake flask, targeting an inoculation cell density of 1x10A6 cells/ml. The production reactor was grown at 36.5 deg C for seven days, using a pH of 7.05 (+/- 0.15), and targeting a C02 saturation of 5 10%. pH is controlled by sodium/potassium bicarbonate for base control, and C02 sparge for acid control. Dissolved oxygen is controlled at a setpoint of 40% using pure oxygen through the sparge. The temperature was adjusted to 31 deg C on day seven. The reactor was fed on day 1 using a feed strategy that adds feed in correlation to the viable cell density, this is achieved by using a feed factor of 0.75 in order to ensure feed components do not run out during the run. The feed is then added continuously to provide the desired volume of feed over the course of the day. The production reactor was harvested on day 13, and the harvest culture was centrifuged and 0.22 pm filtered, prior to downstream processing.

EXAMPLE 35: Immunogenicity of FimH Antigens and 0-antigen Combinations in Mice

E. coli FimH lectin binding domain (FimHLD) and full-length (FimH-DSG or FimCH) variants were evaluated in mouse immunogenicity studies that assessed the ability of elicited antibodies to neutralize the binding of fimbriated E. coli to mannosylated ligands. Wild-type FimH antigens that were secreted and purified at high yield from mammalian cells were similarly immunogenic in mice as analogous native antigens purified at low yield from the E. coli periplasm. By comparison, full-length FimH antigens containing both the lectin and pillin domains were significantly more immunogenic than FimHLD. Use of an adjuvant formulation containing QS21 was required to generate robust functional immune responses to these fimbrial antigens. A follow-up study investigated the immunogenicity of FimH in combination with a four valent O-antigen glycoconjugate mixture and different adjuvants. After a second dose, mice vaccinated with a liposomal QS21 formulation induced consistently higher functional responses to the combined antigens in both FimH neutralization and O-antigen-specific OPA assays compared with groups dosed with no adjuvant or liposomal monophosphoryl lipid A (MPLA). Animal Immunogenicity. 6-8 week old CD-1 mice were obtained from Charles River and groups of 10 or 20 animals were dosed subcutaneously (SC) with 0.1 mL of test antigen or buffer control. Polyclonal anti-FimH rabbit control serum was prepared by immunizing rabbits with three doses of E. coli FimHLD antigen with 100 pg complete Freund's adjuvant (at wk 0) followed by antigen in 100 pg incomplete Freund's adjuvant (at wks 4 and 8) (Covance). Each vaccination administered 0.5 mLs containing 50 pg of antigen at two sites (sub-cutaneous Dorsal). Adjuvant Formulations. Diluent for unadjuvanted antigens was 10 mM phosphate buffer, pH 6.1. For AIPO4, 50 pg (100 pL of a 0.5 mg/ml suspension) was given per mouse dose. The default dose of Quillaja Saponaria-21 (QS21) used in preclinical studies was 20 pg per mouse from a stock containing 5.1 mg/mL QS-21, 5 mM Succinate, 60mM NaCI, 0.1% PS80, pH 5.6. Liposomal formulations of monophosphoryl Lipid A (MPLA, Synthetic, PHAD@, Avanti) and QS21 were prepared with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol (Avanti polar lipids). Liposomal MPLA was used at 5 pg per dose from stock comprising 15 mM phosphate buffer, pH 6.1, 4 mg/mL DOPC, 1 mg/mL Cholesterol, 0.2 mg/mL MPLA (Lot 00714551-0018-2XLipoMPL), having a liposomal particle size of 71 nm determined by dynamic light scattering. A liposomal MPLA/QS21 formulation was used at 5 pg each per dose from stock comprising 15 mM phosphate buffer, pH 6.1, 4 mg/mL DOPC, 1 mg/mL Cholesterol, 0.2 mg/mL MPLA, and 0.2 mg/mL QS-21 (Lot 00714551-0018-2XLipoMQ), having particle size of 75 nm for MPLA-QS21 liposomes determined by dynamic light scattering. Expression Plasmids and Derived FimH Fimbrial Antigens used in Immunogenicity Studies. The following expression plasmids were used to generate recombinant protein for mouse experiments:

>pSB02083 -- FimHLD (mlgK signal pept, N28S, N91S), processed protein sequence: FACKTASGTAIPIGGGSANVYVNLAPCVNVGQNCVVDLSTQIFCHNDYPETITDYVTLQRGSAY GGVLSSFSGTVKYSGSSYPFPTTSETPRVVYNSRTDKPWPVALYLTPVSSAGGVAIKAGSLIAV LILRQTNNYNSDDFQFVWNIYANNDVVVPTGGCDVSARDVTVTLPDYPGSVPIPLTVYCAKSQ NLGYYLSGTTADAGNSIFTNTASFSPAQGVGVQLTRQGTIIPANNTVSLGAVGTSAVSLGLTAN YARTGGQVTAGNVQSIIGVTFVYQGGSSGGGADVTITVNGKVVAKGGHHHHHHHH (SEQ ID NO:110);

>pSB02158 -- FimHLD-LM (mlgK signal pept, N28S N91S V48C L55C), processed protein sequence: FACKTASGTAIPIGGGSANVYVNLAPCVNVGQNCVVDLSTQIFCHNDYPETITDYVTLQRGSAY GGVLSSFSGTVKYSGSSYPFPTTSETPRVVYNSRTDKPWPVALYLTPVSSAGGVAIKAGSLIAV LILRQTNNYNSDDFQFVWNIYANNDVVVPTGGHHHHHHHH (SEQ ID NO:111);

>pSB02307 - FimH-DSG (mlgK signal pept, N28S N91S N249Q 7aa linker FimG A1..K14), processed protein sequence: FACKTASGTAIPIGGGSANVYVNLAPVVNVGQNLVVDLSTQIFCHNDYPETITDYVTLQRGSAY GGVLSSFSGTVKYSGSSYPFPTTSETPRVVYNSRTDKPWPVALYLTPVSSAGGVAIKAGSLIAV LILRQTNNYNSDDFQFVWNIYANNDVVVPTGGCDVSARDVTVTLPDYPGSVPIPLTVYCAKSQ NLGYYLSGTTADAGNSIFTNTASFSPAQGVGVQLTRQGTIIPANNTVSLGAVGTSAVSLGLTAN YARTGGQVTAGNVQSIIGVTFVYQGGSSGGGADVTITVNGKVVAKGGHHHHHHHH (SEQ ID NO:112);

>pSB02198 - FimH-DSG-LM (mlgK signal pept, N28S N91S 249Q V48C L55C 7aa linker FimG A1..K14), processed protein sequence: FACKTASGTAIPIGGGSANVYVNLAPVVNVGQNLVVDLSTQIFCHNDYPETITDYVTLQRGSAY GGVLSSFSGTVKYSGSSYPFPTTSETPRVVYNSRTDKPWPVALYLTPVSSAGGVAIKAGSLIAV LILRQTNNYNSDDFQFVWNIYANNDVVVPTGGHHHHHHHH (SEQ ID NO:113).

Amino acid numbering is based on the full-length amino acid sequence of the J96 strain FimH, whereas academic publications use numbering based on the N-terminal Phenylalanine (in position 21) that is exposed after signal peptide processing. Other mutations remove glycosylation site asparagine residues in the lectin domain (N28S and N91S), in the pilin domain of the full-length FimH-DSG construct (N249Q). The donor-strand G peptide that is added to the C-terminus stabilizes FimH. The 7 amino acid linker length was optimized for mammalian expression as described in the above Examples. FimH Whole Cell Neutralization Assays. FimH neutralization assays were developed to measure the inhibition by serum antibodies of the binding of fimbriated E. colito mannoside ligands. Ligands were either microplate-immobilized yeast mannan or bladder 5637 cells that express the mannosylated uroplakin receptor UPla. For the yeast mannan assay black microtiter 96-well plates (Maxisorb, Nunc) were coated with 20 pg/ml of yeast mannan (Sigma-Aldrich) in PBS buffer. The wells were blocked with 1% bovine serum albumin (BSA, Sigma-Aldrich) in PBS for 20 min. The human bladder epithelial cell line 5637 was obtained from ATCC (ATCC HTB-9). Cells were grown on black tissue-culture microplates (Greiner) in RPMI 1640 (Sigma, St Louis, MO) supplemented with 10% fetal bovine serum (FBS, Sigma), 2.0 g/I sodium bicarbonate (Sigma) and 0.3 g/I I glutamine, grown at 37°C with 5% C02 and utilized between passages 10 and 24. Surface expression of Uroplakin 1a receptor was confirmed by immunofluorescence staining with polyclonal antibody (Novus #NBP214694). E. coliserotype 025b UTI strain PFEEC0547 was serially passaged in 10 mL static LB cultures at 37°C to induce FimH expression. Expression of FimH on the bacterial surface was confirmed by flow cytometry with rabbit immune serum to FimHLD antigen. Specificity of bacterial binding to mannan or bladder cells was established by the inclusion of negative control compound methyl a-D-mannopyranoside (Sigma) which reduced binding by >95% at 50 mM levels. Eight-step two-fold serial dilutions of test sera starting at 1:100 (in PBS, 0.1% BSA) were co-incubated with 1x10 7 E. coli for 1 h at 37C before adding to immobilized yeast mannan or 5637 cell monolayers. Serially diluted anti FimHLD rabbit serum wasused as an internal standard on each plate. Plates were incubated for 1 h at 37°C before washing away unbound bacteria. Bound E. coli were stained for 45 min at RT with 3 pg/ml of an 025b-specific mAb conjugated to Alexafluor 488. The mAb was reconstructed from variable light and heavy chain sequences of 025b antibody 3E9-11 (Nagy E, Nagy G, Szijarto V, et al. Antibodies to multi-drug resistant Escherichia coli: Arsanis Biosciences GmbH, Austria. 2014:76pp). The human pTT5 IgG expression plasmid was used as cloning vector. The fluorescence intensity of individual wells was read on a ClarioStar Plus instrument. IC50 inhibition values were interpolated using sigmoidal dose response variable slope curve fitting (Graphpad Prism). Titers are the reciprocal of the serum dilution at which half-maximal inhibition is observed. A vaccine antigen responder was defined as a neutralizing titer that exceeds 80% inhibition at the starting serum dilution of 1:100. In addition, the serum dilution titration of binding activity must satisfy variable slope sigmoidal curve fitting parameters (R2 >0.95, with interpolated Log IC50 value or trigger an experimental repeat with a broader dilution until resolved). The statistical significance (p-value) of differences in responses between groups was determined using an unpaired t-test with Welch's correction applied to log transformed data. For experiments involving mice vaccinated with multivalent O-antigen conjugates of different serotypes the yeast mannan assay was adapted for use with the Bactiter Glo detection reagent (Promega), to avoid potential interference by elicited anti-O-Ag IgG with bacterial detection. In this case detection is based on photon emission catalyzed by firefly luciferase following release of ATP from permeabilized viable bacteria. Use of this reagent simplified detection by eliminating the wash step and facilitated implementation of a higher throughput 384-well assay format with liquid handling automation (Bravo, Agilent). The miniaturized assay reduced reaction volumes from 100 pL to 30 pL and the required number of bacteria from1x107 per well to 1x106. After diluting 1:1 in PBS, Bactiter Glo was added to all wells and after mixing, read on the ClarioStar reader in luminometer mode. The serotype 06:K2 E. coli UTI strain CFT073 (ATCC@ 700928TM) was selected for use in the semi-automated 384-well assay due to its enhanced sensitivity compared with 025b UTI strain PFEEC0547. Both strains yielded similar results in bridging studies comparing individual serum titers in a subset of mouse vaccination groups (data not shown). E. col0O-Ag Mouse IgG Direct Luminex Immunoassay (dLIA). Long chain E. coli 0 Ag polysaccharides of serotypes 025b, 01a, 02 and 06 produced in-house by eBPD (Pfizer) were conjugated to poly-L-lysine and then to Luminex bead microspheres with EDC/NHS. Use of beads with distinct spectral addresses for each O-antigen enabled four-fold multiplexing. Beads were incubated with serially diluted individual mouse sera or control mAbs with shaking at 4°C for 18h. After washing, bound serotype-specific IgG was detected with a PE-conjugated goat anti-mouse IgG mouse secondary antibody (90 min RT incubation). Microplates were read on a FlexMap 3D instrument (Biorad). Serotype specific mouse IgG mAbs with similar binding properties (generated in-house) were used as internal standards to quantify IgG levels. Plots of standard curves for each mAb yielded overlapping linear slope profiles across 103 serum dilutions (log luminescence vs log serum dilution). A lower limit of quantitation (LLOQ) for the 4 plex IgG dLIA of 0.15259 ug/mL was calculated from standard curve bias, which was the same for each of the four antigens. E. colOpsonophagocytic Assays (OPA). Clinical E. coli invasive blood isolates were obtained from the Pfizer-sponsored Antimicrobial Testing Leadership and Surveillance (ATLAS) database which is maintained by the International Health Management associates (IHMA) clinical lab. Strains were genotypically characterized by WGS using the Illumina Miseq platform, including in-silico serotyping for prediction of O-antigen and K-capsule types. Serotype 01a, 02, 06 and 025b strains that express both O-antigen and K-antigens when grown in Dulbecco's Modified Eagle's medium (DMEM) under cell-banking conditions were used as test strains, as well as multidrug-resistant unencapsulated strains representing 06 and 025b serotypes. O-antigen expression was confirmed by flow cytometry with antigen-specific monoclonal or polyclonal antibodies generated in-house. K1-antigen expression was confirmed using K1-capsule mAb mAbl3D9-151 (obtained from NRC, Canada). K2-antigen expression was detected with K2-typing serum (Statens Serum Institute). Expression of the K5 heparosan capsule was confirmed by increased surface exposure of underlying O-antigen upon treatment with heparinase. The following E. coli clinical strains were developed for OPA assays: O1a:K1 (PFEEC0435, ST-95), 02:K1 (PFEEC0146, ST-95, Ticarcillin/Clavulanic AcidR), 06:K- (PFEEC0412, ST-127, Ticarcillin/Clavulanic AcidR, ESBL),

06:K2 (PFEECO150, ST-127), 025b:K- (PFEECO068, ST-131, ImipenemR, fluoroquinoloneR), 025b:K5 (PFEECO066, ST-131, fluorquinoloneR). Pre-frozen bacterial stocks of were grown in DMEM to an ODoo of between 0.5 and 1.0 and glycerol added to a final concentration 20% prior to freezing. Pre-titered thawed bacteria were diluted to 1 X 105 CFU/ml in OPA buffer (Hanks Balanced Salt Solution (Life Technologies), 0.1% gelatin, 1mM MgCl 2 , 2.5mM CaCl 2) and 20 pL (103 CFU) of the bacterial suspension was opsonized with 10 pL of serially diluted sera for 30min at RT in a 384-well tissue culture microplate. Conditions, such as the need for the opsonization step, the level of baby rabbit complement, the length of assay incubation, and HL60 to bacteria ratio, varied slightly and was optimized according to the assay strain used. Subsequently, 10 pl of 2-6% complement (Baby Rabbit Serum, Pel-Freez) and 10 pL of HL-60 cells (at 100-200:1 ratio) were added to each well and the mixture shaken at 2000 rpm for 45-60 min at 37°C in a 5% C02 incubator. Ten pL of each 50 pL reaction was transferred into the corresponding wells of a prewetted 384-well Millipore MultiScreen HTS HV filter plate containing 50 pL water. After vacuum filtering the liquid, 50 pL of 50% DMEM was applied and filtered and plate incubated overnight at 37°C in a sealed zip-lock bag. The next day the colonies were enumerated after staining with Coomassie dye using an ImmunoSpot@ analyzer and ImmunoCapture software. To establish the specificity of OPA activity, immune sera were preincubated with 20 pg/mL of the homologous serotype purified O-antigen prior to the opsonization step. The OPA assay includes control reactions without HL60 cells or complement, to demonstrate dependence of any observed killing on these components. Individual serum OPA titers were calculated using variable slope curve fitting (Excel). Combined data were plotted using GraphPad Prism to generate GMTs and associated p values for significance (unpaired t- test with Welch's correction from log transformed data). Results Mouse Immunogenicity Studies. A first study was designed to evaluate the immunogenicity of recombinant FimHLD antigensexpressed and purified from the E. coli periplasm at different dose levels and with different adjuvants. Purification of FimHLD was based on a previously described method (Schembri MA, Hasman H, Klemm P. Expression and purification of the mannose recognition domain of the FimH adhesin. FEMS microbiology letters 2000; 188:147-51). A second study compared the activity of E. coli FimHLD with mammalian expressed variants of FimHLD and full-length FimH-DsG. Finally, a third study investigated immunogenicity of combinations of FimH-DsG with a 4-valent long O-antigen glycoconjugate mixture and different adjuvant formulations.

Immunogenicity of FimH Antigens. Induction of functional anti-FimH antibodies induced by the various FimH constructs was assessed using neutralization assays similar to those described previously based on inhibition of binding by E. coli to surface immobilized yeast mannan (Kisiela DI, Avagyan H, Friend D, et al. Inhibition and Reversal of Microbial Attachment by an Antibody with Parasteric Activity against the FimH Adhesin of Uropathogenic E. coli. PLOS Pathogens 2015; 11:e004857) or bladder epithelial cells (Langermann S, Palaszynski S, Barnhart M, et al. Prevention of mucosal Escherichia coli infection by FimH-adhesin-based systemic vaccination. Science 1997; 276:607-11; Starks CM, Miller MM, Broglie PM, et al. Optimization and Qualification of an Assay that Demonstrates that a FimH Vaccine Induces Functional Antibody Responses in Women with Histories of Urinary Tract Infections. Human Vaccines & Immunotherapeutics 2020:1-10). The functional potency of antigen variants was quantified by determining serum neutralization titers that prevent the binding of fimbriated bacteria to the yeast mannoside ligands. The titer is defined as the reciprocal of the serum dilution at which 50% of bacteria are bound to the assay microplate in an eight-point two-fold titration of vaccinated animal sera. Results of an initial study with FimHLD antigens purified from E. coli are shown in FIG. 36A and FIG. 36B and Table 34. The vaccination schedule is shown in FIG. 36A and individual mouse reponses plotted in FIG. 36B. Mouse Immunogenicity Experiment 1 - Comparison of Different E. col Periplasmic Produced FimH Constructs. After vaccination with three 3pg doses of the FimHLD antigen, only 25% of mice in the unadjuvanted group or group adjuvanted with 50 pg of AIPO4 yielded neutralizing antibody titers in the yeast mannan assay. In contrast, 61% of mice in the group adjuvanted with 20 pg QS21/PS80, yielded neutralizing titers. In this case the geometric mean titer (GMT) was 5-fold higher (p value of <0.05) than the other groups. At the 30 pg FimHLD dose level with QS21/PS80, a further threefold increase in GMT and a 78% seroresponder rate was observed. In this adjuvant context, the presence of the disulfide mutation, designed and predicted to enhance functional immunogenicity by locking the FimHLD antigen in an open conformation (Rodriguez VB, Kidd BA, Interlandi G, et al. Allosteric coupling in the bacterial adhesive protein FimH. J Biol Chem 2013; 288:24128-39; Kisiela DI, Rodriguez VB, Tchesnokova V, et al. Conformational inactivation induces immunogenicity of the receptor binding pocket of a bacterial adhesin. Proceedings of the National Academy of Sciences of the United States of America 2013; 110:19089-94) did not improve immunogenicity compared with the wild-type FimHLD construct. An approximately three-fold lower GMT was observed for the lock-mutant compared with wild-type that bordered on statistical significance (p = 0.09). A key observation from this study is that the E. coli FimHLD antigen requires a potent QS21 adjuvant formulation to elicit robust levels of neutralizing antibodies in mice.

Table 34. VAC-2019-PRL-EC-1369 Neutralizing Titer GMTs and Responder Rates

Vaccination Group % Responders # of Responders Geometric mean (ICso) titers 3ug FimHLD no adjuvant 25 3/12 88 3ug FimHLD AIPO 4 25 5/20 93 3ug FimHLD QS21/PS80 61 11/18 495 30ug FimHLD QS21IPS80 78 14/18 1486 30ug FimHLDLMa- QS21 58 11/19 459 a. LM is the dusfide lock mutation V48C L55C

Immuogenicity of Periplasmic Compared to Mammalian Produced FimH Constructs. In the second study the immunogenicity of FimHLD and full-length FimH proteins expressed in E. coliwere compared with analogous variants expressed in Expi293 mammalian cells. E. coi-expressed full-length FimH complexed with its periplasmic chaperone FimC was used as a benchmark. Neutralization data comparing the relative level of functional antibody induction by E. colivs mammalian expressed antigens with or without the lock-mutation after three vaccinations are shown in FIG. 37A and FIG. 37B and Table 35. Antigens were dosed at 10 pg each with 20 pg QS21/PS80 according to the schedule shown in FIG. 37B. The E. coli expressed FimHLD group of mice yielded a neutralization assay GMT of 300 and 55% responder rate, which was not statistically different from the analogous mammalian expressed FimHLD group (with GMT of 194 and 45 % responder rate). The mammalian full-length FimH-DSG was significantly more immunogenic than its FimHLD counterpart, showing a two-fold higher GMT (529 vs 194) and improved seroresponder rate (75% vs 45%). As with the first study, the presence of the lock mutation offered no benefit in terms of generating a functional neutralizing response, either in the context of FimHLD (E. color mammalian expressed) or full-length FimH DSG. Mammalian expressed FimH-DSG (with or without the lock mutation) also presented as more immunogenic than the E. coli FimCH antigen, although the differences were not statistically significant. Taken together these results identified full-length FimH-DSG, as the antigen of choice for subsequent investigation. We speculate that the presence of the FimH pilin domain in this construct, in complex with the C-terminal FimG donor strand peptide, may provide additional functional epitopes and/ or enhanced stability.

Table35. VAC-2019-PRL-EC-1438 Neutralizing Titer GMTs and Responder Rates

Vaccination Group % Responders # of responders Geometric mean (ICso) titers Periplasmic- FimHLD 55 11/20 300

Periplasmic- FimHLD-LM 40 8/20 254 Mammalian- FimHLD 45 9/20 194 Mammalian- FimHLD-LM 10 2/20 73 Mammalian FimH-DSG 75 15/20 529 Mammalian FimH-DSG-LM 80 16/20 579 Periplasmic- FimCH 67 12/18 354

Immunogenicity of Coformulated Mammalian-generated FimH-DSG and 4-valent 0-antigen Glycoconjugate in Mice. The third mouse study was designed to assess the impact of combined O-antigen and adjuvant formulations on FimH-DSG immunogenicity. Long 0 antigen glycoconjugates of CRM 97representing four most prevalent serotypes 025b, 01a, 02 and 06 were mixed and administered at a dose of 2 pg each as a baseline control group (FIG. 38). Other vaccine groups evaluated the impact on immunogenicity of three adjuvants with 10 pg of FimH-DSG, either individually or in combination with the four-valent O-antigens. Adjuvants were the default 20 pg QS21/PS80 per dose formulation (used in FimH studies 1 and 2); a liposomal synthetic MPLA formulation (5 pg per dose); and a liposomal synthetic MPLA /QS21 formulation (at 5 pg each per dose). Sera were tested in functional FimH neutralization and 0 antigen specific opsonophagocytic (OPA) assays at post-dose 2 (PD2) and post-dose 3 (PD3) timepoints. Results of yeast manan FimH binding neutralization assays at PD2 and PD3 timepoints are shown in FIG. 39A and FIG. 39B with GMTs and responder rates summarized in Table 36.

Table 36. VAC-2020-PRL-EC-1679 Combination Study Yeast Mannan Binding Neutralization Assay Responder Rates and GMTs

% Responders Geomean of IC50s # of responders (out of 10) Vaccination Group PD2 PD3 PD2 PD3 PD2 PD3 4plex O-antigen combo - 0 0 50 50 0 0 No adjuvant FimH-DSG (1Opg)- QS21 100 100 3938 1457 10 10 (20pg) FimH-DSG (1Opg)- MPLA 90 90 1243 559 9 9 (5pg) FimH-DSG (1Opg)- 70 90 1075 990 7 9 MPLA/QS21 (5pg/5pg)

% Responders Geomean of IC50s # of responders (out of 10) Vaccination Group PD2 PD3 PD2 PD3 PD2 PD3 FimH-DSG (1Opg) + 4plex 40 100 169 581 4 10 O-antigen combo - No adjuvant FimH-DSG (1Opg) + 4plex 40 70 176 397 4 8 O-antigen combo - MPLA (5pg) FimH-DSG (1Opg) + 4plex 90 90 1332 1223 9 9 O-antigen combo MPLA/QS21 (5pg/5pg)

As antibody responses to individual FimH and monovalent serotype 025b antigens were sub-maximal after two doses in previous experiments, it was anticipated that the PD2 timepoint would best differentiate effects of vaccine antigen and adjuvant composition. Indeed, by PD3 responder rates and FimH neutralizing GMTs were not significantly different between any of the groups (p >0.05). At PD2, the liposomal adjuvant formulations had no significant effect on groups with FimH-DSG alone. In contrast, for the groups vaccinated with combined FimH DSG/0-antigens, significantly lower FimH neutralizing GMTs were observed with the no adjuvant or liposomal MPLA groups, compared with the group of mice vaccinated with liposomal MPLA/QS21 or any of the FimH-DSG alone groups. PD1 sera were also tested but none of the mice showed neutralizing titers. The bladder cell binding neutralization assay was used to test sera pooled from each vaccine group at PD2 and PD3 timepoints. Bladder 5637 cells constitutively express the mannosylated uroplakin receptor UPIa which is a natural ligand for FimH (Thumbikat P, Berry RE, Zhou G, et al. Bacteria-induced uroplakin signaling mediates bladder response to infection. PLoS pathogens 2009; 5:el000415-e; Ktnik-Prastowska I, Lis J, Matejuk A. Glycosylation of uroplakins. Implications for bladder physiopathology. Glycoconjugate journal 2014; 31:623-36). Results shown in FIG. 40A and FIG. 40B and Table 37 were consistent with the findings of the yeast mannan neutralization assay run with individual sera. Again, combined FimH-DSG/0 antigens formulated with liposomal MPLA or no adjuvant generated significantly weaker neutralizing responses than the liposomal MPLA/QS21 group, which responded similarly to the groups of mice vaccinated with QS21/PS80 or liposomal MPLA/QS21 and FimH-DSG without

O-antigens. A minor difference between the mannan and bladder neutralization assay data was a trend in the latter toward weaker responses with the liposomal MPLA formulation for groups vaccinated with either FimH-DSG only or combined FimH-DSG/0-antigens.

Table 37. VAC-2020-PRL-EC-1679 Bladder Cell Neutralization Titers with Mouse Serum Pools Vaccination Group PD2 IC50 PD3 IC50 4V O-Ag Inactive Inactive FimH-DSG QS21 3656 1140 FimH-DSG MPLA 775 750-1000a FimH-DSG MPLA/QS21 2458 1198 FimH-DSG + 4V O-Ag no adj 337 1177 FimH-DSG + 4V O-Ag MPLA Inactive 434 FimH-DSG + 4V O-Ag MPLA/QS21 3447 931 Rabbit serum control 3266 3809 a. IC50titer estimate due to poor fit of biphasic curve with variable slope equation.

Levels of O-antigen specific total IgGs generated with the 4-plex dLIA after vaccination of four groups of mice with the 4-valent O-antigens alone or in combination with FimH-DSG and liposomal adjuvants are shown in FIG. 41 and Table 38. Serotype 025b-specific IgG responses after two doses were markedly lower than those elicited by serotype 01a, 02 and 06 0 antigens, which is consistent with results of previous conjugate chemistry studies with monovalent O-antigens (not shown) and reported preclinical studies with tetravalent bioconjugate O-antigens (van den Dobbelsteen G, Fae KC, Serroyen J, et al. Immunogenicity and safety of a tetravalent E. coliO-antigen bioconjugate vaccine in animal models. Vaccine 2016; 34:4152-60). The lowest level of anti-O-Ag IgG across the 025b groups at PD2 was observed with the unadjuvanted FimH-DSG and O-antigen combination, which yielded only a single titer exceeding the five-fold over baseline IgG responder threshold; in comparison, the unadjuvanted O-antigen only group generated higher responder rates of 56% (5/9), although the difference in GMTs (0.79 vs 0.24 pg/ mL IgG) between the two groups was not statistically significant (p > 0.5). At PD2, the liposomal MPLA/QS21 adjuvant significantly enhanced the immunogenicity of the FimH-DSG and O-antigen combination by inducing a 16-fold increase in GMT (3.84 vs 0.24) and improved responder rate (70% vs 10%); a significant increase in GMT was also observed at PD3 (40.78 vs 3.75). A responder is defined as mouse with serum that yields a complete dilution dependent killing response that meets variable slope curve fitting criteria. Despite higher responder rates and titers across the groups than for serotype 025b, serotype 01a IgG titers for the FimH-DSG and O-antigen combination were also significantly improved at PD2 and PD3 by liposomal MPLA/QS21 by 5.9 and 4.6-fold, respectively. The impact of the MPLA/QS21 on 02 and 06 O-antigens was less pronounced than for 01a and 025b due to smaller differences overall in GMTs between the vaccination groups.

Table 38. VAC-2020-PRL-EC-1679: Influence of Adjuvant and FimH-DSG on 0-antigen Specific Serum IgG GMTs Vaccination Group O1a 02 06 025b PD2 PD3 PD2 PD3 PD2 PD3 PD2 PD3 4v O-Ag 13.86 56.72 21.96 145.99 9.66 74.25 0.79 13.73 FimH-DSG + 4v O-Ag 13.42 39.05 20.56 86.81 19.19 101.48 0.24 3.75 no adjuvant FimH-DSG + 4v O-Ag in 54.48 61.59 89.69 138.18 13.86 35.15 0.68 19.05 liposomal MPLA FimH-DSG + 4v O-Ag 79.59 181.73 52.82 230.35 39.64 94.34 3.84 40.78 in liposomal MPLA/QS21

Functional bactericidal antibodies induced by individual O-antigens of the 4-valent composition were assessed in OPA assays with invasive clinical strains. Selection of OPA strains was based on a combination of factors: expression of both O-antigen and K-capsule under in vitro growth conditions and/or multidrug-resistance; and the ability to identify compatible baby rabbit serum complement capable of facilitating bactericidal killing in the presence of neutrophil-like HL60 cells and O-antigen specific antibodies. While E. coli O-antigens are considered to be the primary determinant for preventing non-specific killing by serum complement, K1, K2 and K5-capsular antigens have also been shown to play a role in resisting phagocytosis in whole blood (Sarkar S, Ulett GC, Totsika M, et al. Role of Capsule and 0 Antigen in the Virulence of Uropathogenic Escherichia coli. PLoS ONE 2014; 9:e94786; Burns SM, Hull SI. Loss of resistance to ingestion and phagocytic killing by 0(-) and K(-) mutants of a uropathogenic Escherichia coli 075:K5 strain. Infection and immunity 1999; 67:3757-62; Buckles EL, Wang X, Lane MC, et al. Role of the K2 Capsule in Escherichia coli Urinary Tract Infection and Serum Resistance. The Journal of infectious diseases 2009; 199:10.1086/598524). Results of OPA assays with four encapsulated 01a, 02, 06 and 025b strains and two multidrug-resistant unencapsulated 06 and 025b strains are shown in FIG. 42 and Table 39.

With the exception of the 06:K- strain assay which exhibited a relatively high baseline titer of 174 for pre-vaccination sera, sera from unvaccinated mice for all other strains generated titers that were at the limit of assay detection (LOD) or 100, the starting serum dilution. Individual OPA responses to the four-valent O-antigens largely parallel the IgG binding antibody titers (FIG. 41, Table 38). For example, the significant positive impact of the liposomal MPLA/QS21 formulation on the immunogenicity of the FimH-DSG 4-valent O-antigen combination was confirmed in OPAs with encapsulated serotype Ola, 02, and 06 strains, which reported responder rates of 70% or greater after two vaccine doses. In contrast, only after a third dose did the 025b:K5 OPA strain report titers reaching this level of functional activity with the MPLA/QS21 formulation. Compared with their encapsulated 06:K1 and 025b:K5 counterparts, unencapsulated 06 and 025b strains generated higher OPA titers and responder rates, especially among non-adjuvanted groups of mice vaccinated with O-antigens alone or the combined FimH-DSG/ 4-valent O-antigens. This greater sensitivity is consistent with the absence of respective K2 or K5 capsules shown previously to confer resistance to phagocytosis (Burns SM, Hull SI. Loss of resistance to ingestion and phagocytic killing by 0(-) and K(-) mutants of a uropathogenic Escherichia coli 075:K5 strain. Infection and immunity 1999; 67:3757-62; Buckles EL, Wang X, Lane MC, et al. Role of the K2 Capsule in Escherichia coli Urinary Tract Infection and Serum Resistance. The Journal of infectious diseases 2009; 199:10.1086/598524).

Table 39. VAC-2020-PRL-EC-1679: Influence of Adjuvant and FimH-DSG on 0 antigen Specific Serum OPA GMTs Vaccination O1a:K1 02:K1 06:K2 06:K- 025b:K5 025b:K Group OPA OPA PD2 PD3 PD2 PD3 PD2 PD3 PD2 PD3 PD2 PD3 PD2 PD3

4v O-Ag 119 348 191 1108 50 155 383 1931 68 461 1516 14260 FimH-DSG + 157 575 161 385 98 1256 760 5375 50 443 223 9000 4v O-Ag no adjuvant FimH-DSG + 675 979 499 1525 510 388 714 1104 91 457 1382 28204 4v O-Ag in liposomal MPLA FimH-DSG + 6978 7714 984 8610 1064 737 1681 1961 110 606 2593 19462 4v O-Ag in liposomal MPLA/QS21

Conclusion Taken together, the results of these studies support the use of mammalian-produced FimH-DSG (or derived mutant variants) in combination with O-antigen glycoconjugates and QS21 adjuvant as a vaccine composition to prevent invasive E. coli infections. Such a formulation may be advantageous for preventing recurrent UTI infections and sepsis in elderly patients. Results confirm the benefit of QS21 adjuvant for enhancing the functional immune response to FimH-DSG and the long 025b O-antigen, which are individually less immunogenic than analogous serotype 01a, 02, and 06 O-antigen glycoconjugates in mice.

EXAMPLE 36: Antibodies Elicited by CRM197 Conjugates of E.coli serotype 08 and 09 O-antigens show cross-protective bactericidal activity against K.pneumoniae serotype 05 and 03 invasive isolates. This Example demonstrates that antibodies elicited by short single-end native CRM1 97 polymannan conjugates of E. coli serotype 08 and 09 O-antigens are bactericidal and cross protective against Klebsiella 05 and 03 strains that express equivalent or related O-antigens.

E. coli and K. pneumoniae share common polymannan O-antigens which are synthesized by enzymes encoded by highly homologous biosynthetic gene clusters. The E. coli 08 and 09 O-antigen polysaccharides are linear mannose homopolymers whose repeat units differ in their monosaccharide linkages and in the number of residues. Their counterparts in K. pneumoniae are the serotype 05 and 03 O-antigens. Biosynthesis of the E. coli and K. pneumoniae polymannan O-antigens involves a different mechanism of O-unit translocation and chain synthesis than other E. coliO-antigens. In this case chain elongation is regulated by the biosynthetic WbdA-WbdD complex (King JD, Berry S, et al. Proceedings of the National Academy of Sciences 2014; 111:6407-12), which is distinct from the Wzx/Wzy-dependent pathway where chain length is controlled by WzzB or FepE enzymes. As a consequence, the native polymannan O-antigens can only be produced in their short form, which requires different bioprocesses methods for purification and carrier protein conjugation than the engineered long E. coliO-antigens. The same mechanism and limitation applies to the predominant K. pneumoniae serotype 01 and 02 O-antigens, which are polygalactans comprised of galactose residues.

The structural relationship between these O-antigens and their subtypes is shown in FIG. 33. The E. coli 08 and K. pneumoniae 05 O-antigens are identical (Vinogradov E, et al. J

Biol Chem 2002; 277:25070-81). E. coli 09 and K. pneumoniae 03 O-antigens share common tetrameric 09a/03a and pentameric 09/03 repeat unit subtypes, while the trimeric 03b subtype is only found in K. pneumoniae. These subtypes can be identified serologically and genotypically (Guachalla LM, et al. Scientific Reports 2017; 7:6635). Serotype 03a loci are distinguished by a single point mutation in wbdA (C80R). An analogous point mutation in the E. coli09 wbdA enzyme (C55R) converts the 09 polysaccharide into 09a (Kido N, Kobayashi H. Journal of bacteriology 2000; 182:2567-73). The 03b subtype has sufficient nucleotide divergence in the sequence of the WbdD enzyme to necessitate a separate reference sequence. The Kaptive web algorithm (Wick RR, et al. J Clin Microbiol 2018; 56), implemented into Pfizer's BigSdb whole genome sequencing (WGS) pipeline, designates 03 loci as either 03/03a (covered by the same reference sequence) or 03b.

Materials and Methods

a. Production of E. coliserotype 08 and 09 CRM19 7 immune sera in rabbits

Two groups of four female New Zealand White rabbits each were used for the study run at Covance. Animals received 1Opg/animal of serotype 08 or 09 CRM 197 conjugate per dose with CFA/IFA as adjuvant. Native 08 and 09 O-antigens were conjugated using single-end chemistry. Each 1 mL dose of 1Opg of antigen was split across two subcutaneous vaccination sites. Vaccinations were given at weeks 0, 6 and 14, with blood draws at weeks 7 and 15, which correspond to post-dose two (PD2) and post-dose three (PD3) timepoints.

b. Bacterial strains

E. coli and K. pneumoniae clinical isolates were obtained from the Pfizer-sponsored Antimicrobial Testing Leadership and Surveillance (ATLAS) collection which is maintained by the International Health Management Associates (IHMA) clinical lab. Strains were genotypically characterized by whole genome sequencing (WGS) using the Miseq platform (Illumina). WGS data was used to generate multi-locus-sequence type (MLST) information using established E. coli and K. pneumoniae schemes integrated into the BigDdb platform (Wirth T, et al. Molecular microbiology 2006; 60:1136-51; Jolley KA, et al. Wellcome Open Res 2018; 3:124; Diancourt L, et al. Journal of clinical microbiology 2005; 43:4178-82). Embedded in silico serotyping algorithms for E. coliand K. pneumoniae were used to predict O-antigen serotype (Wick RR, et al. J Clin Microbiol 2018; 56; Joensen KG, et al. J Clin Microbiol 2015; 53:2410-26).

