JP7664386B2 - E. coli compositions and methods thereof - Google Patents

Description

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

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

FIELD OF THEINVENTION The present invention relates to novel immunogenic compositions for use in vaccines to elicit immune responses against E. coli and Klebsiella pneumoniae serotypes in a subject.

Escherichia coli are one of the most common human bacterial pathogens with clinical manifestations including bloodstream infections, urinary tract infections (catheter and noncatheter associated; surgical site infections, pneumonia, and severe food-related diarrhea. They are classified serologically by differences in the structure of the lipopolysaccharide-associated O antigen (>180 known serotypes), capsular polysaccharide K antigen (>100 serotypes), and flagellar H antigen (>50 serotypes).

Urinary tract infections (UTIs) most often manifest as cystitis that may recur repeatedly after resolution in some individuals. If left untreated, they can progress to pyelonephritis and bloodstream infections. Escherichia coli (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 last resort antibiotics 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 global pandemic clone causing primarily community-acquired infections and exhibiting 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]. BSI- and UTI-infecting strains of E. coli are also known as invasive extraintestinal pathogenic E. coli (ExPEC) or uropathogenic E. coli (UPEC).

After E. coli, Klebsiella spp. (K. pneumoniae and K. oxytoca) are the next most common Gram-negative pathogens associated with invasive infections, including UTIs, pneumonia, intra-abdominal infections, and bloodstream infections (BSIs) [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 American College of Clinical Microbiology, 2013; Association of Physicians 1996;89:933-41; Anderson DJ et al., PLoS One 2014;9:e91713]. Klebsiella maintains a high capacity to acquire antibiotic resistance via horizontally transferred ESBLs and carbapenem resistance-conferring genes [Chen L et al., Trends Microbiol 2014;22:686-96; Iredell J et al., Bmj 2016;352:h6420]. Thus, during the last decade, the prevalence of ESBL-resistant Klebsiella producing extended-spectrum β-lactamases (ESBLs) has increased dramatically at the global level. Klebsiella spp. can express up to eight different O types and more than 80 K types. Although there are numerous K antigens associated with virulent Klebsiella strains, only four O antigen serotypes account for more than 80% of Klebsiella clinical isolates, regardless of the sampling site (blood, urine, saliva), infection status (invasive vs. non-invasive) or nature of acquisition (community vs. hospital) [Follador R et al., Microbial Genomics 2016;2:e000073].

Increasing rates of invasive multidrug-resistant (MDE) E. coli and Klebsiella infections in vulnerable neonatal populations and the elderly highlight the need for vaccine-based approaches as alternatives to antibiotics, the increasingly effective standard of care.

To meet these and other needs, the present invention relates to compositions and methods of use for producing novel immunogenic compositions for use in vaccines to elicit immune responses against E. coli and Klebsiella pneumoniae serotypes in subjects.

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, e.g., an infant. In some other particular embodiments, the human is a female, particularly a pregnant female.

The composition can be administered to a subject with or without an adjuvant. The effective amount administered to a subject is an amount sufficient to elicit an immune response in the subject against E. coli or K. pneumoniae antigens. Subjects that can be selected for treatment include those at risk of developing E. coli or K. pneumoniae infection due to exposure or potential exposure to E. coli or K. pneumoniae. Because humans can become infected with E. coli or K. pneumoniae by age 2, entire birth cohorts are included as suitable populations for immunization. This can be done, for example, by initiating immunization regimens in pregnant women (or women of childbearing age) to protect infants still in utero by passive transfer of antibodies any time from birth to 6 months of age, from 6 months to 5 years of age, and in subjects over 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 comprises a sugar selected from any sugar having a formula in Table 1, preferably formula O1A, formula O1B, formula O2, formula O6, and formula O25B, where n is an integer between 1 and 100, preferably between 31 and 100.

In one aspect, the present invention relates to a composition comprising 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 comprising 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, where n is 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20 or more). In some embodiments, the composition further comprises a sugar selected from any one of the formulas in Table 1, preferably formula O1A, formula O1B, formula O2, formula O6, and formula O25B, where n is an integer from 1 to 100, preferably 31 to 100.