Table 40. Clinical isolates used for0-antigen production or development of bactericidal assays

ID Species MLST Serotype Source ST (subtype) EC0130 E. coli 162 08 Blood EC0423 E. coli 46 09a Blood EC0305 E. coli 448 08 Blood KPO121 K. 279 05 Blood pneumoniae EC0611 E. coli New 09a UTI, Kidney KPOO09 K. 37 03b UTI, pneumoniae Bladder

c. E. coli08 and 09 CRM 197 conjugates

Serotype 08 and 09a O-antigen polysaccharides were extracted and purified from strains EC0130 and EC0423, respectively (Table 40). The conjugation process involves selective activation of the Kdo monosaccharide present on the reducing end of the short native E. coli08 and 09 O-antigens with a disulfide amine linker. Upon unmasking of thiol functional group, it is then conjugated to bromo activated CRM 197 protein as described Example 26 set forth herein.

d. Bactericidal assays

Pre-frozen E. coli and K. pneumoniae stocks were prepared by growing strains in DMEM or LB media to an OD6oo of between 0.5 and 1.0 and glycerol was added to a final concentration of 20% prior to freezing. Specific assay conditions varied according to conditions optimized for each bacterial strain. Pre-titered thawed bacteria were diluted to 1 X 105 CFU/ml in OPA buffer (Hanks Balanced Salt Solution (Life Technologies) and 0.1% gelatin) and 20 pL (103 CFU) of the bacterial suspension was opsonized with 20 pL of serially diluted sera for 30 min at RT in a tissue culture microplate. Subsequently, 10 pl of complement (Baby Rabbit Serum or IgG/IgM depleted human serum, Pel-Freez) and 20 pL of HL-60 cells (at 100-200:1 ratio) were added to each well with OPA buffer to a final volume of 100 pL. The reaction mixture was shaken for 60 min at 37°C in a 5% C02 incubator. In some cases, bacteria were directly combined with complement and HL60s without the pre-opsonization step and shaken for 60 min at 37°C under 5% C02. After the incubation, 10 pL of each reaction was transferred into the corresponding wells of a prewetted Millipore MultiScreen HTS HV filter plate containing 100 pL water. After vacuum filtering the liquid, 100 pL of 50% bacterial growth media was applied and filtered, and the plate incubated overnight at 37°C in a sealed zip-lock bag. The next day microcolonies were enumerated after staining with Coomassie dye using an ImmunoSpot@ analyzer and ImmunoCapture software. In the case of the E. coli serotype 09 assay, the OPA was miniaturized for 50 pL reaction reactions volumes in the 384-well format. To establish the specificity of OPA activity, immune sera were preincubated with purified O-antigen polysaccharide prior to the opsonization step. The OPA assay included control reactions without HL60 cells or complement, to demonstrate dependence of any observed killing on these components. For the Klebsiella serotype 05 assay where presence of HL60s had no impact, serum bactericidal reactions were run in the absence of effector cells.

Results

a. E. coliand K. pneumoniae strain selection for bactericidal assays

Bacterial clinical strains were initially selected after confirming O-antigen expression by LPS profiling (by SDS-PAGE), and O-antigen surface accessibility by flow cytometry with 0 antigen specific rabbit antisera. Next, empiric screening of serum complement was done to identify individual compatible lots across a range of concentrations that provided a suitable balance of low levels of non-specific killing combined with a high degree of susceptibility in the presence of immune sera. Additional assay optimization parameters included adjusting the ratio of HL60 effector cell to bacteria, shaker speed, presence/ absence of plate sealer and inclusion of an opsonization preincubation step.

b. E. coli08 and K. pneumoniae 05 O-antigen immune serum cross protection and specificity

E. coli08 strain EC0305 and K. pneumoniae 05 strain KPO121 were selected for assay development. Both are blood isolates. EC0305 is resistant to cephalosporins and tetracycline while KPO121 is resistant to ampicillin. An OPA assay was developed for the E. coli08 strain EC0305 with conditions including 3.0% BRC, a 1:100 bacteria to HL60 ratio, and a single step 60 min OPA incubation reaction. As the bactericidal activity of the K. pneumoniae 05 strain KP121 in the presence of immune sera was found to be independent of HL60 effector cells, an SBA was developed. In this case, the SBA reaction required the use of 10% depleted human serum as source of complement. Results of bactericidal assays with these E. coli08 and K. pneumoniae 05 strains are shown in FIG. 34A-34B. In the E. coli serotype 08 OPA, rabbit immune serum generated after two doses of 08-CRM1 9 7 conjugate showed potent O-antigen specific killing that was blocked by preadsorption of the immune serum with free 08 O-antigen polysaccharide. Complete killing was observed at serum dilutions of less than 1:1000. Matched pre-immune sera from the same rabbit was inactive. The same rabbit sera was evaluated in the

K. pneumoniae 05 SBA, and found to be similarly bactericidal at a 1:2000 serum dilution. Killing was blocked by free 08 O-antigen and absent with the pre-immune serum. In this case a serum matrix prozone masked SBA activity at serum dilutions of less than 1:1000.

c. E. col09 and K. pneumoniae 03 OPA O-antigen immune serum cross protection and specificity

E. coli09a strain EC0611 and K. pneumoniae 03b strain KPOO09 were selected for assay development. EC0611 is resistant to ampicillin, while KPOO09 is resistant to cephalosporins, fluoroquinolones, and tetracycline. Both are UTI isolates from kidney and bladder infections, respectively. The 09a O-antigen used to generate the CRM 197 conjugate and resulting immune serum has the tetrametic polymannan repeat unit structure and is identical to the 09a EC0611 assay strain O-antigen; however, it is structurally heterologous to the K. pneumoniae 03b O-antigen KPOO09 assay strain, which is predicted to express the shorter trimeric repeat unit based on the sequence of its wbdD gene (See FIG. 33). Results of OPAs with the E. coli09a and K. pneumoniae 03b strains show that anti-E. coli 09a immune serum is potent against both (FIG. 35A-35B). Complete killing in the OPAs was observed at serum dilutions of less than 1:8,000 for the E. coli09a strain and at less than 1:1,600 for the Klebsiella 03b strain. Specificity was demonstrated by the lack of activity upon preadsorption of serum with free 09a O-antigen and with the matched-pre-immune serum.

Conclusion

E. coli serotype 08 and 09 polymannan CRM 197 conjugates elicit functional antibodies that are capable of killing not only homologous E. coli clinical strains but also Klebsiella serotype 05 and 03 strains in bactericidal assays. Results confirm that these conjugates elicit antibodies that are cross-protective against isolates of both species expressing structurally related polymannan O-antigens.

The following clauses describe additional embodiments of the invention:

C1.A composition comprising a polypeptide derived from FimH or a fragment thereof; and a saccharide comprising a structure selected from any one of Formula 01 (e.g., Formula 01A, Formula 01B, and Formula 01C), Formula 02, Formula 03, Formula 04 (e.g., Formula 04:K52 and Formula 04:K6), Formula 05 (e.g., Formula 05ab and Formula 05ac (strain 180/C3)), Formula 06 (e.g., Formula 06:K2; K13; K15 and Formula 06:K54), Formula 07, Formula 08, Formula 09, Formula 010, Formula 011, Formula 012, Formula 013, Formula 014, Formula 015, Formula 016, Formula 017, Formula 018 (e.g., Formula

018A, Formula 018ac, Formula 018A1, Formula 018B, and Formula 018B1), Formula 019, Formula 020, Formula 021, Formula 022, Formula 023 (e.g., Formula 023A), Formula 024, Formula 025 (e.g., Formula 025a and Formula 025b), Formula 026, Formula 027, Formula 028, Formula 029, Formula 030, Formula 032, Formula 033, Formula 034, Formula 035, Formula 036, Formula 037, Formula 038, Formula 039, Formula 040, Formula 041, Formula 042, Formula 043, Formula 044, Formula 045 (e.g., Formula 045 and Formula 045rel), Formula 046, Formula 048, Formula 049, Formula 050, Formula 051, Formula 052, Formula 053, Formula 054, Formula 055, Formula 056, Formula 057, Formula 058, Formula 059, Formula 060, Formula 061, Formula 062, 62 Formula D1, Formula 063, Formula 064, Formula 065, Formula 066, Formula 068, Formula 069, Formula 070, Formula 071, Formula 073 (e.g., Formula 073 (strain 73-1)), Formula 074, Formula 075, Formula 076, Formula 077, Formula 078, Formula 079, Formula 080, Formula 081, Formula 082, Formula 083, Formula 084, Formula 085, Formula 086, Formula 087, Formula 088, Formula 089, Formula 090, Formula 091, Formula 092, Formula 093, Formula 095, Formula 096, Formula 097, Formula 098, Formula 099, Formula 0100, Formula 0101, Formula 0102, Formula 0103, Formula 0104, Formula 0105, Formula 0106, Formula 0107, Formula 0108, Formula 0109, Formula 0110, Formula 0111, Formula 0112, Formula 0113, Formula 0114, Formula 0115, Formula 0116, Formula 0117, Formula 0118, Formula 0119, Formula 0120, Formula 0121, Formula 0123, Formula 0124, Formula 0125, Formula 0126, Formula 0127, Formula 0128, Formula 0129, Formula 0130, Formula 0131, Formula 0132, Formula 0133, Formula 0134, Formula 0135, Formula 0136, Formula 0137, Formula 0138, Formula 0139, Formula 0140, Formula 0141, Formula 0142, Formula 0143, Formula 0144, Formula 0145, Formula 0146, Formula 0147, Formula 0148, Formula 0149, Formula 0150, Formula 0151, Formula 0152, Formula 0153, Formula 0154, Formula 0155, Formula 0156, Formula 0157, Formula 0158, Formula 0159, Formula 0160, Formula 0161, Formula 0162, Formula 0163, Formula 0164, Formula 0165, Formula 0166, Formula 0167, Formula 0168, Formula 0169, Formula 0170, Formula 0171, Formula 0172, Formula 0173, Formula 0174, Formula 0175, Formula 0176, Formula 0177, Formula 0178, Formula 0179, Formula 0180, Formula 0181, Formula 0182, Formula 0183, Formula 0184, Formula 0185, Formula 0186, and Formula 0187, wherein n is an integer from 1 to 100. C2.The composition according to clause C1, wherein the saccharide comprises a structure selected from Formula 01 (e.g., Formula 01A, Formula 01B, and Formula 01C), Formula 02, Formula 03, Formula 04 (e.g., Formula 04:K52 and Formula 04:K6), Formula 05 (e.g., Formula 05ab and Formula 05ac (strain 180/C3)), Formula 06 (e.g., Formula 06:K2;

K13; K15 and Formula 06:K54), Formula 07, Formula 010, Formula 016, Formula 017, Formula 018 (e.g., Formula 018A, Formula 018ac, Formula 018A1, Formula 018B, and Formula 018B1), Formula 021, Formula 023 (e.g., Formula 023A), Formula 024, Formula 025 (e.g., Formula 025a and Formula 025b), Formula 026, Formula 028, Formula 044, Formula 045 (e.g., Formula 045 and Formula 045rel), Formula 055, Formula 056, Formula 058, Formula 064, Formula 069, Formula 073 (e.g., Formula 073 (strain 73-1)), Formula 075, Formula 077, Formula 078, Formula 086, Formula 088, Formula 090, Formula 098, Formula 0104, Formula 0111, Formula 0113, Formula 0114, Formula 0119, Formula 0121, Formula 0124, Formula 0125, Formula 0126, Formula 0127, Formula 0128, Formula 0136, Formula 0138, Formula 0141, Formula 0142, Formula 0143, Formula 0147, Formula 0149, Formula 0152, Formula 0157, Formula 0158, Formula 0159, Formula 0164, Formula 0173, Formula 62D1, Formula 022, Formula 035, Formula 065, Formula 066, Formula 083, Formula 091, Formula 0105, Formula 0116, Formula 0117, Formula 0139, Formula 0153, Formula 0167, and Formula 0172, wherein n is an integer from 20 to 100. C3.The composition according to clause C2, wherein the saccharide comprises a structure selected from Formula 01 (e.g., Formula 01A, Formula 01B, and Formula 01C), Formula 02, Formula 03, Formula 04 (e.g., Formula 04:K52 and Formula 04:K6), Formula 05 (e.g., Formula 05ab and Formula 05ac (strain 180/C3)), Formula 06 (e.g., Formula 06:K2; K13; K15 and Formula 06:K54), Formula 07, Formula 010, Formula 016, Formula 017, Formula 018 (e.g., Formula 018A, Formula 018ac, Formula 018A1, Formula 018B, and Formula 018B1), Formula 021, Formula 023 (e.g., Formula 023A), Formula 024, Formula 025 (e.g., Formula 025a and Formula 025b), Formula 026, Formula 028, Formula 044, Formula 045 (e.g., Formula 045 and Formula 045rel), Formula 055, Formula 056, Formula 058, Formula 064, Formula 069, Formula 073 (e.g., Formula 073 (strain 73-1)), Formula 075, Formula 077, Formula 078, Formula 086, Formula 088, Formula 090, Formula 098, Formula 0104, Formula 0111, Formula 0113, Formula 0114, Formula 0119, Formula 0121, Formula 0124, Formula 0125, Formula 0126, Formula 0127, Formula 0128, Formula 0136, Formula 0138, Formula 0141, Formula 0142, Formula 0143, Formula 0147, Formula 0149, Formula 0152, Formula 0157, Formula 0158, Formula 0159, Formula 0164, Formula 0173, and Formula 62D1, wherein n is an integer from 20 to 100. C4.The composition according to clause C2, comprising a structure selected from Formula 01 (e.g., Formula 01A, Formula 01B, and Formula 01C), Formula 02, Formula 06 (e.g., Formula 06:K2; K13; K15 and Formula 06:K54), Formula 015, Formula 016, Formula 021, Formula 025 (e.g., Formula 025a and Formula 025b), and Formula 075.

C5.The composition according to clause C2, comprising a structure selected from Formula 04, Formula 011, Formula 021, and Formula 075. C6.The composition according to clause C1, wherein the saccharide does not comprise a structure selected from Formula 08, Formula 09a, Formula 09, Formula 020ab, Formula 020ac, Formula 052, Formula 097, and Formula 0101. C7.The composition according to clause C1, wherein the saccharide does not comprise a structure selected from Formula 012. C8.The composition according to clause C4, wherein the saccharide is produced by expressing a wzz family protein in a Gram-negative bacterium to generate said saccharide. C9.The composition according to clause C8, wherein the wzz family protein is selected from the group consisting of wzzB,wzz,wzzSF,wZZST, fepE, wzzfepE, wzz1 and wzz2. C10. The composition according to clause C8, wherein the wzz family protein is wzzB. C11. The composition according to clause C8, wherein the wzz family protein is fepE. C12. The composition according to clause C8, wherein the wzz family protein is wzzB and fepE. C13. The composition according to clause C8, wherein the wzz family protein is derived from Salmonella enterica. C14. The composition according to clause C8, wherein the wzz family protein comprises a sequence selected from any one of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39. C15. The composition according to clause C8, wherein the wzz family protein comprises a sequence having at least 90% sequence identity to any one of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34. C16. The composition according to clause C8, wherein the wzz family protein comprises a sequence selected from any one of SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39. C17. The composition according to clause C1, wherein the saccharide is synthetically synthesized. C18. The composition according to any one of clauses C1 to C17, wherein the saccharide further comprises an E. coli R1 moiety. C19. The composition according to any one of clauses C1 to C17, wherein the saccharide further comprises an E. coli R2 moiety. C20. The composition according to any one of clauses C1 to C17, wherein the saccharide further comprises an E. coli R3 moiety.

C21. The composition according to any one of clauses C1 to C17, wherein the saccharide further comprises an E. coli R4 moiety. C22. The composition according to any one of clauses C1 to C17, wherein the saccharide further comprises an E. coliK-12 moiety. C23. The composition according to any one of clauses C1 to C22, wherein the saccharide further comprises a 3-deoxy-d-manno-oct-2-ulosonic acid (KDO) moiety. C24. The composition according to any one of clauses C1 to C17, wherein the saccharide does not further comprise an E. coli R1 moiety. C25. The composition according to any one of clauses C1 to C17, wherein the saccharide does not further comprise an E. coli R2 moiety. C26. The composition according to any one of clauses C1 to C17, wherein the saccharide does not further comprise an E. coli R3 moiety. C27. The composition according to any one of clauses C1 to C17, wherein the saccharide does not further comprise an E. coli R4 moiety. C28. The composition according to any one of clauses C1 to C17, wherein the saccharide does not further comprise an E. coliK-12 moiety. C29. The composition according to any one of clauses C1 to C22, wherein the saccharide does not further comprise a 3-deoxy-d-manno-oct-2-ulosonic acid (KDO) moiety. C30. The composition according to any one of clauses C1 to C23, wherein the saccharide does not comprise a Lipid A. C31. The composition according to any one of clauses C1 to C30, wherein the polysaccharide has a molecular weight of between 10 kDa and 2,000 kDa, or between 50 kDa and 2,000 kDa. C32. The composition according to any one of clauses C1 to C31, wherein the saccharide has an average molecular weight of 20-40 kDa. C33. The composition according to any one of clauses C1 to C32, wherein the saccharide has an average molecular weight of 40,000 to 60,000 kDa. C34. The composition according to any one of clauses C1 to C33, wherein n is an integer 31 to 90. C35. A composition comprising a polypeptide derived from FimH or fragment thereof; and a conjugate comprising a saccharide covalently bound a carrier protein, wherein the saccharide is derived from E. coli. C36. A composition comprising a polypeptide derived from FimH or fragment thereof; and a conjugate comprising a saccharide according to any one of clause C1 to clause C34, covalently bound a carrier protein.

C37. A composition comprising a polypeptide derived from FimH or fragment thereof; and a conjugate according to any one of clause C35 to clause C36, wherein the carrier protein is selected from any one of poly(L-lysine), CRM 197, diphtheria toxin fragment B (DTFB), DTFB C8, Diphtheria toxoid (DT), tetanus toxoid (TT), fragment C of TT, pertussis toxoid, cholera toxoid, or exotoxin A from Pseudomonas aeruginosa; detoxified Exotoxin A of P. aeruginosa (EPA), maltose binding protein (MBP), detoxified hemolysin A of S. aureus, clumping factor A, clumping factor B, Cholera toxin B subunit (CTB), Streptococcus pneumoniae Pneumolysin and detoxified variants thereof, C. jejuni AcrA, C. jejuni natural glycoproteins and Streptococcal C5a peptidase (SCP). C38. The composition according to any one of clause C35 to clause C37, wherein the carrier protein is CRM 197 .

C39. The composition according to any one of clause C35 to clause C37, wherein the carrier protein is tetanus toxoid (TT). C40. The composition according to any one of clause C35 to clause C37, wherein the carrier protein is poly(L-lysine). C41. The composition according to any one of clause C35 to clause C39, wherein the conjugate is prepared by reductive amination. C42. The composition according to any one of clause C35 to clause C39, wherein the conjugate is prepared by CDAP chemistry. C43. The composition according to any one of clause C35 to clause C39, wherein the conjugate is a single-end linked conjugated saccharide. C44. The composition according to any one of clause C35 to clause C39, wherein the saccharide is conjugated to the carrier protein through a (2-((2 oxoethyl)thio)ethyl)carbamate (eTEC) spacer. C45. The composition according to clause C44, wherein the saccharide is conjugated to the carrier protein through a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer, wherein the saccharide is covalently linked to the eTEC spacer through a carbamate linkage, and wherein the carrier protein is covalently linked to the eTEC spacer through an amide linkage. C46. The composition according to any one of clause C44 to clause C45, wherein the CRM 197 comprises 2 to 20, or 4 to 16, lysine residues covalently linked to the polysaccharide through an eTEC spacer. C47. The composition according to any one of clause C35 to clause C46, wherein the saccharide:carrier protein ratio (w/w) is between 0.2 and 4. C48. The composition according to any one of clause C35 to clause C46, wherein the ratio of saccharide to protein is at least 0.5 and at most 2.

C49. The composition according to any one of clause C35 to clause C46, wherein the ratio of saccharide to protein is between 0.4 and 1.7 C50. The composition according to any one of clause C43 to clause C49, wherein the saccharide is conjugated to the carrier protein through a 3-deoxy-d-manno-oct-2-ulosonic acid (KDO) residue. C51. A composition comprising a polypeptide derived from FimH or fragment thereof; and a conjugate comprising a saccharide covalently bound a carrier protein, wherein the saccharide comprises a structure selected from Formula 08, Formula 09a, Formula 09, Formula 020ab, Formula 020ac, Formula 052, Formula 097, and Formula 0101, wherein n is an integer from 1 to 10. C52. A composition comprising a polypeptide derived from FimH or fragment thereof; and a saccharide according to any one of clause C1 to clause C34, and a pharmaceutically acceptable diluent. C53. A composition comprising a polypeptide derived from FimH or fragment thereof; and a conjugate according to any one of clause C35 to clause C51, and a pharmaceutically acceptable diluent. C54. The composition according to clause C53, comprising at most about 25% free saccharide as compared to the total amount of saccharide in the composition. C55. The composition according to any one of clause C52 to clause C53, further comprising an adjuvant. C56. The composition according to any one of clause C52 to clause C53,further comprising aluminum. C57. The composition according to any one of clause C52 to clause C53, further comprising QS-21. C58. The composition according to any one of clause C52 to clause C53, further comprising a CpG oligonucleotide. C59. The composition according to any one of clause C52 to clause C53, wherein the composition does not include an adjuvant. C60. A composition comprising a polypeptide derived from FimH or fragment thereof; and a saccharide derived from E. coli, conjugated to a carrier protein through a (2-((2 oxoethyl)thio)ethyl)carbamate (eTEC) spacer, wherein the polysaccharide is covalently linked to the eTEC spacer through a carbamate linkage, and wherein the carrier protein is covalently linked to the eTEC spacer through an amide linkage. C61. The composition according to clause C60, wherein the saccharide is an 0-antigen derived from E. coli.

C62. The composition according to clause C60, further comprising a pharmaceutically acceptable excipient, carrier or diluent. C63. The composition according to clause C60, wherein the saccharide is an 0-antigen derived from E. coli. C64. A composition comprising a polypeptide derived from FimH or fragment thereof; and a saccharide according to any one of clause C1 to clause C17, conjugated to a carrier protein through a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer, wherein the polysaccharide is covalently linked to the eTEC spacer through a carbamate linkage, and wherein the carrier protein is covalently linked to the eTEC spacer through an amide linkage. C65. A composition comprising a polypeptide derived from FimH or fragment thereof; and (i) a conjugate of an E. coli025B antigen covalently coupled to a carrier protein, (ii) a conjugate of an E. coliO1A antigen covalently coupled to a carrier protein, (iii) a conjugate of an E. coli02 antigen covalently coupled to a carrier protein, and (iv) a conjugate of an 06 antigen covalently coupled to a carrier protein, wherein the E. coli025B antigen comprises the structure of Formula 025B, wherein n is an integer greater than 30. C66. The composition of clause C65, wherein the carrier protein is selected from any one of poly(L-lysine), CRM 197, diphtheria toxin fragment B (DTFB), DTFB C8, Diphtheria toxoid (DT), tetanus toxoid (TT), fragment C of TT, pertussis toxoid, cholera toxoid, or exotoxin A from Pseudomonas aeruginosa; detoxified Exotoxin A of P. aeruginosa (EPA), maltose binding protein (MBP), detoxified hemolysin A of S. aureus, clumping factor A, clumping factor B, Cholera toxin B subunit (CTB), Streptococcus pneumoniae Pneumolysin and detoxified variants thereof, C. jejuni AcrA, C. jejuni natural glycoproteins and Streptococcal C5a peptidase (SCP). C67. A composition comprising a polypeptide derived from FimH or fragment thereof; and (i) a conjugate of an E. coli025B antigen covalently coupled to a carrier protein, (ii) a conjugate of an E. coli04 antigen covalently coupled to a carrier protein, (iii) a conjugate of an E. coli 011 antigen covalently coupled to a carrier protein, and (iv) a conjugate of an 021 antigen covalently coupled to a carrier protein, wherein the E. coli025B antigen comprises the structure of Formula 075, wherein n is an integer greater than 30. C68. The composition of clause C67, wherein the carrier protein is selected from any one of poly(L-lysine), CRM 197, diphtheria toxin fragment B (DTFB), DTFB C8, Diphtheria toxoid (DT), tetanus toxoid (TT), fragment C of TT, pertussis toxoid, cholera toxoid, or exotoxin A from Pseudomonas aeruginosa; detoxified Exotoxin A of P. aeruginosa (EPA), maltose binding protein (MBP), detoxified hemolysin A of S. aureus, clumping factor A, clumping factor B, Cholera toxin B subunit (CTB), Streptococcus pneumoniae Pneumolysin and detoxified variants thereof, C. jejuni AcrA, C. jejuni natural glycoproteins and Streptococcal C5a peptidase (SCP). C69. A method of making a composition comprising a polypeptide derived from FimH or fragment thereof; and a conjugate comprising a saccharide conjugated to a carrier protein through a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer, comprising the steps of a) reacting a saccharide with 1,1'-carbonyl-di-(1,2,4-triazole) (CDT) or 1,1'-carbonyldiimidazole (CDI), in an organic solvent to produce an activated saccharide; b) reacting the activated saccharide with cystamine or cysteamine or a salt thereof, to produce a thiolated saccharide; c) reacting the thiolated saccharide with a reducing agent to produce an activated thiolated saccharide comprising one or more free sulfhydryl residues; d) reacting the activated thiolated saccharide with an activated carrier protein comprising one or more a-haloacetamide groups, to produce a thiolated saccharide-carrier protein conjugate; and e) reacting the thiolated saccharide-carrier protein conjugate with (i) a first capping reagent capable of capping unconjugated a-haloacetamide groups of the activated carrier protein; and/or (ii) a second capping reagent capable of capping unconjugated free sulfhydryl residues; whereby an eTEC linked glycoconjugate is produced, wherein the saccharide is derived from E. coli; further comprising expressing a polynucleotide encoding a polypeptide derived from FimH or fragment thereof in a recombinant mammalian cell, and isolating said polypeptide or fragment thereof. C70. The method according to clause C69, comprising making the composition according to any one of clause C1 to clause C34. C71. The method according to any of one clause C69 to clause C70, wherein the capping step e) comprises reacting the thiolated saccharide-carrier protein conjugate with (i) N acetyl-L-cysteine as a first capping reagent, and/or (ii) iodoacetamide as a second capping reagent. C72. The method according to any of one clause C69 to clause C71, further comprising a step of compounding the saccharide by reaction with triazole or imidazole to provide a compounded saccharide, wherein the compounded saccharide is shell frozen,lyophilized and reconstituted in an organic solvent prior to step a). C73. The method according to any of one clause C69 to clause C72, further comprising purification of the thiolated polysaccharide produced in step c), wherein the purification step comprises diafiltration. C74. The method according to any of one clause C69 to clause C73, wherein the method further comprises purification of the eTEC linked glycoconjugate by diafiltration. C75. The method according to any of one clause C69 to clause C74, wherein the organic solvent in step a) is a polar aprotic solvent selected from any one of dimethyl sulfoxide

(DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), N-methyl-2-pyrrolidone (NMP), acetonitrile, 1,3-Dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU) and hexamethylphosphoramide (HMPA), or a mixture thereof. C76. A medium comprising KH 2PO 4 , K 2 HPO 4 , (NH 4) 2SO 4 , sodium citrate, Na 2 SO4 , aspartic acid, glucose, MgSO 4 , FeSO 4-7H 20, Na 2MoO 4-2H 20, H 3BO 3, CoC1 2 -6H 2 0, CuC1 2-2H 20, MnCl 2-4H 20, ZnCl2 and CaC1 2-2H 20. C77. The medium according to clause C76, wherein the medium is used for culturing E. coli. C78. A method for producing a saccharide according to any one of clause C1 to clause C34, comprising culturing a recombinant E. coli in a medium; producing said saccharide by culturing said cell in said medium; whereby said cell produces said saccharide. C79. The method according to clause C78, wherein the medium comprises an element selected from any one of KH 2 PO 4 , K2 HPO 4 , (NH 4) 2 SO 4 , sodium citrate, Na 2 SO 4 , aspartic acid, glucose, MgSO 4 , FeSO 4-7H 20, Na 2MoO 4-2H 20, H 3 BO 3 , CoC1 2 -6H 2 0, CuC1 2-2H 20, MnCl2-4H20, ZnCl2 and CaC12-2H20. C80. The method according to clause C78, wherein the medium comprises soy hydrolysate. C81. The method according to clause C78, wherein the medium comprises yeast extract. C82. The method according to clause C78, wherein the medium does not further comprise soy hydrolysate and yeast extract. C83. The method according to clause C78, wherein the E. coli cell comprises a heterologous wzz family protein selected from any one of wzzB, wzz, wzzSF, wZZST, fepE, wzzfepE, wZZ1 and wzz2. C84. The method according to clause C78, wherein the E. coli cell comprises a Salmonella enterica wzz family protein selected from any one of wzzB, wzz, wzzSF,wZZST, fepE, wzzfepE, wzz1 and wzz2. C85. The method according to clause C84, wherein the wzz family protein comprises a sequence selected from any one of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19. C86. The method according to clause C78, wherein the culturing produces a yield of > 120 ODeoo/mL. C87. The method according to clause C78, further comprising purifying the saccharide. C88. The method according to clause C78, wherein the purifying step comprises any one of the following: dialysis, concentration operations, diafiltration operations, tangential flow filtration, precipitation, elution, centrifugation, precipitation, ultra-filtration, depth filtration, and column chromatography (ion exchange chromatography, multimodal ion exchange chromatography, DEAE, and hydrophobic interaction chromatography).

C89. A method for inducing an immune response in a mammal comprising administering to the subject a composition according to any one of clause C1 to clause C68. C90. The method according to clause C89, wherein the immune response comprises induction of an anti-E. coliO-specific polysaccharide serum antibody. C91. The method according to clause C89, wherein the immune response comprises induction of an anti-E. coillgG antibody. C92. The method according to clause C89, wherein the immune response comprises induction of bactericidal activity against E. coli. C93. The method according to clause C89, wherein the immune response comprises induction of opsonophagocytic antibodies against E. coli. C94. The method according to clause C89, wherein the immune response comprises a geometric mean titer (GMT) level of at least 1,000 to 200,000 after initial dosing. C95. The method according to clause C89, wherein the composition comprises a saccharide comprising the Formula 025, wherein n is an integer 40 to 100, wherein the immune response comprises a geometric mean titer (GMT) level of at least 1,000 to 200,000 after initial dosing. C96. The method according to clause C89, wherein the mammal is at risk of any one of the conditions selected from urinary tract infection, cholecystitis, cholangitis, diarrhea, hemolytic uremic syndrome, neonatal meningitis, urosepsis, intra-abdominal infection, meningitis, complicated pneumonia, wound infection, post-prostate biopsy-related infection, neonatal/infant sepsis, neutropenic fever, and other blood stream infection; pneumonia, bacteremia, and sepsis. C97. The method according to clause C89, wherein the mammal is has any one of the conditions selected from urinary tract infection, cholecystitis, cholangitis, diarrhea, hemolytic uremic syndrome, neonatal meningitis, urosepsis, intra-abdominal infection, meningitis, complicated pneumonia, wound infection, post-prostate biopsy-related infection, neonatal/infant sepsis, neutropenic fever, and other blood stream infection; pneumonia, bacteremia, and sepsis. C98. A method for (i) inducing an immune response in a subject against extra-intestinal pathogenic Escherichia coli, (ii) inducing an immune response in a subject against extra intestinal pathogenic Escherichia coli, or (iii) inducing the production of opsonophagocytic antibodies in a subject that are specific to extra-intestinal pathogenic Escherichia coli, wherein the method comprises administering to the subject an effective amount of the composition according to any one of clause C1 to clause C68. C99. The method of clause C98, wherein the subject is at risk of developing a urinary tract infection.

C100. The method of clause C98, wherein the subject is at risk of developing bacteremia. C101. The method of clause C98, wherein the subject is at risk of developing sepsis. C102. A composition comprising a polypeptide derived from FimH or fragment thereof; and a (i) a conjugate of an an E. coli025B antigen covalently coupled to a carrier protein, (ii) a conjugate of an E. coliO1A antigen covalently coupled to a carrier protein, (iii) a conjugate of an E. coli02 antigen covalently coupled to a carrier protein, and (iv) a conjugate of an 06 antigen covalently coupled to a carrier protein, wherein the E. coli025B antigen comprises the structure of Formula 025B, wherein n is an integer greater than 30. C103. The composition of clause C102, wherein the carrier protein is selected from the group consisting of poly(L-lysine), detoxified Exotoxin A of P. aeruginosa (EPA), CRM197, maltose binding protein (MBP). Diphtheria toxoid, Tetanus toxoid, detoxified hemolysin A of S. aureus, clumping factor A, clumping factor B, Cholera toxin B subunit (CTB), cholera toxin, detoxified variants of cholera toxin, Streptococcus pneumoniae Pneumolysin and detoxified variants thereof, C. jejuni AcrA, C. jejuni natural glycoproteins and Streptococcal C5a peptidase (SCP). C104. A method for (i) inducing an immune response in a subject against extra-intestinal pathogenic Escherichia coli, (ii) inducing an immune response in a subject against extra intestinal pathogenic Escherichia coli, or (iii) inducing the production of opsonophagocytic antibodies in a subject that are specific to extra-intestinal pathogenic Escherichia coli, wherein the method comprises administering to the subject an effective amount of the composition of clause C1. C105. The method of clause C104, wherein the subject is at risk of developing a urinary tract infection. C106. The method of clause C104, wherein the subject is at risk of developing bacteremia. C107. The method of clause C104, wherein the subject is at risk of developing sepsis. C108. A composition comprising a polypeptide derived from FimH or fragment thereof; and a saccharide comprising an increase of at least 5 repeating units, compared to the corresponding wild-type 0-polysaccharide of an E. coli. C109. The composition according to clause C108, wherein the saccharide comprises Formula 025a and the E. coli is an E. coli serotype 025a. C110. The composition according to clause C108, wherein the saccharide comprises Formula 025b and the E. coli is an E. coli serotype 025b. C111. The composition according to clause C108, wherein the saccharide comprises Formula 02 and the E. coli is an E. coli serotype 02. C112. The composition according to clause C108, wherein the saccharide comprises Formula 06 and the E. coli is an E. coli serotype 06.

C113. The composition according to clause C108, wherein the saccharide comprises Formula 01 and the E. coli is an E. coli serotype 01. C114. The composition according to clause C108, wherein the saccharide comprises Formula 017 and the E. coliis an E. coliserotype 017. C115. The composition according to clause C108, wherein the saccharide comprises a structure selected from: Formula 01, Formula 02, Formula 03, Formula 04, Formula 05, Formula 06, Formula 07, Formula 08, Formula 09, Formula 010, Formula 011, Formula 012, Formula 013, Formula 014, Formula 015, Formula 016, Formula 017, Formula 018, Formula 019, Formula 020, Formula 021, Formula 022, Formula 023, Formula 024, Formula 025, Formula 025b, Formula 026, Formula 027, Formula 028, Formula 029, Formula 030, Formula 032, Formula 033, Formula 034, Formula 035, Formula 036, Formula 037, Formula 038, Formula 039, Formula 040, Formula 041, Formula 042, Formula 043, Formula 044, Formula 045, Formula 046, Formula 048, Formula 049, Formula 050, Formula 051, Formula 052, Formula 053, Formula 054, Formula 055, Formula 056, Formula 057, Formula 058, Formula 059, Formula 060, Formula 061, Formula 062, Formula 063, Formula 064, Formula 065, Formula 066, Formula 068, Formula 069, Formula 070, Formula 071, Formula 073, Formula 074, Formula 075, Formula 076, Formula 077, Formula 078, Formula 079, Formula 080, Formula 081, Formula 082, Formula 083, Formula 084, Formula 085, Formula 086, Formula 087, Formula 088, Formula 089, Formula 090, Formula 091, Formula 092, Formula 093, Formula 095, Formula 096, Formula 097, Formula 098, Formula 099, Formula 0100, Formula 0101, Formula 0102, Formula 0103, Formula 0104, Formula 0105, Formula 0106, Formula 0107, Formula 0108, Formula 0109, Formula 0110, Formula 0111, Formula 0112, Formula 0113, Formula 0114, Formula 0115, Formula 0116, Formula 0117, Formula 0118, Formula 0119, Formula 0120, Formula 0121, Formula 0123, Formula 0124, Formula 0125, Formula 0126, Formula 0127, Formula 0128, Formula 0129, Formula 0130, Formula 0131, Formula 0132, Formula 0133, Formula 0134, Formula 0135, Formula 0136, Formula 0137, Formula 0138, Formula 0139, Formula 0140, Formula 0141, Formula 0142, Formula 0143, Formula 0144,0145, Formula 0146, Formula 0147, Formula 0148, Formula 0149, Formula 0150, Formula 0151, Formula 0152, Formula 0153, Formula 0154, Formula 0155, Formula 0156, Formula 0157, Formula 0158, Formula 0159, Formula 0160, Formula 0161, Formula 0162, Formula 0163, Formula 0164, Formula 0165, Formula 0166, Formula 0167, Formula 0168, Formula 0169, Formula 0170, Formula 0171, Formula 0172, Formula 0173, Formula 0174, Formula 0175, Formula 0176, Formula 0177, Formula 0178, Formula 0179,

Formula 0180, Formula 0181, Formula 0182, Formula 0183, Formula 0184, Formula 0185, Formula 0186, and Formula 0187, wherein n is an integer from 5 to 1000. C116. The composition according to clause C108, wherein the E. coli is E. coliserotype selected from the group consisting of: 01, 02, 03, 04, 05, 06, 07, 08, 09, 010, 011, 012, 013, 014, 015, 016, 017, 018, 019, 020, 021, 022, 023, 024,025, 025b, 026, 027, 028, 029, 030, 032, 033, 034, 035, 036, 037, 038, 039, 040, 041, 042, 043, 044, 045, 046, 048, 049, 050, 051, 052, 053, 054, 055, 056, 057, 058, 059, 060, 061, 062, 063, 064, 065, 066, 068, 069, 070, 071, 073, 074, 075, 076, 077, 078, 079, 080, 081, 082, 083, 084, 085, 086, 087, 088, 089, 090, 091, 092, 093, 095, 096, 097, 098, 099, 0100, 0101, 0102, 0103, 0104, 0105, 0106, 0107, 0108, 0109, 0110, 0111, 0112, 0113, 0114, 0115, 0116, 0117, 0118, 0119, 0120, 0121, 0123, 0124, 0125, 0126, 0127, 0128, 0129, 0130, 0131, 0132, 0133, 0134, 0135, 0136, 0137, 0138, 0139, 0140, 0141, 0142, 0143, 0144,0145, 0146, 0147, 0148, 0149, 0150, 0151, 0152, 0153, 0154, 0155, 0156, 0157, 0158, 0159, 0160, 0161, 0162, 0163, 0164, 0165, 0166, 0167, 0168, 0169, 0170, 0171, 0172, 0173, 0174, 0175, 0176, 0177, 0178, 0179, 0180, 0181, 0182, 0183, 0184, 0185, 0186, and 0187. C117. The composition according to clause C108, wherein the saccharide is produced by increasing repeating units of 0-polysaccharides produced by a Gram- negative bacterium in culture comprising overexpressing wzz family proteins in a Gram-negative bacterium to generate said saccharide. C118. The composition according to clause C117, wherein the overexpressed wzz family protein is selected from the group consisting of wzzB, wzz, wzzSF, wZZST, fepE, wzzfepE, wzz1 and wzz2. C119. The composition according to clause C117, wherein the overexpressed wzz family protein is wzzB. C120. The composition according to clause C117, wherein the overexpressed wzz family protein is fepE. C121. The composition according to clause C117, wherein the overexpressed wzz family protein is wzzB and fepE. C122. The composition according to clause C108, wherein the saccharide is synthetically synthesized. C123. A composition comprising a polypeptide derived from FimH or fragment thereof; and a conjugate comprising a saccharide according to clause C108, covalently bound to a carrier protein. C124. The composition according to clause C123, wherein the carrier protein is CRM197 .