1A-H depict amino acid sequences, including the amino acid sequence of an exemplary polypeptide or fragment thereof derived from E. coli; and the amino acid sequence of an exemplary wzzB sequence. 2A-T depict maps of exemplary expression vectors. FIG. 3 depicts the expression and purification results. FIG. 4 depicts the expression and purification results. FIG. 5 depicts the expression results. Figures 6A-C depict pSB02083 and pSB02158 SEC pools and affinities, including yields. FIG. 7 depicts the results of expression of the pSB2198 FimH dscG Lock mutant construct. FIG. 8 depicts the results of expression of pSB2307 FimH dscG wild type. 9A-C depict the structures of O antigens synthesized by the polymerase-dependent pathway that contain four or fewer residues in the backbone. FIG. 10A depicts the structure of an O antigen synthesized by the polymerase-dependent pathway that contains five or six residues in the backbone; FIG. 10B depicts an O antigen thought to be synthesized by the ABC transporter-dependent pathway. FIG. 11 depicts computational mutagenesis scanning of Phe1 with other amino acids with aliphatic hydrophobic side chains that may stabilize the FimH protein and accommodate mannose binding, such as Ile, Leu, and Val. 12A-12B depict the plasmids: pUC replicon plasmid, 500-700 copies per cell, chain length regulator (FIG. 12A); and P15a replicon plasmid, 10-12 copies per cell, O antigen operon (FIG. 12B). Figures 13A-13B depict modulation of O-antigen chain length in serotype O25a and O25b 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 O25K5H1 (O25a) and GAR2401 (O25b) is shown. On the left side of Figure 13A, the LPS profile of plasmid transformants of O25a O25K5HΔwzzB is shown; on the right side, a similar profile of O25b GAR 2401ΔwzzB transformants is shown. Immunoblots of replicate gels probed with O25-specific serum (Statens Serum Institute) are shown in Figure 13B. Lanes 1-7 correspond to O25aΔwxxB (knockout) background; lanes 8-15 correspond to O25b 2401ΔwzzB (knockout) background. FIG. 14 depicts long O antigen expression mediated by E. coli and Salmonella fepE plasmids in host O25K5H1ΔwzzB. FIG. 15 depicts Salmonella fepE expression produces long O-antigen LPS in various clinical isolates. Figures 16A-16B depict plasmid-mediated arabinose-inducible expression of O25b long chain O antigen LPS in an O25b O antigen knockout host strain. Results from SPS PAGE are shown in Figure 16A and results from an O25 immunoblot are shown in Figure 16B, where in both Figures 16A and 16B, 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 an O55 E. coli LPS standard; and lane 6 is from an O111 E. coli LPS standard. FIG. 17 shows plasmid-mediated arabinose-inducible expression of long-chain O-antigen LPS in a common host strain. FIG. 18 depicts expression of O25 O-antigen LPS in exploratory bioprocess strains. 19A-19B depict the SEC profiles and characteristics of short (FIG. 19A, strain 1, O25b wt2831) and long O25b O antigens (FIG. 19B, strain 2, O25b 2401ΔwzzB/LT2 FepE) purified from GAR2831 and '2401ΔwzzB/fepE strains. 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. Figures 21A-21C depict O25b glycoconjugate IgG responses. -●- represents pre-bleed results; -■- bleed 1 (week 6); -A- bleed 2 (week 8); -◆- bleed 3 (week 12). Figure 21A depicts the results for rabbit 1-3 (medium activation); Figure 21B depicts the results for rabbit 2-3 (low activation); Figure 21C depicts the results for rabbit 3-1 (high activation). Figures 22A-22F depict IgG responses to O25b long chain O antigen saccharide conjugates, i.e., low activated O25b-CRM ₁₉₇ conjugates (in Figures 22D-F, -●- represents pre-bleed results from rabbit 2-1, -■- represents week 12 antiserum results from rabbit 2-1) and non-conjugated polysaccharides, i.e., free O25b polysaccharides (in Figures 22A-C, -●- represents pre-bleed results from rabbit A-1, -■- represents week 6 antiserum results from rabbit A-1, -▲- represents week 8 antiserum results from rabbit A-1). Note that MFIs are plotted on a logarithmic scale to highlight the differences between pre-immune and immune antibodies in the MFI range below 1000. FIG. 22A depicts the results for rabbit A-1 (unconjugated poly); FIG. 22B depicts the results for rabbit A-3 (unconjugated poly); FIG. 22C depicts the results for rabbit A-4 (unconjugated poly); FIG. 22D depicts the results for rabbit 2-1 (low activation); FIG. 22E depicts the results for rabbit 2-2 (low activation); and FIG. 22F depicts the results for rabbit 2-3 (low activation). Figures 23A-23C depict surface expression of native and long chain O25b O antigens detected using O25b antisera. Figure 23A depicts results where -●- depicts results for O25b 2831 vs. PD3 antisera, -■- represents results for O25b 2831 wt vs. pre-bleed, -▲- represents results for O25b 2831/fepE vs. PD3 antisera, and -▼- represents results for O25b 2831/fepE vs. pre-bleed. Figure 23B depicts results where -●- represents results for O25b 2401 vs. PD3 antiserum, -■- represents results for O25b 2401 vs. pre-bleeding, -▲- represents results for O25b 2401/fepE vs. PD3 antiserum, -▼- represents results for O25b 2401/fepE vs. pre-bleeding. Figure 23C depicts results where -●- represents results for E. coli K12 vs. PD3 antiserum, -■- represents results for E. coli K12 vs. pre-bleeding. Figure 24 depicts the generalized structures of the carbohydrate backbones of the outer core oligosaccharides of the five known chemotypes. All glycoses are in the α-anomeric configuration unless otherwise noted. The genes whose products catalyze the formation of each bond are indicated with dashed arrows. An asterisk indicates the residue of the core oligosaccharide at which attachment of the O-antigen occurs. FIG. 25 depicts that unconjugated free O25b polysaccharide is not immunogenic (dLIA), where -●- represents the results of antisera from 4-1 at week 18 (1 week=PD4); -■- represents the results of antisera from 4-2 at week 18 (1 week=PD4); -▲- represents the results of antisera from 5-1 at week 18 (1 week=PD4); -▼- represents the results of antisera from 5-2 at week 18 (1 week=PD4); -*- represents the results of antisera from 6-1 at week 18 (1 week=PD4); -^- represents the results of antisera from 6-2 at week 18 (1 week=PD4). 26A-26C- depict graphs showing specificity of BRC rabbit O25b RAC conjugate immune serum OPA titers. FIG. 26A shows OPA titers of rabbit 2-3 with pre-immune serum -●- and post-immune serum week 13 -■-. FIG. 26B shows OPA titers of rabbit 1-2 with pre-immune serum -●- and post-immune serum week 19 -■-. FIG. 26C shows OPA titer specificity of rabbit 1-2 week 19, where OPA activity of rabbit 1-2 immune serum is blocked by pre-incubation with 100 μg/mL purified unconjugated O25b long chain O antigen polysaccharide. -■- represents rabbit 1-2 immune serum week 19 results, and -▼- represents rabbit 1-2 w/R1 long chain OAg results week 19. Figure 27A shows an illustration of an exemplary dosing schedule. Figures 27B and 27C show graphs depicting O antigen O25b IgG levels elicited by unconjugated O25b long chain O antigen polysaccharide (Figure 27B, O25b free poly(2 μg)) and induced O25b RAC/DMSO long chain O antigen saccharide conjugate (Figure 27C, O25b-CRM ₁₉₇ RAC long chain(2 μg)), where -...