C125. The composition according to clause C123, wherein the saccharide comprises a structure selected from: Formula 01, Formula 02, Formula 03, Formula 04, Formula 05, Formula 06, Formula 07, Formula 08, Formula 09, Formula 010, Formula 011, Formula 012, Formula 013, Formula 014, Formula 015, Formula 016, Formula 017, Formula 018, Formula 019, Formula 020, Formula 021, Formula 022, Formula 023, Formula 024, Formula 025, Formula 025b, Formula 026, Formula 027, Formula 028, Formula 029, Formula 030, Formula 032, Formula 033, Formula 034, Formula 035, Formula 036, Formula 037, Formula 038, Formula 039, Formula 040, Formula 041, Formula 042, Formula 043, Formula 044, Formula 045, Formula 046, Formula 048, Formula 049, Formula 050, Formula 051, Formula 052, Formula 053, Formula 054, Formula 055, Formula 056, Formula 057, Formula 058, Formula 059, Formula 060, Formula 061, Formula 062, Formula 063, Formula 064, Formula 065, Formula 066, Formula 068, Formula 069, Formula 070, Formula 071, Formula 073, Formula 074, Formula 075, Formula 076, Formula 077, Formula 078, Formula 079, Formula 080, Formula 081, Formula 082, Formula 083, Formula 084, Formula 085, Formula 086, Formula 087, Formula 088, Formula 089, Formula 090, Formula 091, Formula 092, Formula 093, Formula 095, Formula 096, Formula 097, Formula 098, Formula 099, Formula 0100, Formula 0101, Formula 0102, Formula 0103, Formula 0104, Formula 0105, Formula 0106, Formula 0107, Formula 0108, Formula 0109, Formula 0110, Formula 0111, Formula 0112, Formula 0113, Formula 0114, Formula 0115, Formula 0116, Formula 0117, Formula 0118, Formula 0119, Formula 0120, Formula 0121, Formula 0123, Formula 0124, Formula 0125, Formula 0126, Formula 0127, Formula 0128, Formula 0129, Formula 0130, Formula 0131, Formula 0132, Formula 0133, Formula 0134, Formula 0135, Formula 0136, Formula 0137, Formula 0138, Formula 0139, Formula 0140, Formula 0141, Formula 0142, Formula 0143, Formula 0144,0145, Formula 0146, Formula 0147, Formula 0148, Formula 0149, Formula 0150, Formula 0151, Formula 0152, Formula 0153, Formula 0154, Formula 0155, Formula 0156, Formula 0157, Formula 0158, Formula 0159, Formula 0160, Formula 0161, Formula 0162, Formula 0163, Formula 0164, Formula 0165, Formula 0166, Formula 0167, Formula 0168, Formula 0169, Formula 0170, Formula 0171, Formula 0172, Formula 0173, Formula 0174, Formula 0175, Formula 0176, Formula 0177, Formula 0178, Formula 0179, Formula 0180, Formula 0181, Formula 0182, Formula 0183, Formula 0184, Formula 0185, Formula 0186, and Formula 0187, wherein n is an integer from 5 to 1000. C126. The composition according to clause C123, wherein said saccharide comprises an increase of at least 5 repeating units, compared to the corresponding wild-type 0 polysaccharide.

C127. The composition according to clause C1, further comprising a pharmaceutically acceptable diluent. C128. The composition according to clause C127, further comprising an adjuvant. C129. The composition according to clause C127, further comprising aluminum. C130. The composition according to clause C127, further comprising QS-21. C131. The composition according to clause C127, wherein the composition does not include an adjuvant. C132. A method for inducing an immune response in a subject comprising administering to the subject a composition according to clause C127. C133. The composition according to clause C123, further comprising a pharmaceutically acceptable diluent. C134. A method for inducing an immune response in a subject comprising administering to the subject a composition according to clause C133. C135. The method according to clauses C132 orC134, wherein the immune response comprises induction of an anti-E. coliO-specific polysaccharide serum antibody. C136. The method according to clause C135, wherein the anti-E. coli0-specific polysaccharide serum antibody is an IgG antibody. C137. The method according to clause C135, wherein the anti-E. coli0-specific polysaccharide serum antibody is an IgG antibody has bactericidal activity against E. coli. C138. An immunogenic composition comprising a polypeptide derived from FimH or fragment thereof; and a saccharide derived from E. coli, conjugated to a carrier protein through a (2 ((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer, wherein the polysaccharide is covalently linked to the eTEC spacer through a carbamate linkage, and wherein the carrier protein is covalently linked to the eTEC spacer through an amide linkage. C139. The immunogenic composition according to clause C138, further comprising a pharmaceutically acceptable excipient, carrier or diluent. C140. The immunogenic composition according to clause C138, wherein the saccharide is an 0-antigen derived from E. coli. C141. The immunogenic composition according to clause C138, wherein the saccharide comprises a structure selected from: Formula 01, Formula 02, Formula 03, Formula 04, Formula 05, Formula 06, Formula 07, Formula 08, Formula 09, Formula 010, Formula 011, Formula 012, Formula 013, Formula 014, Formula 015, Formula 016, Formula 017, Formula 018, Formula 019, Formula 020, Formula 021, Formula 022, Formula 023, Formula 024, Formula 025, Formula 025b, Formula 026, Formula 027, Formula 028, Formula 029, Formula 030, Formula 032, Formula 033, Formula 034, Formula 035, Formula 036, Formula 037, Formula 038, Formula 039, Formula 040, Formula 041,

Formula 042, Formula 043, Formula 044, Formula 045, Formula 046, Formula 048, Formula 049, Formula 050, Formula 051, Formula 052, Formula 053, Formula 054, Formula 055, Formula 056, Formula 057, Formula 058, Formula 059, Formula 060, Formula 061, Formula 062, Formula 063, Formula 064, Formula 065, Formula 066, Formula 068, Formula 069, Formula 070, Formula 071, Formula 073, Formula 074, Formula 075, Formula 076, Formula 077, Formula 078, Formula 079, Formula 080, Formula 081, Formula 082, Formula 083, Formula 084, Formula 085, Formula 086, Formula 087, Formula 088, Formula 089, Formula 090, Formula 091, Formula 092, Formula 093, Formula 095, Formula 096, Formula 097, Formula 098, Formula 099, Formula 0100, Formula 0101, Formula 0102, Formula 0103, Formula 0104, Formula 0105, Formula 0106, Formula 0107, Formula 0108, Formula 0109, Formula 0110, Formula 0111, Formula 0112, Formula 0113, Formula 0114, Formula 0115, Formula 0116, Formula 0117, Formula 0118, Formula 0119, Formula 0120, Formula 0121, Formula 0123, Formula 0124, Formula 0125, Formula 0126, Formula 0127, Formula 0128, Formula 0129, Formula 0130, Formula 0131, Formula 0132, Formula 0133, Formula 0134, Formula 0135, Formula 0136, Formula 0137, Formula 0138, Formula 0139, Formula 0140, Formula 0141, Formula 0142, Formula 0143, Formula 0144,0145, Formula 0146, Formula 0147, Formula 0148, Formula 0149, Formula 0150, Formula 0151, Formula 0152, Formula 0153, Formula 0154, Formula 0155, Formula 0156, Formula 0157, Formula 0158, Formula 0159, Formula 0160, Formula 0161, Formula 0162, Formula 0163, Formula 0164, Formula 0165, Formula 0166, Formula 0167, Formula 0168, Formula 0169, Formula 0170, Formula 0171, Formula 0172, Formula 0173, Formula 0174, Formula 0175, Formula 0176, Formula 0177, Formula 0178, Formula 0179, Formula 0180, Formula 0181, Formula 0182, Formula 0183, Formula 0184, Formula 0185, Formula 0186, and Formula 0187, wherein n is an integer from 5 to 1000. C142. The immunogenic composition according to clause C138, wherein the saccharide has a degree of 0-acetylation between 75-100%. C143. The immunogenic composition according to clause C138, wherein the carrier protein is CRM197. C144. The immunogenic composition according to clause C143, wherein the CRM197 comprises 2 to 20 lysine residues covalently linked to the polysaccharide through an eTEC spacer. C145. The immunogenic composition according to clause C143, wherein the CRM197 comprises 4 to 16 lysine residues covalently linked to the polysaccharide through an eTEC spacer.

C146. The immunogenic composition according to clause C138, further comprising an additional antigen. C147. The immunogenic composition according to clause C138, further comprising an adjuvant. C148. The immunogenic composition according to clause C147, wherein the adjuvant is an aluminum-based adjuvant selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide. C149. The immunogenic composition according to clause C138, wherein the composition does not comprise an adjuvant. C150. An immunogenic composition comprising a polypeptide derived from FimH or fragment thereof; and a glycoconjugate comprising a saccharide derived from E. coli conjugated to a carrier protein, wherein the glycoconjugate is prepared using reductive amination. C151. The immunogenic composition according to clause C150, further comprising a pharmaceutically acceptable excipient, carrier or diluent. C152. The immunogenic composition according to clause C150, wherein the saccharide is an O-antigen derived from E. coli. C153. The immunogenic composition according to clause C150, wherein the saccharide comprises a structure selected from: Formula 01, Formula 02, Formula 03, Formula 04, Formula 05, Formula 06, Formula 07, Formula 08, Formula 09, Formula 010, Formula 011, Formula 012, Formula 013, Formula 014, Formula 015, Formula 016, Formula 017, Formula 018, Formula 019, Formula 020, Formula 021, Formula 022, Formula 023, Formula 024, Formula 025, Formula 025b, Formula 026, Formula 027, Formula 028, Formula 029, Formula 030, Formula 032, Formula 033, Formula 034, Formula 035, Formula 036, Formula 037, Formula 038, Formula 039, Formula 040, Formula 041, Formula 042, Formula 043, Formula 044, Formula 045, Formula 046, Formula 048, Formula 049, Formula 050, Formula 051, Formula 052, Formula 053, Formula 054, Formula 055, Formula 056, Formula 057, Formula 058, Formula 059, Formula 060, Formula 061, Formula 062, Formula 063, Formula 064, Formula 065, Formula 066, Formula 068, Formula 069, Formula 070, Formula 071, Formula 073, Formula 074, Formula 075, Formula 076, Formula 077, Formula 078, Formula 079, Formula 080, Formula 081, Formula 082, Formula 083, Formula 084, Formula 085, Formula 086, Formula 087, Formula 088, Formula 089, Formula 090, Formula 091, Formula 092, Formula 093, Formula 095, Formula 096, Formula 097, Formula 098, Formula 099, Formula 0100, Formula 0101, Formula 0102, Formula 0103, Formula 0104, Formula 0105, Formula 0106, Formula 0107, Formula 0108, Formula 0109, Formula 0110, Formula 0111, Formula 0112, Formula 0113, Formula 0114, Formula 0115, Formula

0116, Formula 0117, Formula 0118, Formula 0119, Formula 0120, Formula 0121, Formula 0123, Formula 0124, Formula 0125, Formula 0126, Formula 0127, Formula 0128, Formula 0129, Formula 0130, Formula 0131, Formula 0132, Formula 0133, Formula 0134, Formula 0135, Formula 0136, Formula 0137, Formula 0138, Formula 0139, Formula 0140, Formula 0141, Formula 0142, Formula 0143, Formula 0144,0145, Formula 0146, Formula 0147, Formula 0148, Formula 0149, Formula 0150, Formula 0151, Formula 0152, Formula 0153, Formula 0154, Formula 0155, Formula 0156, Formula 0157, Formula 0158, Formula 0159, Formula 0160, Formula 0161, Formula 0162, Formula 0163, Formula 0164, Formula 0165, Formula 0166, Formula 0167, Formula 0168, Formula 0169, Formula 0170, Formula 0171, Formula 0172, Formula 0173, Formula 0174, Formula 0175, Formula 0176, Formula 0177, Formula 0178, Formula 0179, Formula 0180, Formula 0181, Formula 0182, Formula 0183, Formula 0184, Formula 0185, Formula 0186, and Formula 0187, wherein n is an integer from 5 to 1000. C154. The immunogenic composition according to clause C150, wherein the saccharide has a degree of O-acetylation between 75-100%. C155. The immunogenic composition according to clause C150, wherein the carrier protein is CRM197. C156. The immunogenic composition according to clause C150, further comprising an additional antigen. C157. The immunogenic composition according to clause C150, further comprising an adjuvant. C158. The immunogenic composition according to clause C157, wherein the adjuvant is an aluminum-based adjuvant selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide. C159. The immunogenic composition according to clause C150, wherein the composition does not comprise an adjuvant. C160. A method for inducing an immune response in a subject comprising administering to the subject a composition according to any one of clauses C138-C159. C161. The method according to clause C160, wherein the immune response comprises induction of an anti-E. coliO-specific polysaccharide serum antibody. C162. The method according to clause C135, wherein the anti-E. coliO-specific polysaccharide serum antibody is an IgG antibody. C163. The method according to clause C135, wherein the anti-E. coliO-specific polysaccharide serum antibody is an IgG antibody has bactericidal activity against E. coli.

C164. A composition comprising a polypeptide derived from FimH or fragment thereof; and a saccharide comprising a structure selected from any one of Formula 01, Formula 01A, Formula 01B, Formula 01C, Formula 02, Formula 03, Formula 04, Formula 04:K52, Formula 04:K6, Formula 05, Formula 05ab, Formula 05ac, Formula 06, Formula 06:K2; K13; K15, Formula 06:K54, Formula 07, Formula 08, Formula 09, Formula 010, Formula 011, Formula 012, Formula 013, Formula 014, Formula 015, Formula 016, Formula 017, Formula 018, Formula 018A, Formula 018ac, Formula 018A1, Formula 018B, Formula 018B1, Formula 019, Formula 020, Formula 021, Formula 022, Formula 023, Formula 023A, Formula 024, Formula 025, Formula 025a, Formula 025b, Formula 026, Formula 027, Formula 028, Formula 029, Formula 030, Formula 032, Formula 033, Formula 034, Formula 035, Formula 036, Formula 037, Formula 038, Formula 039, Formula 040, Formula 041, Formula 042, Formula 043, Formula 044, Formula 045, Formula 045, Formula 045rel, Formula 046, Formula 048, Formula 049, Formula 050, Formula 051, Formula 052, Formula 053, Formula 054, Formula 055, Formula 056, Formula 057, Formula 058, Formula 059, Formula 060, Formula 061, Formula 062, Formula 62D1, Formula 063, Formula 064, Formula 065, Formula 066, Formula 068, Formula 069, Formula 070, Formula 071, Formula 073, Formula 073, Formula 074, Formula 075, Formula 076, Formula 077, Formula 078, Formula 079, Formula 080, Formula 081, Formula 082, Formula 083, Formula 084, Formula 085, Formula 086, Formula 087, Formula 088, Formula 089, Formula 090, Formula 091, Formula 092, Formula 093, Formula 095, Formula 096, Formula 097, Formula 098, Formula 099, Formula 0100, Formula 0101, Formula 0102, Formula 0103, Formula 0104, Formula 0105, Formula 0106, Formula 0107, Formula 0108, Formula 0109, Formula 0110, Formula 0111, Formula 0112, Formula 0113, Formula 0114, Formula 0115, Formula 0116, Formula 0117, Formula 0118, Formula 0119, Formula 0120, Formula 0121, Formula 0123, Formula 0124, Formula 0125, Formula 0126, Formula 0127, Formula 0128, Formula 0129, Formula 0130, Formula 0131, Formula 0132, Formula 0133, Formula 0134, Formula 0135, Formula 0136, Formula 0137, Formula 0138, Formula 0139, Formula 0140, Formula 0141, Formula 0142, Formula 0143, Formula 0144, Formula 0145, Formula 0146, Formula 0147, Formula 0148, Formula 0149, Formula 0150, Formula 0151, Formula 0152, Formula 0153, Formula 0154, Formula 0155, Formula 0156, Formula 0157, Formula 0158, Formula 0159, Formula 0160, Formula 0161, Formula 0162, Formula 0163, Formula 0164, Formula 0165, Formula 0166, Formula 0167, Formula 0168, Formula 0169, Formula 0170, Formula 0171, Formula 0172, Formula 0173, Formula 0174, Formula 0175, Formula 0176, Formula 0177, Formula 0178, Formula 0179, Formula 0180, Formula 0181, Formula 0182, Formula 0183, Formula

0184, Formula 0185, Formula 0186, Formula 0187, wherein n is greater than the number of repeat units in the corresponding wild-type E. coli polysaccharide. C165. The composition according to clause C164, wherein n is an integer from 31 to 100. C166. The composition according to clause C164, wherein the saccharide comprises a structure according to any one of Formula 01A, Formula 01B, and Formula 01C, Formula 02, Formula 06, and Formula 025B. C167. The composition according to clause C164, wherein the saccharide is produced in a recombinant host cell that expresses a wzz family protein having at least 90% sequence identity to any one of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39. C168. The composition according to clause C167, wherein the protein comprises any one of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34. C169. The saccharide according to clause C164, wherein the saccharide is synthetically synthesized. C170. A composition comprising a polypeptide derived from FimH or fragment thereof; and a conjugate comprising a carrier protein covalently bound to a saccharide, said saccharide comprising a structure selected from any one of Formula 01, Formula 01A, Formula 01B, Formula 01C, Formula 02, Formula 03, Formula 04, Formula 04:K52, Formula 04:K6, Formula 05, Formula 05ab, Formula 05ac, Formula 06, Formula 06:K2; K13; K15, Formula 06:K54, Formula 07, Formula 08, Formula 09, Formula 010, Formula 011, Formula 012, Formula 013, Formula 014, Formula 015, Formula 016, Formula 017, Formula 018, Formula 018A, Formula 018ac, Formula 018A1, Formula 018B, Formula 018B1, Formula 019, Formula 020, Formula 021, Formula 022, Formula 023, Formula 023A, Formula 024, Formula 025, Formula 025a, Formula 025b, Formula 026, Formula 027, Formula 028, Formula 029, Formula 030, Formula 032, Formula 033, Formula 034, Formula 035, Formula 036, Formula 037, Formula 038, Formula 039, Formula 040, Formula 041, Formula 042, Formula 043, Formula 044, Formula 045, Formula 045, Formula 045rel, Formula 046, Formula 048, Formula 049, Formula 050, Formula 051, Formula 052, Formula 053, Formula 054, Formula 055, Formula 056, Formula 057, Formula 058, Formula 059, Formula 060, Formula 061, Formula 062, Formula 62D1, Formula 063, Formula 064, Formula 065, Formula 066, Formula 068, Formula 069, Formula 070, Formula 071, Formula 073, Formula 073, Formula 074, Formula 075, Formula 076, Formula 077, Formula 078, Formula 079, Formula 080, Formula 081, Formula 082, Formula 083, Formula 084, Formula 085, Formula 086, Formula 087, Formula 088, Formula 089, Formula 090, Formula 091, Formula 092, Formula 093,

Formula 095, Formula 096, Formula 097, Formula 098, Formula 099, Formula 0100, Formula 0101, Formula 0102, Formula 0103, Formula 0104, Formula 0105, Formula 0106, Formula 0107, Formula 0108, Formula 0109, Formula 0110, Formula 0111, Formula 0112, Formula 0113, Formula 0114, Formula 0115, Formula 0116, Formula 0117, Formula 0118, Formula 0119, Formula 0120, Formula 0121, Formula 0123, Formula 0124, Formula 0125, Formula 0126, Formula 0127, Formula 0128, Formula 0129, Formula 0130, Formula 0131, Formula 0132, Formula 0133, Formula 0134, Formula 0135, Formula 0136, Formula 0137, Formula 0138, Formula 0139, Formula 0140, Formula 0141, Formula 0142, Formula 0143, Formula 0144, Formula 0145, Formula 0146, Formula 0147, Formula 0148, Formula 0149, Formula 0150, Formula 0151, Formula 0152, Formula 0153, Formula 0154, Formula 0155, Formula 0156, Formula 0157, Formula 0158, Formula 0159, Formula 0160, Formula 0161, Formula 0162, Formula 0163, Formula 0164, Formula 0165, Formula 0166, Formula 0167, Formula 0168, Formula 0169, Formula 0170, Formula 0171, Formula 0172, Formula 0173, Formula 0174, Formula 0175, Formula 0176, Formula 0177, Formula 0178, Formula 0179, Formula 0180, Formula 0181, Formula 0182, Formula 0183, Formula 0184, Formula 0185, Formula 0186, or Formula 0187, wherein n is an integer from 1 to 100. C171. The composition according to clause C170, wherein the saccharide comprises any one of the following Formula 025b, Formula 01A, Formula 02, and Formula 06. C172. The composition according to clause C170, wherein the saccharide further comprises any one of an E. coli R1 moiety, E. coli R2 moiety, E. coli R3 moiety, E. coli R4 moiety, and E. coli K-12 moiety. C173. The composition according to clause C170, wherein the saccharide does not further comprise any one of an E. coli R1 moiety, E. coli R2 moiety, E. coli R3 moiety, E. coli R4 moiety, and E. coliK-12 moiety.The composition according to clause C170, wherein the saccharide does not further comprise an E. coli R2 moiety. C174. The composition according to clause C170, wherein the saccharide further comprises a 3-deoxy-d-manno-oct-2-ulosonic acid (KDO) moiety. C175. The composition according to clause C170, wherein the carrier protein is selected from any one of CRM19 7 , diphtheria toxin fragment B (DTFB), DTFB C8, Diphtheria toxoid (DT), tetanus toxoid (TT), fragment C of TT, pertussis toxoid, cholera toxoid, or exotoxin A from Pseudomonas aeruginosa; detoxified Exotoxin A of P. aeruginosa (EPA), maltose binding protein (MBP), detoxified hemolysin A of S. aureus, clumping factor A, clumping factor B, Cholera toxin B subunit (CTB), Streptococcus pneumoniae Pneumolysin and detoxified variants thereof, C. jejuni AcrA, C. jejuni natural glycoproteins and Streptococcal C5a peptidase (SCP). C176. The composition according to clause C170, wherein the carrier protein is CRM1 97

. C177. The composition according to clause C170, wherein the carrier protein is tetanus toxoid. C178. The composition according to clause C170, wherein the ratio of saccharide to protein is at least 0.5 to at most 2. C179. The composition according to clause C170, wherein the conjugate is prepared via reductive amination. C180. The composition according to clause C170, wherein the saccharide is conjugated to the carrier protein through a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. C181. The composition according to clause C170, wherein the saccharide is a single-end linked conjugated saccharide. C182. The composition according to clause C174, wherein the saccharide is conjugated to the carrier protein through a 3-deoxy-d-manno-oct-2-ulosonic acid (KDO) residue. C183. The composition according to clause C170, wherein the conjugate is prepared via CDAP chemistry. C184. A composition comprising a polypeptide derived from FimH or fragment thereof; and (a) a conjugate comprising a carrier protein covalently bound to a saccharide comprising Formula 025b, wherein n is an integer from 31 to 90, (b) a conjugate comprising a carrier protein covalently bound to a saccharide comprising Formula 01A, wherein n is an integer from 31 to 90, (c) a conjugate comprising a carrier protein covalently bound to a saccharide comprising Formula 02, wherein n is an integer from 31 to 90, and (d) a conjugate comprising a carrier protein covalently bound to a saccharide comprising and Formula 06, wherein n is an integer from 31 to 90. C185. The composition according to clause C184, further comprising a conjugate comprising a carrier protein covalently bound to a saccharide comprising a structure selected from any one of the following: Formula 015, Formula 016, Formula 017, Formula 018 and Formula 075, wherein n is an integer from 31 to 90. C186. The composition according to clause C184, comprising at most 25% free saccharide as compared to the total amount of saccharide in the composition. C187. A method of eliciting an immune response against Escherichiacoli in a mammal, comprising administering to the mammal an effective amount of the composition according to any one of clauses C184 to C186. C188. The method according to clause C187, wherein the immune response comprises opsonophagocytic antibodies against E. coli.

C189. The method according to clause C187, wherein the immune response protects the mammal from an E. coli infection. C190. A mammalian cell comprising (a) a first gene of interest encoding a polypeptide derived from E. color a fragment thereof, wherein the gene is integrated between at least two recombination target sites (RTS). C191. The embodiment of clause C190, wherein the two RTS are chromosomally-integrated within the NL1 locus or the NL2 locus. C192. The embodiment of clause C190, wherein the first gene of interest further comprises a reporter gene, a gene encoding a difficult to express protein, an ancillary gene or a combination thereof. C193. The embodiment of clause C190, further comprising a second gene of interest that is integrated within a second chromosomal locus distinct from the locus of (a), wherein the second gene of interest comprises a reporter gene, a gene encoding a difficult to express protein, an ancillary gene or a combination thereof. C194. A recombinant mammalian cell, comprising a polynucleotide encoding a polypeptide derived from E. color a fragment thereof. C195. The recombinant cell according to C194, wherein the polypeptide is derived from E. coli fimbrial H (FimH). C196. The recombinant cell according to C195, wherein the polypeptide comprises a phenylalanine residue at the N-terminus of the polypeptide. C197. The recombinant cell according to C195, wherein the polypeptide comprises a phenylalanine residue within the first 20 residue positions of the N-terminus. C198. The recombinant cell according to C195, wherein the polypeptide comprises a phenylalanine residue at position 1 of the polypeptide. C199. The recombinant cell according to C198, wherein the polypeptide does not comprise a glycine residue immediately before the phenylalanine residue at position 1 of the polypeptide. C200. The recombinant cell according to C195, wherein the polypeptide does not comprise an N-glycosylation site at position 7 of the polypeptide. C201. The recombinant cell according to C199, wherein the polypeptide does not comprise an Asn residue at position 7 of the polypeptide. C202. The recombinant cell according to C201, wherein the polypeptide comprises a residue selected from the group consisting of Ser, Asp, Thr, and Gln at position 7. C203. The recombinant cell according to C198, wherein the polypeptide does not comprise an N-glycosylation site at position 70 of the polypeptide.

C204. The recombinant cell according to C203, wherein the polypeptide does not comprise an Asn residue at position 70 of the polypeptide. C205. The recombinant cell according to C203, wherein the polypeptide does not comprise a Ser residue at position 70 of the polypeptide. C206. The recombinant cell according to C194, wherein the polypeptide comprises a residue substitution selected from the group consisting of Ser, Asp, Thr, and GIn at an N glycosylation site of the polypeptide. C207. The recombinant cell according to C206, wherein the N-glycosylation site comprises position N235 of the polypeptide. C208. The recombinant cell according to C206, wherein the N-glycosylation site comprises position N228 of the polypeptide. C209. The recombinant cell according to C206, wherein the N-glycosylation site comprises position N235 and position N228 of the polypeptide. C210. The recombinant cell according to C195, wherein the polypeptide comprises SEQ ID NO: 3. C211. The recombinant cell according to C195, wherein the polypeptide comprises SEQ ID NO: 2. C212. The recombinant cell according to C194, wherein the polypeptide comprises an aliphatic hydrophobic amino acid residue at position 1 of the polypeptide. C213. The recombinant cell according to C212, wherein the aliphatic hydrophobic amino acid residue is selected from the group consisting of Ile, Leu, and Val. C214. The recombinant cell according to C194, wherein the polypeptide comprises a fragment of FimH. C215. The recombinant cell according to C214, wherein the polypeptide comprises a lectin domain of FimH. C216. The recombinant cell according to C215, wherein the lectin domain comprises a mass of about 17022 Daltons. C217. The recombinant cell according to C194, wherein the polypeptide is complexed with a FimC polypeptide or a fragment thereof. C218. The recombinant cell according to C217, wherein the FimC polypeptide or a fragment thereof comprises a glycine residue at position 37 of the FimC polypeptide or a fragment thereof. C219. The recombinant cell according to C195, wherein the polypeptide is in the low affinity conformation. C220. The recombinant cell according to C195, wherein the polypeptide is stabilized by FimG.

C221. The recombinant cell according to C195, wherein the polypeptide is stabilized by a donor-strand peptide of FimG (DsG). C222. The recombinant cell according to C221, wherein the polynucleotide sequence further encodes a linker sequence. C223. The recombinant cell according to C222, wherein the linker comprises at least 4 amino acid residues and at most 15 amino acid residues. C224. The recombinant cell according to C222, wherein the linker comprises at least 5 amino acid residues and at most 10 amino acid residues. C225. The recombinant cell according to C222, wherein the linker comprises 7 amino acid residues. C226. The recombinant cell according to C194, wherein the polypeptide does not comprise a signal peptide selected from the group consisting of a native FimH leader peptide, influenza hemagglutinin signal peptide, and a human respiratory syncytial virus A (strain A2) fusion glycoprotein FO signal peptide. C227. The recombinant cell according to C194, wherein the polypeptide comprises a murine IgK signal peptide sequence. C228. The recombinant cell according to C194, wherein the polypeptide comprises anyone signal peptide sequence selected from human IgG receptor FcRn large subunit p51 signal peptide and a human IL10 protein signal peptide. C229. The recombinant cell according to C195, wherein the polypeptide comprises a mutation of arginine to proline at amino acid position 60 (R60P), according to the numbering of SEQ ID NO: 3. C230. The recombinant cell according to C194, wherein the expression level of the polypeptide is greater than the expression level of the corresponding wild-type polypeptide expressed in the periplasm of a wild-type E. coli cell. C231. The recombinant cell according to C194, wherein the expression level of the polypeptide is greater than 10 mg/L. C232. The recombinant cell according to C194, wherein the polynucleotide sequence is integrated into the genomic DNA of said mammalian cell. C233. The recombinant cell according to C194, wherein the polynucleotide sequence is codon optimized for expression in the cell. C234. The recombinant cell according to C194, wherein the cell is a human embryonic kidney cell. C235. The recombinant cell according to C234, wherein the human embryonic kidney cell comprises a HEK293 cell.

C236. The recombinant cell according to C235, wherein the HEK293 cell is selected from any one of HEK293T cells, HEK293TS cells, and HEK293E cells. C237. The recombinant cell according to C195, wherein the cell is a CHO cell. C238. The recombinant cell according to C237, wherein said CHO cell is a CHO-K1 cell, CHO DUXB11, CHO-DG44 cell, or CHO-S cell. C239. The recombinant cell according to C194, wherein the polypeptide is soluble. C240. The recombinant cell according to C194, wherein the polypeptide is secreted from the cell. C241. The recombinant cell according to C195, wherein the polypeptide comprises a N28Q substitution, according to the numbering of SEQ ID NO: 1. C242. The recombinant cell according to C195, wherein the polypeptide comprises a N28D substitution, according to the numbering of SEQ ID NO: 1. C243. The recombinant cell according to C195, wherein the polypeptide comprises a N28S substitution, according to the numbering of SEQ ID NO: 1. C244. The recombinant cell according to C195, wherein the polypeptide comprises a substitution selected from any one of N28Q, V48C, and L55C, according to the numbering of SEQ ID NO: 1. C245. The recombinant cell according to C195, wherein the polypeptide comprises a substitution N92S according to the numbering of SEQ ID NO: 1. C246. The recombinant cell according to C194, wherein the polypeptide derived from FimH or fragment thereof comprises a substation selected from any one of V48C and L55C, according to the numbering of SEQ ID NO: 1. C247. A culture comprising the recombinant cell of C194, wherein said culture is at least 5 liter in size. C248. The culture according to C242, wherein the yield of the polypeptide or fragment thereof is at least 0.05 g/L. C249. The culture according to C248, wherein the yield of the polypeptide or fragment thereof is at least 0.10 g/L. C250. A method for producing a polypeptide derived from E. color a fragment thereof, comprising culturing a recombinant mammalian cell according to C194 under a suitable condition, thereby expressing the polypeptide or fragment thereof; and harvesting the polypeptide or fragment thereof. C251. The method according to C250, further comprising purifying the polypeptide or fragment thereof. C252. The method according to C250, wherein the cell comprises a nucleic acid encoding any one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 27.

C253. The method according to C250, wherein the yield of the polypeptide or fragment thereof is at least 0.05 g/L. C254. The method according to C250, wherein the yield of the polypeptide or fragment thereof is at least 0.10 g/L. C255. A composition comprising a polypeptide having at least 70% identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, and SEQ ID NO: 29. C256. The composition according to C255, further comprising a saccharide comprising a structure selected from any one Formula in Table 1. C257. The composition according to C256, wherein the saccharide is covalently bound a carrier protein. C258. The composition according to C257, wherein the carrier protein is selected from any one of poly(L-lysine), CRM 197, diphtheria toxin fragment B (DTFB), DTFB C8, Diphtheria toxoid (DT), tetanus toxoid (TT), fragment C of TT, pertussis toxoid, cholera toxoid, or exotoxin A from Pseudomonas aeruginosa; detoxified Exotoxin A of P. aeruginosa (EPA), maltose binding protein (MBP), detoxified hemolysin A of S. aureus, clumping factor A, clumping factor B, Cholera toxin B subunit (CTB), Streptococcus pneumoniae Pneumolysin and detoxified variants thereof, C. jejuniAcrA, C. jejuni natural glycoproteins and Streptococcal C5a peptidase (SCP). C259. The composition according to C257, wherein the carrier protein is CRM 197

. C260. The composition according to C257, wherein the carrier protein is tetanus toxoid (TT). C261. The composition according to C257, wherein the carrier protein is poly(L-lysine). C262. The composition according to C257, wherein the saccharide is covalently bound a carrier protein by reductive amination. C263. The composition according to C257, wherein the saccharide is covalently bound a carrier protein by CDAP chemistry. C264. The composition according to C257, wherein the saccharide is covalently bound a carrier protein by single-end linked conjugation. C265. The composition according to C257, wherein the saccharide is covalently bound a carrier protein through a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. C266. A polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 27. C267. A composition comprising a polypeptide derived from FimH or a fragment thereof; and a saccharide comprising a structure selected from any one of Formula 01, Formula 01A, Formula 01B, Formula 01C, Formula 02, Formula 03, Formula 04, Formula 04:K52, Formula 04:K6, Formula 05, Formula 05ab, Formula 05ac, Formula 06, Formula 06:K2;

K13; K15, Formula 06:K54, Formula 07, Formula 08, Formula 09, Formula 010, Formula 011, Formula 012, Formula 013, Formula 014, Formula 015, Formula 016, Formula 017, Formula 018, Formula 018A, Formula 018ac, Formula 018A1, Formula 018B, Formula 018B1, Formula 019, Formula 020, Formula 021, Formula 022, Formula 023, Formula 023A, Formula 024, Formula 025, Formula 025a, Formula 025b, Formula 026, Formula 027, Formula 028, Formula 029, Formula 030, Formula 032, Formula 033, Formula 034, Formula 035, Formula 036, Formula 037, Formula 038, Formula 039, Formula 040, Formula 041, Formula 042, Formula 043, Formula 044, Formula 045, Formula 045, Formula 045rel, Formula 046, Formula 048, Formula 049, Formula 050, Formula 051, Formula 052, Formula 053, Formula 054, Formula 055, Formula 056, Formula 057, Formula 058, Formula 059, Formula 060, Formula 061, Formula 062, Formula 62D1, Formula 063, Formula 064, Formula 065, Formula 066, Formula 068, Formula 069, Formula 070, Formula 071, Formula 073, Formula 073, Formula 074, Formula 075, Formula 076, Formula 077, Formula 078, Formula 079, Formula 080, Formula 081, Formula 082, Formula 083, Formula 084, Formula 085, Formula 086, Formula 087, Formula 088, Formula 089, Formula 090, Formula 091, Formula 092, Formula 093, Formula 095, Formula 096, Formula 097, Formula 098, Formula 099, Formula 0100, Formula 0101, Formula 0102, Formula 0103, Formula 0104, Formula 0105, Formula 0106, Formula 0107, Formula 0108, Formula 0109, Formula 0110, Formula 0111, Formula 0112, Formula 0113, Formula 0114, Formula 0115, Formula 0116, Formula 0117, Formula 0118, Formula 0119, Formula 0120, Formula 0121, Formula 0123, Formula 0124, Formula 0125, Formula 0126, Formula 0127, Formula 0128, Formula 0129, Formula 0130, Formula 0131, Formula 0132, Formula 0133, Formula 0134, Formula 0135, Formula 0136, Formula 0137, Formula 0138, Formula 0139, Formula 0140, Formula 0141, Formula 0142, Formula 0143, Formula 0144, Formula 0145, Formula 0146, Formula 0147, Formula 0148, Formula 0149, Formula 0150, Formula 0151, Formula 0152, Formula 0153, Formula 0154, Formula 0155, Formula 0156, Formula 0157, Formula 0158, Formula 0159, Formula 0160, Formula 0161, Formula 0162, Formula 0163, Formula 0164, Formula 0165, Formula 0166, Formula 0167, Formula 0168, Formula 0169, Formula 0170, Formula 0171, Formula 0172, Formula 0173, Formula 0174, Formula 0175, Formula 0176, Formula 0177, Formula 0178, Formula 0179, Formula 0180, Formula 0181, Formula 0182, Formula 0183, Formula 0184, Formula 0185, Formula 0186, Formula 0187. C268. The composition according to Clause C267, further comprising at least one saccharide derived from any one K. pneumoniae type selected from the group consisting of 01, 02, 03, and 05.