- (dotted line) represents naive CD1 O25b IgG levels. Figures 28A-28B depict graphs depicting OPA immunogenicity of RAC, eTEC O25b long chain glycoconjugates, and single-end glycoconjugates after dose 2 (Figure 28A) and dose 3 (Figure 28B), where -○- represents single-end short 2 μg results, -●- represents single-end long 2 μg results, -▲- represents RAC/DMSO long 2 μg results, -▼- represents eTEC long 2 μg results, * represents background control (n=20). †Response rate is % of mice with titers greater than 2-fold above unvaccinated baseline. Figure 29 depicts a graph showing OPA immunogenicity and altered levels of polysaccharide activation of eTEC chemistry. † Response rate is % of mice with titers greater than 2x unvaccinated baseline. 30A-30B show an illustration of an exemplary dosing schedule (FIG. 30A); and a graph depicting protection of mice immunized with doses of E. coli eTEC conjugate from a lethal challenge with an O25b isolate (FIG. 30B), where -◇- represents 17% activation of eTEC long chains, -△- represents 10% activation of eTEC long chains, -▽- represents 4% activation of eTEC long chains, -□- represents O25b polysaccharide, and -○- represents unvaccinated controls. 31 depicts a schematic diagram showing an exemplary preparation of a single-terminated conjugate, where the conjugation process involves selective activation of 2-keto-3-deoxyoctanoic acid (KDO) with a disulfide amine linker upon unmasking of the thiol functional group. KDO is then conjugated to bromo-activated CRM ₁₉₇ protein as depicted in FIG. 31 (Preparation of a Single-Terminated Conjugate). 32A-32B depict exemplary process flow diagrams for the activation (FIG. 32A) and conjugation (FIG. 32B) processes used in the preparation of E. coli glycoconjugates to CRM ₁₉₇ . Figure 33 depicts the repeating unit (RU) structures of E. coli and K. pneumoniae polymannan O antigens. Legend: Trimeric E. coli O8 and K. pneumoniae O5 are identical, as are tetrameric E. coli O9A/K. pneumoniae O3a and pentameric E. coli O9/K. pneumoniae O3. Differences between K. pneumoniae O3 subtypes at the level of biosynthetic enzyme sequences are described in Guachalla LM et al. (Scientific Reports 2017;7:6635). Figures 34A-34B depict that E. coli serotype O8 immune sera are bactericidal against invasive K. pneumoniae serotype O5 strains. Legend: Rabbit immune sera elicited by E. coli serotype O8 O antigen CRM ₁₉₇ conjugate were evaluated in bactericidal assays with E. coli O8 strains (Figure 34A) and K. pneumoniae O5 strains (Figure 34B). Strong opsonophagocytosis assay (OPA) activity against E. coli O8 strains was observed after two vaccine doses (week 15), but was not present after preadsorption with unconjugated O8 polysaccharide (O8-OAg) or with matched pre-immune sera (week 0). The same rabbit immune serum showed antigen-specific serum bactericidal activity (SBA) against K. pneumoniae strain O5. BRC-baby rabbit complement, hC-IgG/IgM depleted human serum as a complement source. 35A-35B depict E. coli serotype O9 O-antigen immune sera are bactericidal against invasive K. pneumoniae O3 isolates. Legend: Rabbit immune sera raised with E. coli serotype O9a O-antigen CRM ₁₉₇ conjugate was evaluated in an opsonophagocytosis assay (OPA) with an E. coli O9a strain (FIG. 35A) and a K. pneumoniae O3b strain (FIG. 35B). OPA activity against E. coli O9 strains was observed after two vaccine doses (week 15), but was absent after preadsorption with unconjugated O9 polysaccharide (O9-OAg) or with matched preimmune serum (week 0). The same rabbit immune serum also showed potent antigen-specific serum bactericidal activity (SBA) against K. pneumoniae O3b strains. BRC-juvenile rabbit complement, hC-IgG/IgM depleted human serum as a complement source. Figures 36A-36B depict that E. coli FimH _LD antigen requires a strong adjuvant to elicit neutralizing antibodies. Figure 36A depicts the dosing schedule for test VAC-2019-PRL-EC-1369: 12-20 CD-1 mice per group were vaccinated with a 3 μg or 30 μg dose of E. coli FimH _LD antigen with or without 20 μg QS21/PS80 or 50 μg _AlPO4 adjuvant. Figure 36B depicts post-dose 3 titers of individual mice vaccinated with wild-type FimH _LD or FimH _LD lock mutant antigen, shown as closed or open symbols, respectively. p-values from t-tests (unpaired Welch's correction) of log-transformed neutralization titer data are shown. Figures 37A-37B show that the FimH-DSG variant is more immunogenic than the FimH _LD construct. Figure 37A depicts the dosing schedule for test VAC-2019-PRL-EC-1438: 20 CD-1 mice per group were vaccinated with 10 μg or 30 μg of E. coli FimH _LD or FimH-DSG antigen variants along with 20 μg of QS21/PS80. Figure 37B depicts post-dose 3 titers of individual mice vaccinated with wild-type FimH _LD or FimH _LD lock mutant antigens, shown as closed or open symbols, respectively. p values derived from t-tests (unpaired Welch's correction) of log-transformed neutralization titer data are shown. FIG. 38 depicts the schedule and dosing for the FimH-DSG and O-antigen combination and adjuvant formulation study (Study VAC-2020-PRL-EC-1679). 39A-39B depict study VAC-2020-PRL-EC-1679 demonstrating the impact of adjuvant and FimH-DSG and tetravalent O antigen combination on FimH neutralization. Yeast mannan assay binding neutralization titers at PD2 (FIG. 39A) and PD3 (FIG. 39B). Closed symbols reflect mice vaccinated with O antigen or FimH-DSG antigen only; open symbols are mice from the group vaccinated with the FimH-DSG O antigen combination labeled on the x-axis. Figures 40A-40B depict that the results of the bladder assay with a mouse serum pool corroborate the yeast mannan assay data for individual mouse sera. Bladder cell binding inhibition curves from pooled mouse sera (n=10). Details are the same as in the legend for Figures 39A-39B. A rabbit anti-FimH positive control serum titration is also shown as an invariant assay reference standard. FIG. 41 depicts the effect of adjuvants and FimH-DSG on O antigen-specific serum IgG levels. Closed and open symbols represent values at PD2 and PD3 time points, respectively. Ten pre-vaccinated CD-1 mice per group were pooled to determine baseline IgG levels for each antigen. The GMT of each pre-vaccinated pool was below the lower limit of quantification (0.15 μg/mL, dotted line), determined from standard curve bias analysis. Responder rates are shown as the percentage of mice per group showing a greater than 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. Figure 42 depicts the effect of adjuvants and FimH-DSG on O antigen-specific serum OPA titers. Details are the same as in Figure 41. The dotted line reflects baseline titers (1/2xLOD or 50, n=10) from mouse pre-vaccination serum pools. Closed and open symbols represent values at PD2 and PD3 time points, respectively.

SEQ ID NO:1 sets forth the amino acid sequence of the wild-type type 1 fimbria D-mannose specific adhesin [E. coli FimH J96].
SEQ ID NO:2 sets forth the amino acid sequence of a fragment of FimH corresponding to aa residues 22-300 of SEQ ID NO:1 (the mature FimH protein).
SEQ ID NO:3 sets forth the amino acid sequence of the FimH lectin domain.
SEQ ID NO:4 sets forth the amino acid sequence of the FimH pilin domain.
SEQ ID NO:5 sets forth the amino acid sequence of 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 the amino acid sequence of 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 the amino acid sequence of a fragment of a polypeptide derived from E. coli FimH (pSB02083 FimH lectin domain wild-type construct).
SEQ ID NO:8 sets forth the amino acid sequence of a fragment of a polypeptide derived from E. coli FimH (pSB02158 FimH lectin domain Lock mutant).
SEQ ID NO:9 sets forth the amino acid sequence of a fragment of a polypeptide derived from E. coli FimG (FimG A1..K14).
SEQ ID NO:10 sets forth the amino acid sequence of a fragment of a polypeptide derived from E. coli FimC.
SEQ ID NO:11 sets forth the amino acid sequence of the 4aa linker.
SEQ ID NO:12 sets forth the amino acid sequence of the 5aa linker.
SEQ ID NO:13 sets forth the amino acid sequence of the 6aa linker.
SEQ ID NO:14 sets forth the amino acid sequence of the 7aa linker.
SEQ ID NO:15 sets forth the amino acid sequence of the 8aa linker.
SEQ ID NO:16 sets forth the amino acid sequence of the 9aa linker.
SEQ ID NO:17 sets forth the amino acid sequence of the 10aa linker.
SEQ ID NO:18 sets forth the amino acid sequence of the FimH J96 signal sequence.
SEQ ID NO:19 sets forth the amino acid sequence of 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 A1..K14/GGHis8 in pcDNA3.1(+).
SEQ ID NO:20 sets forth the amino acid sequence of a polypeptide derived from E. coli FimH according to SEQ ID NO:5 (mature protein of pSB02198 in pcDNA3.1(+)-FimH mlgK signal pept / F22..Q300 J96 FimH N28S V48C L55C N91S N249Q / 7 AA linker / FimG A1..K14 / GGHis8).
SEQ ID NO:21 sets forth the amino acid sequence of a polypeptide derived from E. coli FimG.
SEQ ID NO:22 sets forth the amino acid sequence of 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 the amino acid sequence of 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 the amino acid sequence of a polypeptide derived from E. coli FimH according to SEQ ID NO:7 (mature protein of the pSB02083 FimH lectin domain wild-type construct).
SEQ ID NO:25 sets forth the amino acid sequence of the His tag.
SEQ ID NO:26 sets forth the amino acid sequence of a polypeptide derived from E. coli FimH according to SEQ ID NO:8 (mature protein of the pSB02158 FimH lectin domain Lock mutant).
SEQ ID NO:27 sets forth the amino acid sequence of a polypeptide derived from E. coli FimH (pSB01878).
SEQ ID NO:28 sets forth the amino acid sequence of a polypeptide derived from E. coli FimH (K12).
SEQ ID NO:29 sets forth the amino acid sequence of a polypeptide derived from E. coli FimH (UTI89).
SEQ ID NO:30 sets forth the O25b 2401 WzzB amino acid sequence.
SEQ ID NO:31 sets forth the O25a:K5:H1 WzzB amino acid sequence.
SEQ ID NO:32 sets forth the O25a ETEC ATCC WzzB amino acid sequence.
SEQ ID NO:33 sets forth the K12 W3110 WzzB amino acid sequence.
SEQ ID NO:34 sets forth the Salmonella LT2 WzzB amino acid sequence.
SEQ ID NO:35 sets forth the O25b 2401 FepE amino acid sequence.
SEQ ID NO:36 sets forth the O25a:K5:H1 FepE amino acid sequence.
SEQ ID NO:37 sets forth the O25a ETEC ATCC FepE amino acid sequence.
SEQ ID NO:38 sets forth the O157 FepE amino acid sequence.
SEQ ID NO:39 sets forth the Salmonella LT2 FepE amino acid sequence.
SEQ ID NO:40 sets forth the primer sequence of LT2wzzB_S.
SEQ ID NO:41 sets forth the primer sequence of LT2wzzB_AS.
SEQ ID NO:42 sets forth the primer sequence of O25bFepE_S.
SEQ ID NO:43 sets forth the primer sequence for O25bFepE_A.
SEQ ID NO:44 sets forth the primer sequence of wzzB P1_S.
SEQ ID NO:45 sets forth the primer sequence of wzzB P2_AS.
SEQ ID NO:46 sets forth the primer sequence of wzzB P3_S.
SEQ ID NO:47 sets forth the primer sequence of wzzB P4_AS.
SEQ ID NO:48 sets forth the primer sequence for O157 FepE_S.
SEQ ID NO:49 sets forth the primer sequence for O157 FepE_AS.
SEQ ID NO:50 sets forth the primer sequence of pBAD33_adaptor_S.
SEQ ID NO:51 sets forth the primer sequence of pBAD33_adaptor_AS.
SEQ ID NO:52 sets forth the primer sequence of JUMPSTART_r.
SEQ ID NO:53 sets forth the primer sequence of gnd_f.
SEQ ID NO:54 sets forth the amino acid sequence of a mouse IgK signal sequence.
SEQ ID NO:55 sets forth the amino acid sequence of the human IgG receptor FcRn large subunit p51 signal peptide.
SEQ ID NO:56 sets forth the amino acid sequence of the human IL10 protein signal peptide.
SEQ ID NO:57 sets forth the amino acid sequence of the human respiratory syncytial virus A (A2 strain) fusion glycoprotein F0 signal peptide.
SEQ ID NO:58 sets forth the amino acid sequence of the influenza A hemagglutinin signal peptide.
SEQ ID NOs:59-101 set forth the amino acid and nucleic acid sequences of nanostructure-associated polypeptides or fragments thereof.
SEQ ID NOs:102-109 set forth Signal P 4.1 (DTU Bioinformatics) sequences from various species used for signal peptide prediction.
SEQ ID NO:110 sets forth the amino acid sequence of a polypeptide derived from E. coli FimH (pSB02083-FimH _LD (mIgK signal pept, N28S, N91S)).
SEQ ID NO:111 sets forth the amino acid sequence of a polypeptide derived from E. coli FimH (pSB02158-FimH _LD -LM (mIgK signal pept, N28S N91S V48C L55C)).
SEQ ID NO:112 sets forth the amino acid sequence of a polypeptide derived from E. coli FimH (pSB02307 - FimH-DSG (mIgK signal pept, N28S N91S N249Q 7aa linker FimG A1..K14)).
SEQ ID NO:113 sets forth the amino acid sequence of a polypeptide derived from E. coli FimH (pSB02198 - FimH-DSG-LM (mIgK signal pept, N28S N91S 249Q V48C L55C 7aa linker FimG A1..K14)).