C269. The composition according to Clause C267, further comprising a saccharide derived from Klebsiella pneumoniae type 01. C270. The composition according to Clause C267, further comprising a saccharide derived from K. pneumoniae type 02. C271. The composition according to Clause C267, further comprising a saccharide derived from K. pneumoniae type 03. C272. The composition according to Clause C267, further comprising a saccharide derived from K. pneumoniae type 05. C273. The composition according to Clause C267, further comprising a saccharide derived from K. pneumoniae type 01 and a saccharide derived from K. pneumoniae type 02. C274. The composition according to Clause C268, wherein the saccharide derived from K. pneumoniae is conjugated to a carrier protein; and the saccharide derived from E. coli is conjugated to a carrier protein. C275. The composition according to Clause C267, further comprising a polypeptide derived from K. pneumoniae. C276. A composition comprising a polypeptide derived from FimH or a fragment thereof; and at least one saccharide derived from any one K. pneumoniae type selected from the group consisting of 01, 02, 03, and 05. C277. The composition according to Clause C276, further comprising at least one saccharide comprising a structure selected from any one of Formula 01, Formula 01A, Formula 01B, Formula 01C, Formula 02, Formula 03, Formula 04, Formula 04:K52, Formula 04:K6, Formula 05, Formula 05ab, Formula 05ac, Formula 06, Formula 06:K2; K13; K15, Formula 06:K54, Formula 07, Formula 08, Formula 09, Formula 010, Formula 011, Formula 012, Formula 013, Formula 014, Formula 015, Formula 016, Formula 017, Formula 018, Formula 018A, Formula 018ac, Formula 018A1, Formula 018B, Formula 018B1, Formula 019, Formula 020, Formula 021, Formula 022, Formula 023, Formula 023A, Formula 024, Formula 025, Formula 025a, Formula 025b, Formula 026, Formula 027, Formula 028, Formula 029, Formula 030, Formula 032, Formula 033, Formula 034, Formula 035, Formula 036, Formula 037, Formula 038, Formula 039, Formula 040, Formula 041, Formula 042, Formula 043, Formula 044, Formula 045, Formula 045, Formula 045rel, Formula 046, Formula 048, Formula 049, Formula 050, Formula 051, Formula 052, Formula 053, Formula 054, Formula 055, Formula 056, Formula 057, Formula 058, Formula 059, Formula 060, Formula 061, Formula 062, Formula 62D1, Formula 063, Formula 064, Formula 065, Formula 066, Formula 068, Formula 069, Formula 070, Formula 071, Formula 073, Formula 073, Formula 074, Formula 075, Formula 076, Formula 077, Formula 078, Formula 079, Formula 080, Formula 081,

Formula 082, Formula 083, Formula 084, Formula 085, Formula 086, Formula 087, Formula 088, Formula 089, Formula 090, Formula 091, Formula 092, Formula 093, Formula 095, Formula 096, Formula 097, Formula 098, Formula 099, Formula 0100, Formula 0101, Formula 0102, Formula 0103, Formula 0104, Formula 0105, Formula 0106, Formula 0107, Formula 0108, Formula 0109, Formula 0110, Formula 0111, Formula 0112, Formula 0113, Formula 0114, Formula 0115, Formula 0116, Formula 0117, Formula 0118, Formula 0119, Formula 0120, Formula 0121, Formula 0123, Formula 0124, Formula 0125, Formula 0126, Formula 0127, Formula 0128, Formula 0129, Formula 0130, Formula 0131, Formula 0132, Formula 0133, Formula 0134, Formula 0135, Formula 0136, Formula 0137, Formula 0138, Formula 0139, Formula 0140, Formula 0141, Formula 0142, Formula 0143, Formula 0144, Formula 0145, Formula 0146, Formula 0147, Formula 0148, Formula 0149, Formula 0150, Formula 0151, Formula 0152, Formula 0153, Formula 0154, Formula 0155, Formula 0156, Formula 0157, Formula 0158, Formula 0159, Formula 0160, Formula 0161, Formula 0162, Formula 0163, Formula 0164, Formula 0165, Formula 0166, Formula 0167, Formula 0168, Formula 0169, Formula 0170, Formula 0171, Formula 0172, Formula 0173, Formula 0174, Formula 0175, Formula 0176, Formula 0177, Formula 0178, Formula 0179, Formula 0180, Formula 0181, Formula 0182, Formula 0183, Formula 0184, Formula 0185, Formula 0186, Formula 0187. C278. The composition according to Clause C277, wherein the saccharide derived from K. pneumoniae is conjugated to a carrier protein; and the saccharide derived from E. coli is conjugated to a carrier protein. C279. The composition according to Clause C277, further comprising a polypeptide derived from K. pneumoniae. C280. A composition comprising at least one saccharide derived from any one K. pneumoniae type selected from the group consisting of 01, 02, 03, and 05; and at least one saccharide comprising a structure selected from any one of Formula 01, Formula 01A, Formula 01B, Formula 01C, Formula 02, Formula 03, Formula 04, Formula 04:K52, Formula 04:K6, Formula 05, Formula 05ab, Formula 05ac, Formula 06, Formula 06:K2; K13; K15, Formula 06:K54, Formula 07, Formula 08, Formula 09, Formula 010, Formula 011, Formula 012, Formula 013, Formula 014, Formula 015, Formula 016, Formula 017, Formula 018, Formula 018A, Formula 018ac, Formula 018A1, Formula 018B, Formula 018B1, Formula 019, Formula 020, Formula 021, Formula 022, Formula 023, Formula 023A, Formula 024, Formula 025, Formula 025a, Formula 025b, Formula 026, Formula 027, Formula 028, Formula 029, Formula 030, Formula 032, Formula 033, Formula 034, Formula 035, Formula 036, Formula 037, Formula 038, Formula 039, Formula 040,

Formula 041, Formula 042, Formula 043, Formula 044, Formula 045, Formula 045, Formula 045rel, Formula 046, Formula 048, Formula 049, Formula 050, Formula 051, Formula 052, Formula 053, Formula 054, Formula 055, Formula 056, Formula 057, Formula 058, Formula 059, Formula 060, Formula 061, Formula 062, Formula 62D1, Formula 063, Formula 064, Formula 065, Formula 066, Formula 068, Formula 069, Formula 070, Formula 071, Formula 073, Formula 073, Formula 074, Formula 075, Formula 076, Formula 077, Formula 078, Formula 079, Formula 080, Formula 081, Formula 082, Formula 083, Formula 084, Formula 085, Formula 086, Formula 087, Formula 088, Formula 089, Formula 090, Formula 091, Formula 092, Formula 093, Formula 095, Formula 096, Formula 097, Formula 098, Formula 099, Formula 0100, Formula 0101, Formula 0102, Formula 0103, Formula 0104, Formula 0105, Formula 0106, Formula 0107, Formula 0108, Formula 0109, Formula 0110, Formula 0111, Formula 0112, Formula 0113, Formula 0114, Formula 0115, Formula 0116, Formula 0117, Formula 0118, Formula 0119, Formula 0120, Formula 0121, Formula 0123, Formula 0124, Formula 0125, Formula 0126, Formula 0127, Formula 0128, Formula 0129, Formula 0130, Formula 0131, Formula 0132, Formula 0133, Formula 0134, Formula 0135, Formula 0136, Formula 0137, Formula 0138, Formula 0139, Formula 0140, Formula 0141, Formula 0142, Formula 0143, Formula 0144, Formula 0145, Formula 0146, Formula 0147, Formula 0148, Formula 0149, Formula 0150, Formula 0151, Formula 0152, Formula 0153, Formula 0154, Formula 0155, Formula 0156, Formula 0157, Formula 0158, Formula 0159, Formula 0160, Formula 0161, Formula 0162, Formula 0163, Formula 0164, Formula 0165, Formula 0166, Formula 0167, Formula 0168, Formula 0169, Formula 0170, Formula 0171, Formula 0172, Formula 0173, Formula 0174, Formula 0175, Formula 0176, Formula 0177, Formula 0178, Formula 0179, Formula 0180, Formula 0181, Formula 0182, Formula 0183, Formula 0184, Formula 0185, Formula 0186, Formula 0187. C281. The composition according to Clause C280, further comprising a polypeptide derived from FimH or a fragment thereof. C282. The composition according to Clause C280, wherein the E. coli saccharide comprises Formula 08. C283. The composition according to Clause C280, wherein the E. coli saccharide comprises Formula 09. C284. The composition according to Clause C280, further comprising a polypeptide derived from K. pneumoniae. C285. The composition according to any of Clauses C267-C284, wherein the saccharide is covalently bound to a carrier protein.

C286. The composition according to Clause C285, wherein the saccharide further comprises a 3-deoxy-d-manno-oct-2-ulosonic acid (KDO) moiety. C287. The composition according to Clause C285, wherein the saccharide comprises Lipid A. C288. The composition according to any one of clauses C285-C287, wherein the saccharide is synthetically synthesized. C289. The composition according to Clause C285, wherein the carrier protein is selected from any one of CRM197, diphtheria toxin fragment B (DTFB), DTFB C8, Diphtheria toxoid (DT), tetanus toxoid (TT), fragment C of TT, pertussis toxoid, cholera toxoid, or exotoxin A from Pseudomonas aeruginosa; detoxified Exotoxin A of P. aeruginosa (EPA), maltose binding protein (MBP), detoxified hemolysin A of S. aureus, clumping factor A, clumping factor B, Cholera toxin B subunit (CTB), Streptococcus pneumoniae Pneumolysin and detoxified variants thereof, C. jejuni AcrA, C. jejuni natural glycoproteins and Streptococcal C5a peptidase (SCP). C290. A method of eliciting an immune response against Escherichia coli in a mammal, comprising administering to the mammal an effective amount of the composition according to any one of Clauses C267-C289. C291. The method according to Clause C290, wherein the immune response comprises opsonophagocytic antibodies against E. coli. C292. The method according to Clause C290, wherein the immune response protects the mammal from an E. coli infection. C293. A method of eliciting an immune response against Klebsiella pneumoniae in a mammal, comprising administering to the mammal an effective amount of the composition according to any one of Clauses C267-C289. C294. The method according to Clause C293, wherein the immune response comprises opsonophagocytic antibodies against Klebsiella pneumoniae. C295. The method according to Clause C293, wherein the immune response protects the mammal from a Klebsiella pneumoniae infection. C296. The compositions and methods according to any one of Clauses C1-C266, further comprising at least one saccharide derived from any one of K. pneumoniae type selected from the group consisting of 01, 02, 03, and 05. C297. The compositions and methods of Clause C296, wherein the K. pneumoniae serotype 01 comprises variant 01V1 or 01V2. C298. The compositions and methods of Clause C296, wherein the K. pneumoniae serotype 02 comprises variant 02V1 or 02V2. C299. Use of the compositions set forth in any one of Clauses C1-C298 as set forth herein. C300. A composition comprising

(i) a polypeptide derived from fimbrial H (FimH) or a fragment thereof; and (ii) one or more conjugates, wherein the conjugate comprises a carrierprotein covalently bound to a saccharide, said saccharide comprising a structure selected from the group consisting of Formula 01, Formula 01A, Formula 01B, Formula 01C, Formula 02, Formula 03, Formula 04, Formula 04:K52, Formula 04:K6, Formula 05, Formula 05ab, Formula 05ac, Formula 06, Formula 06:K2; K13; K15, Formula 06:K54, Formula 07, Formula 08, Formula 09, Formula 010, Formula 011, Formula 012, Formula 013, Formula 014, Formula 015, Formula 016, Formula 017, Formula 018, Formula 018A, Formula 018ac, Formula 018A1, Formula 018B, Formula 018B1, Formula 019, Formula 020, Formula 021, Formula 022, Formula 023, Formula 023A, Formula 024, Formula 025, Formula 025a, Formula 025b, Formula 026, Formula 027, Formula 028, Formula 029, Formula 030, Formula 032, Formula 033, Formula 034, Formula 035, Formula 036, Formula 037, Formula 038, Formula 039, Formula 040, Formula 041, Formula 042, Formula 043, Formula 044, Formula 045, Formula 045, Formula 045rel, Formula 046, Formula 048, Formula 049, Formula 050, Formula 051, Formula 052, Formula 053, Formula 054, Formula 055, Formula 056, Formula 057, Formula 058, Formula 059, Formula 060, Formula 061, Formula 062, Formula 62D1, Formula 063, Formula 064, Formula 065, Formula 066, Formula 068, Formula 069, Formula 070, Formula 071, Formula 073, Formula 073, Formula 074, Formula 075, Formula 076, Formula 077, Formula 078, Formula 079, Formula 080, Formula 081, Formula 082, Formula 083, Formula 084, Formula 085, Formula 086, Formula 087, Formula 088, Formula 089, Formula 090, Formula 091, Formula 092, Formula 093, Formula 095, Formula 096, Formula 097, Formula 098, Formula 099, Formula 0100, Formula 0101, Formula 0102, Formula 0103, Formula 0104, Formula 0105, Formula 0106, Formula 0107, Formula 0108, Formula 0109, Formula 0110, Formula 0111, Formula 0112, Formula 0113, Formula 0114, Formula 0115, Formula 0116, Formula 0117, Formula 0118, Formula 0119, Formula 0120, Formula 0121, Formula 0123, Formula 0124, Formula 0125, Formula 0126, Formula 0127, Formula 0128, Formula 0129, Formula 0130, Formula 0131, Formula 0132, Formula 0133, Formula 0134, Formula 0135, Formula 0136, Formula 0137, Formula 0138, Formula 0139, Formula 0140, Formula 0141, Formula 0142, Formula 0143, Formula 0144, Formula 0145, Formula 0146, Formula 0147, Formula 0148, Formula 0149, Formula 0150, Formula 0151, Formula 0152, Formula 0153, Formula 0154, Formula 0155, Formula 0156, Formula 0157, Formula 0158, Formula 0159, Formula 0160, Formula 0161, Formula 0162, Formula 0163, Formula 0164, Formula 0165, Formula 0166, Formula 0167, Formula 0168, Formula 0169, Formula 0170, Formula 0171, Formula 0172, Formula 0173, Formula 0174, Formula 0175, Formula

0176, Formula 0177, Formula 0178, Formula 0179, Formula 0180, Formula 0181, Formula 0182, Formula 0183, Formula 0184, Formula 0185, Formula 0186, and Formula 0187, wherein n is an integer from 1 to 100. C301. The composition according to clause C300, wherein the saccharide is selected from the group consisting of Formula 025b, Formula 01A, Formula 02, Formula 06, Formula 08 and Formula 09. C302. The composition according to clause C301, wherein the saccharide is selected from the group consisting of Formula 025b, Formula 01A, Formula 02, and Formula 06. C303. A composition comprising a polypeptide derived from Fimbrial antigen H (FimH) or a fragment thereof; and (a) a conjugate comprising a carrier protein covalently bound to a saccharide comprising Formula 025b, wherein n is an integer from 31 to 90, (b) a conjugate comprising a carrier protein covalently bound to a saccharide comprising Formula 01A, wherein n is an integer from 31 to 90, (c) a conjugate comprising a carrier protein covalently bound to a saccharide comprising Formula 02, wherein n is an integer from 31 to 90, (d) a conjugate comprising a carrier protein covalently bound to a saccharide comprising and Formula 06, wherein n is an integer from 31 to 90, (e) a conjugate comprising a carrier protein covalently bound to a saccharide comprising and Formula 08, wherein n is an integer from 31 to 90, and (f) a conjugate comprising a carrier protein covalently bound to a saccharide comprising and Formula 09, wherein n is an integer from 31 to 90. C304. The composition of clause C303 comprising a polypeptide derived from Fimbrial antigen H (FimH) or a fragment thereof; and (a) a conjugate comprising a carrier protein covalently bound to a saccharide comprising Formula 025b, wherein n is an integer from 31 to 90, (b) a conjugate comprising a carrier protein covalently bound to a saccharide comprising Formula 01A, wherein n is an integer from 31 to 90, (c) a conjugate comprising a carrier protein covalently bound to a saccharide comprising Formula 02, wherein n is an integer from 31 to 90, and (d) a conjugate comprising a carrier protein covalently bound to a saccharide comprising and Formula 06, wherein n is an integer from 31 to 90. C305. The composition according to clauses C303 or C304, further comprising one or more conjugates having a saccharide selected from the group consisting of Formula 015, Formula 016, Formula 017, Formula 018 and Formula 075, wherein n is an integer from

31 to 90.7. The composition according to any one of clauses 1-6, wherein the saccharide further comprises any one of an E. coli R1 moiety, E. coli R2 moiety, E. coli R3 moiety, E. coli R4 moiety, and E. coli K-12 moiety. C306. The composition according to any one of clauses C300-C304, wherein the saccharide does not further comprise any one of an E. coli R1 moiety, E. coli R2 moiety, E. coli R3 moiety, E. coli R4 moiety, and E. coli K-12 moiety. C307. The composition according to clause C306, wherein the saccharide does not further comprise an E. coli R2 moiety. C308. The composition according to any one of clauses C300-C307, wherein the saccharide further comprises a 3-deoxy-d-manno-oct-2-ulosonic acid (KDO) moiety. C309. The composition according to any one of clauses C300-C308, wherein the carrier protein is selected from the group consisting of CRM197, diphtheria toxin fragment B (DTFB), DTFB C8, Diphtheria toxoid (DT), tetanus toxoid (TT), fragment C of TT, pertussis toxoid, cholera toxoid, or exotoxin A from Pseudomonas aeruginosa; detoxified Exotoxin A of P. aeruginosa (EPA), maltose binding protein (MBP), detoxified hemolysin A of S. aureus, clumping factor A, clumping factor B, Cholera toxin B subunit (CTB), Streptococcus pneumoniae Pneumolysin and detoxified variants thereof, C. jejuni AcrA, C. jejuni natural glycoproteins, and Streptococcal C5a peptidase (SCP) or a variant thereof. C310. The composition according to clause C309, wherein the carrier protein is CRM197. C311. The composition according to clause C309, wherein the carrier protein is tetanus toxoid. C312. The composition according to any one of clauses C300-C311, wherein the ratio of saccharide to protein is at least 0.5 to at most 2. C313. The composition according to any one of clauses C300-C312, wherein the conjugate is prepared via reductive amination. C314. The composition according to any one of clauses C300-C312, wherein the saccharide is conjugated to the carrier protein through a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. C315. The composition according to any one of clauses C300-C314, wherein the saccharide is a single-end linked conjugated saccharide. C316. The composition according to clause C308, wherein the saccharide is conjugated to the carrier protein through a 3-deoxy-d-manno-oct-2-ulosonic acid (KDO) residue. C317. The composition according to any one of clauses C300-C313, wherein the conjugate is prepared via CDAP chemistry. C318. The composition according to any one of clauses C300-C317, wherein the polypeptide is derived from E. coli fimbrial H (FimH).

C319. The composition according to clause C318, wherein the polypeptide comprises a phenylalanine residue at the N-terminus of the polypeptide. C320. The composition according to clause C318, wherein the polypeptide comprises a phenylalanine residue within the first 20 residue positions of the N-terminus. C321. The composition according to clause C314, wherein the polypeptide comprises a phenylalanine residue at position 1 of the polypeptide. C322. The composition according to clause C317, wherein the polypeptide does not comprise a glycine residue immediately before the phenylalanine residue at position 1 of the polypeptide. C323. The composition according to clause C314, wherein the polypeptide does not comprise an N-glycosylation site at position 7 of the polypeptide. C324. The composition according to clause C318, wherein the polypeptide does not comprise an Asn residue at position 7 of the polypeptide. C325. The composition according to clause C320, wherein the polypeptide comprises a residue selected from the group consisting of Ser, Asp, Thr, and Gln at position 7. C326. The composition according to clause C317, wherein the polypeptide does not comprise an N-glycosylation site at position 70 of the polypeptide. C327. The composition according to clause C322, wherein the polypeptide does not comprise an Asn residue at position 70 of the polypeptide. C328. The composition according to clause C322, wherein the polypeptide does not comprise a Ser residue at position 70 of the polypeptide. C329. The composition according to clause C314, wherein the polypeptide comprises a residue substitution selected from the group consisting of Ser, Asp, Thr, and Gln at an N glycosylation site of the polypeptide. C330. The composition according to clause C325, wherein the N-glycosylation site comprises position N235 of the polypeptide. C331. The composition according to clause C325, wherein the N-glycosylation site comprises position N228 of the polypeptide. C332. The composition according to clause C325, wherein the N-glycosylation site comprises position N235 and position N228 of the polypeptide. C333. The composition according to clause C314, wherein the polypeptide comprises SEQ ID NO: 2. C334. The composition according to clause C314, wherein the polypeptide comprises SEQ ID NO: 3. C335. The composition according to clause C314, wherein the polypeptide comprises amino acids having an amino acid sequence selected from the group consisting of SEQ ID NO: 5,

SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 27, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, and SEQ ID NO: 113. C336. The composition of cliam C314, wherein the polypeptide comprises amino acids having an amino acid sequence having at least 70% identity to the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112 and SEQ ID NO: 113. C337. The composition according to clause C314, wherein the polypeptide comprises an aliphatic hydrophobic amino acid residue at position 1 of the polypeptide. C338. The composition according to clause C333, wherein the aliphatic hydrophobic amino acid residue is selected from the group consisting of Ile, Leu, and Val. C339. The composition according to clause C314, wherein the polypeptide comprises a fragment of FimH. C340. The composition according to clause C335, wherein the polypeptide comprises a lectin domain of FimH. C341. The composition according to clause C336, wherein the lectin domain comprises a mass of about 17022 Daltons. C342. The composition according to clause C314, wherein the polypeptide is complexed with a FimC polypeptide or a fragment thereof. C343. The composition according to clause C338, wherein the FimC polypeptide or a fragment thereof comprises a glycine residue at position 37 of the FimC polypeptide or a fragment thereof. C344. The composition according to clause C314, wherein the polypeptide is in the low affinity conformation. C345. The composition according to clause C314, wherein the polypeptide is stabilized by FimG. C346. The composition according to clause C314, wherein the polypeptide is stabilized by a donor-strand peptide of FimG (DsG). C347. The composition according to clause C346, wherein the polypeptide further comprises a linker. C348. The composition according to clause C343, wherein the linker comprises at least 4 amino acid residues and at most 15 amino acid residues. C349. The composition according to clause C343, wherein the linker comprises at least 5 amino acid residues and at most 10 amino acid residues. C350. The composition according to clause C343, wherein the linker comprises 7 amino acid residues.

C351. The composition according to clause C314, wherein the polypeptide does not comprise a signal peptide selected from the group consisting of a native FimH leader peptide, influenza hemagglutinin signal peptide, and a human respiratory syncytial virus A (strain A2) fusion glycoprotein FO signal peptide. C352. The composition according to clause C314, wherein the polypeptide comprises a murine IgK signal peptide sequence. C353. The composition according to clause C314, wherein the polypeptide comprises any one signal peptide sequence selected from human IgG receptor FcRn large subunit p51 signal peptide and a human IL10 protein signal peptide. C354. The composition according to clause C314, wherein the polypeptide comprises a mutation of arginine to proline at amino acid position 60 (R60P), according to the numbering of SEQ ID NO: 3. C355. The composition according to clauses C300-C354, comprising at most 25% free saccharide as compared to the total amount of saccharide in the composition. C356. The composition of any one of clauses C300-C354, further comprising one or more conjugates, wherein the conjugate comprises a carrier protein covalently bound to a K. pneumoniae O-antigen selected from 01 and 02. C357. The composition of clause C356, wherein the K. pneumoniae O-antigen is selected from the group consisting of a) serotype 01 subtype v1 (Olvi), b) serotype 01 subtype v2 (01v2), c) serotype 02 subtype v1 (02v1), and d) serotype 02 subtype v2 (O2v2). C358. A composition comprising (i) one or more conjugates comprising a carrier protein covalently bound to a K. pneumoniae O-antigen selected from the group consisting of serotype 01 subtype v1 (Olvi), serotype 01 subtype v2 (01v2), serotype 02 subtype v1 (02v1), and serotype 02 subtype v2 (02v2); and (ii) one or more conjugates, wherein the conjugate comprises a carrier protein covalently bound to a saccharide, said saccharide comprising a structure selected from the group consisting of Formula 01, Formula 01A, Formula 01B, Formula 01C, Formula 02, Formula 03, Formula 04, Formula 04:K52, Formula 04:K6, Formula 05, Formula 05ab, Formula 05ac, Formula 06, Formula 06:K2; K13; K15, Formula 06:K54, Formula 07, Formula 08, Formula 09, Formula 010, Formula 011, Formula 012, Formula 013, Formula 014, Formula 015, Formula 016, Formula 017, Formula 018, Formula 018A, Formula 018ac, Formula 018A1, Formula 018B, Formula 018B1, Formula 019, Formula 020, Formula 021, Formula 022, Formula 023, Formula 023A, Formula 024, Formula 025, Formula 025a, Formula 025b, Formula 026, Formula 027, Formula 028, Formula 029, Formula 030, Formula 032, Formula 033, Formula 034, Formula 035, Formula 036, Formula 037,

Formula 038, Formula 039, Formula 040, Formula 041, Formula 042, Formula 043, Formula 044, Formula 045, Formula 045, Formula 045rel, Formula 046, Formula 048, Formula 049, Formula 050, Formula 051, Formula 052, Formula 053, Formula 054, Formula 055, Formula 056, Formula 057, Formula 058, Formula 059, Formula 060, Formula 061, Formula 062, Formula 62D1, Formula 063, Formula 064, Formula 065, Formula 066, Formula 068, Formula 069, Formula 070, Formula 071, Formula 073, Formula 073, Formula 074, Formula 075, Formula 076, Formula 077, Formula 078, Formula 079, Formula 080, Formula 081, Formula 082, Formula 083, Formula 084, Formula 085, Formula 086, Formula 087, Formula 088, Formula 089, Formula 090, Formula 091, Formula 092, Formula 093, Formula 095, Formula 096, Formula 097, Formula 098, Formula 099, Formula 0100, Formula 0101, Formula 0102, Formula 0103, Formula 0104, Formula 0105, Formula 0106, Formula 0107, Formula 0108, Formula 0109, Formula 0110, Formula 0111, Formula 0112, Formula 0113, Formula 0114, Formula 0115, Formula 0116, Formula 0117, Formula 0118, Formula 0119, Formula 0120, Formula 0121, Formula 0123, Formula 0124, Formula 0125, Formula 0126, Formula 0127, Formula 0128, Formula 0129, Formula 0130, Formula 0131, Formula 0132, Formula 0133, Formula 0134, Formula 0135, Formula 0136, Formula 0137, Formula 0138, Formula 0139, Formula 0140, Formula 0141, Formula 0142, Formula 0143, Formula 0144, Formula 0145, Formula 0146, Formula 0147, Formula 0148, Formula 0149, Formula 0150, Formula 0151, Formula 0152, Formula 0153, Formula 0154, Formula 0155, Formula 0156, Formula 0157, Formula 0158, Formula 0159, Formula 0160, Formula 0161, Formula 0162, Formula 0163, Formula 0164, Formula 0165, Formula 0166, Formula 0167, Formula 0168, Formula 0169, Formula 0170, Formula 0171, Formula 0172, Formula 0173, Formula 0174, Formula 0175, Formula 0176, Formula 0177, Formula 0178, Formula 0179, Formula 0180, Formula 0181, Formula 0182, Formula 0183, Formula 0184, Formula 0185, Formula 0186, and Formula 0187, wherein n is an integer from 1 to 100. C359. The composition according to clause C358, wherein the saccharide is selected from the group consisting of Formula 025b, Formula 01A, Formula 02, Formula 06, Formula 08 and Formula 09. C360. The composition according to clause C359, wherein the saccharide is selected from the group consisting of Formula 025b, Formula 01A, Formula 02, and Formula 06. C361. A composition comprising one or more conjugates comprising a carrier protein covalently bound to a K. pneumoniae 0-antigen selected from the group consisting of serotype 01 subtype v1 (01v1), serotype 01 subtype v2 (01v2), serotype 02 subtype v1 (02v1), and serotype 02 subtype v2 (02v2); and (a) a conjugate comprising a carrier protein covalently bound to a saccharide comprising Formula 025b, wherein n is an integer from 31 to 90, (b) a conjugate comprising a carrier protein covalently bound to a saccharide comprising Formula 01A, wherein n is an integer from 31 to 90, (c) a conjugate comprising a carrier protein covalently bound to a saccharide comprising Formula 02, wherein n is an integer from 31 to 90, (d) a conjugate comprising a carrier protein covalently bound to a saccharide comprising and Formula 06, wherein n is an integer from 31 to 90, (e) a conjugate comprising a carrier protein covalently bound to a saccharide comprising and Formula 08, wherein n is an integer from 31 to 90, and (f) a conjugate comprising a carrier protein covalently bound to a saccharide comprising and Formula 09, wherein n is an integer from 31 to 90. C362. The composition of clause C361 comprising (i) one or more conjugates comprising a carrier protein covalently bound to a K. pneumoniae 0-antigen selected from the group consisting of serotype 01 subtype v1 (01v1), serotype 01 subtype v2 (01v2), serotype 02 subtype v1 (02v1), and serotype 02 subtype v2 (02v2); and (a) a conjugate comprising a carrier protein covalently bound to a saccharide comprising Formula 025b, wherein n is an integer from 31 to 90, (b) a conjugate comprising a carrier protein covalently bound to a saccharide comprising Formula 01A, wherein n is an integer from 31 to 90, (c) a conjugate comprising a carrier protein covalently bound to a saccharide comprising Formula 02, wherein n is an integer from 31 to 90, and (d) a conjugate comprising a carrier protein covalently bound to a saccharide comprising and Formula 06, wherein n is an integer from 31 to 90. C363. The composition according to clauses C361 or C362, further comprising one or more conjugates having a saccharide selected from the group consisting of Formula 015, Formula 016, Formula 017, Formula 018 and Formula 075, wherein n is an integer from 31 to 90. C364. The composition according to any one of clauses C358-C363, wherein the saccharide further comprises any one of an E. coli R1 moiety, E. coli R2 moiety, E. coli R3 moiety, E. coli R4 moiety, and E. coli K-12 moiety. C365. The composition according to any one of clauses C358-C363, wherein the saccharide does not further comprise any one of an E. coli R1 moiety, E. coli R2 moiety, E. coli R3 moiety, E. coli R4 moiety, and E. coli K-12 moiety.

C366. The composition according to clause 67, wherein the saccharide does not further comprise an E. coli R2 moiety. C367. The composition according to any one of clauses C358-C366, wherein the saccharide further comprises a 3-deoxy-d-manno-oct-2-ulosonic acid (KDO) moiety. C368. The composition according to any one of clauses C358-C367, wherein the carrier protein is selected from the group consisting of CRM197, diphtheria toxin fragment B (DTFB), DTFB C8, Diphtheria toxoid (DT), tetanus toxoid (TT), fragment C of TT, pertussis toxoid, cholera toxoid, or exotoxin A from Pseudomonas aeruginosa; detoxified Exotoxin A of P. aeruginosa (EPA), maltose binding protein (MBP), detoxified hemolysin A of S. aureus, clumping factor A, clumping factor B, Cholera toxin B subunit (CTB), Streptococcus pneumoniae Pneumolysin and detoxified variants thereof, C. jejuni AcrA, C. jejuni natural glycoproteins, and Streptococcal C5a peptidase (SCP) or a variant thereof. C369. The composition according to clause C368, wherein the carrier protein is CRM197. C370. The composition according to clause C368, wherein the carrier protein is tetanus toxoid. C371. The composition according to any one of clauses C358-C370, wherein the ratio of saccharide to protein is at least 0.5 to at most 2. C372. The composition according to any one of clauses C358-C371, wherein the conjugate is prepared via reductive amination. C373. The composition according to any one of clauses C358-C371, wherein the saccharide is conjugated to the carrier protein through a(2(2-2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. C374. The composition according to any one of clauses C358-C373, wherein the saccharide is a single-end linked conjugated saccharide. C375. The composition according to clause C367, wherein the saccharide is conjugated to the carrier protein through a 3-deoxy-d-manno-oct-2-ulosonic acid (KDO) residue. C376. The composition according to any one of clauses C358-C372, wherein the conjugate is prepared via CDAP chemistry. C377. The composition according to clauses C358-C376, comprising at most 25% free saccharide as compared to the total amount of saccharide in the composition. C378. The composition of any one of clauses C300-C377 further comprising an adjuvant. C379. The composition of clause C378, wherein the adjuvant comprises aluminum. C380. The composition of clause C378, wherein the adjuvant comprises QS21. C381. The composition of clause C378, wherein the adjuvant comprises MPLA. C382. The composition of clause C380 or C381, wherein the adjuvant is a liposomal adjuvant.

C383. A method of eliciting an immune response against Escherichia coli in a mammal, comprising administering to the mammal an effective amount of the composition according to any one of clauses C300-C382. C384. The method according to clause C383, wherein the immune response comprises opsonophagocytic antibodies against E. coli. C385. The method according to clause C383, wherein the immune response protects the mammal from an E. coli infection. C386. Use of the composition of any one of clauses C300-C382 for inducing an immune response against E. coli. C387. Use of the composition of any one of clauses C300-C382 in the manufacture of a medicament for inducing an immune response against E. coli. C388. The use of clause C386 or C387, wherein the immune response comprises opsonophagocytic antibodies against E. coli. C389. The use of clause C386 or C387, wherein the immune response protects the mammal from an E. coli infection. C390. A method of eliciting an immune response against K. pneumoniae in a mammal, comprising administering to the mammal an effective amount of the composition according to any one of clauses C300-C382. C391. The method according to clause C390, wherein the immune response comprises opsonophagocytic antibodies against K. pneumoniae. C392. The method according to clause C390, wherein the immune response protects the mammal from a K. pneumoniae infection. C393. Use of the composition of any one of clauses C300 - C382 for inducing an immune response against K. pneumoniae. C394. Use of the composition of any one of clauses C300 - C382 in the manufacture of a medicament for inducing an immune response against K. pneumoniae. C395. The use of clause C386 or C387, wherein the immune response comprises opsonophagocytic antibodies against K. pneumoniae. C396. The use of clause C386 or C387, wherein the immune response protects the mammal from an K. pneumoniae infection. C397. A nucleic acid comprising nucleotides encoding the polypeptides of any one of clauses C1-C382. C398. The nucleic acid of clause C397, wherein the nucleic acid is RNA. C399. A nanoparticle comprising the nucleic acid of clause C397 or C398. C400. The composition according to any one of clauses 303, 304, 305, 359, 360, 361, 362 or 363, further comprising one or more conjugates having a saccharide selected from the group consisting of Formula 04, Formula 011, Formula 013, Formula 021 and Formula 086, wherein n is an integer from 1 to 100, preferably from 31 to 90. Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

SEQUENCE LISTING SEQUENCE LISTING

<110> Pfizer Inc. <110> Pfizer Inc. Chorro, Laurent O. Chorro, Laurent 0. <120> ESCHERICHIA COLI COMPOSITIONS AND METHODS THEREOF <120> ESCHERICHIA COLI COMPOSITIONS AND METHODS THEREOF

<130> PC72671 <130> PC72671

<160> 113 <160> 113

<170> PatentIn version 3.5 <170> PatentIn version 3.5

<210> 1 <210> 1 <211> 300 <211> 300 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 1 <400> 1

Met Lys Arg Val Ile Thr Leu Phe Ala Val Leu Leu Met Gly Trp Ser Met Lys Arg Val Ile Thr Leu Phe Ala Val Leu Leu Met Gly Trp Ser 1 5 10 15 1 5 10 15

Val Asn Ala Trp Ser Phe Ala Cys Lys Thr Ala Asn Gly Thr Ala Ile Val Asn Ala Trp Ser Phe Ala Cys Lys Thr Ala Asn Gly Thr Ala Ile 20 25 30 20 25 30

Pro Ile Gly Gly Gly Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Pro Ile Gly Gly Gly Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val 35 40 45 35 40 45

Val Asn Val Gly Gln Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Val Asn Val Gly Gln Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe 50 55 60 50 55 60

Cys His Asn Asp Tyr Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Cys His Asn Asp Tyr Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln 65 70 75 80 70 75 80

Arg Gly Ser Ala Tyr Gly Gly Val Leu Ser Asn Phe Ser Gly Thr Val Arg Gly Ser Ala Tyr Gly Gly Val Leu Ser Asn Phe Ser Gly Thr Val 85 90 95 85 90 95

Lys Tyr Ser Gly Ser Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Lys Tyr Ser Gly Ser Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro 100 105 110 100 105 110

Arg Val Val Tyr Asn Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Arg Val Val Tyr Asn Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu 115 120 125 115 120 125

Tyr Leu Thr Pro Val Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Tyr Leu Thr Pro Val Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly 130 135 140 130 135 140

Ser Leu Ile Ala Val Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Ser Leu Ile Ala Val Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser 145 150 155 160 145 150 155 160

Asp Asp Phe Gln Phe Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Asp Asp Phe Gln Phe Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val 165 170 175 165 170 175

Val Pro Thr Gly Gly Cys Asp Val Ser Ala Arg Asp Val Thr Val Thr Val Pro Thr Gly Gly Cys Asp Val Ser Ala Arg Asp Val Thr Val Thr 180 185 190 180 185 190

Leu Pro Asp Tyr Pro Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys Leu Pro Asp Tyr Pro Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys 195 200 205 195 200 205

Ala Lys Ser Gln Asn Leu Gly Tyr Tyr Leu Ser Gly Thr Thr Ala Asp Ala Lys Ser Gln Asn Leu Gly Tyr Tyr Leu Ser Gly Thr Thr Ala Asp 210 215 220 210 215 220

Ala Gly Asn Ser Ile Phe Thr Asn Thr Ala Ser Phe Ser Pro Ala Gln Ala Gly Asn Ser Ile Phe Thr Asn Thr Ala Ser Phe Ser Pro Ala Gln 225 230 235 240 225 230 235 240

Gly Val Gly Val Gln Leu Thr Arg Asn Gly Thr Ile Ile Pro Ala Asn Gly Val Gly Val Gln Leu Thr Arg Asn Gly Thr Ile Ile Pro Ala Asn 245 250 255 245 250 255

Asn Thr Val Ser Leu Gly Ala Val Gly Thr Ser Ala Val Ser Leu Gly Asn Thr Val Ser Leu Gly Ala Val Gly Thr Ser Ala Val Ser Leu Gly 260 265 270 260 265 270

Leu Thr Ala Asn Tyr Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn Leu Thr Ala Asn Tyr Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn 275 280 285 275 280 285

Val Gln Ser Ile Ile Gly Val Thr Phe Val Tyr Gln Val Gln Ser Ile Ile Gly Val Thr Phe Val Tyr Gln 290 295 300 290 295 300

<210> 2 <210> 2 <211> 279 <211> 279 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 2 <400> 2

Phe Ala Cys Lys Thr Ala Asn Gly Thr Ala Ile Pro Ile Gly Gly Gly Phe Ala Cys Lys Thr Ala Asn Gly Thr Ala Ile Pro Ile Gly Gly Gly 1 5 10 15 1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln 20 25 30 20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr 35 40 45 35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr 50 55 60 50 55 60

Gly Gly Val Leu Ser Asn Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser Gly Gly Val Leu Ser Asn Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser 65 70 75 80 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn 85 90 95 85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val 100 105 110 100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val 115 120 125 115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe 130 135 140 130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly 145 150 155 160 145 150 155 160

Cys Asp Val Ser Ala Arg Asp Val Thr Val Thr Leu Pro Asp Tyr Pro Cys Asp Val Ser Ala Arg Asp Val Thr Val Thr Leu Pro Asp Tyr Pro 165 170 175 165 170 175

Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys Ala Lys Ser Gln Asn Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys Ala Lys Ser Gln Asn 180 185 190 180 185 190

Leu Gly Tyr Tyr Leu Ser Gly Thr Thr Ala Asp Ala Gly Asn Ser Ile Leu Gly Tyr Tyr Leu Ser Gly Thr Thr Ala Asp Ala Gly Asn Ser Ile 195 200 205 195 200 205

Phe Thr Asn Thr Ala Ser Phe Ser Pro Ala Gln Gly Val Gly Val Gln Phe Thr Asn Thr Ala Ser Phe Ser Pro Ala Gln Gly Val Gly Val Gln

210 215 220 210 215 220

Leu Thr Arg Asn Gly Thr Ile Ile Pro Ala Asn Asn Thr Val Ser Leu Leu Thr Arg Asn Gly Thr Ile Ile Pro Ala Asn Asn Thr Val Ser Leu 225 230 235 240 225 230 235 240

Gly Ala Val Gly Thr Ser Ala Val Ser Leu Gly Leu Thr Ala Asn Tyr Gly Ala Val Gly Thr Ser Ala Val Ser Leu Gly Leu Thr Ala Asn Tyr 245 250 255 245 250 255

Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn Val Gln Ser Ile Ile Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn Val Gln Ser Ile Ile 260 265 270 260 265 270

Gly Val Thr Phe Val Tyr Gln Gly Val Thr Phe Val Tyr Gln 275 275

<210> 3 <210> 3 <211> 156 <211> 156 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 3 <400> 3

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110 100 105 110

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val 145 150 155 145 150 155

<210> 4 <210> 4 <211> 120 <211> 120 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 4 <400> 4

Gly Cys Asp Val Ser Ala Arg Asp Val Thr Val Thr Leu Pro Asp Tyr Gly Cys Asp Val Ser Ala Arg Asp Val Thr Val Thr Leu Pro Asp Tyr 1 5 10 15 1 5 10 15

Pro Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys Ala Lys Ser Gln Pro Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys Ala Lys Ser Gln 20 25 30 20 25 30

Asn Leu Gly Tyr Tyr Leu Ser Gly Thr Thr Ala Asp Ala Gly Asn Ser Asn Leu Gly Tyr Tyr Leu Ser Gly Thr Thr Ala Asp Ala Gly Asn Ser 35 40 45 35 40 45

Ile Phe Thr Asn Thr Ala Ser Phe Ser Pro Ala Gln Gly Val Gly Val Ile Phe Thr Asn Thr Ala Ser Phe Ser Pro Ala Gln Gly Val Gly Val 50 55 60 50 55 60

Gln Leu Thr Arg Asn Gly Thr Ile Ile Pro Ala Asn Asn Thr Val Ser Gln Leu Thr Arg Asn Gly Thr Ile Ile Pro Ala Asn Asn Thr Val Ser 65 70 75 80 70 75 80

Leu Gly Ala Val Gly Thr Ser Ala Val Ser Leu Gly Leu Thr Ala Asn Leu Gly Ala Val Gly Thr Ser Ala Val Ser Leu Gly Leu Thr Ala Asn 85 90 95 85 90 95

Tyr Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn Val Gln Ser Ile Tyr Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn Val Gln Ser Ile 100 105 110 100 105 110

Ile Gly Val Thr Phe Val Tyr Gln Ile Gly Val Thr Phe Val Tyr Gln

115 120 115 120

<210> 5 <210> 5 <211> 330 <211> 330 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 5 <400> 5

Val Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro Val Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 1 5 10 15

Gly Ser Thr Gly Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Gly Ser Thr Gly Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro 20 25 30 20 25 30

Ile Gly Gly Gly Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Cys Val Ile Gly Gly Gly Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Cys Val 35 40 45 35 40 45

Asn Val Gly Gln Asn Cys Val Val Asp Leu Ser Thr Gln Ile Phe Cys Asn Val Gly Gln Asn Cys Val Val Asp Leu Ser Thr Gln Ile Phe Cys 50 55 60 50 55 60

His Asn Asp Tyr Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg His Asn Asp Tyr Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg 65 70 75 80 70 75 80

Gly Ser Ala Tyr Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Gly Ser Ala Tyr Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys 85 90 95 85 90 95

Tyr Ser Gly Ser Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Tyr Ser Gly Ser Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg 100 105 110 100 105 110

Val Val Tyr Asn Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Val Val Tyr Asn Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr 115 120 125 115 120 125

Leu Thr Pro Val Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Thr Pro Val Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser 130 135 140 130 135 140

Leu Ile Ala Val Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Leu Ile Ala Val Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp 145 150 155 160 145 150 155 160

Asp Phe Gln Phe Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Asp Phe Gln Phe Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val

165 170 175 165 170 175

Pro Thr Gly Gly Cys Asp Val Ser Ala Arg Asp Val Thr Val Thr Leu Pro Thr Gly Gly Cys Asp Val Ser Ala Arg Asp Val Thr Val Thr Leu 180 185 190 180 185 190

Pro Asp Tyr Pro Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys Ala Pro Asp Tyr Pro Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys Ala 195 200 205 195 200 205

Lys Ser Gln Asn Leu Gly Tyr Tyr Leu Ser Gly Thr Thr Ala Asp Ala Lys Ser Gln Asn Leu Gly Tyr Tyr Leu Ser Gly Thr Thr Ala Asp Ala 210 215 220 210 215 220

Gly Asn Ser Ile Phe Thr Asn Thr Ala Ser Phe Ser Pro Ala Gln Gly Gly Asn Ser Ile Phe Thr Asn Thr Ala Ser Phe Ser Pro Ala Gln Gly 225 230 235 240 225 230 235 240

Val Gly Val Gln Leu Thr Arg Gln Gly Thr Ile Ile Pro Ala Asn Asn Val Gly Val Gln Leu Thr Arg Gln Gly Thr Ile Ile Pro Ala Asn Asn 245 250 255 245 250 255

Thr Val Ser Leu Gly Ala Val Gly Thr Ser Ala Val Ser Leu Gly Leu Thr Val Ser Leu Gly Ala Val Gly Thr Ser Ala Val Ser Leu Gly Leu 260 265 270 260 265 270

Thr Ala Asn Tyr Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn Val Thr Ala Asn Tyr Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn Val 275 280 285 275 280 285

Gln Ser Ile Ile Gly Val Thr Phe Val Tyr Gln Gly Gly Ser Ser Gly Gln Ser Ile Ile Gly Val Thr Phe Val Tyr Gln Gly Gly Ser Ser Gly 290 295 300 290 295 300

Gly Gly Ala Asp Val Thr Ile Thr Val Asn Gly Lys Val Val Ala Lys Gly Gly Ala Asp Val Thr Ile Thr Val Asn Gly Lys Val Val Ala Lys 305 310 315 320 305 310 315 320

Gly Gly His His His His His His His His Gly Gly His His His His His His His His 325 330 325 330

<210> 6 <210> 6 <211> 330 <211> 330 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 6 <400> 6

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15 1 5 10 15

Ile Gly Gly Gly Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Ile Gly Gly Gly Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val 35 40 45 35 40 45

Asn Val Gly Gln Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys Asn Val Gly Gln Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys 50 55 60 50 55 60

Asp Phe Gln Phe Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Asp Phe Gln Phe Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val 165 170 175 165 170 175

<210> 7 <210> 7 <211> 188 <211> 188 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 7 <400> 7

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 1 5 10 15

Pro Thr Gly Gly His His His His His His His His Pro Thr Gly Gly His His His His His His His His 180 185 180 185

<210> 8 <210> 8 <211> 188 <211> 188 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 8 <400> 8

<210> 9 < 210> 9 <211> 14 <211> 14 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 9 <400> 9

Ala Asp Val Thr Ile Thr Val Asn Gly Lys Val Val Ala Lys Ala Asp Val Thr Ile Thr Val Asn Gly Lys Val Val Ala Lys 1 5 10 1 5 10

<210> 10 <210> 10 <211> 241 <211> 241 <212> PRT <212> PRT

<213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 10 <400> 10

Met Ser Asn Lys Asn Val Asn Val Arg Lys Ser Gln Glu Ile Thr Phe Met Ser Asn Lys Asn Val Asn Val Arg Lys Ser Gln Glu Ile Thr Phe 1 5 10 15 1 5 10 15

Cys Leu Leu Ala Gly Ile Leu Met Phe Met Ala Met Met Val Ala Gly Cys Leu Leu Ala Gly Ile Leu Met Phe Met Ala Met Met Val Ala Gly 20 25 30 20 25 30

Arg Ala Glu Ala Gly Val Ala Leu Gly Ala Thr Arg Val Ile Tyr Pro Arg Ala Glu Ala Gly Val Ala Leu Gly Ala Thr Arg Val Ile Tyr Pro 35 40 45 35 40 45

Ala Gly Gln Lys Gln Val Gln Leu Ala Val Thr Asn Asn Asp Glu Asn Ala Gly Gln Lys Gln Val Gln Leu Ala Val Thr Asn Asn Asp Glu Asn 50 55 60 50 55 60

Ser Thr Tyr Leu Ile Gln Ser Trp Val Glu Asn Ala Asp Gly Val Lys Ser Thr Tyr Leu Ile Gln Ser Trp Val Glu Asn Ala Asp Gly Val Lys 65 70 75 80 70 75 80

Asp Gly Arg Phe Ile Val Thr Pro Pro Leu Phe Ala Met Lys Gly Lys Asp Gly Arg Phe Ile Val Thr Pro Pro Leu Phe Ala Met Lys Gly Lys 85 90 95 85 90 95

Lys Glu Asn Thr Leu Arg Ile Leu Asp Ala Thr Asn Asn Gln Leu Pro Lys Glu Asn Thr Leu Arg Ile Leu Asp Ala Thr Asn Asn Gln Leu Pro 100 105 110 100 105 110

Gln Asp Arg Glu Ser Leu Phe Trp Met Asn Val Lys Ala Ile Pro Ser Gln Asp Arg Glu Ser Leu Phe Trp Met Asn Val Lys Ala Ile Pro Ser 115 120 125 115 120 125

Met Asp Lys Ser Lys Leu Thr Glu Asn Thr Leu Gln Leu Ala Ile Ile Met Asp Lys Ser Lys Leu Thr Glu Asn Thr Leu Gln Leu Ala Ile Ile 130 135 140 130 135 140

Ser Arg Ile Lys Leu Tyr Tyr Arg Pro Ala Lys Leu Ala Leu Pro Pro Ser Arg Ile Lys Leu Tyr Tyr Arg Pro Ala Lys Leu Ala Leu Pro Pro 145 150 155 160 145 150 155 160

Asp Gln Ala Ala Glu Lys Leu Arg Phe Arg Arg Ser Ala Asn Ser Leu Asp Gln Ala Ala Glu Lys Leu Arg Phe Arg Arg Ser Ala Asn Ser Leu 165 170 175 165 170 175

Thr Leu Ile Asn Pro Thr Pro Tyr Tyr Leu Thr Val Thr Glu Leu Asn Thr Leu Ile Asn Pro Thr Pro Tyr Tyr Leu Thr Val Thr Glu Leu Asn 180 185 190 180 185 190

Ala Gly Thr Arg Val Leu Glu Asn Ala Leu Val Pro Pro Met Gly Glu Ala Gly Thr Arg Val Leu Glu Asn Ala Leu Val Pro Pro Met Gly Glu 195 200 205 195 200 205

Ser Thr Val Lys Leu Pro Ser Asp Ala Gly Ser Asn Ile Thr Tyr Arg Ser Thr Val Lys Leu Pro Ser Asp Ala Gly Ser Asn Ile Thr Tyr Arg 210 215 220 210 215 220

Thr Ile Asn Asp Tyr Gly Ala Leu Thr Pro Lys Met Thr Gly Val Met Thr Ile Asn Asp Tyr Gly Ala Leu Thr Pro Lys Met Thr Gly Val Met 225 230 235 240 225 230 235 240

Glu Glu

<210> 11 <210> 11 <211> 4 <211> 4 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 11 <400> 11

Asp Asn Lys Gln Asp Asn Lys Gln 1 1

<210> 12 <210> 12 <211> 5 <211> 5 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 12 <400> 12

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 1 5 1 5

<210> 13 <210> 13 <211> 6 <211> 6 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 13 <400> 13

Gly Gly Ser Ser Gly Gly Gly Gly Ser Ser Gly Gly 1 5 1 5

<210> 14 <210> 14 <211> 7 <211> 7 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 14 <400> 14

Gly Gly Ser Ser Gly Gly Gly Gly Gly Ser Ser Gly Gly Gly 1 5 1 5

<210> 15 <210> 15 <211> 8 <211> 8 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 15 <400> 15

Gly Gly Gly Ser Ser Gly Gly Gly Gly Gly Gly Ser Ser Gly Gly Gly 1 5 1 5

<210> 16 <210> 16 <211> 9 <211> 9 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 16 <400> 16

Gly Gly Gly Ser Gly Ser Gly Gly Gly Gly Gly Gly Ser Gly Ser Gly Gly Gly 1 5 1 5

<210> 17 <210> 17 <211> 10 <211> 10 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 17 <400> 17

Gly Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Gly Gly Ser Gly Gly Ser Gly Gly Gly 1 5 10 1 5 10

<210> 18 <210> 18 <211> 21 <211> 21 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 18 <400> 18

Val Asn Ala Trp Ser Val Asn Ala Trp Ser 20 20

<210> 19 <210> 19 <211> 20 <211> 20 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 19 <400> 19

Gly Ser Thr Gly Gly Ser Thr Gly 20 20

<210> 20 <210> 20 <211> 279 <211> 279 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 20 <400> 20

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15 1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Cys Val Asn Val Gly Gln Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Cys Val Asn Val Gly Gln 20 25 30 20 25 30

Asn Cys Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr Asn Cys Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr 35 40 45 35 40 45

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser 65 70 75 80 70 75 80

Phe Thr Asn Thr Ala Ser Phe Ser Pro Ala Gln Gly Val Gly Val Gln Phe Thr Asn Thr Ala Ser Phe Ser Pro Ala Gln Gly Val Gly Val Gln 210 215 220 210 215 220

Leu Thr Arg Gln Gly Thr Ile Ile Pro Ala Asn Asn Thr Val Ser Leu Leu Thr Arg Gln Gly Thr Ile Ile Pro Ala Asn Asn Thr Val Ser Leu 225 230 235 240 225 230 235 240

Gly Val Thr Phe Val Tyr Gln Gly Val Thr Phe Val Tyr Gln 275 275

<210> 21 <210> 21 <211> 24 <211> 24 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 21 <400> 21

Ala Asp Val Thr Ile Thr Val Asn Gly Lys Val Val Ala Lys Gly Gly Ala Asp Val Thr Ile Thr Val Asn Gly Lys Val Val Ala Lys Gly Gly 1 5 10 15 1 5 10 15

His His His His His His His His His His His His His His His His 20 20

<210> 22 <210> 22 <211> 20 <211> 20 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 22 <400> 22

Gly Ser Thr Gly Gly Ser Thr Gly 20 20

<210> 23 <210> 23

<211> 279 <211> 279 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 23 <400> 23

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly 1 5 10 15 1 5 10 15

Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys Ala Lys Ser Gln Asn Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys Ala Lys Ser Gln Asn

180 185 190 180 185 190

Gly Val Thr Phe Val Tyr Gln Gly Val Thr Phe Val Tyr Gln 275 275

<210> 24 <210> 24 <211> 160 <211> 160 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 24 <400> 24

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80 70 75 80

<210> 25 <210> 25 <211> 8 <211> 8 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 25 <400> 25

His His His His His His His His His His His His His His His His 1 5 1 5

<210> 26 <210> 26 <211> 160 <211> 160 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 26 <400> 26

<210> 27 < 220 27 <211> 168 <211> 168 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 27 <400> 27

His His His His His His His His His His His His His His His His 165 165

<210> 28 <210> 28 <211> 279 <211> 279 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 28 <400> 28

Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn Val Gln Ser Ile Ile Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn Val Gln Ser Ile Ile

260 265 270 260 265 270

Gly Val Thr Phe Val Tyr Gln Gly Val Thr Phe Val Tyr Gln 275 275

<210> 29 <210> 29 <211> 279 <211> 279 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 29 <400> 29

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Ala Val Asn Val Gly Gln Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Ala Val Asn Val Gly Gln 20 25 30 20 25 30

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ala Ala Tyr Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ala Ala Tyr 50 55 60 50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Asn Gly Ser Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Asn Gly Ser 65 70 75 80 70 75 80

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160 145 150 155 160

Gly Val Thr Phe Val Tyr Gln Gly Val Thr Phe Val Tyr Gln 275 275

<210> 30 <210> 30 <211> 325 <211> 325 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 30 <400> 30

Met Arg Val Glu Asn Asn Asn Val Ser Gly Gln Asn His Asp Pro Glu Met Arg Val Glu Asn Asn Asn Val Ser Gly Gln Asn His Asp Pro Glu 1 5 10 15 1 5 10 15

Gln Ile Asp Leu Ile Asp Leu Leu Val Gln Leu Trp Arg Gly Lys Met Gln Ile Asp Leu Ile Asp Leu Leu Val Gln Leu Trp Arg Gly Lys Met 20 25 30 20 25 30

Thr Ile Ile Ile Ser Val Ile Val Ala Ile Ala Leu Ala Ile Gly Tyr Thr Ile Ile Ile Ser Val Ile Val Ala Ile Ala Leu Ala Ile Gly Tyr

35 40 45 35 40 45

Leu Ala Val Ala Lys Glu Lys Trp Thr Ser Thr Ala Ile Ile Thr Gln Leu Ala Val Ala Lys Glu Lys Trp Thr Ser Thr Ala Ile Ile Thr Gln 50 55 60 50 55 60

Pro Asp Val Gly Gln Ile Ala Gly Tyr Asn Asn Ala Met Asn Val Ile Pro Asp Val Gly Gln Ile Ala Gly Tyr Asn Asn Ala Met Asn Val Ile 65 70 75 80 70 75 80

Tyr Gly Gln Ala Ala Pro Lys Val Ser Asp Leu Gln Glu Thr Leu Ile Tyr Gly Gln Ala Ala Pro Lys Val Ser Asp Leu Gln Glu Thr Leu Ile 85 90 95 85 90 95

Gly Arg Phe Ser Ser Ala Phe Ser Ala Leu Ala Glu Thr Leu Asp Asn Gly Arg Phe Ser Ser Ala Phe Ser Ala Leu Ala Glu Thr Leu Asp Asn 100 105 110 100 105 110

Gln Glu Glu Pro Glu Lys Leu Thr Ile Glu Pro Ser Val Lys Asn Gln Gln Glu Glu Pro Glu Lys Leu Thr Ile Glu Pro Ser Val Lys Asn Gln 115 120 125 115 120 125

Gln Leu Pro Leu Thr Val Ser Tyr Val Gly Gln Thr Ala Glu Gly Ala Gln Leu Pro Leu Thr Val Ser Tyr Val Gly Gln Thr Ala Glu Gly Ala 130 135 140 130 135 140

Gln Met Lys Leu Ala Gln Tyr Ile Gln Gln Val Asp Asp Lys Val Asn Gln Met Lys Leu Ala Gln Tyr Ile Gln Gln Val Asp Asp Lys Val Asn 145 150 155 160 145 150 155 160

Gln Glu Leu Glu Lys Asp Leu Lys Asp Asn Ile Ala Leu Gly Arg Lys Gln Glu Leu Glu Lys Asp Leu Lys Asp Asn Ile Ala Leu Gly Arg Lys 165 170 175 165 170 175

Asn Leu Gln Asp Ser Leu Arg Thr Gln Glu Val Val Ala Gln Glu Gln Asn Leu Gln Asp Ser Leu Arg Thr Gln Glu Val Val Ala Gln Glu Gln 180 185 190 180 185 190

Lys Asp Leu Arg Ile Arg Gln Ile Gln Glu Ala Leu Gln Tyr Ala Asn Lys Asp Leu Arg Ile Arg Gln Ile Gln Glu Ala Leu Gln Tyr Ala Asn 195 200 205 195 200 205

Gln Glu Gln Val Thr Lys Pro Gln Val Gln Gln Thr Glu Asp Val Thr Gln Glu Gln Val Thr Lys Pro Gln Val Gln Gln Thr Glu Asp Val Thr 210 215 220 210 215 220

Gln Asp Thr Leu Phe Leu Leu Gly Ser Glu Ala Leu Glu Ser Met Ile Gln Asp Thr Leu Phe Leu Leu Gly Ser Glu Ala Leu Glu Ser Met Ile 225 230 235 240 225 230 235 240

Lys His Glu Ala Thr Arg Pro Leu Val Phe Ser Ser Asn Tyr Tyr Gln Lys His Glu Ala Thr Arg Pro Leu Val Phe Ser Ser Asn Tyr Tyr Gln 245 250 255 245 250 255

Thr Arg Gln Asn Leu Leu Asp Ile Glu Ser Leu Lys Val Asp Asp Leu Thr Arg Gln Asn Leu Leu Asp Ile Glu Ser Leu Lys Val Asp Asp Leu 260 265 270 260 265 270

Asp Ile His Ala Tyr Arg Tyr Val Met Lys Pro Thr Leu Pro Ile Arg Asp Ile His Ala Tyr Arg Tyr Val Met Lys Pro Thr Leu Pro Ile Arg 275 280 285 275 280 285

Arg Asp Ser Pro Lys Lys Ala Ile Thr Leu Ile Leu Ala Val Leu Leu Arg Asp Ser Pro Lys Lys Ala Ile Thr Leu Ile Leu Ala Val Leu Leu 290 295 300 290 295 300

Gly Gly Met Val Gly Ala Gly Ile Val Leu Gly Arg Asn Ala Leu Arg Gly Gly Met Val Gly Ala Gly Ile Val Leu Gly Arg Asn Ala Leu Arg 305 310 315 320 305 310 315 320

Asn Tyr Asn Ala Lys Asn Tyr Asn Ala Lys 325 325

<210> 31 <210> 31 <211> 326 <211> 326 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 31 <400> 31

Met Arg Val Glu Asn Asn Asn Val Ser Gly Gln Asn Asn Asp Pro Glu Met Arg Val Glu Asn Asn Asn Val Ser Gly Gln Asn Asn Asp Pro Glu 1 5 10 15 1 5 10 15

Thr Ile Ile Ile Ser Val Ile Val Ala Ile Ala Leu Ala Ile Gly Tyr Thr Ile Ile Ile Ser Val Ile Val Ala Ile Ala Leu Ala Ile Gly Tyr 35 40 45 35 40 45

Gln Asp Glu Pro Glu Lys Leu Thr Ile Glu Pro Ser Val Lys Asn Gln Gln Asp Glu Pro Glu Lys Leu Thr Ile Glu Pro Ser Val Lys Asn Gln 115 120 125 115 120 125

Gln Ala Gln Val Thr Lys Pro Gln Ile Gln Gln Thr Gly Glu Asp Ile Gln Ala Gln Val Thr Lys Pro Gln Ile Gln Gln Thr Gly Glu Asp Ile 210 215 220 210 215 220

Thr Gln Asp Thr Leu Phe Leu Leu Gly Ser Glu Ala Leu Glu Ser Met Thr Gln Asp Thr Leu Phe Leu Leu Gly Ser Glu Ala Leu Glu Ser Met 225 230 235 240 225 230 235 240

Ile Lys His Glu Ala Thr Arg Pro Leu Val Phe Ser Pro Asn Tyr Tyr Ile Lys His Glu Ala Thr Arg Pro Leu Val Phe Ser Pro Asn Tyr Tyr 245 250 255 245 250 255

Gln Thr Arg Gln Asn Leu Leu Asp Ile Glu Ser Leu Lys Val Asp Asp Gln Thr Arg Gln Asn Leu Leu Asp Ile Glu Ser Leu Lys Val Asp Asp 260 265 270 260 265 270

Leu Asp Ile His Ala Tyr Arg Tyr Val Met Lys Pro Thr Leu Pro Ile Leu Asp Ile His Ala Tyr Arg Tyr Val Met Lys Pro Thr Leu Pro Ile 275 280 285 275 280 285

Arg Arg Asp Ser Pro Lys Lys Ala Ile Thr Leu Ile Leu Ala Val Leu Arg Arg Asp Ser Pro Lys Lys Ala Ile Thr Leu Ile Leu Ala Val Leu 290 295 300 290 295 300

Leu Gly Gly Met Val Gly Ala Gly Ile Val Leu Gly Arg Asn Ala Leu Leu Gly Gly Met Val Gly Ala Gly Ile Val Leu Gly Arg Asn Ala Leu

305 310 315 320 305 310 315 320

Arg Asn Tyr Asn Ala Lys Arg Asn Tyr Asn Ala Lys 325 325

<210> 32 <210> 32 <211> 326 <211> 326 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 32 <400> 32

Thr Ile Ile Ile Ser Val Val Val Ala Ile Ala Leu Ala Ile Gly Tyr Thr Ile Ile Ile Ser Val Val Val Ala Ile Ala Leu Ala Ile Gly Tyr 35 40 45 35 40 45

Gly Arg Phe Ser Phe Ala Phe Ser Ala Leu Ala Glu Thr Leu Asp Asn Gly Arg Phe Ser Phe Ala Phe Ser Ala Leu Ala Glu Thr Leu Asp Asn 100 105 110 100 105 110

Gln Lys Glu Pro Glu Lys Leu Thr Ile Glu Pro Ser Val Lys Asn Gln Gln Lys Glu Pro Glu Lys Leu Thr Ile Glu Pro Ser Val Lys Asn Gln 115 120 125 115 120 125

Gln Leu Pro Leu Thr Val Ser Tyr Val Gly Gln Thr Ala Glu Asp Ala Gln Leu Pro Leu Thr Val Ser Tyr Val Gly Gln Thr Ala Glu Asp Ala 130 135 140 130 135 140

Gln Met Lys Leu Ala Gln Tyr Ile Gln Gln Val Asp Asp Lys Val Asn Gln Met Lys Leu Ala Gln Tyr Ile Gln Gln Val Asp Asp Lys Val Asn

145 150 155 160 145 150 155 160

Gln Glu Leu Glu Lys Asp Leu Lys Asp Asn Leu Ala Leu Gly Arg Lys Gln Glu Leu Glu Lys Asp Leu Lys Asp Asn Leu Ala Leu Gly Arg Lys 165 170 175 165 170 175

Gln Thr Arg Gln Asn Leu Leu Asp Ile Glu Asn Leu Lys Val Asp Asp Gln Thr Arg Gln Asn Leu Leu Asp Ile Glu Asn Leu Lys Val Asp Asp 260 265 270 260 265 270

Leu Gly Gly Met Val Gly Ala Gly Ile Val Leu Gly Arg Asn Ala Leu Leu Gly Gly Met Val Gly Ala Gly Ile Val Leu Gly Arg Asn Ala Leu 305 310 315 320 305 310 315 320

Arg Asn Tyr Asn Ser Lys Arg Asn Tyr Asn Ser Lys 325 325

<210> 33 <210> 33 <211> 326 <211> 326 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 33 <400> 33

Gln Glu Glu Arg Glu Lys Leu Thr Ile Glu Pro Ser Val Lys Asn Gln Gln Glu Glu Arg Glu Lys Leu Thr Ile Glu Pro Ser Val Lys Asn Gln 115 120 125 115 120 125

Leu Asp Ile His Ala Tyr Arg Tyr Val Met Lys Pro Met Leu Pro Ile Leu Asp Ile His Ala Tyr Arg Tyr Val Met Lys Pro Met Leu Pro Ile 275 280 285 275 280 285

Arg Asn Tyr Asn Ala Lys Arg Asn Tyr Asn Ala Lys 325 325

<210> 34 <210> 34 <211> 327 <211> 327 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 34 <400> 34

Met Thr Val Asp Ser Asn Thr Ser Ser Gly Arg Gly Asn Asp Pro Glu Met Thr Val Asp Ser Asn Thr Ser Ser Gly Arg Gly Asn Asp Pro Glu 1 5 10 15 1 5 10 15

Gln Ile Asp Leu Ile Glu Leu Leu Leu Gln Leu Trp Arg Gly Lys Met Gln Ile Asp Leu Ile Glu Leu Leu Leu Gln Leu Trp Arg Gly Lys Met 20 25 30 20 25 30

Thr Ile Ile Val Ala Val Ile Ile Ala Ile Leu Leu Ala Val Gly Tyr Thr Ile Ile Val Ala Val Ile Ile Ala Ile Leu Leu Ala Val Gly Tyr 35 40 45 35 40 45

Leu Met Ile Ala Lys Glu Lys Trp Thr Ser Thr Ala Ile Ile Thr Gln Leu Met Ile Ala Lys Glu Lys Trp Thr Ser Thr Ala Ile Ile Thr Gln 50 55 60 50 55 60

Pro Asp Ala Ala Gln Val Ala Thr Tyr Thr Asn Ala Leu Asn Val Leu Pro Asp Ala Ala Gln Val Ala Thr Tyr Thr Asn Ala Leu Asn Val Leu 65 70 75 80 70 75 80

Tyr Gly Gly Asn Ala Pro Lys Ile Ser Glu Val Gln Ala Asn Phe Ile Tyr Gly Gly Asn Ala Pro Lys Ile Ser Glu Val Gln Ala Asn Phe Ile 85 90 95 85 90 95

Ser Arg Phe Ser Ser Ala Phe Ser Ala Leu Ser Glu Val Leu Asp Asn Ser Arg Phe Ser Ser Ala Phe Ser Ala Leu Ser Glu Val Leu Asp Asn 100 105 110 100 105 110

Gln Lys Glu Arg Glu Lys Leu Thr Ile Glu Gln Ser Val Lys Gly Gln Gln Lys Glu Arg Glu Lys Leu Thr Ile Glu Gln Ser Val Lys Gly Gln 115 120 125 115 120 125

Ala Leu Pro Leu Ser Val Ser Tyr Val Ser Thr Thr Ala Glu Gly Ala Ala Leu Pro Leu Ser Val Ser Tyr Val Ser Thr Thr Ala Glu Gly Ala 130 135 140 130 135 140

Gln Arg Arg Leu Ala Glu Tyr Ile Gln Gln Val Asp Glu Glu Val Ala Gln Arg Arg Leu Ala Glu Tyr Ile Gln Gln Val Asp Glu Glu Val Ala 145 150 155 160 145 150 155 160

Lys Glu Leu Glu Val Asp Leu Lys Asp Asn Ile Thr Leu Gln Thr Lys Lys Glu Leu Glu Val Asp Leu Lys Asp Asn Ile Thr Leu Gln Thr Lys 165 170 175 165 170 175

Thr Leu Gln Glu Ser Leu Glu Thr Gln Glu Val Val Ala Gln Glu Gln Thr Leu Gln Glu Ser Leu Glu Thr Gln Glu Val Val Ala Gln Glu Gln 180 185 190 180 185 190

Lys Asp Leu Arg Ile Lys Gln Ile Glu Glu Ala Leu Arg Tyr Ala Asp Lys Asp Leu Arg Ile Lys Gln Ile Glu Glu Ala Leu Arg Tyr Ala Asp 195 200 205 195 200 205

Glu Ala Lys Ile Thr Gln Pro Gln Ile Gln Gln Thr Gln Asp Val Thr Glu Ala Lys Ile Thr Gln Pro Gln Ile Gln Gln Thr Gln Asp Val Thr 210 215 220 210 215 220

Gln Asp Thr Met Phe Leu Leu Gly Ser Asp Ala Leu Lys Ser Met Ile Gln Asp Thr Met Phe Leu Leu Gly Ser Asp Ala Leu Lys Ser Met Ile 225 230 235 240 225 230 235 240

Gln Asn Glu Ala Thr Arg Pro Leu Val Phe Ser Pro Ala Tyr Tyr Gln Gln Asn Glu Ala Thr Arg Pro Leu Val Phe Ser Pro Ala Tyr Tyr Gln 245 250 255 245 250 255

Thr Lys Gln Thr Leu Leu Asp Ile Lys Asn Leu Lys Val Thr Ala Asp Thr Lys Gln Thr Leu Leu Asp Ile Lys Asn Leu Lys Val Thr Ala Asp

260 265 270 260 265 270

Thr Val His Val Tyr Arg Tyr Val Met Lys Pro Thr Leu Pro Val Arg Thr Val His Val Tyr Arg Tyr Val Met Lys Pro Thr Leu Pro Val Arg 275 280 285 275 280 285

Arg Asp Ser Pro Lys Thr Ala Ile Thr Leu Val Leu Ala Val Leu Leu Arg Asp Ser Pro Lys Thr Ala Ile Thr Leu Val Leu Ala Val Leu Leu 290 295 300 290 295 300

Gly Gly Met Ile Gly Ala Gly Ile Val Leu Gly Arg Asn Ala Leu Arg Gly Gly Met Ile Gly Ala Gly Ile Val Leu Gly Arg Asn Ala Leu Arg 305 310 315 320 305 310 315 320

Ser Tyr Lys Pro Lys Ala Leu Ser Tyr Lys Pro Lys Ala Leu 325 325

<210> 35 <210> 35 <211> 377 <211> 377 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 35 <400> 35

Met Ser Ser Leu Asn Ile Lys Gln Gly Ser Asp Ala His Phe Pro Asp Met Ser Ser Leu Asn Ile Lys Gln Gly Ser Asp Ala His Phe Pro Asp 1 5 10 15 1 5 10 15

Tyr Pro Leu Ala Ser Pro Ser Asn Asn Glu Ile Asp Leu Leu Asn Leu Tyr Pro Leu Ala Ser Pro Ser Asn Asn Glu Ile Asp Leu Leu Asn Leu 20 25 30 20 25 30

Ile Ser Val Leu Trp Arg Ala Lys Lys Thr Val Met Ala Val Val Phe Ile Ser Val Leu Trp Arg Ala Lys Lys Thr Val Met Ala Val Val Phe 35 40 45 35 40 45

Ala Phe Ala Cys Ala Gly Leu Leu Ile Ser Phe Ile Leu Pro Gln Lys Ala Phe Ala Cys Ala Gly Leu Leu Ile Ser Phe Ile Leu Pro Gln Lys 50 55 60 50 55 60

Trp Thr Ser Ala Ala Val Val Thr Pro Pro Glu Pro Val Gln Trp Gln Trp Thr Ser Ala Ala Val Val Thr Pro Pro Glu Pro Val Gln Trp Gln 65 70 75 80 70 75 80

Glu Leu Glu Lys Ser Phe Thr Lys Leu Arg Val Leu Asp Leu Asp Ile Glu Leu Glu Lys Ser Phe Thr Lys Leu Arg Val Leu Asp Leu Asp Ile 85 90 95 85 90 95

Lys Ile Asp Arg Thr Glu Ala Phe Asn Leu Phe Ile Lys Lys Phe Gln Lys Ile Asp Arg Thr Glu Ala Phe Asn Leu Phe Ile Lys Lys Phe Gln

100 105 110 100 105 110

Ser Val Ser Leu Leu Glu Glu Tyr Leu Arg Ser Ser Pro Tyr Val Met Ser Val Ser Leu Leu Glu Glu Tyr Leu Arg Ser Ser Pro Tyr Val Met 115 120 125 115 120 125

Asp Gln Leu Lys Glu Ala Lys Ile Asp Glu Leu Asp Leu His Arg Ala Asp Gln Leu Lys Glu Ala Lys Ile Asp Glu Leu Asp Leu His Arg Ala 130 135 140 130 135 140

Ile Val Ala Leu Ser Glu Lys Met Lys Ala Val Asp Asp Asn Ala Ser Ile Val Ala Leu Ser Glu Lys Met Lys Ala Val Asp Asp Asn Ala Ser 145 150 155 160 145 150 155 160

Lys Lys Lys Asp Glu Pro Ser Leu Tyr Thr Ser Trp Thr Leu Ser Phe Lys Lys Lys Asp Glu Pro Ser Leu Tyr Thr Ser Trp Thr Leu Ser Phe 165 170 175 165 170 175

Thr Ala Pro Thr Ser Glu Glu Ala Gln Thr Val Leu Ser Gly Tyr Ile Thr Ala Pro Thr Ser Glu Glu Ala Gln Thr Val Leu Ser Gly Tyr Ile 180 185 190 180 185 190

Asp Tyr Ile Ser Thr Leu Val Val Lys Glu Ser Leu Glu Asn Val Arg Asp Tyr Ile Ser Thr Leu Val Val Lys Glu Ser Leu Glu Asn Val Arg 195 200 205 195 200 205

Asn Lys Leu Glu Ile Lys Thr Gln Phe Glu Lys Glu Lys Leu Ala Gln Asn Lys Leu Glu Ile Lys Thr Gln Phe Glu Lys Glu Lys Leu Ala Gln 210 215 220 210 215 220

Asp Arg Ile Lys Thr Lys Asn Gln Leu Asp Ala Asn Ile Gln Arg Leu Asp Arg Ile Lys Thr Lys Asn Gln Leu Asp Ala Asn Ile Gln Arg Leu 225 230 235 240 225 230 235 240

Asn Tyr Ser Leu Asp Ile Ala Asn Ala Ala Gly Ile Lys Lys Pro Val Asn Tyr Ser Leu Asp Ile Ala Asn Ala Ala Gly Ile Lys Lys Pro Val 245 250 255 245 250 255

Tyr Ser Asn Gly Gln Ala Val Lys Asp Asp Pro Asp Phe Ser Ile Ser Tyr Ser Asn Gly Gln Ala Val Lys Asp Asp Pro Asp Phe Ser Ile Ser 260 265 270 260 265 270

Leu Gly Ala Asp Gly Ile Glu Arg Lys Leu Glu Ile Glu Lys Ala Val Leu Gly Ala Asp Gly Ile Glu Arg Lys Leu Glu Ile Glu Lys Ala Val 275 280 285 275 280 285

Thr Asp Val Ala Glu Leu Asn Gly Glu Leu Arg Asn Arg Gln Tyr Leu Thr Asp Val Ala Glu Leu Asn Gly Glu Leu Arg Asn Arg Gln Tyr Leu 290 295 300 290 295 300

Val Glu Gln Leu Thr Lys Ala His Val Asn Asp Val Asn Phe Thr Pro Val Glu Gln Leu Thr Lys Ala His Val Asn Asp Val Asn Phe Thr Pro 305 310 315 320 305 310 315 320

Phe Lys Tyr Gln Leu Ser Pro Ser Leu Pro Val Lys Lys Asp Gly Pro Phe Lys Tyr Gln Leu Ser Pro Ser Leu Pro Val Lys Lys Asp Gly Pro 325 330 335 325 330 335

Gly Lys Ala Ile Ile Val Ile Leu Ser Ala Leu Ile Gly Gly Met Val Gly Lys Ala Ile Ile Val Ile Leu Ser Ala Leu Ile Gly Gly Met Val 340 345 350 340 345 350

Ala Cys Gly Gly Val Leu Leu Arg Tyr Ala Met Ala Ser Arg Lys Gln Ala Cys Gly Gly Val Leu Leu Arg Tyr Ala Met Ala Ser Arg Lys Gln 355 360 365 355 360 365

Asp Ala Met Met Ala Asp His Leu Val Asp Ala Met Met Ala Asp His Leu Val 370 375 370 375

<210> 36 <210> 36 <211> 377 <211> 377 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 36 <400> 36

Met Ser Ser Leu Asn Ile Lys Gln Gly Ser Glu Ala His Phe Pro Glu Met Ser Ser Leu Asn Ile Lys Gln Gly Ser Glu Ala His Phe Pro Glu 1 5 10 15 1 5 10 15

Ile Glu Val Leu Trp Arg Ala Lys Lys Thr Val Met Ala Val Val Phe Ile Glu Val Leu Trp Arg Ala Lys Lys Thr Val Met Ala Val Val Phe 35 40 45 35 40 45

Glu Leu Glu Lys Thr Phe Thr Lys Leu Arg Val Leu Asp Leu Asp Ile Glu Leu Glu Lys Thr Phe Thr Lys Leu Arg Val Leu Asp Leu Asp Ile 85 90 95 85 90 95

Lys Ile Asp Arg Thr Glu Ala Phe Asn Leu Phe Ile Lys Lys Phe Gln Lys Ile Asp Arg Thr Glu Ala Phe Asn Leu Phe Ile Lys Lys Phe Gln 100 105 110 100 105 110

Asp Gln Leu Lys Glu Ala Lys Ile Asp Pro Leu Asp Leu His Arg Ala Asp Gln Leu Lys Glu Ala Lys Ile Asp Pro Leu Asp Leu His Arg Ala 130 135 140 130 135 140

Lys Lys Lys Asp Glu Ser Ala Leu Tyr Thr Ser Trp Thr Leu Ser Phe Lys Lys Lys Asp Glu Ser Ala Leu Tyr Thr Ser Trp Thr Leu Ser Phe 165 170 175 165 170 175

Thr Ala Pro Thr Ser Glu Glu Ala Gln Lys Val Leu Ala Gly Tyr Ile Thr Ala Pro Thr Ser Glu Glu Ala Gln Lys Val Leu Ala Gly Tyr Ile 180 185 190 180 185 190

Asp Tyr Ile Ser Ala Leu Val Val Lys Glu Ser Ile Glu Asn Val Arg Asp Tyr Ile Ser Ala Leu Val Val Lys Glu Ser Ile Glu Asn Val Arg 195 200 205 195 200 205

Val Glu Gln Leu Thr Lys Thr Asn Ile Asn Asp Val Asn Phe Thr Pro Val Glu Gln Leu Thr Lys Thr Asn Ile Asn Asp Val Asn Phe Thr Pro 305 310 315 320 305 310 315 320