DETAILED DESCRIPTION OF THE DISCLOSURE The present disclosure relates to compositions comprising E. coli FimH polypeptides and O-antigen glycoconjugates, methods for producing and purifying said compositions, and methods of using said compositions.

In one aspect, the invention includes compositions comprising a FimH polypeptide or fragment thereof as described herein. The compositions may comprise a polypeptide or fragment thereof suitable for administration in vivo. For example, the polypeptide or fragment thereof in such compositions 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 certain embodiments, the polypeptide in such compositions may have a purity of at least 95% by mass. In certain embodiments, the polypeptide in such compositions may have a purity of at least 97% by mass. In certain embodiments, the polypeptide in such compositions may have a purity of at least 98% by mass. In certain embodiments, the polypeptide in such compositions may have a purity of at least 99% by mass. The compositions may further comprise an adjuvant.

In a further aspect, the invention includes compositions for use in inducing an immune response to E. coli or E. coli infection. Use of the compositions described herein for inducing an immune response to E. coli or E. coli infection and use of the compositions described herein in the manufacture of a medicament for inducing an immune response to E. coli or E. coli infection are also disclosed.

The recitation of ranges of values herein is merely intended to serve as a shorthand method of individually referring to each separate value within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually set forth 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 example language (e.g., "etc.") provided herein is intended merely to better illustrate the disclosure and does not impose limitations 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 body of this disclosure. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such disclosure.

DEFINITIONS As used herein, the term "about" means approximately or near, and in the context of numerical values or ranges described herein, means in one embodiment ±20%, ±10%, ±5%, or ±3% of the stated or claimed numerical value or range.

As used in the context of describing this disclosure (particularly in the context of the claims), the terms "a" and "an" and "the" and similar references should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

A "fragment" referring to an amino acid sequence (peptide or protein) relates to a portion of the amino acid sequence, i.e. a sequence representing an amino acid sequence truncated at the N-terminus and/or C-terminus. A fragment truncated at the C-terminus (N-terminal fragment) is obtained, for example, by translation of a truncated open reading frame lacking the 3' end of the open reading frame. A fragment truncated at the N-terminus (C-terminal fragment) is obtained, for example, by translation of a truncated open reading frame lacking the 5' end of the open reading frame, as long as the truncated open reading frame contains an initiation codon serving to initiate translation. A fragment of an amino acid sequence comprises, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the amino acid residues derived from the 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 derived from the amino acid sequence.

As used herein, the terms "wild-type" or "WT" or "native" refer to an amino acid sequence 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, a reference to a "variant" of an amino acid sequence (peptide, protein or polypeptide), or a "mutant" or "mutated" polypeptide includes 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 variants, splice variants, post-translationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, particularly those that occur naturally. The term "variant" or "mutant" specifically includes fragments of an amino acid sequence.

Amino acid insertion variants include the insertion of a single or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted at a specific site in the amino acid sequence, but random insertion is also possible with appropriate screening of the resulting product. Amino acid addition variants include 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 the removal of 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. The deletion may be at any position in the protein. Amino acid deletion variants that include deletions at the N-terminus and/or C-terminus of the protein are also called N-terminus and/or C-terminus truncation variants. Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. Preferably, the modification is at a position in the amino acid sequence that is not conserved between homologous proteins or peptides and/or replaces an amino acid with another amino acid with similar properties. Preferably, the amino acid changes in the peptides and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes include substitutions of members of a family of amino acids that are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartic acid, glutamic acid), 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 together as aromatic amino acids. In one embodiment, conservative amino acid substitutions are made in the following groups:
Glycine, Alanine;
Valine, isoleucine, leucine;
Aspartic acid, glutamic acid;
Asparagine, Glutamine;
Serine, Threonine;
Includes substitutions within lysine, arginine; and phenylalanine, tyrosine.

Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that 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 preferably given for an amino acid region that 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 full 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 preferably given for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments, consecutive amino acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be performed using means known in the art, preferably using best sequence alignment, e.g., using Align, using standard settings, preferably EMBOSS:needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5.

As used herein, "sequence similarity" refers to the percentage of amino acids that are identical or that are conservative amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are identical between the sequences.

The term "% identical", "% identity" or similar terms is intended to refer in particular to the percentage of nucleotides or amino acids that are identical in optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be randomly distributed, but not necessarily, over the entire length of the sequences to be compared. Comparison of two sequences is usually carried out by comparing the sequences after optimal alignment over a segment or "comparison window" in order to identify partial regions of the corresponding sequences. Optimal alignment for comparison is performed manually or by using the partial homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the partial homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the partial homology algorithm of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 88, 2444, or a computer program using said algorithm (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.) can be used. In some embodiments, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, available on the United States National Center for Biotechnology Information (NCBI) website (e.g., blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq). In some embodiments, the algorithm parameters used for the BLASTN algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max match in the query range set to 0; (iv) Match/Mismatch Score set to 1, -2; (v) Gap Cost set to Linear; and (vi) use of a filter for low complexity regions. In some embodiments, the algorithm parameters used for the BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max match in query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Cost set to Existence: 11 Extension: 1; and (vi) Conditional Compositional Score Matrix Adjustment.

The percentage identity is obtained by determining the number of identical positions where the sequences being compared match, dividing this number by the number of positions being 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 that 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 of 200 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, contiguous nucleotides. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence.

Homologous amino acid sequences, according to the present disclosure, exhibit at least 40% identity of amino acid residues, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably at least 95%, at least 98% or at least 99%. The skilled artisan can easily prepare the amino acid sequence variants/mutants described herein, for example, by recombinant DNA manipulation. The manipulation of DNA sequences to prepare peptides or proteins with substitutions, additions, insertions or deletions is described in detail, for example, in Sambrook et al. (1989). Furthermore, the peptides and amino acid variants described herein can be easily prepared with the aid of known peptide synthesis techniques, 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 that exhibits 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 an antigen or antigen sequence, one particular function is one or more immunogenic activities exhibited by the amino acid sequence from which the fragment or variant is derived. As used herein, the term "functional fragment" or "functional variant/mutant" refers in particular to a variant/mutant molecule or sequence that comprises an amino acid sequence that has been altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence and that still fulfills one or more functions of the parent molecule or sequence, e.g., is capable of inducing an immune response. In one aspect, the alterations 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 functionality of the functional fragment or variant may be reduced but still significantly present, for example, the 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, the immunogenicity of the functional fragment or 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 peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-native environment, such as, for example, a host cell.

I. Polypeptides and Fragments thereof derived from E. coli In one aspect, disclosed herein is a mammalian cell comprising a polynucleotide encoding a polypeptide or fragment thereof derived from E. coli. The term "derived from" as used herein refers to a polypeptide comprising the amino acid sequence of a FimH polypeptide or FimCH polypeptide complex, or a fragment thereof, as described herein, which has been altered by the introduction of substitutions, deletions, or additions of amino acid residues. Preferably, the polypeptide or fragment thereof derived from E. coli comprises 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 or fragment thereof derived from E. coli has at least 85% identity to the sequence of the corresponding wild-type E. coli FimH polypeptide or fragment. In some embodiments, the polypeptide or fragment thereof derived from E. coli has at least 90% identity to the sequence of the corresponding wild-type E. coli FimH polypeptide or fragment. In some embodiments, the polypeptide or fragment thereof derived from E. coli has at least 95% identity to the sequence of the corresponding wild-type E. coli FimH polypeptide or fragment. In some embodiments, the polypeptide or fragment thereof derived from E. coli has at least 98% identity to the sequence of the corresponding wild-type E. coli FimH polypeptide or fragment. In some embodiments, the polypeptide or fragment thereof derived from E. coli has at least 99% identity to the sequence of the corresponding wild-type E. coli FimH polypeptide or fragment. In some embodiments, the polypeptides or fragments thereof derived from E. coli have the same overall amino acid sequence as the corresponding wild-type FimH polypeptide or FimCH polypeptide complex or fragment thereof. In some embodiments, the polypeptides or fragments thereof derived from E. coli have the same overall amino acid sequence as the corresponding wild-type FimH polypeptide or FimCH polypeptide complex.