Phe Lys Tyr Gln Leu Arg Pro Ser Leu Pro Val Lys Lys Asp Gly Gln Phe Lys Tyr Gln Leu Arg Pro Ser Leu Pro Val Lys Lys Asp Gly Gln

325 330 335 325 330 335

Gly Lys Ala Ile Ile Val Ile Leu Ser Ala Leu Val Gly Gly Met Val Gly Lys Ala Ile Ile Val Ile Leu Ser Ala Leu Val Gly Gly Met Val 340 345 350 340 345 350

Ala Cys Gly Gly Val Leu Leu Arg His Ala Met Ala Ser Arg Lys Gln Ala Cys Gly Gly Val Leu Leu Arg His Ala Met Ala Ser Arg Lys Gln 355 360 365 355 360 365

<210> 37 <210> 37 <211> 377 <211> 377 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 37 :400> 37

Ser Val Ser Leu Leu Glu Glu Tyr Leu Arg Ser Ser Pro Tyr Val Met Ser Val Ser Leu Leu Glu Glu Tyr Leu Arg Ser Ser Pro Tyr Val Met

115 120 125 115 120 125

<210> 38 <210> 38 <211> 377 <211> 377 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 38 <400> 38

Asp Arg Ile Lys Met Lys Asn Gln Leu Asp Ala Asn Ile Gln Arg Leu Asp Arg Ile Lys Met Lys Asn Gln Leu Asp Ala Asn Ile Gln Arg Leu 225 230 235 240 225 230 235 240

Val Glu Gln Leu Thr Lys Ala Asn Ile Asn Asp Val Asn Phe Thr Pro Val Glu Gln Leu Thr Lys Ala Asn Ile Asn Asp Val Asn Phe Thr Pro 305 310 315 320 305 310 315 320

Gly Lys Ala Ile Ile Val Ile Leu Ser Ala Leu Ile Gly Gly Met Val Gly Lys Ala Ile Ile Val Ile Leu Ser Ala Leu Ile Gly Gly Met Val

340 345 350 340 345 350

Ala Cys Gly Ser Val Leu Leu Arg Tyr Ala Met Ala Ser Arg Lys Gln Ala Cys Gly Ser Val Leu Leu Arg Tyr Ala Met Ala Ser Arg Lys Gln 355 360 365 355 360 365

<210> 39 <210> 39 <211> 378 <211> 378 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 39 <400> 39

Met Pro Ser Leu Asn Val Lys Gln Glu Lys Asn Gln Ser Phe Ala Gly Met Pro Ser Leu Asn Val Lys Gln Glu Lys Asn Gln Ser Phe Ala Gly 1 5 10 15 1 5 10 15

Tyr Ser Leu Pro Pro Ala Asn Ser His Glu Ile Asp Leu Phe Ser Leu Tyr Ser Leu Pro Pro Ala Asn Ser His Glu Ile Asp Leu Phe Ser Leu 20 25 30 20 25 30

Ile Glu Val Leu Trp Gln Ala Lys Arg Arg Ile Leu Ala Thr Val Phe Ile Glu Val Leu Trp Gln Ala Lys Arg Arg Ile Leu Ala Thr Val Phe 35 40 45 35 40 45

Ala Phe Ala Cys Val Gly Leu Leu Leu Ser Phe Leu Leu Pro Gln Lys Ala Phe Ala Cys Val Gly Leu Leu Leu Ser Phe Leu Leu Pro Gln Lys 50 55 60 50 55 60

Trp Thr Ser Gln Ala Ile Val Thr Pro Ala Glu Ser Val Gln Trp Gln Trp Thr Ser Gln Ala Ile Val Thr Pro Ala Glu Ser Val Gln Trp Gln 65 70 75 80 70 75 80

Gly Leu Glu Arg Thr Leu Thr Ala Leu Arg Val Leu Asp Met Glu Val Gly Leu Glu Arg Thr Leu Thr Ala Leu Arg Val Leu Asp Met Glu Val 85 90 95 85 90 95

Ser Val Asp Arg Gly Ser Val Phe Asn Leu Phe Ile Lys Lys Phe Ser Ser Val Asp Arg Gly Ser Val Phe Asn Leu Phe Ile Lys Lys Phe Ser 100 105 110 100 105 110

Ser Pro Ser Leu Leu Glu Glu Tyr Leu Arg Ser Ser Pro Tyr Val Met Ser Pro Ser Leu Leu Glu Glu Tyr Leu Arg Ser Ser Pro Tyr Val Met 115 120 125 115 120 125

Asp Gln Leu Lys Gly Ala Gln Ile Asp Glu Gln Asp Leu His Arg Ala Asp Gln Leu Lys Gly Ala Gln Ile Asp Glu Gln Asp Leu His Arg Ala

130 135 140 130 135 140

Ile Val Leu Leu Ser Glu Lys Met Lys Ala Val Asp Ser Asn Val Gly Ile Val Leu Leu Ser Glu Lys Met Lys Ala Val Asp Ser Asn Val Gly 145 150 155 160 145 150 155 160

Lys Lys Asn Glu Thr Ser Leu Phe Thr Ser Trp Thr Leu Ser Phe Thr Lys Lys Asn Glu Thr Ser Leu Phe Thr Ser Trp Thr Leu Ser Phe Thr 165 170 175 165 170 175

Ala Pro Thr Arg Glu Glu Ala Gln Lys Val Leu Ala Gly Tyr Ile Gln Ala Pro Thr Arg Glu Glu Ala Gln Lys Val Leu Ala Gly Tyr Ile Gln 180 185 190 180 185 190

Tyr Ile Ser Asp Ile Val Val Lys Glu Thr Leu Glu Asn Ile Arg Asn Tyr Ile Ser Asp Ile Val Val Lys Glu Thr Leu Glu Asn Ile Arg Asn 195 200 205 195 200 205

Gln Leu Glu Ile Lys Thr Arg Tyr Glu Gln Glu Lys Leu Ala Met Asp Gln Leu Glu Ile Lys Thr Arg Tyr Glu Gln Glu Lys Leu Ala Met Asp 210 215 220 210 215 220

Arg Val Arg Leu Lys Asn Gln Leu Asp Ala Asn Ile Gln Arg Leu His Arg Val Arg Leu Lys Asn Gln Leu Asp Ala Asn Ile Gln Arg Leu His 225 230 235 240 225 230 235 240

Tyr Ser Leu Glu Ile Ala Asn Ala Ala Gly Ile Lys Arg Pro Val Tyr Tyr Ser Leu Glu Ile Ala Asn Ala Ala Gly Ile Lys Arg Pro Val Tyr 245 250 255 245 250 255

Ser Asn Gly Gln Ala Val Lys Asp Asp Pro Asp Phe Ser Ile Ser Leu Ser Asn Gly Gln Ala Val Lys Asp Asp Pro Asp Phe Ser Ile Ser Leu 260 265 270 260 265 270

Gly Ala Asp Gly Ile Ser Arg Lys Leu Glu Ile Glu Lys Gly Val Thr Gly Ala Asp Gly Ile Ser Arg Lys Leu Glu Ile Glu Lys Gly Val Thr 275 280 285 275 280 285

Asp Val Ala Glu Ile Asp Gly Asp Leu Arg Asn Arg Gln Tyr His Val Asp Val Ala Glu Ile Asp Gly Asp Leu Arg Asn Arg Gln Tyr His Val 290 295 300 290 295 300

Glu Gln Leu Ala Ala Met Asn Val Ser Asp Val Lys Phe Thr Pro Phe Glu Gln Leu Ala Ala Met Asn Val Ser Asp Val Lys Phe Thr Pro Phe 305 310 315 320 305 310 315 320

Lys Tyr Gln Leu Ser Pro Ser Leu Pro Val Lys Lys Asp Gly Pro Gly Lys Tyr Gln Leu Ser Pro Ser Leu Pro Val Lys Lys Asp Gly Pro Gly 325 330 335 325 330 335

Lys Ala Ile Ile Ile Ile Leu Ala Ala Leu Ile Gly Gly Met Met Ala Lys Ala Ile Ile Ile Ile Leu Ala Ala Leu Ile Gly Gly Met Met Ala 340 345 350 340 345 350

Cys Gly Gly Val Leu Leu Arg His Ala Met Val Ser Arg Lys Met Glu Cys Gly Gly Val Leu Leu Arg His Ala Met Val Ser Arg Lys Met Glu 355 360 365 355 360 365

Asn Ala Leu Ala Ile Asp Glu Arg Leu Val Asn Ala Leu Ala Ile Asp Glu Arg Leu Val 370 375 370 375

<210> 40 <210> 40 <211> 22 <211> 22 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 40 <400> 40 gaagcaaacc gtacgcgtaa ag 22 gaagcaaacc gtacgcgtaa ag 22

<210> 41 <210> 41 <211> 20 <211> 20 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 41 <400> 41 cgaccagctc ttacacggcg 20 cgaccagctc ttacacggcg 20

<210> 42 <210> 42 <211> 36 <211> 36 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 42 <400> 42 gaaataggac cactaataaa tacacaaatt aataac 36 gaaataggac cactaataaa tacacaaatt aataac 36

<210> 43 <210> 43 <211> 21 <211> 21 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 43 <400> 43 ataattgacg atccggttgc c 21 ataattgacg atccggttgc C 21

<210> 44 <210> 44 <211> 27 <211> 27 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 44 <400> 44 gctatttacg ccctgattgt cttttgt 27 gctatttacg ccctgattgt cttttgt 27

<210> 45 <210> 45 <211> 22 <211> 22 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 45 <400> 45 attgagaacc tgcgtaaacg gc 22 attgagaacc tgcgtaaacg gc 22

<210> 46 <210> 46 <211> 24 <211> 24 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 46 <400> 46 tgaagagcgg ttcagataac ttcc 24 tgaagagcgg ttcagataac ttcc 24

<210> 47 <210> 47 <211> 21 <211> 21 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 47 <400> 47 cgatccggaa acctcctaca c 21 cgatccggaa acctcctaca C 21

<210> 48 <210> 48 <211> 26 <211> 26 <212> DNA <212> DNA

<213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 48 <400> 48 gattattcgc gcaacgctaa acagat 26 gattattcgc gcaacgctaa acagat 26

<210> 49 <210> 49 <211> 23 <211> 23 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 49 <400> 49 tgatcattga cgatccggta gcc 23 tgatcattga cgatccggta gcc 23

<210> 50 <210> 50 <211> 70 <211> 70 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 50 <400> 50 cggtagctgt aaagccaggg gcggtagcgt ggtttaaacc caagcaacag atcggcgtcg 60 cggtagctgt aaagccaggg gcggtagcgt ggtttaaacc caagcaacag atcggcgtcg 60

tcggtatgga 70 tcggtatgga 70

<210> 51 <210> 51 <211> 78 <211> 78 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 51 <400> 51 agcttccata ccgacgacgc cgatctgttg cttgggttta aaccacgcta ccgcccctgg 60 agcttccata ccgacgacgc cgatctgttg cttgggttta aaccacgcta ccgcccctgg 60

ctttacagct accgagct 78 ctttacagct accgagct 78

<210> 52 <210> 52 <211> 30 <211> 30 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 52 <400> 52 ggtagctgta aagccagggg cggtagcgtg 30 ggtagctgta aagccagggg cggtagcgtg 30

<210> 53 <210> 53 <211> 30 <211> 30 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 53 <400> 53 ccataccgac gacgccgatc tgttgcttgg 30 ccataccgac gacgccgatc tgttgcttgg 30

<210> 54 <210> 54 <211> 19 <211> 19 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 54 <400> 54

Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro Gly Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro Gly 1 5 10 15 1 5 10 15

Ser Thr Gly Ser Thr Gly

<210> 55 <210> 55 <211> 23 <211> 23 <212> PRT <212> PRT <213> Homo sapiens <213> Homo sapiens

<400> 55 <400> 55

Met Gly Val Pro Arg Pro Gln Pro Trp Ala Leu Gly Leu Leu Leu Phe Met Gly Val Pro Arg Pro Gln Pro Trp Ala Leu Gly Leu Leu Leu Phe 1 5 10 15 1 5 10 15

Leu Leu Pro Gly Ser Leu Gly Leu Leu Pro Gly Ser Leu Gly 20 20

<210> 56 <210> 56 <211> 18 <211> 18

<212> PRT <212> PRT <213> Homo sapiens <213> Homo sapiens

<400> 56 <400> 56

Met His Ser Ser Ala Leu Leu Cys Cys Leu Val Leu Leu Thr Gly Val Met His Ser Ser Ala Leu Leu Cys Cys Leu Val Leu Leu Thr Gly Val 1 5 10 15 1 5 10 15

Arg Ala Arg Ala

<210> 57 <210> 57 <211> 25 <211> 25 <212> PRT <212> PRT <213> Human respiratory syncytial virus A (strain A2) <213> Human respiratory syncytial virus A (strain A2)

<400> 57 <400> 57

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr 1 5 10 15 1 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Ala Val Thr Phe Cys Phe Ala Ser Gly 20 25 20 25

<210> 58 <210> 58 <211> 15 <211> 15 <212> PRT <212> PRT <213> Influenza A virus (strain A/Japan/305/1957 H2N2) <213> Influenza A virus (strain A/Japan/305/1957 H2N2)

<400> 58 <400> 58

Met Ala Ile Ile Tyr Leu Ile Leu Leu Phe Thr Ala Val Arg Gly Met Ala Ile Ile Tyr Leu Ile Leu Leu Phe Thr Ala Val Arg Gly 1 5 10 15 1 5 10 15

<210> 59 <210> 59 <211> 207 <211> 207 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 59 <400> 59

Met Glu Gly Met Asp Pro Leu Ala Val Leu Ala Glu Ser Arg Leu Leu Met Glu Gly Met Asp Pro Leu Ala Val Leu Ala Glu Ser Arg Leu Leu 1 5 10 15 1 5 10 15

Pro Leu Leu Thr Val Arg Gly Gly Glu Asp Leu Ala Gly Leu Ala Thr Pro Leu Leu Thr Val Arg Gly Gly Glu Asp Leu Ala Gly Leu Ala Thr

20 25 30 20 25 30

Val Leu Glu Leu Met Gly Val Gly Ala Leu Glu Ile Thr Leu Arg Thr Val Leu Glu Leu Met Gly Val Gly Ala Leu Glu Ile Thr Leu Arg Thr 35 40 45 35 40 45

Glu Lys Gly Leu Glu Ala Leu Lys Ala Leu Arg Lys Ser Gly Leu Leu Glu Lys Gly Leu Glu Ala Leu Lys Ala Leu Arg Lys Ser Gly Leu Leu 50 55 60 50 55 60

Leu Gly Ala Gly Thr Val Arg Ser Pro Lys Glu Ala Glu Ala Ala Leu Leu Gly Ala Gly Thr Val Arg Ser Pro Lys Glu Ala Glu Ala Ala Leu 65 70 75 80 70 75 80

Glu Ala Gly Ala Ala Phe Leu Val Ser Pro Gly Leu Leu Glu Glu Val Glu Ala Gly Ala Ala Phe Leu Val Ser Pro Gly Leu Leu Glu Glu Val 85 90 95 85 90 95

Ala Ala Leu Ala Gln Ala Arg Gly Val Pro Tyr Leu Pro Gly Val Leu Ala Ala Leu Ala Gln Ala Arg Gly Val Pro Tyr Leu Pro Gly Val Leu 100 105 110 100 105 110

Thr Pro Thr Glu Val Glu Arg Ala Leu Ala Leu Gly Leu Ser Ala Leu Thr Pro Thr Glu Val Glu Arg Ala Leu Ala Leu Gly Leu Ser Ala Leu 115 120 125 115 120 125

Lys Phe Phe Pro Ala Glu Pro Phe Gln Gly Val Arg Val Leu Arg Ala Lys Phe Phe Pro Ala Glu Pro Phe Gln Gly Val Arg Val Leu Arg Ala 130 135 140 130 135 140

Tyr Ala Glu Val Phe Pro Glu Val Arg Phe Leu Pro Thr Gly Gly Ile Tyr Ala Glu Val Phe Pro Glu Val Arg Phe Leu Pro Thr Gly Gly Ile 145 150 155 160 145 150 155 160

Lys Glu Glu His Leu Pro His Tyr Ala Ala Leu Pro Asn Leu Leu Ala Lys Glu Glu His Leu Pro His Tyr Ala Ala Leu Pro Asn Leu Leu Ala 165 170 175 165 170 175

Val Gly Gly Ser Trp Leu Leu Gln Gly Asp Leu Ala Ala Val Met Lys Val Gly Gly Ser Trp Leu Leu Gln Gly Asp Leu Ala Ala Val Met Lys 180 185 190 180 185 190

Lys Val Lys Ala Ala Lys Ala Leu Leu Ser Pro Gln Ala Pro Gly Lys Val Lys Ala Ala Lys Ala Leu Leu Ser Pro Gln Ala Pro Gly 195 200 205 195 200 205

<210> 60 <210> 60 <211> 156 <211> 156 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 60 <400> 60

Met Thr Lys Lys Val Gly Ile Val Asp Thr Thr Phe Ala Arg Val Asp Met Thr Lys Lys Val Gly Ile Val Asp Thr Thr Phe Ala Arg Val Asp 1 5 10 15 1 5 10 15

Met Ala Glu Ala Ala Ile Arg Thr Leu Lys Ala Leu Ser Pro Asn Ile Met Ala Glu Ala Ala Ile Arg Thr Leu Lys Ala Leu Ser Pro Asn Ile 20 25 30 20 25 30

Lys Ile Ile Arg Lys Thr Val Pro Gly Ile Lys Asp Leu Pro Val Ala Lys Ile Ile Arg Lys Thr Val Pro Gly Ile Lys Asp Leu Pro Val Ala 35 40 45 35 40 45

Cys Lys Lys Leu Leu Glu Glu Glu Gly Cys Asp Ile Val Met Ala Leu Cys Lys Lys Leu Leu Glu Glu Glu Gly Cys Asp Ile Val Met Ala Leu 50 55 60 50 55 60

Gly Met Pro Gly Lys Ala Glu Lys Asp Lys Val Cys Ala His Glu Ala Gly Met Pro Gly Lys Ala Glu Lys Asp Lys Val Cys Ala His Glu Ala 65 70 75 80 70 75 80

Ser Leu Gly Leu Met Leu Ala Gln Leu Met Thr Asn Lys His Ile Ile Ser Leu Gly Leu Met Leu Ala Gln Leu Met Thr Asn Lys His Ile Ile 85 90 95 85 90 95

Glu Val Phe Val His Glu Asp Glu Ala Lys Asp Asp Asp Glu Leu Asp Glu Val Phe Val His Glu Asp Glu Ala Lys Asp Asp Asp Glu Leu Asp 100 105 110 100 105 110

Ile Leu Ala Leu Val Arg Ala Ile Glu His Ala Ala Asn Val Tyr Tyr Ile Leu Ala Leu Val Arg Ala Ile Glu His Ala Ala Asn Val Tyr Tyr 115 120 125 115 120 125

Leu Leu Phe Lys Pro Glu Tyr Leu Thr Arg Met Ala Gly Lys Gly Leu Leu Leu Phe Lys Pro Glu Tyr Leu Thr Arg Met Ala Gly Lys Gly Leu 130 135 140 130 135 140

Arg Gln Gly Arg Glu Asp Ala Gly Pro Ala Arg Glu Arg Gln Gly Arg Glu Asp Ala Gly Pro Ala Arg Glu 145 150 155 145 150 155

<210> 61 <210> 61 <211> 156 <211> 156 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 61 <400> 61

Met Thr Lys Lys Val Gly Ile Val Asp Thr Thr Phe Ala Arg Val Asp Met Thr Lys Lys Val Gly Ile Val Asp Thr Thr Phe Ala Arg Val Asp

1 5 10 15 1 5 10 15

Met Ala Ser Ala Ala Ile Leu Thr Leu Lys Met Glu Ser Pro Asn Ile Met Ala Ser Ala Ala Ile Leu Thr Leu Lys Met Glu Ser Pro Asn Ile 20 25 30 20 25 30

Glu Val Phe Val His Glu Asp Glu Ala Lys Asp Asp Ala Glu Leu Lys Glu Val Phe Val His Glu Asp Glu Ala Lys Asp Asp Ala Glu Leu Lys 100 105 110 100 105 110

Ile Leu Ala Ala Arg Arg Ala Ile Glu His Ala Leu Asn Val Tyr Tyr Ile Leu Ala Ala Arg Arg Ala Ile Glu His Ala Leu Asn Val Tyr Tyr 115 120 125 115 120 125

Arg Gln Gly Phe Glu Asp Ala Gly Pro Ala Arg Glu Arg Gln Gly Phe Glu Asp Ala Gly Pro Ala Arg Glu 145 150 155 145 150 155

<210> 62 < 210> 62 <211> 209 <211> 209 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 62 <400> 62

Met Ser Thr Ile Asn Asn Gln Leu Lys Ala Leu Lys Val Ile Pro Val Met Ser Thr Ile Asn Asn Gln Leu Lys Ala Leu Lys Val Ile Pro Val 1 5 10 15 1 5 10 15

Ile Ala Ile Asp Asn Ala Glu Asp Ile Ile Pro Leu Gly Lys Val Leu Ile Ala Ile Asp Asn Ala Glu Asp Ile Ile Pro Leu Gly Lys Val Leu

20 25 30 20 25 30

Ala Glu Asn Gly Leu Pro Ala Ala Glu Ile Thr Phe Arg Ser Ser Ala Ala Glu Asn Gly Leu Pro Ala Ala Glu Ile Thr Phe Arg Ser Ser Ala 35 40 45 35 40 45

Ala Val Lys Ala Ile Met Leu Leu Arg Ser Ala Gln Pro Glu Met Leu Ala Val Lys Ala Ile Met Leu Leu Arg Ser Ala Gln Pro Glu Met Leu 50 55 60 50 55 60

Ile Gly Ala Gly Thr Ile Leu Asn Gly Val Gln Ala Leu Ala Ala Lys Ile Gly Ala Gly Thr Ile Leu Asn Gly Val Gln Ala Leu Ala Ala Lys 65 70 75 80 70 75 80

Glu Ala Gly Ala Thr Phe Val Val Ser Pro Gly Phe Asn Pro Asn Thr Glu Ala Gly Ala Thr Phe Val Val Ser Pro Gly Phe Asn Pro Asn Thr 85 90 95 85 90 95

Val Arg Ala Cys Gln Ile Ile Gly Ile Asp Ile Val Pro Gly Val Asn Val Arg Ala Cys Gln Ile Ile Gly Ile Asp Ile Val Pro Gly Val Asn 100 105 110 100 105 110

Asn Pro Ser Thr Val Glu Ala Ala Leu Glu Met Gly Leu Thr Thr Leu Asn Pro Ser Thr Val Glu Ala Ala Leu Glu Met Gly Leu Thr Thr Leu 115 120 125 115 120 125

Lys Phe Phe Pro Ala Glu Ala Ser Gly Gly Ile Ser Met Val Lys Ser Lys Phe Phe Pro Ala Glu Ala Ser Gly Gly Ile Ser Met Val Lys Ser 130 135 140 130 135 140

Leu Val Gly Pro Tyr Gly Asp Ile Arg Leu Met Pro Thr Gly Gly Ile Leu Val Gly Pro Tyr Gly Asp Ile Arg Leu Met Pro Thr Gly Gly Ile 145 150 155 160 145 150 155 160

Thr Pro Ser Asn Ile Asp Asn Tyr Leu Ala Ile Pro Gln Val Leu Ala Thr Pro Ser Asn Ile Asp Asn Tyr Leu Ala Ile Pro Gln Val Leu Ala 165 170 175 165 170 175

Cys Gly Gly Thr Trp Met Val Asp Lys Lys Leu Val Thr Asn Gly Glu Cys Gly Gly Thr Trp Met Val Asp Lys Lys Leu Val Thr Asn Gly Glu 180 185 190 180 185 190

Trp Asp Glu Ile Ala Arg Leu Thr Arg Glu Ile Val Glu Gln Val Asn Trp Asp Glu Ile Ala Arg Leu Thr Arg Glu Ile Val Glu Gln Val Asn 195 200 205 195 200 205

Pro Pro

<210> 63 <210> 63 <211> 114 <211> 114 <212> PRT <212> PRT

<213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 63 <400> 63

Met Pro Ile Phe Thr Leu Asn Thr Asn Ile Lys Ala Thr Asp Val Pro Met Pro Ile Phe Thr Leu Asn Thr Asn Ile Lys Ala Thr Asp Val Pro 1 5 10 15 1 5 10 15

Ser Asp Phe Leu Ser Leu Thr Ser Arg Leu Val Gly Leu Ile Leu Ser Ser Asp Phe Leu Ser Leu Thr Ser Arg Leu Val Gly Leu Ile Leu Ser 20 25 30 20 25 30

Lys Pro Gly Ser Tyr Val Ala Val His Ile Asn Thr Asp Gln Gln Leu Lys Pro Gly Ser Tyr Val Ala Val His Ile Asn Thr Asp Gln Gln Leu 35 40 45 35 40 45

Ser Phe Gly Gly Ser Thr Asn Pro Ala Ala Phe Gly Thr Leu Met Ser Ser Phe Gly Gly Ser Thr Asn Pro Ala Ala Phe Gly Thr Leu Met Ser 50 55 60 50 55 60

Ile Gly Gly Ile Glu Pro Ser Lys Asn Arg Asp His Ser Ala Val Leu Ile Gly Gly Ile Glu Pro Ser Lys Asn Arg Asp His Ser Ala Val Leu 65 70 75 80 70 75 80

Phe Asp His Leu Asn Ala Met Leu Gly Ile Pro Lys Asn Arg Met Tyr Phe Asp His Leu Asn Ala Met Leu Gly Ile Pro Lys Asn Arg Met Tyr 85 90 95 85 90 95

Ile His Phe Val Asn Leu Asn Gly Asp Asp Val Gly Trp Asn Gly Thr Ile His Phe Val Asn Leu Asn Gly Asp Asp Val Gly Trp Asn Gly Thr 100 105 110 100 105 110

Thr Phe Thr Phe

<210> 64 <210> 64 <211> 157 <211> 157 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 64 <400> 64

Met Asn Gln His Ser His Lys Asp Tyr Glu Thr Val Arg Ile Ala Val Met Asn Gln His Ser His Lys Asp Tyr Glu Thr Val Arg Ile Ala Val 1 5 10 15 1 5 10 15

Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala

20 25 30 20 25 30

Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 35 40 45

Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 50 55 60

Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 70 75 80

Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 85 90 95

Asp Gly Met Met Asn Val Gln Leu Ser Thr Gly Val Pro Val Leu Ser Asp Gly Met Met Asn Val Gln Leu Ser Thr Gly Val Pro Val Leu Ser 100 105 110 100 105 110

Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Ala Glu His His Arg Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Ala Glu His His Arg 115 120 125 115 120 125

Phe Phe Ala Ala His Phe Ala Val Lys Gly Val Glu Ala Ala Arg Ala Phe Phe Ala Ala His Phe Ala Val Lys Gly Val Glu Ala Ala Arg Ala 130 135 140 130 135 140

Cys Ile Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala Cys Ile Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 145 150 155

<210> 65 < :210> 65 <211> 205 <211> 205 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 65 <400> 65

Met Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu Met Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu 1 5 10 15 1 5 10 15

Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe 20 25 30 20 25 30

Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala

35 40 45 35 40 45

Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile 50 55 60 50 55 60

Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val 65 70 75 80 70 75 80

Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile 85 90 95 85 90 95

Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met 100 105 110 100 105 110

Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Thr Ile Leu Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Thr Ile Leu 115 120 125 115 120 125

Lys Leu Phe Pro Gly Glu Val Val Gly Pro Gln Phe Val Lys Ala Met Lys Leu Phe Pro Gly Glu Val Val Gly Pro Gln Phe Val Lys Ala Met 130 135 140 130 135 140

Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asn Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asn 145 150 155 160 145 150 155 160

Leu Asp Asn Val Cys Glu Trp Phe Lys Ala Gly Val Leu Ala Val Gly Leu Asp Asn Val Cys Glu Trp Phe Lys Ala Gly Val Leu Ala Val Gly 165 170 175 165 170 175

Val Gly Ser Ala Leu Val Lys Gly Thr Pro Asp Glu Val Arg Glu Lys Val Gly Ser Ala Leu Val Lys Gly Thr Pro Asp Glu Val Arg Glu Lys 180 185 190 180 185 190

Ala Lys Ala Phe Val Glu Lys Ile Arg Gly Cys Thr Glu Ala Lys Ala Phe Val Glu Lys Ile Arg Gly Cys Thr Glu 195 200 205 195 200 205

<210> 66 <210> 66 <211> 157 <211> 157 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 66 <400> 66

Met Asn Gln His Ser His Lys Asp Tyr Glu Thr Val Arg Ile Ala Val Met Asn Gln His Ser His Lys Asp Tyr Glu Thr Val Arg Ile Ala Val

1 5 10 15 1 5 10 15

Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala 20 25 30 20 25 30

Phe Glu Ala Ala Met Ala Asp Ile Gly Gly Asp Arg Phe Ala Val Asp Phe Glu Ala Ala Met Ala Asp Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 35 40 45

Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Asp Ala His Thr Leu Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Asp Ala His Thr Leu 115 120 125 115 120 125

Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala 130 135 140 130 135 140

Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 145 150 155

<210> 67 <210> 67 <211> 177 <211> 177 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 67 <400> 67

Met Phe Thr Lys Ser Gly Asp Asp Gly Asn Thr Asn Val Ile Asn Lys Met Phe Thr Lys Ser Gly Asp Asp Gly Asn Thr Asn Val Ile Asn Lys 1 5 10 15 1 5 10 15

Arg Val Gly Lys Asp Ser Pro Leu Val Asn Phe Leu Gly Asp Leu Asp Arg Val Gly Lys Asp Ser Pro Leu Val Asn Phe Leu Gly Asp Leu Asp

20 25 30 20 25 30

Glu Leu Asn Ser Phe Ile Gly Phe Ala Ile Ser Lys Ile Pro Trp Glu Glu Leu Asn Ser Phe Ile Gly Phe Ala Ile Ser Lys Ile Pro Trp Glu 35 40 45 35 40 45

Asp Met Lys Lys Asp Leu Glu Arg Val Gln Val Glu Leu Phe Glu Ile Asp Met Lys Lys Asp Leu Glu Arg Val Gln Val Glu Leu Phe Glu Ile 50 55 60 50 55 60

Gly Glu Asp Leu Ser Thr Gln Ser Ser Lys Lys Lys Ile Asp Glu Ser Gly Glu Asp Leu Ser Thr Gln Ser Ser Lys Lys Lys Ile Asp Glu Ser 65 70 75 80 70 75 80

Tyr Val Leu Trp Leu Leu Ala Ala Thr Ala Ile Tyr Arg Ile Glu Ser Tyr Val Leu Trp Leu Leu Ala Ala Thr Ala Ile Tyr Arg Ile Glu Ser 85 90 95 85 90 95

Gly Pro Val Lys Leu Phe Val Ile Pro Gly Gly Ser Glu Glu Ala Ser Gly Pro Val Lys Leu Phe Val Ile Pro Gly Gly Ser Glu Glu Ala Ser 100 105 110 100 105 110

Val Leu His Val Thr Arg Ser Val Ala Arg Arg Val Glu Arg Asn Ala Val Leu His Val Thr Arg Ser Val Ala Arg Arg Val Glu Arg Asn Ala 115 120 125 115 120 125

Val Lys Tyr Thr Lys Glu Leu Pro Glu Ile Asn Arg Met Ile Ile Val Val Lys Tyr Thr Lys Glu Leu Pro Glu Ile Asn Arg Met Ile Ile Val 130 135 140 130 135 140

Tyr Leu Asn Arg Leu Ser Ser Leu Leu Phe Ala Met Ala Leu Val Ala Tyr Leu Asn Arg Leu Ser Ser Leu Leu Phe Ala Met Ala Leu Val Ala 145 150 155 160 145 150 155 160

Asn Lys Arg Arg Asn Gln Ser Glu Lys Ile Tyr Glu Ile Gly Lys Ser Asn Lys Arg Arg Asn Gln Ser Glu Lys Ile Tyr Glu Ile Gly Lys Ser 165 170 175 165 170 175

Trp Trp

<210> 68 <210> 68 <211> 157 <211> 157 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 68 <400> 68

1 5 10 15 1 5 10 15

Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Gln Cys Val Arg Ala Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Gln Cys Val Arg Ala 20 25 30 20 25 30

Phe Glu Glu Ala Met Ala Asp Ala Gly Gly Asp Arg Phe Ala Val Asp Phe Glu Glu Ala Met Ala Asp Ala Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 35 40 45

Ala Val Leu Thr Pro His Arg Tyr Arg Ser Ser Arg Glu His His Glu Ala Val Leu Thr Pro His Arg Tyr Arg Ser Ser Arg Glu His His Glu 115 120 125 115 120 125

Phe Phe Arg Glu His Phe Met Val Lys Gly Val Glu Ala Ala Ala Ala Phe Phe Arg Glu His Phe Met Val Lys Gly Val Glu Ala Ala Ala Ala 130 135 140 130 135 140

Cys Ile Thr Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala Cys Ile Thr Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 145 150 155

<210> 69 <210> 69 <211> 201 <211> 201 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 69 <400> 69

Met Gly His Thr Lys Gly Pro Thr Pro Gln Gln His Asp Gly Ser Ala Met Gly His Thr Lys Gly Pro Thr Pro Gln Gln His Asp Gly Ser Ala 1 5 10 15 1 5 10 15

Leu Arg Ile Gly Ile Val His Ala Arg Trp Asn Lys Thr Ile Ile Met Leu Arg Ile Gly Ile Val His Ala Arg Trp Asn Lys Thr Ile Ile Met

20 25 30 20 25 30

Pro Leu Leu Ile Gly Thr Ile Ala Lys Leu Leu Glu Cys Gly Val Lys Pro Leu Leu Ile Gly Thr Ile Ala Lys Leu Leu Glu Cys Gly Val Lys 35 40 45 35 40 45

Ala Ser Asn Ile Val Val Gln Ser Val Pro Gly Ser Trp Glu Leu Pro Ala Ser Asn Ile Val Val Gln Ser Val Pro Gly Ser Trp Glu Leu Pro 50 55 60 50 55 60

Ile Ala Val Gln Arg Leu Tyr Ser Ala Ser Gln Leu Gln Thr Pro Ser Ile Ala Val Gln Arg Leu Tyr Ser Ala Ser Gln Leu Gln Thr Pro Ser 65 70 75 80 70 75 80

Ser Gly Pro Ser Leu Ser Ala Gly Asp Leu Leu Gly Ser Ser Thr Thr Ser Gly Pro Ser Leu Ser Ala Gly Asp Leu Leu Gly Ser Ser Thr Thr 85 90 95 85 90 95

Asp Leu Thr Ala Leu Pro Thr Thr Thr Ala Ser Ser Thr Gly Pro Phe Asp Leu Thr Ala Leu Pro Thr Thr Thr Ala Ser Ser Thr Gly Pro Phe 100 105 110 100 105 110

Asp Ala Leu Ile Ala Ile Gly Val Leu Ile Lys Gly Glu Thr Met His Asp Ala Leu Ile Ala Ile Gly Val Leu Ile Lys Gly Glu Thr Met His 115 120 125 115 120 125

Phe Glu Tyr Ile Ala Asp Ser Val Ser His Gly Leu Met Arg Val Gln Phe Glu Tyr Ile Ala Asp Ser Val Ser His Gly Leu Met Arg Val Gln 130 135 140 130 135 140

Leu Asp Thr Gly Val Pro Val Ile Phe Gly Val Leu Thr Val Leu Thr Leu Asp Thr Gly Val Pro Val Ile Phe Gly Val Leu Thr Val Leu Thr 145 150 155 160 145 150 155 160

Asp Asp Gln Ala Lys Ala Arg Ala Gly Val Ile Glu Gly Ser His Asn Asp Asp Gln Ala Lys Ala Arg Ala Gly Val Ile Glu Gly Ser His Asn 165 170 175 165 170 175

His Gly Glu Asp Trp Gly Leu Ala Ala Val Glu Met Gly Val Arg Arg His Gly Glu Asp Trp Gly Leu Ala Ala Val Glu Met Gly Val Arg Arg 180 185 190 180 185 190

Arg Asp Trp Ala Ala Gly Lys Thr Glu Arg Asp Trp Ala Ala Gly Lys Thr Glu 195 200 195 200

<210> 70 <210> 70 <211> 237 <211> 237 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 70 <400> 70

Met Tyr Glu Val Asp His Ala Asp Val Tyr Asp Leu Phe Tyr Leu Gly Met Tyr Glu Val Asp His Ala Asp Val Tyr Asp Leu Phe Tyr Leu Gly 1 5 10 15 1 5 10 15

Arg Gly Lys Asp Tyr Ala Ala Glu Ala Ser Asp Ile Ala Asp Leu Val Arg Gly Lys Asp Tyr Ala Ala Glu Ala Ser Asp Ile Ala Asp Leu Val 20 25 30 20 25 30

Arg Ser Arg Thr Pro Glu Ala Ser Ser Leu Leu Asp Val Ala Cys Gly Arg Ser Arg Thr Pro Glu Ala Ser Ser Leu Leu Asp Val Ala Cys Gly 35 40 45 35 40 45

Thr Gly Thr His Leu Glu His Phe Thr Lys Glu Phe Gly Asp Thr Ala Thr Gly Thr His Leu Glu His Phe Thr Lys Glu Phe Gly Asp Thr Ala 50 55 60 50 55 60

Gly Leu Glu Leu Ser Glu Asp Met Leu Thr His Ala Arg Lys Arg Leu Gly Leu Glu Leu Ser Glu Asp Met Leu Thr His Ala Arg Lys Arg Leu 65 70 75 80 70 75 80

Pro Asp Ala Thr Leu His Gln Gly Asp Met Arg Asp Phe Gln Leu Gly Pro Asp Ala Thr Leu His Gln Gly Asp Met Arg Asp Phe Gln Leu Gly 85 90 95 85 90 95

Arg Lys Phe Ser Ala Val Val Ser Met Phe Ser Ser Val Gly Tyr Leu Arg Lys Phe Ser Ala Val Val Ser Met Phe Ser Ser Val Gly Tyr Leu 100 105 110 100 105 110

Lys Thr Val Ala Glu Leu Gly Ala Ala Val Ala Ser Phe Ala Glu His Lys Thr Val Ala Glu Leu Gly Ala Ala Val Ala Ser Phe Ala Glu His 115 120 125 115 120 125

Leu Glu Pro Gly Gly Val Val Val Val Glu Pro Trp Trp Phe Pro Glu Leu Glu Pro Gly Gly Val Val Val Val Glu Pro Trp Trp Phe Pro Glu 130 135 140 130 135 140

Thr Phe Ala Asp Gly Trp Val Ser Ala Asp Val Val Arg Arg Asp Gly Thr Phe Ala Asp Gly Trp Val Ser Ala Asp Val Val Arg Arg Asp Gly 145 150 155 160 145 150 155 160

Arg Thr Val Ala Arg Val Ser His Ser Val Arg Glu Gly Asn Ala Thr Arg Thr Val Ala Arg Val Ser His Ser Val Arg Glu Gly Asn Ala Thr 165 170 175 165 170 175

Arg Met Glu Val His Phe Thr Val Ala Asp Pro Gly Lys Gly Val Arg Arg Met Glu Val His Phe Thr Val Ala Asp Pro Gly Lys Gly Val Arg 180 185 190 180 185 190