The fragment should comprise at least n consecutive amino acids from the sequence, where n is 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20 or more), depending on the particular sequence. Preferably, the fragment comprises an epitope from the sequence. In some embodiments, the fragment comprises 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 the polypeptide derived from E. coli. In some embodiments, the fragment comprises an amino acid sequence of at least 50 consecutive amino acid residues of the amino acid sequence of the polypeptide derived from E. coli. In some embodiments, the fragment comprises an amino acid sequence of at least 100 consecutive amino acid residues of the amino acid sequence of the polypeptide derived from E. coli. In some embodiments, the fragment comprises an amino acid sequence of at least 150 contiguous amino acid residues of the amino acid sequence of the polypeptide derived from E. coli. In some embodiments, the fragment comprises an amino acid sequence of at least 200 contiguous amino acid residues of the amino acid sequence of the polypeptide derived from E. coli. In some embodiments, the fragment comprises an amino acid sequence of at least 250 contiguous amino acid residues of the amino acid sequence of the polypeptide derived from E. coli.

In some embodiments, the E. coli derived polypeptide or fragment thereof comprises one or more non-classical amino acids when compared to the corresponding wild-type E. coli FimH polypeptide or fragment.

As used herein, the term "FimH polypeptide" refers to any FimH polypeptide described herein or a fragment thereof that has been altered by the introduction of amino acid substitutions, deletions, or additions, any FimH domain of a full-length wild-type E. coli FimH polypeptide, any combination of domains of a full-length wild-type E. coli FimH polypeptide, or a full-length E. coli FimH polypeptide. For example, in one embodiment, the disclosure provides a FimH polypeptide that is a FimH _LD polypeptide, or a FimH-DSG polypeptide.

In some embodiments, the E. coli derived polypeptide or fragment thereof has a similar or identical function to the corresponding wild-type FimH polypeptide or fragment thereof.

In a preferred embodiment, the polypeptide or polypeptide complex of the present invention or a fragment thereof is isolated or purified.

In some embodiments, a polynucleotide encoding a polypeptide or fragment thereof derived from E. coli is integrated into the genomic DNA of a mammalian cell, such that the E. coli derived polypeptide or fragment thereof is expressed by the mammalian cell when cultured under suitable conditions.

In a preferred embodiment, the polypeptide or fragment thereof derived from E. coli is soluble.

In some embodiments, the E. coli derived polypeptide or fragment thereof is secreted from a mammalian host cell.

In some embodiments, the polypeptide or fragment thereof derived from E. coli may include additional amino acid residues, such as an N-terminal or C-terminal extension. 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 allowing purification on nickel-chelating resins) of the polypeptide or fragment thereof. In some embodiments, the tag comprises an 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, for example, His tags (e.g., hexahistidine, which may bind to metal ions), maltose binding protein (MBP), which may bind to amylose, glutathione-S-transferase (GST), which may bind to glutathione, FLAG tags, which may bind to anti-flag antibodies, and Strep tags, which may bind to streptavidin or derivatives thereof. In preferred embodiments, the E. coli derived polypeptide or fragment thereof does not include additional amino acid residues, such as N-terminal or C-terminal extensions. In some embodiments, the E. coli derived polypeptide or fragment thereof described herein does not include an exogenous tag sequence.

Although specific strains of E. coli may be mentioned herein, it should be understood that the polypeptides or fragments thereof derived from E. coli are not limited to specific strains unless specified.

In some embodiments, the polypeptide or fragment thereof derived from E. coli FimH comprises a phenylalanine residue at the N-terminus of the polypeptide. In some embodiments, the polypeptide or fragment thereof derived from FimH comprises 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 or fragment thereof derived from E. coli FimH does not comprise an additional glycine residue at the N-terminus of the polypeptide or fragment thereof derived from E. coli FimH.

In some embodiments, the phenylalanine residue at position 1 of wild-type mature E. coli FimH is replaced with an aliphatic hydrophobic amino acid, such as, for example, any one of Ile, Leu, and Val residues.

In some embodiments, a signal peptide can be used to express a polypeptide or fragment thereof from E. coli. 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. Signal peptides generally contain a long stretch of hydrophobic amino acids that have 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 enforce the correct 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 a signal peptidase, which can cleave to generate a free signal peptide and a mature protein, either during or after the translocation is complete. In some embodiments, the signal peptide comprises an 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 comprises an 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 an 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 E. coli derived polypeptide or fragment thereof described herein may include a cleavable linker. Such a linker allows 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 by acid-catalyzed hydrolysis. Another example of a cleavable linker includes a polypeptide linker that includes a protease recognition site and can be cleaved by the addition of a suitable protease enzyme.

In some embodiments, the polypeptide or fragment thereof derived from E. coli comprises a modification compared to the corresponding wild-type E. coli FimH polypeptide or fragment. The modification may comprise a covalent attachment of a molecule to the polypeptide. For example, such modifications may comprise glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, and the like. In some embodiments, the polypeptide or fragment thereof derived from E. coli comprises a modification compared to the corresponding wild-type E. coli FimH polypeptide or fragment, such as by chemical modification using techniques known to those of skill in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In another embodiment, the modification may comprise a covalent attachment of a lipid molecule to the polypeptide. In some embodiments, the polypeptide does not comprise a covalent attachment of a molecule to the polypeptide compared to the corresponding wild-type E. coli FimH polypeptide or fragment.