His Phe Ser Asp Val His Leu Ile Thr Leu Phe His Gln Arg Glu Tyr His Phe Ser Asp Val His Leu Ile Thr Leu Phe His Gln Arg Glu Tyr 195 200 205 195 200 205

Glu Ala Ala Phe Met Ala Ala Gly Leu Arg Val Glu Tyr Leu Glu Gly Glu Ala Ala Phe Met Ala Ala Gly Leu Arg Val Glu Tyr Leu Glu Gly 210 215 220 210 215 220

Gly Pro Ser Gly Arg Gly Leu Phe Val Gly Val Pro Ala Gly Pro Ser Gly Arg Gly Leu Phe Val Gly Val Pro Ala 225 230 235 225 230 235

<210> 71 <210> 71 <211> 138 <211> 138 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 71 <400> 71

Met Gly Met Lys Glu Lys Phe Val Leu Ile Ile Thr His Gly Asp Phe Met Gly Met Lys Glu Lys Phe Val Leu Ile Ile Thr His Gly Asp Phe 1 5 10 15 1 5 10 15

Gly Lys Gly Leu Leu Ser Gly Ala Glu Val Ile Ile Gly Lys Gln Glu Gly Lys Gly Leu Leu Ser Gly Ala Glu Val Ile Ile Gly Lys Gln Glu 20 25 30 20 25 30

Asn Val His Thr Val Gly Leu Asn Leu Gly Asp Asn Ile Glu Lys Val Asn Val His Thr Val Gly Leu Asn Leu Gly Asp Asn Ile Glu Lys Val 35 40 45 35 40 45

Ala Lys Glu Val Met Arg Ile Ile Ile Ala Lys Leu Ala Glu Asp Lys Ala Lys Glu Val Met Arg Ile Ile Ile Ala Lys Leu Ala Glu Asp Lys 50 55 60 50 55 60

Glu Ile Ile Ile Val Val Asp Leu Phe Gly Gly Ser Pro Phe Asn Ile Glu Ile Ile Ile Val Val Asp Leu Phe Gly Gly Ser Pro Phe Asn Ile 65 70 75 80 70 75 80

Ala Leu Glu Met Met Lys Thr Phe Asp Val Lys Val Ile Thr Gly Ile Ala Leu Glu Met Met Lys Thr Phe Asp Val Lys Val Ile Thr Gly Ile 85 90 95 85 90 95

Asn Met Pro Met Leu Val Glu Leu Leu Thr Ser Ile Asn Val Tyr Asp Asn Met Pro Met Leu Val Glu Leu Leu Thr Ser Ile Asn Val Tyr Asp 100 105 110 100 105 110

Thr Thr Glu Leu Leu Glu Asn Ile Ser Lys Ile Gly Lys Asp Gly Ile Thr Thr Glu Leu Leu Glu Asn Ile Ser Lys Ile Gly Lys Asp Gly Ile 115 120 125 115 120 125

Lys Val Ile Glu Lys Ser Ser Leu Lys Met Lys Val Ile Glu Lys Ser Ser Leu Lys Met 130 135 130 135

<210> 72 <210> 72 <211> 154 <211> 154 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 72 <400> 72

Met Lys Tyr Asp Gly Ser Lys Leu Arg Ile Gly Ile Leu His Ala Arg Met Lys Tyr Asp Gly Ser Lys Leu Arg Ile Gly Ile Leu His Ala Arg 1 5 10 15 1 5 10 15

Trp Asn Leu Glu Ile Ile Ala Ala Leu Val Ala Gly Ala Ile Lys Arg Trp Asn Leu Glu Ile Ile Ala Ala Leu Val Ala Gly Ala Ile Lys Arg 20 25 30 20 25 30

Leu Gln Glu Phe Gly Val Lys Ala Glu Asn Ile Ile Ile Glu Thr Val Leu Gln Glu Phe Gly Val Lys Ala Glu Asn Ile Ile Ile Glu Thr Val 35 40 45 35 40 45

Pro Gly Ser Phe Glu Leu Pro Tyr Gly Ser Lys Leu Phe Val Glu Lys Pro Gly Ser Phe Glu Leu Pro Tyr Gly Ser Lys Leu Phe Val Glu Lys 50 55 60 50 55 60

Gln Lys Arg Leu Gly Lys Pro Leu Asp Ala Ile Ile Pro Ile Gly Val Gln Lys Arg Leu Gly Lys Pro Leu Asp Ala Ile Ile Pro Ile Gly Val 65 70 75 80 70 75 80

Leu Ile Lys Gly Ser Thr Met His Phe Glu Tyr Ile Cys Asp Ser Thr Leu Ile Lys Gly Ser Thr Met His Phe Glu Tyr Ile Cys Asp Ser Thr 85 90 95 85 90 95

Thr His Gln Leu Met Lys Leu Asn Phe Glu Leu Gly Ile Pro Val Ile Thr His Gln Leu Met Lys Leu Asn Phe Glu Leu Gly Ile Pro Val Ile 100 105 110 100 105 110

Phe Gly Val Leu Thr Cys Leu Thr Asp Glu Gln Ala Glu Ala Arg Ala Phe Gly Val Leu Thr Cys Leu Thr Asp Glu Gln Ala Glu Ala Arg Ala 115 120 125 115 120 125

Gly Leu Ile Glu Gly Lys Met His Asn His Gly Glu Asp Trp Gly Ala Gly Leu Ile Glu Gly Lys Met His Asn His Gly Glu Asp Trp Gly Ala 130 135 140 130 135 140

Ala Ala Val Glu Met Ala Thr Lys Phe Asn Ala Ala Val Glu Met Ala Thr Lys Phe Asn 145 150 145 150

<210> 73 <210> 73 <211> 164 <211> 164 <212> PRT <212> PRT

<213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 73 <400> 73

Met Ala Val Lys Gly Leu Gly Glu Val Asp Gln Lys Tyr Asp Gly Ser Met Ala Val Lys Gly Leu Gly Glu Val Asp Gln Lys Tyr Asp Gly Ser 1 5 10 15 1 5 10 15

Lys Leu Arg Ile Gly Ile Leu His Ala Arg Trp Asn Arg Lys Ile Ile Lys Leu Arg Ile Gly Ile Leu His Ala Arg Trp Asn Arg Lys Ile Ile 20 25 30 20 25 30

Leu Ala Leu Val Ala Gly Ala Val Leu Arg Leu Leu Glu Phe Gly Val Leu Ala Leu Val Ala Gly Ala Val Leu Arg Leu Leu Glu Phe Gly Val 35 40 45 35 40 45

Lys Ala Glu Asn Ile Ile Ile Glu Thr Val Pro Gly Ser Phe Glu Leu Lys Ala Glu Asn Ile Ile Ile Glu Thr Val Pro Gly Ser Phe Glu Leu 50 55 60 50 55 60

Pro Tyr Gly Ser Lys Leu Phe Val Glu Lys Gln Lys Arg Leu Gly Lys Pro Tyr Gly Ser Lys Leu Phe Val Glu Lys Gln Lys Arg Leu Gly Lys 65 70 75 80 70 75 80

Pro Leu Asp Ala Ile Ile Pro Ile Gly Val Leu Ile Lys Gly Ser Thr Pro Leu Asp Ala Ile Ile Pro Ile Gly Val Leu Ile Lys Gly Ser Thr 85 90 95 85 90 95

Met His Phe Glu Tyr Ile Cys Asp Ser Thr Thr His Gln Leu Met Lys Met His Phe Glu Tyr Ile Cys Asp Ser Thr Thr His Gln Leu Met Lys 100 105 110 100 105 110

Leu Asn Phe Glu Leu Gly Ile Pro Val Ile Phe Gly Val Leu Thr Cys Leu Asn Phe Glu Leu Gly Ile Pro Val Ile Phe Gly Val Leu Thr Cys 115 120 125 115 120 125

Leu Thr Asp Glu Gln Ala Glu Ala Arg Ala Gly Leu Ile Glu Gly Lys Leu Thr Asp Glu Gln Ala Glu Ala Arg Ala Gly Leu Ile Glu Gly Lys 130 135 140 130 135 140

Met His Asn His Gly Glu Asp Trp Gly Ala Ala Ala Val Glu Met Ala Met His Asn His Gly Glu Asp Trp Gly Ala Ala Ala Val Glu Met Ala 145 150 155 160 145 150 155 160

Thr Lys Phe Asn Thr Lys Phe Asn

<210> 74 <210> 74 <211> 175 <211> 175 <212> PRT <212> PRT

<213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 74 <400> 74

Met Gly Ala Asn Trp Tyr Leu Asp Asn Glu Ser Ser Arg Leu Ser Phe Met Gly Ala Asn Trp Tyr Leu Asp Asn Glu Ser Ser Arg Leu Ser Phe 1 5 10 15 1 5 10 15

Thr Ser Thr Lys Asn Ala Asp Ile Ala Glu Val His Arg Phe Leu Val Thr Ser Thr Lys Asn Ala Asp Ile Ala Glu Val His Arg Phe Leu Val 20 25 30 20 25 30

Leu His Gly Lys Val Asp Pro Lys Gly Leu Ala Glu Val Glu Val Glu Leu His Gly Lys Val Asp Pro Lys Gly Leu Ala Glu Val Glu Val Glu 35 40 45 35 40 45

Thr Glu Ser Ile Ser Thr Gly Ile Pro Leu Arg Asp Met Leu Leu Arg Thr Glu Ser Ile Ser Thr Gly Ile Pro Leu Arg Asp Met Leu Leu Arg 50 55 60 50 55 60

Val Leu Val Phe Gln Val Ser Lys Phe Pro Val Ala Gln Ile Asn Ala Val Leu Val Phe Gln Val Ser Lys Phe Pro Val Ala Gln Ile Asn Ala 65 70 75 80 70 75 80

Gln Leu Asp Met Arg Pro Ile Asn Asn Leu Ala Pro Gly Ala Gln Leu Gln Leu Asp Met Arg Pro Ile Asn Asn Leu Ala Pro Gly Ala Gln Leu 85 90 95 85 90 95

Glu Leu Arg Leu Pro Leu Thr Val Ser Leu Arg Gly Lys Ser His Ser Glu Leu Arg Leu Pro Leu Thr Val Ser Leu Arg Gly Lys Ser His Ser 100 105 110 100 105 110

Tyr Asn Ala Glu Leu Leu Ala Thr Arg Leu Asp Glu Arg Arg Phe Gln Tyr Asn Ala Glu Leu Leu Ala Thr Arg Leu Asp Glu Arg Arg Phe Gln 115 120 125 115 120 125

Val Val Thr Leu Glu Pro Leu Val Ile His Ala Gln Asp Phe Asp Met Val Val Thr Leu Glu Pro Leu Val Ile His Ala Gln Asp Phe Asp Met 130 135 140 130 135 140

Val Arg Ala Phe Asn Ala Leu Arg Leu Val Ala Gly Leu Ser Ala Val Val Arg Ala Phe Asn Ala Leu Arg Leu Val Ala Gly Leu Ser Ala Val 145 150 155 160 145 150 155 160

Ser Leu Ser Val Pro Val Gly Ala Val Leu Ile Phe Thr Ala Arg Ser Leu Ser Val Pro Val Gly Ala Val Leu Ile Phe Thr Ala Arg 165 170 175 165 170 175

<210> 75 <210> 75 <211> 208 <211> 208 <212> PRT <212> PRT

<213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 75 <400> 75

Met Thr Asp Tyr Ile Arg Asp Gly Ser Ala Ile Lys Ala Leu Ser Phe Met Thr Asp Tyr Ile Arg Asp Gly Ser Ala Ile Lys Ala Leu Ser Phe 1 5 10 15 1 5 10 15

Ala Ile Ile Leu Ala Glu Ala Asp Leu Arg His Ile Pro Gln Asp Leu Ala Ile Ile Leu Ala Glu Ala Asp Leu Arg His Ile Pro Gln Asp Leu 20 25 30 20 25 30

Gln Arg Leu Ala Val Arg Val Ile His Ala Cys Gly Met Val Asp Val Gln Arg Leu Ala Val Arg Val Ile His Ala Cys Gly Met Val Asp Val 35 40 45 35 40 45

Ala Asn Asp Leu Ala Phe Ser Glu Gly Ala Gly Lys Ala Gly Arg Asn Ala Asn Asp Leu Ala Phe Ser Glu Gly Ala Gly Lys Ala Gly Arg Asn 50 55 60 50 55 60

Ala Leu Leu Ala Gly Ala Pro Ile Leu Cys Asp Ala Arg Met Val Ala Ala Leu Leu Ala Gly Ala Pro Ile Leu Cys Asp Ala Arg Met Val Ala 65 70 75 80 70 75 80

Glu Gly Ile Thr Arg Ser Arg Leu Pro Ala Asp Asn Arg Val Ile Tyr Glu Gly Ile Thr Arg Ser Arg Leu Pro Ala Asp Asn Arg Val Ile Tyr 85 90 95 85 90 95

Thr Leu Ser Asp Pro Ser Val Pro Glu Leu Ala Lys Lys Ile Gly Asn Thr Leu Ser Asp Pro Ser Val Pro Glu Leu Ala Lys Lys Ile Gly Asn 100 105 110 100 105 110

Thr Arg Ser Ala Ala Ala Leu Asp Leu Trp Leu Pro His Ile Glu Gly Thr Arg Ser Ala Ala Ala Leu Asp Leu Trp Leu Pro His Ile Glu Gly 115 120 125 115 120 125

Ser Ile Val Ala Ile Gly Asn Ala Pro Thr Ala Leu Phe Arg Leu Phe Ser Ile Val Ala Ile Gly Asn Ala Pro Thr Ala Leu Phe Arg Leu Phe 130 135 140 130 135 140

Glu Leu Leu Asp Ala Gly Ala Pro Lys Pro Ala Leu Ile Ile Gly Met Glu Leu Leu Asp Ala Gly Ala Pro Lys Pro Ala Leu Ile Ile Gly Met 145 150 155 160 145 150 155 160

Pro Val Gly Phe Val Gly Ala Ala Glu Ser Lys Asp Glu Leu Ala Ala Pro Val Gly Phe Val Gly Ala Ala Glu Ser Lys Asp Glu Leu Ala Ala 165 170 175 165 170 175

Asn Ser Arg Gly Val Pro Tyr Val Ile Val Arg Gly Arg Arg Gly Gly Asn Ser Arg Gly Val Pro Tyr Val Ile Val Arg Gly Arg Arg Gly Gly 180 185 190 180 185 190

Ser Ala Met Thr Ala Ala Ala Val Asn Ala Leu Ala Ser Glu Arg Glu Ser Ala Met Thr Ala Ala Ala Val Asn Ala Leu Ala Ser Glu Arg Glu 195 200 205 195 200 205

<210> 76 <210> 76 <211> 128 <211> 128 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 76 <400> 76

Met Ile Thr Val Phe Gly Leu Lys Ser Lys Leu Ala Pro Arg Arg Glu Met Ile Thr Val Phe Gly Leu Lys Ser Lys Leu Ala Pro Arg Arg Glu 1 5 10 15 1 5 10 15

Lys Leu Ala Glu Val Ile Tyr Ser Ser Leu His Leu Gly Leu Asp Ile Lys Leu Ala Glu Val Ile Tyr Ser Ser Leu His Leu Gly Leu Asp Ile 20 25 30 20 25 30

Pro Lys Gly Lys His Ala Ile Arg Phe Leu Cys Leu Glu Lys Glu Asp Pro Lys Gly Lys His Ala Ile Arg Phe Leu Cys Leu Glu Lys Glu Asp 35 40 45 35 40 45

Phe Tyr Tyr Pro Phe Asp Arg Ser Asp Asp Tyr Thr Val Ile Glu Ile Phe Tyr Tyr Pro Phe Asp Arg Ser Asp Asp Tyr Thr Val Ile Glu Ile 50 55 60 50 55 60

Asn Leu Met Ala Gly Arg Ser Glu Glu Thr Lys Met Leu Leu Ile Phe Asn Leu Met Ala Gly Arg Ser Glu Glu Thr Lys Met Leu Leu Ile Phe 65 70 75 80 70 75 80

Leu Leu Phe Ile Ala Leu Glu Arg Lys Leu Gly Ile Arg Ala His Asp Leu Leu Phe Ile Ala Leu Glu Arg Lys Leu Gly Ile Arg Ala His Asp 85 90 95 85 90 95

Val Glu Ile Thr Ile Lys Glu Gln Pro Ala His Cys Trp Gly Phe Arg Val Glu Ile Thr Ile Lys Glu Gln Pro Ala His Cys Trp Gly Phe Arg 100 105 110 100 105 110

Gly Arg Thr Gly Asp Ser Ala Arg Asp Leu Asp Tyr Asp Ile Tyr Val Gly Arg Thr Gly Asp Ser Ala Arg Asp Leu Asp Tyr Asp Ile Tyr Val 115 120 125 115 120 125

<210> 77 <210> 77 <211> 235 <211> 235 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 77 <400> 77

Met Gly Ser Asp Leu Gln Lys Leu Gln Arg Phe Ser Thr Cys Asp Ile Met Gly Ser Asp Leu Gln Lys Leu Gln Arg Phe Ser Thr Cys Asp Ile 1 5 10 15 1 5 10 15

Ser Asp Gly Leu Leu Asn Val Tyr Asn Ile Pro Thr Gly Gly Tyr Phe Ser Asp Gly Leu Leu Asn Val Tyr Asn Ile Pro Thr Gly Gly Tyr Phe 20 25 30 20 25 30

Pro Asn Leu Thr Ala Ile Ser Pro Pro Gln Asn Ser Ser Ile Val Gly Pro Asn Leu Thr Ala Ile Ser Pro Pro Gln Asn Ser Ser Ile Val Gly 35 40 45 35 40 45

Thr Ala Tyr Thr Val Leu Phe Ala Pro Ile Asp Asp Pro Arg Pro Ala Thr Ala Tyr Thr Val Leu Phe Ala Pro Ile Asp Asp Pro Arg Pro Ala 50 55 60 50 55 60

Val Asn Tyr Ile Asp Ser Val Pro Pro Asn Ser Ile Leu Val Leu Ala Val Asn Tyr Ile Asp Ser Val Pro Pro Asn Ser Ile Leu Val Leu Ala 65 70 75 80 70 75 80

Leu Glu Pro His Leu Gln Ser Gln Phe His Pro Phe Ile Lys Ile Thr Leu Glu Pro His Leu Gln Ser Gln Phe His Pro Phe Ile Lys Ile Thr 85 90 95 85 90 95

Gln Ala Met Tyr Gly Gly Leu Met Ser Thr Arg Ala Gln Tyr Leu Lys Gln Ala Met Tyr Gly Gly Leu Met Ser Thr Arg Ala Gln Tyr Leu Lys 100 105 110 100 105 110

Ser Asn Gly Thr Val Val Phe Gly Arg Ile Arg Asp Val Asp Glu His Ser Asn Gly Thr Val Val Phe Gly Arg Ile Arg Asp Val Asp Glu His 115 120 125 115 120 125

Arg Thr Leu Asn His Pro Val Phe Ala Tyr Gly Val Gly Ser Cys Ala Arg Thr Leu Asn His Pro Val Phe Ala Tyr Gly Val Gly Ser Cys Ala 130 135 140 130 135 140

Pro Lys Ala Val Val Lys Ala Val Gly Thr Asn Val Gln Leu Lys Ile Pro Lys Ala Val Val Lys Ala Val Gly Thr Asn Val Gln Leu Lys Ile 145 150 155 160 145 150 155 160

Leu Thr Ser Asp Gly Val Thr Gln Thr Ile Cys Pro Gly Asp Tyr Ile Leu Thr Ser Asp Gly Val Thr Gln Thr Ile Cys Pro Gly Asp Tyr Ile 165 170 175 165 170 175

Ala Gly Asp Asn Asn Gly Ile Val Arg Ile Pro Val Gln Glu Thr Asp Ala Gly Asp Asn Asn Gly Ile Val Arg Ile Pro Val Gln Glu Thr Asp 180 185 190 180 185 190

Ile Ser Lys Leu Val Thr Tyr Ile Glu Lys Ser Ile Glu Val Asp Arg Ile Ser Lys Leu Val Thr Tyr Ile Glu Lys Ser Ile Glu Val Asp Arg 195 200 205 195 200 205

Leu Val Ser Glu Ala Ile Lys Asn Gly Leu Pro Ala Lys Ala Ala Gln Leu Val Ser Glu Ala Ile Lys Asn Gly Leu Pro Ala Lys Ala Ala Gln 210 215 220 210 215 220

Thr Ala Arg Arg Met Val Leu Lys Asp Tyr Ile Thr Ala Arg Arg Met Val Leu Lys Asp Tyr Ile 225 230 235 225 230 235

<210> 78 <210> 78 <211> 162 <211> 162 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 78 <400> 78

Met Ser Gly Met Arg Val Tyr Leu Gly Ala Asp His Ala Gly Tyr Glu Met Ser Gly Met Arg Val Tyr Leu Gly Ala Asp His Ala Gly Tyr Glu 1 5 10 15 1 5 10 15

Leu Lys Gln Ala Ile Ile Ala Phe Leu Lys Met Thr Gly His Glu Pro Leu Lys Gln Ala Ile Ile Ala Phe Leu Lys Met Thr Gly His Glu Pro 20 25 30 20 25 30

Ile Asp Cys Gly Ala Leu Arg Tyr Asp Ala Asp Asp Asp Tyr Pro Ala Ile Asp Cys Gly Ala Leu Arg Tyr Asp Ala Asp Asp Asp Tyr Pro Ala 35 40 45 35 40 45

Phe Cys Ile Ala Ala Ala Thr Arg Thr Val Ala Asp Pro Gly Ser Leu Phe Cys Ile Ala Ala Ala Thr Arg Thr Val Ala Asp Pro Gly Ser Leu 50 55 60 50 55 60

Gly Ile Val Leu Gly Gly Ser Gly Asn Gly Glu Gln Ile Ala Ala Asn Gly Ile Val Leu Gly Gly Ser Gly Asn Gly Glu Gln Ile Ala Ala Asn 65 70 75 80 70 75 80

Lys Val Pro Gly Ala Arg Cys Ala Leu Ala Trp Ser Val Gln Thr Ala Lys Val Pro Gly Ala Arg Cys Ala Leu Ala Trp Ser Val Gln Thr Ala 85 90 95 85 90 95

Ala Leu Ala Arg Glu His Asn Asn Ala Gln Leu Ile Gly Ile Gly Gly Ala Leu Ala Arg Glu His Asn Asn Ala Gln Leu Ile Gly Ile Gly Gly 100 105 110 100 105 110

Arg Met His Thr Leu Glu Glu Ala Leu Arg Ile Val Lys Ala Phe Val Arg Met His Thr Leu Glu Glu Ala Leu Arg Ile Val Lys Ala Phe Val 115 120 125 115 120 125

Thr Thr Pro Trp Ser Lys Ala Gln Arg His Gln Arg Arg Ile Asp Ile Thr Thr Pro Trp Ser Lys Ala Gln Arg His Gln Arg Arg Ile Asp Ile 130 135 140 130 135 140

Leu Ala Glu Tyr Glu Arg Thr His Glu Ala Pro Pro Val Pro Gly Ala Leu Ala Glu Tyr Glu Arg Thr His Glu Ala Pro Pro Val Pro Gly Ala 145 150 155 160 145 150 155 160

Pro Ala Pro Ala

<210> 79 <210> 79 <211> 157 <211> 157 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 79 <400> 79

Met Gly Asp Asp Ala Arg Ile Ala Ala Ile Gly Asp Val Asp Glu Leu Met Gly Asp Asp Ala Arg Ile Ala Ala Ile Gly Asp Val Asp Glu Leu 1 5 10 15 1 5 10 15

Asn Ser Gln Ile Gly Val Leu Leu Ala Glu Pro Leu Pro Asp Asp Val Asn Ser Gln Ile Gly Val Leu Leu Ala Glu Pro Leu Pro Asp Asp Val 20 25 30 20 25 30

Arg Ala Ala Leu Ser Ala Ile Gln His Asp Leu Phe Asp Leu Gly Gly Arg Ala Ala Leu Ser Ala Ile Gln His Asp Leu Phe Asp Leu Gly Gly 35 40 45 35 40 45

Glu Leu Cys Ile Pro Gly His Ala Ala Ile Thr Glu Asp His Leu Leu Glu Leu Cys Ile Pro Gly His Ala Ala Ile Thr Glu Asp His Leu Leu 50 55 60 50 55 60

Arg Leu Ala Leu Trp Leu Val His Tyr Asn Gly Gln Leu Pro Pro Leu Arg Leu Ala Leu Trp Leu Val His Tyr Asn Gly Gln Leu Pro Pro Leu 65 70 75 80 70 75 80

Glu Glu Phe Ile Leu Pro Gly Gly Ala Arg Gly Ala Ala Leu Ala His Glu Glu Phe Ile Leu Pro Gly Gly Ala Arg Gly Ala Ala Leu Ala His 85 90 95 85 90 95

Val Cys Arg Thr Val Cys Arg Arg Ala Glu Arg Ser Ile Lys Ala Leu Val Cys Arg Thr Val Cys Arg Arg Ala Glu Arg Ser Ile Lys Ala Leu 100 105 110 100 105 110

Gly Ala Ser Glu Pro Leu Asn Ile Ala Pro Ala Ala Tyr Val Asn Leu Gly Ala Ser Glu Pro Leu Asn Ile Ala Pro Ala Ala Tyr Val Asn Leu 115 120 125 115 120 125

Leu Ser Asp Leu Leu Phe Val Leu Ala Arg Val Leu Asn Arg Ala Ala Leu Ser Asp Leu Leu Phe Val Leu Ala Arg Val Leu Asn Arg Ala Ala 130 135 140 130 135 140

Gly Gly Ala Asp Val Leu Trp Asp Arg Thr Arg Ala His Gly Gly Ala Asp Val Leu Trp Asp Arg Thr Arg Ala His 145 150 155 145 150 155

<210> 80 <210> 80 <211> 157 <211> 157 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 80 <400> 80

Met Ile Leu Ser Ala Glu Gln Ser Phe Thr Leu Arg His Pro His Gly Met Ile Leu Ser Ala Glu Gln Ser Phe Thr Leu Arg His Pro His Gly 1 5 10 15 1 5 10 15

Gln Ala Ala Ala Leu Ala Phe Val Arg Glu Pro Ala Ala Ala Leu Ala Gln Ala Ala Ala Leu Ala Phe Val Arg Glu Pro Ala Ala Ala Leu Ala 20 25 30 20 25 30

Gly Val Gln Arg Leu Arg Gly Leu Asp Ser Asp Gly Glu Gln Val Trp Gly Val Gln Arg Leu Arg Gly Leu Asp Ser Asp Gly Glu Gln Val Trp 35 40 45 35 40 45

Gly Glu Leu Leu Val Arg Val Pro Leu Leu Gly Glu Val Asp Leu Pro Gly Glu Leu Leu Val Arg Val Pro Leu Leu Gly Glu Val Asp Leu Pro 50 55 60 50 55 60

Phe Arg Ser Glu Ile Val Arg Thr Pro Gln Gly Ala Glu Leu Arg Pro Phe Arg Ser Glu Ile Val Arg Thr Pro Gln Gly Ala Glu Leu Arg Pro 65 70 75 80 70 75 80

Leu Thr Leu Thr Gly Glu Arg Ala Trp Val Ala Val Ser Gly Gln Ala Leu Thr Leu Thr Gly Glu Arg Ala Trp Val Ala Val Ser Gly Gln Ala 85 90 95 85 90 95

Thr Ala Ala Glu Gly Gly Glu Met Ala Phe Ala Phe Gln Phe Gln Ala Thr Ala Ala Glu Gly Gly Glu Met Ala Phe Ala Phe Gln Phe Gln Ala 100 105 110 100 105 110

His Leu Ala Thr Pro Glu Ala Glu Gly Glu Gly Gly Ala Ala Phe Glu His Leu Ala Thr Pro Glu Ala Glu Gly Glu Gly Gly Ala Ala Phe Glu 115 120 125 115 120 125

Val Met Val Gln Ala Ala Ala Gly Val Thr Leu Leu Leu Val Ala Met Val Met Val Gln Ala Ala Ala Gly Val Thr Leu Leu Leu Val Ala Met 130 135 140 130 135 140

Ala Leu Pro Gln Gly Leu Ala Ala Gly Leu Pro Pro Ala Ala Leu Pro Gln Gly Leu Ala Ala Gly Leu Pro Pro Ala 145 150 155 145 150 155

<210> 81 <210> 81 <211> 156 <211> 156 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 81 <400> 81

Gly Met Pro Gly Lys Lys Glu Lys Asp Lys Val Cys Ala His Glu Ala Gly Met Pro Gly Lys Lys Glu Lys Asp Lys Val Cys Ala His Glu Ala 65 70 75 80 70 75 80

<210> 82 <210> 82 <211> 209 <211> 209 <212> PRT <212> PRT

<213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 82 <400> 82

Met Asp Asp Ile Asn Asn Gln Leu Lys Arg Leu Lys Val Ile Pro Val Met Asp Asp Ile Asn Asn Gln Leu Lys Arg Leu Lys Val Ile Pro Val 1 5 10 15 1 5 10 15

Ile Ala Ile Asp Asn Ala Glu Asp Ile Ile Pro Leu Gly Lys Val Leu Ile Ala Ile Asp Asn Ala Glu Asp Ile Ile Pro Leu Gly Lys Val Leu 20 25 30 20 25 30

Glu Ala Gly Ala Asp Phe Val Val Ser Pro Gly Phe Asn Pro Asn Thr Glu Ala Gly Ala Asp Phe Val Val Ser Pro Gly Phe Asn Pro Asn Thr 85 90 95 85 90 95

Asn Pro Ser Thr Val Glu Gln Ala Leu Glu Met Gly Leu Thr Thr Leu Asn Pro Ser Thr Val Glu Gln Ala Leu Glu Met Gly Leu Thr Thr Leu 115 120 125 115 120 125

Thr Pro Asp Asn Ile Asp Asn Tyr Leu Ala Ile Pro Gln Val Leu Ala Thr Pro Asp Asn Ile Asp Asn Tyr Leu Ala Ile Pro Gln Val Leu Ala 165 170 175 165 170 175

Cys Gly Gly Thr Trp Met Val Asp Lys Lys Leu Val Arg Asn Gly Glu Cys Gly Gly Thr Trp Met Val Asp Lys Lys Leu Val Arg Asn Gly Glu 180 185 190 180 185 190

Pro Pro

<210> 83 <210> 83 <211> 114 <211> 114 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 83 <400> 83

Met Pro Ile Phe Thr Leu Asn Thr Asn Ile Lys Ala Asp Asp Val Pro Met Pro Ile Phe Thr Leu Asn Thr Asn Ile Lys Ala Asp Asp Val Pro 1 5 10 15 1 5 10 15

Ile Gly Gly Ile Glu Pro Asp Lys Asn Arg Asp His Ser Ala Val Leu Ile Gly Gly Ile Glu Pro Asp Lys Asn Arg Asp His Ser Ala Val Leu 65 70 75 80 70 75 80

Thr Phe Thr Phe

<210> 84 <210> 84 <211> 114 <211> 114 <212> PRT <212> PRT

<213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 84 <400> 84

Glu Pro Gly Ser Tyr Val Ala Val His Ile Asn Thr Asp Gln Gln Leu Glu Pro Gly Ser Tyr Val Ala Val His Ile Asn Thr Asp Gln Gln Leu 35 40 45 35 40 45

Ile Gly Gly Ile Glu Pro Asp Lys Asn Glu Asp His Ser Ala Val Leu Ile Gly Gly Ile Glu Pro Asp Lys Asn Glu Asp His Ser Ala Val Leu 65 70 75 80 70 75 80

Ile His Phe Val Asp Leu Asp Gly Asp Asp Val Gly Trp Asn Gly Thr Ile His Phe Val Asp Leu Asp Gly Asp Asp Val Gly Trp Asn Gly Thr 100 105 110 100 105 110

Thr Phe Thr Phe

<210> 85 <210> 85 <211> 157 <211> 157 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 85 <400> 85

Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val 1 5 10 15 1 5 10 15

20 25 30 20 25 30

Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser 100 105 110 100 105 110

Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Asp Glu His His Arg Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Asp Glu His His Arg 115 120 125 115 120 125

Cys Ile Glu Ile Leu Asn Ala Arg Glu Lys Ile Ala Ala Cys Ile Glu Ile Leu Asn Ala Arg Glu Lys Ile Ala Ala 145 150 155 145 150 155

<210> 86 < 210> 86 <211> 157 <211> 157 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 86 <400> 86

Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala 20 25 30 20 25 30

Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp

35 40 45 35 40 45

Val Asp Gly Gly Ile Tyr Asp His Glu Phe Val Ala Ser Ala Val Ile Val Asp Gly Gly Ile Tyr Asp His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 85 90 95

Ala Val Leu Thr Pro His Glu Tyr Glu Asp Ser Asp Glu Asp His Glu Ala Val Leu Thr Pro His Glu Tyr Glu Asp Ser Asp Glu Asp His Glu 115 120 125 115 120 125

<210> 87 <210> 87 <211> 205 <211> 205 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 87 <400> 87

Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala 35 40 45 35 40 45

Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile

50 55 60 50 55 60

Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Asp Ile Leu Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Asp Ile Leu 115 120 125 115 120 125

Val Gly Asp Ala Leu Val Lys Gly Asp Pro Asp Glu Val Arg Glu Lys Val Gly Asp Ala Leu Val Lys Gly Asp Pro Asp Glu Val Arg Glu Lys 180 185 190 180 185 190

Ala Lys Lys Phe Val Glu Lys Ile Arg Gly Cys Thr Glu Ala Lys Lys Phe Val Glu Lys Ile Arg Gly Cys Thr Glu 195 200 205 195 200 205

<210> 88 <210> 88 <211> 205 <211> 205 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 88 <400> 88

Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe

20 25 30 20 25 30

Lys Leu Phe Pro Gly Glu Val Val Gly Pro Glu Phe Val Glu Ala Met Lys Leu Phe Pro Gly Glu Val Val Gly Pro Glu Phe Val Glu Ala Met 130 135 140 130 135 140

Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asp Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asp 145 150 155 160 145 150 155 160

Leu Asp Asp Val Cys Glu Trp Phe Asp Ala Gly Val Leu Ala Val Gly Leu Asp Asp Val Cys Glu Trp Phe Asp Ala Gly Val Leu Ala Val Gly 165 170 175 165 170 175

Val Gly Asp Ala Leu Val Glu Gly Asp Pro Asp Glu Val Arg Glu Asp Val Gly Asp Ala Leu Val Glu Gly Asp Pro Asp Glu Val Arg Glu Asp 180 185 190 180 185 190

Ala Lys Glu Phe Val Glu Glu Ile Arg Gly Cys Thr Glu Ala Lys Glu Phe Val Glu Glu Ile Arg Gly Cys Thr Glu 195 200 205 195 200 205

<210> 89 <210> 89 <211> 205 <211> 205 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 89 <400> 89

Leu Asp Asn Val Cys Lys Trp Phe Lys Ala Gly Val Leu Ala Val Gly Leu Asp Asn Val Cys Lys Trp Phe Lys Ala Gly Val Leu Ala Val Gly 165 170 175 165 170 175

Val Gly Lys Ala Leu Val Lys Gly Lys Pro Asp Glu Val Arg Glu Lys Val Gly Lys Ala Leu Val Lys Gly Lys Pro Asp Glu Val Arg Glu Lys 180 185 190 180 185 190

Ala Lys Lys Phe Val Lys Lys Ile Arg Gly Cys Thr Glu Ala Lys Lys Phe Val Lys Lys Ile Arg Gly Cys Thr Glu 195 200 205 195 200 205

<210> 90 <210> 90 <211> 157 <211> 157 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 90 <400> 90

Phe Glu Ala Ala Met Arg Asp Ile Gly Gly Asp Arg Phe Ala Val Asp Phe Glu Ala Ala Met Arg Asp Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 35 40 45

<210> 91 <210> 91 <211> 157 <211> 157 <212> PRT <212> PRT

<213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 91 <400> 91

Ala Val Leu Thr Pro His Glu Tyr Glu Asp Ser Asp Ala Asp Thr Leu Ala Val Leu Thr Pro His Glu Tyr Glu Asp Ser Asp Ala Asp Thr Leu 115 120 125 115 120 125

<210> 92 <210> 92 <211> 157 <211> 157 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 92 <400> 92 Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val 1 5 10 15 1 5 10 15

Asn Gly Met Met Asn Val Gln Leu Asn Thr Gly Val Pro Val Leu Ser Asn Gly Met Met Asn Val Gln Leu Asn Thr Gly Val Pro Val Leu Ser 100 105 110 100 105 110

Ala Val Leu Thr Pro His Asn Tyr Asp Lys Ser Lys Ala His Thr Leu Ala Val Leu Thr Pro His Asn Tyr Asp Lys Ser Lys Ala His Thr Leu 115 120 125 115 120 125

<210> 93 < 210> 93 <211> 156 <211> 156 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<220> <220> <221> MISC_FEATURE <221> MISC_FEATURE

<222> (70)..(70) <222> (70) . (70) <223> Xaa is A or K <223> Xaa is A or K

<400> 93 <400> 93

Gly Met Pro Gly Lys Xaa Glu Lys Asp Lys Val Cys Ala His Glu Ala Gly Met Pro Gly Lys Xaa Glu Lys Asp Lys Val Cys Ala His Glu Ala 65 70 75 80 70 75 80

<210> 94 <210> 94 <211> 209 <211> 209 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE <222> (2)..(2) <222> (2) -(2) <223> Xaa is S or D <223> Xaa is S or D

<220> <220> <221> MISC_FEATURE <221> MISC_FEATURE <222> (3)..(3) <222> (3) .(3) <223> Xaa is T or D <223> Xaa is T or D

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE <222> (10)..(10) <222> (10)- (10) <223> Xaa is A or R <223> Xaa is A or R

<220> <220> <221> MISC_FEATURE <221> MISC_FEATURE <222> (85)..(85) <222> (85) .-. (85) <223> Xaa is T or D <223> Xaa is T or D

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE <222> (119)..(119) <222> (119)..(119) <223> Xaa is A or Q <223> Xaa is A or Q

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE <222> (163)..(163) <222> - (163)..(163) <223> Xaa is S or D <223> Xaa is S or D

<220> <220> <221> MISC_FEATURE <221> MISC_FEATURE <222> (189)..(189) <222> (189) (189) <223> Xaa is T or R <223> Xaa is T or R

<400> 94 <400> 94

Met Xaa Xaa Ile Asn Asn Gln Leu Lys Xaa Leu Lys Val Ile Pro Val Met Xaa Xaa Ile Asn Asn Gln Leu Lys Xaa Leu Lys Val Ile Pro Val 1 5 10 15 1 5 10 15

Ile Gly Ala Gly Thr Ile Leu Asn Gly Val Gln Ala Leu Ala Ala Lys Ile Gly Ala Gly Thr Ile Leu Asn Gly Val Gln Ala Leu Ala Ala Lys

65 70 75 80 70 75 80

Glu Ala Gly Ala Xaa Phe Val Val Ser Pro Gly Phe Asn Pro Asn Thr Glu Ala Gly Ala Xaa Phe Val Val Ser Pro Gly Phe Asn Pro Asn Thr 85 90 95 85 90 95

Asn Pro Ser Thr Val Glu Xaa Ala Leu Glu Met Gly Leu Thr Thr Leu Asn Pro Ser Thr Val Glu Xaa Ala Leu Glu Met Gly Leu Thr Thr Leu 115 120 125 115 120 125

Thr Pro Xaa Asn Ile Asp Asn Tyr Leu Ala Ile Pro Gln Val Leu Ala Thr Pro Xaa Asn Ile Asp Asn Tyr Leu Ala Ile Pro Gln Val Leu Ala 165 170 175 165 170 175

Cys Gly Gly Thr Trp Met Val Asp Lys Lys Leu Val Xaa Asn Gly Glu Cys Gly Gly Thr Trp Met Val Asp Lys Lys Leu Val Xaa Asn Gly Glu 180 185 190 180 185 190

Pro Pro

<210> 95 <210> 95 <211> 114 <211> 114 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE <222> (13)..(13) <222> (13)- (13) <223> Xaa is T or D <223> Xaa is T or D

<220> <220>

<221> MISC_FEATURE <221> MISC_FEATURE <222> (33)..(33) <222> (33)- (33) <223> Xaa is K or E <223> Xaa is K or E

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE <222> (71)..(71) <222> (71)- (71) <223> Xaa is S or D <223> Xaa is S or D

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE <222> (74)..(74) <222> (74)- (74) <223> Xaa is R or E <223> Xaa is R or E

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE <222> (101)..(101) <222> (101)- . (101) <223> Xaa is N or D <223> Xaa is N or D

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE <222> (103)..(103) <222> (103)-. (103) <223> Xaa is N or D <223> Xaa is N or D

<400> 95 <400> 95

Met Pro Ile Phe Thr Leu Asn Thr Asn Ile Lys Ala Xaa Asp Val Pro Met Pro Ile Phe Thr Leu Asn Thr Asn Ile Lys Ala Xaa Asp Val Pro 1 5 10 15 1 5 10 15

Xaa Pro Gly Ser Tyr Val Ala Val His Ile Asn Thr Asp Gln Gln Leu Xaa Pro Gly Ser Tyr Val Ala Val His Ile Asn Thr Asp Gln Gln Leu 35 40 45 35 40 45

Ile Gly Gly Ile Glu Pro Xaa Lys Asn Xaa Asp His Ser Ala Val Leu Ile Gly Gly Ile Glu Pro Xaa Lys Asn Xaa Asp His Ser Ala Val Leu 65 70 75 80 70 75 80

Ile His Phe Val Xaa Leu Xaa Gly Asp Asp Val Gly Trp Asn Gly Thr Ile His Phe Val Xaa Leu Xaa Gly Asp Asp Val Gly Trp Asn Gly Thr 100 105 110 100 105 110

Thr Phe Thr Phe

<210> 96 <210> 96 <211> 157 <211> 157 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE <222> (9)..(9) <222> (9) . (9) <223> Xaa is Y or H <223> Xaa is Y or H

<220> <220> <221> MISC_FEATURE <221> MISC_FEATURE <222> (82)..(82) <222> (82)..(82) <223> Xaa is N or D <223> Xaa is N or D

<220> <220> <221> MISC_FEATURE <221> MISC_FEATURE <222> (87)..(87) <222> (87)..(87) <223> Xaa is R or D <223> Xaa is R or D

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE <222> (105)..(105) <222> (105)..(105) <223> Xaa is S or D <223> Xaa is S or D

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE <222> (119)..(119) <222> (119) (119) <223> Xaa is R or E <223> Xaa is R or E

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE <222> (121)..(121) <222> (121) (121) <223> Xaa is R or E <223> Xaa is R or E

<220> <220> <221> MISC_FEATURE <221> MISC_FEATURE <222> (124)..(124) <222> (124)..(124) <223> Xaa is A or D <223> Xaa is A or D

<220> <220> <221> MISC_FEATURE <221> MISC_FEATURE <222> (126)..(126) <222> - (126)..(126) <223> Xaa is H or D <223> Xaa is H or D

<220> <220>

<221> MISC_FEATURE <221> MISC FEATURE <222> (128)..(128) <222> (128) . (128) <223> Xaa is R or E <223> Xaa is R or E

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE <222> (150)..(150) <222> - (150) (150) <223> Xaa is A or N <223> Xaa is A or N

<400> 96 <400> 96

Met Asn Gln His Ser His Lys Asp Xaa Glu Thr Val Arg Ile Ala Val Met Asn Gln His Ser His Lys Asp Xaa Glu Thr Val Arg Ile Ala Val 1 5 10 15 1 5 10 15

Val Xaa Gly Gly Ile Tyr Xaa His Glu Phe Val Ala Ser Ala Val Ile Val Xaa Gly Gly Ile Tyr Xaa His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 85 90 95

Asp Gly Met Met Asn Val Gln Leu Xaa Thr Gly Val Pro Val Leu Ser Asp Gly Met Met Asn Val Gln Leu Xaa Thr Gly Val Pro Val Leu Ser 100 105 110 100 105 110

Ala Val Leu Thr Pro His Xaa Tyr Xaa Asp Ser Xaa Glu Xaa His Xaa Ala Val Leu Thr Pro His Xaa Tyr Xaa Asp Ser Xaa Glu Xaa His Xaa 115 120 125 115 120 125

Cys Ile Glu Ile Leu Xaa Ala Arg Glu Lys Ile Ala Ala Cys Ile Glu Ile Leu Xaa Ala Arg Glu Lys Ile Ala Ala 145 150 155 145 150 155

<210> 97 <210> 97 <211> 205 <211> 205 <212> PRT <212> PRT

<213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<220> <220> <221> MISC_FEATURE <221> MISC_FEATURE <222> (126)..(126) <222> (126) ..(126) <223> Xaa is T or D <223> Xaa is T or D

<220> <220> <221> MISC_FEATURE <221> MISC_FEATURE <222> (139)..(139) <222> (139)..(139) <223> Xaa is Q or E <223> Xaa is Q or E

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE <222> (142)..(142) <222> (142)..(142) <223> Xaa is K or E <223> Xaa is K or E

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE <222> (160)..(160) <222> (160) . (160) <223> Xaa is N or D <223> Xaa is N or D

<220> <220> <221> MISC_FEATURE <221> MISC_FEATURE <222> (163)..(163) <222> (163) (163) <223> Xaa is N or D <223> Xaa is N or D

<220> <220> <221> MISC_FEATURE <221> MISC_FEATURE <222> (166)..(166) <222> (166)..(166) <223> Xaa is E or K <223> Xaa is E or K

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE <222> (169)..(169) <222> - (169)..(169) <223> Xaa is D or K <223> Xaa is D or K

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE <222> (179)..(179) <222> (179)..(179) <223> Xaa is S, D or K <223> Xaa is S, D or K

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE <222> (183)..(183) <222> (183) (183) <223> Xaa is K or E <223> Xaa is K or E

<220> <220> <221> MISC_FEATURE <221> MISC_FEATURE <222> (185)..(185) <222> (185)..(185)

<223> Xaa is T, D, or K <223> Xaa is T, D, or K

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE (192)- . (192) <222> (192)..(192) <222> <223> Xaa is D or K <223> Xaa is D or K

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE <222> (195)..(195) <222> - (195) (195) <223> Xaa is A, E, or K <223> Xaa is A, E, or K

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE <222> (198)..(198) <222> - (198) (198) <223> Xaa is E or K <223> Xaa is E or K

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE <222> (199)..(199) <222> - (199) (199) <223> Xaa is E or K <223> Xaa is E or K

<400> 97 <400> 97

Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Xaa Ile Leu Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Xaa Ile Leu 115 120 125 115 120 125

Lys Leu Phe Pro Gly Glu Val Val Gly Pro Xaa Phe Val Xaa Ala Met Lys Leu Phe Pro Gly Glu Val Val Gly Pro Xaa Phe Val Xaa Ala Met 130 135 140 130 135 140

Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Xaa Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Xaa 145 150 155 160 145 150 155 160

Leu Asp Xaa Val Cys Xaa Trp Phe Xaa Ala Gly Val Leu Ala Val Gly Leu Asp Xaa Val Cys Xaa Trp Phe Xaa Ala Gly Val Leu Ala Val Gly 165 170 175 165 170 175

Val Gly Xaa Ala Leu Val Xaa Gly Xaa Pro Asp Glu Val Arg Glu Xaa Val Gly Xaa Ala Leu Val Xaa Gly Xaa Pro Asp Glu Val Arg Glu Xaa 180 185 190 180 185 190

Ala Lys Xaa Phe Val Xaa Xaa Ile Arg Gly Cys Thr Glu Ala Lys Xaa Phe Val Xaa Xaa Ile Arg Gly Cys Thr Glu 195 200 205 195 200 205

<210> 98 <210> 98 <211> 157 <211> 157 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE <222> (9)..(9) <222> (9) -(9) <223> Xaa is Y or H <223> Xaa is Y or H

<220> <220> <221> MISC_FEATURE <221> MISC_FEATURE <222> (38)..(38) <222> (38) - . (38) <223> Xaa is A or R <223> Xaa is A or R

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE <222> (82)..(82) <222> (82)- (82) <223> Xaa is N or D <223> Xaa is N or D

<220> <220> <221> MISC_FEATURE <221> MISC_FEATURE <222> (87)..(87) <222> (87)- (87) <223> Xaa is R or D <223> Xaa is R or D

<220> <220> <221> MISC_FEATURE <221> MISC_FEATURE <222> (97)..(97) <222> (97)..(97)

<223> Xaa is N or D <223> Xaa is N or D

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE <222> (105)..(105) <222> (105)-. (105) <223> Xaa is S, N, or D <223> Xaa is S, N, or D

<220> <220> <221> MISC_FEATURE <221> MISC_FEATURE <222> (119)..(119) <222> - (119)..(119) <223> Xaa is R, E, or N <223> Xaa is R, E, or N

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE <222> (121)..(121) <222> (121)..(121) <223> Xaa is R, E, or D <223> Xaa is R, E, or D

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE <222> (122)..(122) <222> (122) (122) <223> Xaa is K or D <223> Xaa is K or D

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE <222> (124)..(124) <222> (124)-. (124) <223> Xaa is K or D <223> Xaa is K or D

<220> <220> <221> MISC_FEATURE <221> MISC FEATURE <222> (126)..(126) <222> - (126) (126) <223> Xaa is H or D <223> Xaa is H or D

<400> 98 <400> 98

Phe Glu Ala Ala Met Xaa Asp Ile Gly Gly Asp Arg Phe Ala Val Asp Phe Glu Ala Ala Met Xaa Asp Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 35 40 45

Xaa Gly Met Met Asn Val Gln Leu Xaa Thr Gly Val Pro Val Leu Ser Xaa Gly Met Met Asn Val Gln Leu Xaa Thr Gly Val Pro Val Leu Ser 100 105 110 100 105 110

Ala Val Leu Thr Pro His Xaa Tyr Xaa Xaa Ser Xaa Ala Xaa Thr Leu Ala Val Leu Thr Pro His Xaa Tyr Xaa Xaa Ser Xaa Ala Xaa Thr Leu 115 120 125 115 120 125

<210> 99 <210> 99 <211> 142 <211> 142 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 99 <400> 99 taatgcttaa gtcgaacaga aagtaatcgt attgtacacg gccgcataat cgaaattaat 60 taatgcttaa gtcgaacaga aagtaatcgt attgtacacg gccgcataat cgaaattaat 60

acgactcact ataggggaat tgtgagcgga taacaattcc ccatcttagt atattagtta 120 acgactcact ataggggaat tgtgagcgga taacaattcc ccatcttagt atattagtta 120

agtataagaa ggagatatac tt 142 agtataagaa ggagatatad tt 142

<210> 100 <210> 100 <211> 18 <211> 18 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 100 <400> 100 taaagaagga gatatcat 18 taaagaagga gatatcat 18

<210> 101 <210> 101 <211> 17 <211> 17 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220>

<223> Synthetic <223> Synthetic

<400> 101 <400> 101 tgagaaggag atatcat 17 tgagaaggag atatcat 17

<210> 102 <210> 102 <211> 365 <211> 365 <212> PRT <212> PRT <213> Homo sapiens <213> Homo sapiens

<400> 102 <400> 102

Leu Leu Pro Gly Ser Leu Gly Ala Glu Ser His Leu Ser Leu Leu Tyr Leu Leu Pro Gly Ser Leu Gly Ala Glu Ser His Leu Ser Leu Leu Tyr 20 25 30 20 25 30

His Leu Thr Ala Val Ser Ser Pro Ala Pro Gly Thr Pro Ala Phe Trp His Leu Thr Ala Val Ser Ser Pro Ala Pro Gly Thr Pro Ala Phe Trp 35 40 45 35 40 45

Val Ser Gly Trp Leu Gly Pro Gln Gln Tyr Leu Ser Tyr Asn Ser Leu Val Ser Gly Trp Leu Gly Pro Gln Gln Tyr Leu Ser Tyr Asn Ser Leu 50 55 60 50 55 60

Arg Gly Glu Ala Glu Pro Cys Gly Ala Trp Val Trp Glu Asn Gln Val Arg Gly Glu Ala Glu Pro Cys Gly Ala Trp Val Trp Glu Asn Gln Val 65 70 75 80 70 75 80

Ser Trp Tyr Trp Glu Lys Glu Thr Thr Asp Leu Arg Ile Lys Glu Lys Ser Trp Tyr Trp Glu Lys Glu Thr Thr Asp Leu Arg Ile Lys Glu Lys 85 90 95 85 90 95

Leu Phe Leu Glu Ala Phe Lys Ala Leu Gly Gly Lys Gly Pro Tyr Thr Leu Phe Leu Glu Ala Phe Lys Ala Leu Gly Gly Lys Gly Pro Tyr Thr 100 105 110 100 105 110

Leu Gln Gly Leu Leu Gly Cys Glu Leu Gly Pro Asp Asn Thr Ser Val Leu Gln Gly Leu Leu Gly Cys Glu Leu Gly Pro Asp Asn Thr Ser Val 115 120 125 115 120 125

Pro Thr Ala Lys Phe Ala Leu Asn Gly Glu Glu Phe Met Asn Phe Asp Pro Thr Ala Lys Phe Ala Leu Asn Gly Glu Glu Phe Met Asn Phe Asp 130 135 140 130 135 140

Leu Lys Gln Gly Thr Trp Gly Gly Asp Trp Pro Glu Ala Leu Ala Ile Leu Lys Gln Gly Thr Trp Gly Gly Asp Trp Pro Glu Ala Leu Ala Ile 145 150 155 160 145 150 155 160

Ser Gln Arg Trp Gln Gln Gln Asp Lys Ala Ala Asn Lys Glu Leu Thr Ser Gln Arg Trp Gln Gln Gln Asp Lys Ala Ala Asn Lys Glu Leu Thr

165 170 175 165 170 175

Phe Leu Leu Phe Ser Cys Pro His Arg Leu Arg Glu His Leu Glu Arg Phe Leu Leu Phe Ser Cys Pro His Arg Leu Arg Glu His Leu Glu Arg 180 185 190 180 185 190

Gly Arg Gly Asn Leu Glu Trp Lys Glu Pro Pro Ser Met Arg Leu Lys Gly Arg Gly Asn Leu Glu Trp Lys Glu Pro Pro Ser Met Arg Leu Lys 195 200 205 195 200 205

Ala Arg Pro Ser Ser Pro Gly Phe Ser Val Leu Thr Cys Ser Ala Phe Ala Arg Pro Ser Ser Pro Gly Phe Ser Val Leu Thr Cys Ser Ala Phe 210 215 220 210 215 220

Ser Phe Tyr Pro Pro Glu Leu Gln Leu Arg Phe Leu Arg Asn Gly Leu Ser Phe Tyr Pro Pro Glu Leu Gln Leu Arg Phe Leu Arg Asn Gly Leu 225 230 235 240 225 230 235 240

Ala Ala Gly Thr Gly Gln Gly Asp Phe Gly Pro Asn Ser Asp Gly Ser Ala Ala Gly Thr Gly Gln Gly Asp Phe Gly Pro Asn Ser Asp Gly Ser 245 250 255 245 250 255

Phe His Ala Ser Ser Ser Leu Thr Val Lys Ser Gly Asp Glu His His Phe His Ala Ser Ser Ser Leu Thr Val Lys Ser Gly Asp Glu His His 260 265 270 260 265 270

Tyr Cys Cys Ile Val Gln His Ala Gly Leu Ala Gln Pro Leu Arg Val Tyr Cys Cys Ile Val Gln His Ala Gly Leu Ala Gln Pro Leu Arg Val 275 280 285 275 280 285

Glu Leu Glu Ser Pro Ala Lys Ser Ser Val Leu Val Val Gly Ile Val Glu Leu Glu Ser Pro Ala Lys Ser Ser Val Leu Val Val Gly Ile Val 290 295 300 290 295 300

Ile Gly Val Leu Leu Leu Thr Ala Ala Ala Val Gly Gly Ala Leu Leu Ile Gly Val Leu Leu Leu Thr Ala Ala Ala Val Gly Gly Ala Leu Leu 305 310 315 320 305 310 315 320

Trp Arg Arg Met Arg Ser Gly Leu Pro Ala Pro Trp Ile Ser Leu Arg Trp Arg Arg Met Arg Ser Gly Leu Pro Ala Pro Trp Ile Ser Leu Arg 325 330 335 325 330 335

Gly Asp Asp Thr Gly Val Leu Leu Pro Thr Pro Gly Glu Ala Gln Asp Gly Asp Asp Thr Gly Val Leu Leu Pro Thr Pro Gly Glu Ala Gln Asp 340 345 350 340 345 350

Ala Asp Leu Lys Asp Val Asn Val Ile Pro Ala Thr Ala Ala Asp Leu Lys Asp Val Asn Val Ile Pro Ala Thr Ala 355 360 365 355 360 365

<210> 103 <210> 103 <211> 62 <211> 62 <212> PRT <212> PRT

<213> Homo sapiens <213> Homo sapiens

<400> 103 <400> 103

Leu Leu Pro Gly Ser Leu Gly Phe Ala Cys Lys Thr Ala Asn Gly Thr Leu Leu Pro Gly Ser Leu Gly Phe Ala Cys Lys Thr Ala Asn Gly Thr 20 25 30 20 25 30

Ala Ile Pro Ile Gly Gly Gly Ser Ala Asn Val Tyr Val Asn Leu Ala Ala Ile Pro Ile Gly Gly Gly Ser Ala Asn Val Tyr Val Asn Leu Ala 35 40 45 35 40 45

Pro Val Val Asn Val Gly Gln Asn Leu Val Val Asp Leu Ser Pro Val Val Asn Val Gly Gln Asn Leu Val Val Asp Leu Ser 50 55 60 50 55 60

<210> 104 <210> 104 <211> 574 <211> 574 <212> PRT <212> PRT <213> Human respiratory syncytial virus A (strain A2) <213> Human respiratory syncytial virus A (strain A2)

<400> 104 <400> 104

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe 20 25 30 20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu 35 40 45 35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys 65 70 75 80 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 85 90 95

Met Gln Ser Thr Pro Pro Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro Met Gln Ser Thr Pro Pro Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro 100 105 110 100 105 110

Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr 115 120 125 115 120 125

Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val 130 135 140 130 135 140

Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu 145 150 155 160 145 150 155 160

Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys 165 170 175 165 170 175

Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val 180 185 190 180 185 190

Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn 195 200 205 195 200 205

Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 210 215 220

Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 225 230 235 240

Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 245 250 255

Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 260 265 270

Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 275 280 285

Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 290 295 300

Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 305 310 315 320

Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg

325 330 335 325 330 335

Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 340 345 350

Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 355 360 365

Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Ile Asn Leu Cys Asn Val Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Ile Asn Leu Cys Asn Val 370 375 380 370 375 380

Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 385 390 395 400

Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 405 410 415

Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 420 425 430

Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Met Asp Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Met Asp 435 440 445 435 440 445

Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly 450 455 460 450 455 460

Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro 465 470 475 480 465 470 475 480

Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 485 490 495

Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu 500 505 510 500 505 510

Leu His Asn Val Asn Ala Gly Lys Ser Thr Thr Asn Ile Met Ile Thr Leu His Asn Val Asn Ala Gly Lys Ser Thr Thr Asn Ile Met Ile Thr 515 520 525 515 520 525

Thr Ile Ile Ile Val Ile Ile Val Ile Leu Leu Ser Leu Ile Ala Val Thr Ile Ile Ile Val Ile Ile Val Ile Leu Leu Ser Leu Ile Ala Val 530 535 540 530 535 540

Gly Leu Leu Leu Tyr Cys Lys Ala Arg Ser Thr Pro Val Thr Leu Ser Gly Leu Leu Leu Tyr Cys Lys Ala Arg Ser Thr Pro Val Thr Leu Ser 545 550 555 560 545 550 555 560

Lys Asp Gln Leu Ser Gly Ile Asn Asn Ile Ala Phe Ser Asn Lys Asp Gln Leu Ser Gly Ile Asn Asn Ile Ala Phe Ser Asn 565 570 565 570

<210> 105 <210> 105 <211> 64 <211> 64 <212> PRT <212> PRT <213> Human respiratory syncytial virus A (strain A2) <213> Human respiratory syncytial virus A (strain A2)

<400> 105 <400> 105

Ala Val Thr Phe Cys Phe Ala Ser Gly Phe Ala Cys Lys Thr Ala Asn Ala Val Thr Phe Cys Phe Ala Ser Gly Phe Ala Cys Lys Thr Ala Asn 20 25 30 20 25 30

Gly Thr Ala Ile Pro Ile Gly Gly Gly Ser Ala Asn Val Tyr Val Asn Gly Thr Ala Ile Pro Ile Gly Gly Gly Ser Ala Asn Val Tyr Val Asn 35 40 45 35 40 45

Leu Ala Pro Val Val Asn Val Gly Gln Asn Leu Val Val Asp Leu Ser Leu Ala Pro Val Val Asn Val Gly Gln Asn Leu Val Val Asp Leu Ser 50 55 60 50 55 60

<210> 106 <210> 106 <211> 178 <211> 178 <212> PRT <212> PRT <213> Homo sapiens <213> Homo sapiens

<400> 106 <400> 106

Arg Ala Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Arg Ala Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His 20 25 30 20 25 30

Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe 35 40 45 35 40 45

Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu 50 55 60 50 55 60

Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys 65 70 75 80 70 75 80

Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro 85 90 95 85 90 95

Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu 100 105 110 100 105 110

Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg 115 120 125 115 120 125

Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn 130 135 140 130 135 140

Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu 145 150 155 160 145 150 155 160

Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile 165 170 175 165 170 175

Arg Asn Arg Asn

<210> 107 <210> 107 <211> 57 <211> 57 <212> PRT <212> PRT <213> Homo sapiens <213> Homo sapiens

<400> 107 <400> 107

Arg Ala Phe Ala Cys Lys Thr Ala Asn Gly Thr Ala Ile Pro Ile Gly Arg Ala Phe Ala Cys Lys Thr Ala Asn Gly Thr Ala Ile Pro Ile Gly 20 25 30 20 25 30

Gly Gly Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gly Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val 35 40 45 35 40 45

Gly Gln Asn Leu Val Val Asp Leu Ser Gly Gln Asn Leu Val Val Asp Leu Ser 50 55 50 55

<210> 108 <210> 108 <211> 562 <211> 562 <212> PRT <212> PRT <213> Influenza A virus (strain A/Japan/305/1957 H2N2) <213> Influenza A virus (strain A/Japan/305/1957 H2N2)

<400> 108 <400> 108

Met Ala Ile Ile Tyr Leu Ile Leu Leu Phe Thr Ala Val Arg Gly Asp Met Ala Ile Ile Tyr Leu Ile Leu Leu Phe Thr Ala Val Arg Gly Asp 1 5 10 15 1 5 10 15

Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Lys Val Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Lys Val Asp 20 25 30 20 25 30

Thr Asn Leu Glu Arg Asn Val Thr Val Thr His Ala Lys Asp Ile Leu Thr Asn Leu Glu Arg Asn Val Thr Val Thr His Ala Lys Asp Ile Leu 35 40 45 35 40 45

Glu Lys Thr His Asn Gly Lys Leu Cys Lys Leu Asn Gly Ile Pro Pro Glu Lys Thr His Asn Gly Lys Leu Cys Lys Leu Asn Gly Ile Pro Pro 50 55 60 50 55 60

Leu Glu Leu Gly Asp Cys Ser Ile Ala Gly Trp Leu Leu Gly Asn Pro Leu Glu Leu Gly Asp Cys Ser Ile Ala Gly Trp Leu Leu Gly Asn Pro 65 70 75 80 70 75 80

Glu Cys Asp Arg Leu Leu Ser Val Pro Glu Trp Ser Tyr Ile Met Glu Glu Cys Asp Arg Leu Leu Ser Val Pro Glu Trp Ser Tyr Ile Met Glu 85 90 95 85 90 95

Lys Glu Asn Pro Arg Asp Gly Leu Cys Tyr Pro Gly Ser Phe Asn Asp Lys Glu Asn Pro Arg Asp Gly Leu Cys Tyr Pro Gly Ser Phe Asn Asp 100 105 110 100 105 110

Tyr Glu Glu Leu Lys His Leu Leu Ser Ser Val Lys His Phe Glu Lys Tyr Glu Glu Leu Lys His Leu Leu Ser Ser Val Lys His Phe Glu Lys 115 120 125 115 120 125

Val Lys Ile Leu Pro Lys Asp Arg Trp Thr Gln His Thr Thr Thr Gly Val Lys Ile Leu Pro Lys Asp Arg Trp Thr Gln His Thr Thr Thr Gly 130 135 140 130 135 140

Gly Ser Arg Ala Cys Ala Val Ser Gly Asn Pro Ser Phe Phe Arg Asn Gly Ser Arg Ala Cys Ala Val Ser Gly Asn Pro Ser Phe Phe Arg Asn 145 150 155 160 145 150 155 160

Met Val Trp Leu Thr Lys Glu Gly Ser Asp Tyr Pro Val Ala Lys Gly Met Val Trp Leu Thr Lys Glu Gly Ser Asp Tyr Pro Val Ala Lys Gly 165 170 175 165 170 175

Ser Tyr Asn Asn Thr Ser Gly Glu Gln Met Leu Ile Ile Trp Gly Val Ser Tyr Asn Asn Thr Ser Gly Glu Gln Met Leu Ile Ile Trp Gly Val

180 185 190 180 185 190

His His Pro Ile Asp Glu Thr Glu Gln Arg Thr Leu Tyr Gln Asn Val His His Pro Ile Asp Glu Thr Glu Gln Arg Thr Leu Tyr Gln Asn Val 195 200 205 195 200 205

Gly Thr Tyr Val Ser Val Gly Thr Ser Thr Leu Asn Lys Arg Ser Thr Gly Thr Tyr Val Ser Val Gly Thr Ser Thr Leu Asn Lys Arg Ser Thr 210 215 220 210 215 220

Pro Glu Ile Ala Thr Arg Pro Lys Val Asn Gly Gln Gly Gly Arg Met Pro Glu Ile Ala Thr Arg Pro Lys Val Asn Gly Gln Gly Gly Arg Met 225 230 235 240 225 230 235 240

Glu Phe Ser Trp Thr Leu Leu Asp Met Trp Asp Thr Ile Asn Phe Glu Glu Phe Ser Trp Thr Leu Leu Asp Met Trp Asp Thr Ile Asn Phe Glu 245 250 255 245 250 255

Ser Thr Gly Asn Leu Ile Ala Pro Glu Tyr Gly Phe Lys Ile Ser Lys Ser Thr Gly Asn Leu Ile Ala Pro Glu Tyr Gly Phe Lys Ile Ser Lys 260 265 270 260 265 270

Arg Gly Ser Ser Gly Ile Met Lys Thr Glu Gly Thr Leu Glu Asn Cys Arg Gly Ser Ser Gly Ile Met Lys Thr Glu Gly Thr Leu Glu Asn Cys 275 280 285 275 280 285

Glu Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Thr Thr Leu Pro Glu Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Thr Thr Leu Pro 290 295 300 290 295 300

Phe His Asn Val His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val Phe His Asn Val His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val 305 310 315 320 305 310 315 320

Lys Ser Glu Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Val Pro Gln Lys Ser Glu Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Val Pro Gln 325 330 335 325 330 335

Ile Glu Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Ile Glu Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly 340 345 350 340 345 350

Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Ser Asn Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Ser Asn 355 360 365 355 360 365

Asp Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys Ala Asp Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys Ala 370 375 380 370 375 380

Phe Asp Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Phe Asp Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn 385 390 395 400 385 390 395 400

Thr Gln Phe Glu Ala Val Gly Lys Glu Phe Gly Asn Leu Glu Arg Arg Thr Gln Phe Glu Ala Val Gly Lys Glu Phe Gly Asn Leu Glu Arg Arg 405 410 415 405 410 415

Leu Glu Asn Leu Asn Lys Arg Met Glu Asp Gly Phe Leu Asp Val Trp Leu Glu Asn Leu Asn Lys Arg Met Glu Asp Gly Phe Leu Asp Val Trp 420 425 430 420 425 430

Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu 435 440 445 435 440 445

Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val Arg Met Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val Arg Met 450 455 460 450 455 460

Gln Leu Arg Asp Asn Val Lys Glu Leu Gly Asn Gly Cys Phe Glu Phe Gln Leu Arg Asp Asn Val Lys Glu Leu Gly Asn Gly Cys Phe Glu Phe 465 470 475 480 465 470 475 480

Tyr His Lys Cys Asp Asp Glu Cys Met Asn Ser Val Lys Asn Gly Thr Tyr His Lys Cys Asp Asp Glu Cys Met Asn Ser Val Lys Asn Gly Thr 485 490 495 485 490 495

Tyr Asp Tyr Pro Lys Tyr Glu Glu Glu Ser Lys Leu Asn Arg Asn Glu Tyr Asp Tyr Pro Lys Tyr Glu Glu Glu Ser Lys Leu Asn Arg Asn Glu 500 505 510 500 505 510

Ile Lys Gly Val Lys Leu Ser Ser Met Gly Val Tyr Gln Ile Leu Ala Ile Lys Gly Val Lys Leu Ser Ser Met Gly Val Tyr Gln Ile Leu Ala 515 520 525 515 520 525

Ile Tyr Ala Thr Val Ala Gly Ser Leu Ser Leu Ala Ile Met Met Ala Ile Tyr Ala Thr Val Ala Gly Ser Leu Ser Leu Ala Ile Met Met Ala 530 535 540 530 535 540

Gly Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Gly Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile 545 550 555 560 545 550 555 560

Cys Ile Cys Ile

<210> 109 <210> 109 <211> 54 <211> 54 <212> PRT <212> PRT <213> Influenza A virus (strain A/Japan/305/1957 H2N2) <213> Influenza A virus (strain A/Japan/305/1957 H2N2)

<400> 109 <400> 109

Met Ala Ile Ile Tyr Leu Ile Leu Leu Phe Thr Ala Val Arg Gly Phe Met Ala Ile Ile Tyr Leu Ile Leu Leu Phe Thr Ala Val Arg Gly Phe 1 5 10 15 1 5 10 15

Ala Cys Lys Thr Ala Asn Gly Thr Ala Ile Pro Ile Gly Gly Gly Ser Ala Cys Lys Thr Ala Asn Gly Thr Ala Ile Pro Ile Gly Gly Gly Ser 20 25 30 20 25 30

Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln Asn Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln Asn 35 40 45 35 40 45

Leu Val Val Asp Leu Ser Leu Val Val Asp Leu Ser 50 50

<210> 110 <210> 110 <211> 310 <211> 310 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 110 <400> 110

Gly Val Thr Phe Val Tyr Gln Gly Gly Ser Ser Gly Gly Gly Ala Asp Gly Val Thr Phe Val Tyr Gln Gly Gly Ser Ser Gly Gly Gly Ala Asp 275 280 285 275 280 285

Val Thr Ile Thr Val Asn Gly Lys Val Val Ala Lys Gly Gly His His Val Thr Ile Thr Val Asn Gly Lys Val Val Ala Lys Gly Gly His His 290 295 300 290 295 300

His His His His His His His His His His His His 305 310 305 310

<210> 111 <210> 111 <211> 168 <211> 168 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 111 <400> 111

His His His His His His His His His His His His His His His His 165 165

<210> 112 <210> 112 <211> 310 <211> 310 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 112 <400> 112

His His His His His His His His His His His His 305 310 305 310

<210> 113 <210> 113 <211> 168 <211> 168 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic <223> Synthetic

<400> 113 <400> 113

His His His His His His His His His His His His His His His His 165

Claims

1. A composition comprising (i) a polypeptide derived from fimbrial H (FimH), or a functional fragment thereof, or a nucleic acid molecule that encodes a polypeptide derived from FimH, or a functional fragment thereof, and (ii) each of the following conjugates: a conjugate comprising a carrier protein covalently bound to a saccharide comprising Formula 025b comprising the following structure:

p-Glcp

-Rhap-(1 w3)-a-Glcp-(1-3)*Rhap20Ac-(13)GlcpNAc-In

wherein n is an integer from 31 to 100 in the formula thereof, a conjugate comprising a carrier protein covalently bound to a saccharide comprising Formula 01A comprising the following structure:

[-*3)-a-l-Rha-(1-*3)-a-l-Rha-(1--3)-p-l-Rha-(1-4)-p-d-GcNAc-(1-| j3-d-ManNAc (1-*2) ], wherein n is an integer from 31 to 100 in the formula thereof, a conjugate comprising a carrier protein covalently bound to a saccharide comprising Formula 02 comprising the following structure:

[--3)-a-L-Rha-(1-*2)-a-L-Rha-(1-*3)-p-L-Rha-(1-4)-p-D-GcNAc-(1-l+ca-D Fuc3NAc-(1-*2) ]n, wherein n is an integer from 31 to 100 in the formula thereof, and a conjugate comprising a carrier protein covalently bound to a saccharide comprising Formula 06:K2, Formula 06:K13, Formula 06:K15, or Formula 06:K54 comprising a structure selected from the group consisting of:

[-4)-a-D-GalNAc-(1-*3)-p-D-Man-(l-4)-p-D-Man-(1--3)-a-D-GcNAc-(-Hl -D Glc-(1-2) ],, and

[-4)-a-D-GalNAc-(1-*3)-p-D-Man-(l-4)-p-D-Man-(1--3)-a-D-GlcNAc-(1-l D-GlcNAc-(1-2) ]n, wherein n is an integer from 31 to 100 in the formula thereof.

2. The composition of claim 1, wherein n is an integer from 31 to 90 in the Formula thereof for each of said saccharides, and wherein the composition further comprises: a conjugate comprising a carrier protein covalently bound to a saccharide comprising Formula 08 comprising the following structure:

[ ->2)-a-D-Man-(1->2)-a-D-Man-(1->3)-p-D-Man-(1-> 1, wherein n is an integer from 31 to 90 in the Formula thereof for said saccharide, a conjugate comprising a carrier protein covalently bound to a saccharide comprising Formula 09 comprising the following structure:

[ -o>2)-[a-D-Man-(o->2)]a-D-Man-(1->3)-a-D-Man-(1->3)-a-D-Man-(1-> ], or a conjugate comprising a carrier protein covalently bound to a saccharide comprising Formula 09a comprising the following structure:

[ ->2)-a-D-Man-(1->2)-a-D-Man-(1s->3)-a-D-Man-(1->3)-a-D-Man-(1-> ], wherein n is an integer from 31 to 90 in the Formula thereof for said saccharide.

3. The composition of claim 1, wherein n is an integer from 31 to 90 in the formula thereof for each of said saccharides.

4. The composition according to claim 2 or 3, further comprising one or more conjugates having a saccharide selected from the group consisting of Formula 016 comprising the following structure: [->2)-j-D-Galf (1->6)-a-D-Glc-(1->3)-a-L-Rha2Ac-(1->3)-a-D-GlcNAc (1->],, Formula 017 comprising the following structure: [a-D-Glc-(1->6) I ->6)-a-D-Man (1->2)-a-D-Man-(1->2)- r-D-Man-(1->3)-a-D-GlcNAc(1-> ],, Formula 018A or Formula 018ac comprising the following structure: [->2)-a-L-Rha-(1->6)-a-D-Glc-(1->4)-a-D-Gal (1->3)-a-D-GlcNAc-(1-> | P-D-GlcNAc-(1->3) ],, Formula 018A1 comprising the following structure: [a-D-Glc-(1->6) I ->2)-a-L-Rha-(1->6)-a-D -Glc-(1->4)-a-D -Gal-(1->3)-a-D GlcNAc-(1-> IP-D-GlcNAc-(1->3) ],, Formula 018B comprising the following structure:

[->3) -a-L-Rha-(1->6)-a-D-Glc-(1->4) -a-D-Gal-(1-> 3) -a-D-GlcNAc-(1-> | P-D-Glc-(1->3)]n, Formula 018B1 comprising the following structure: [a-D-Glc-(1->4) I ->3)-a-L-Rha (1->6)-a-D-Glc-(1->4) -a-D-Gal-(1->3) -a-D-GlcNAc-(1-> | P-D-Glc-(1->3) ],, and Formula

075 comprising the following structure: [ P-D-Man-(4) | -3)-a-D-Gal-(1l-4)-a-L-Rha (1-*3)-r-D-GlcNAc-(1->], wherein n is an integer from 31 to 90 in the formula thereof for each saccharide.

5. The composition according to any one of claims 1-4, wherein the carrier protein is selected from the group consisting of CRM 197, diphtheria toxin fragment B (DTFB), DTFB C8, Diphtheria toxoid (DT), tetanus toxoid (TT), fragment C of TT, pertussis toxoid, cholera toxoid, exotoxin A from Pseudomonas aeruginosa, detoxified Exotoxin A of P. aeruginosa(EPA), maltose binding protein (MBP), detoxified hemolysin A of S. aureus, clumping factor A, clumping factor B, Cholera toxin B subunit (CTB), Streptococcuspneumoniae Pneumolysin or detoxified variants thereof, C. jejuni AcrA, C.

jejuni natural glycoproteins, and Streptococcal C5a peptidase (SCP) or a variant thereof.

6. The composition according to any one of claims 1-5, wherein the polypeptide is derived from E. coli fimbrial H (FimH).

7. The composition according to claim 6, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 2.

8. The composition according to claim 6, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 3

9. The composition according to claim 6, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 27, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, and SEQ ID NO: 113.

10. The composition of claim 6, wherein the polypeptide comprises an amino acid sequence having at least 70% identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 23,

SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, and SEQ ID NO: 113.

11. The composition according to claim 6, wherein the polypeptide comprises a fragment of FimH.

12. The composition according to claim 11, wherein the polypeptide comprises a lectin domain of FimH.

13. The composition according to claim 6, wherein the polypeptide is complexed with a FimC polypeptide or a fragment thereof.

14. The composition according to claim 6, wherein the polypeptide is stabilized by a donor strand peptide of FimG (DsG).

15. The composition according to any one of claims 1-14, wherein the polypeptide further comprises a linker.

16. The composition according to any one of claims 1-15, comprising a nucleic acid molecule that encodes a polypeptide derived from FimH, or a functional fragment thereof, wherein the nucleic acid is RNA.

17. The composition according to any one of claims 1-16, wherein the composition further comprises an adjuvant.

18. A method of eliciting an immune response against Escherichiacoli in a mammal, comprising administering to the mammal an effective amount of the composition according to any one of claims 1-17.

19. Use of the composition according to any one of claims 1-17 in the manufacture of a medicament for eliciting an immune response against Escherichiacoli in a mammal.