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 attached to the protein through either N- or O-linkages. The oligosaccharide chains may represent a significant portion of the mass of the glycoprotein. Generally, N-linked oligosaccharides are attached to the amino group on the side chain of an asparagine residue within the target consensus sequence of Asn-X-Ser/Thr, where X can be any amino acid except proline. In some embodiments, the glycosylation site comprises 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 E. coli derived polypeptide or fragment thereof produced in a mammalian cell may be glycosylated. Glycosylation may occur at the N-linked glycosylation signal Asn-Xaa-Ser/Thr in the sequence of the E. coli derived polypeptide or fragment thereof. "N-linked glycosylation" refers to the attachment of a carbohydrate moiety via GIcNAc to an asparagine residue in the polypeptide chain. N-linked carbohydrates include the consensus Man 1-6 (Man 1-3) Manβ1-4GlcNAcβ1-4GlcNAcβ-R core structure, where R represents an asparagine residue in the E. coli derived produced polypeptide or fragment thereof.

In some embodiments, the glycosylation site in the E. coli derived polypeptide or fragment thereof is removed by a mutation in the sequence of the E. coli derived polypeptide or fragment thereof. For example, in some embodiments, the Asn residue of the glycosylation motif (Asn-Xaa-Ser/Thr) may be mutated, preferably by substitution. In some embodiments, the residue substitution is selected from any one of Ser, Asp, Thr, and Gln.

In some embodiments, the Ser residue of the glycosylation motif may be mutated, preferably by substitution. In some embodiments, the residue substitution is selected from any one of Asp, Thr, and Gln.

In some embodiments, the Thr residue of the glycosylation motif may be mutated, preferably by substitution. In some embodiments, the residue substitution is selected from any one of Ser, Asp, and Gln.

In some embodiments, glycosylation sites (such as Asn-Xaa-Ser/Thr) in a polypeptide or fragment thereof derived from E. coli are not removed or modified. In some embodiments, a compound that reduces or inhibits glycosylation may be added to the cell culture medium. In such embodiments, the polypeptide or protein contains at least one more non-glycosylated (i.e., aglycosylated) site, i.e., a completely unoccupied glycan site that does not contain an attached carbohydrate moiety, or contains at least one less carbohydrate moiety at the same potential glycosylation site than an otherwise identical polypeptide or protein produced by a cell under identical conditions but in the absence of a glycosylation-inhibiting compound. Such compounds are known in the art and include, but are not limited to, tunicamycin, tunicamycin homologs, streptovirudin, mycosposidin, amphomycin, tsushimycin, antibiotic 24010, antibiotic MM19290, bacitracin, corynetoxin, showdomycin, duimycin, ), 1-deoxymannonojirimycin, deoxynojirimycin, N-methyl-1-deoxymannojirimycin, 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 inhibitor, 25-hydroxycholesterol, hydroxycholesterol, swainsonine, cycloheximide, puromycin, actinomycin D, monensin, m-chlorocarbonyl-cyanide phenyl Hydrazone (CCCP), compactin, dolichyl-phosphoryl-deoxyglucose, N-acetyl-D-glucosamine, hygoxanthine, thymidine, cholesterol, glucosamine, mannosamine, castanospermine, glutamine, bromoconduritol, conduritol epoxide
epoxide) and conduritol derivatives, glycosylmethyl-p-nitrophenyltriazenes, β-hydroxynorvaline, threo-β-fluoroasparagine, D-(+)-gluconic acid δ-lactone, di(2-ethylhexyl) phosphate, tributyl phosphate, dodecyl phosphate, 2-dimethylaminoethyl ester of (diphenylmethyl)-phosphate, [2-(diphenylphosphinyloxy)ethyl]trimethylammonium iodide, iodoacetate, 2-deoxy-D-glucose, and fluoroacetate. One of skill in the art will readily recognize or be able to determine, without undue experimentation, glycosylation inhibitors that can be used in accordance with the methods and compositions of the invention. In such embodiments, glycosylation of a polypeptide or fragment thereof can be controlled without introducing amino acid mutations into the polypeptide or fragment thereof.

In some embodiments, the level of glycosylation (e.g., the number of glycan sites occupied on the polypeptide or fragment thereof, the size and/or complexity of the glycoform at those sites, etc.) of the polypeptide or fragment thereof produced by the mammalian cell is lower than the level of glycosylation of the polypeptide or fragment thereof produced in the same medium under otherwise identical conditions but without the glycolysis inhibitor compound and/or mutation, etc.

In some embodiments, the sequence of the polypeptide or fragment thereof derived from E. coli does not include a site for N-linked protein glycosylation. In some embodiments, the sequence of the polypeptide or fragment thereof derived from E. coli does not include at least one site for N-linked protein glycosylation. In some embodiments, the sequence of the polypeptide or fragment thereof derived from E. coli does not include any site for N-linked protein glycosylation. In some embodiments, the sequence of the polypeptide or fragment thereof derived from E. coli includes a site for N-linked protein glycosylation. In some embodiments, the sequence of the polypeptide or fragment thereof derived from E. coli includes at most one site for N-linked protein glycosylation. In some embodiments, the sequence of the polypeptide or fragment thereof derived from E. coli includes at most two sites for N-linked protein glycosylation.

E. coli derived polypeptides or fragments thereof expressed by different cell lines or in transgenic animals may have different glycan site occupancy, glycotypes and/or glycosylation patterns compared to each other. In some embodiments, the invention encompasses E. coli derived polypeptides or fragments thereof, regardless of the glycosylation, glycan occupancy or glycotype of the E. coli derived polypeptides or fragments thereof produced in mammalian cells.

In some embodiments, the polypeptide or fragment thereof derived from E. coli may be derived from an E. coli FimH polypeptide, where the amino acid residue at position 1 of the polypeptide is phenylalanine rather than methionine, e.g., a polypeptide having an amino acid sequence of 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 an amino acid sequence of SEQ ID NO:3, where the residue at position 1 of the amino acid sequence of the polypeptide derived from E. coli is a phenylalanine. In some embodiments, the polypeptide derived from E. coli may comprise an amino acid sequence of SEQ ID NO:4, which may be derived from an E. coli FimH polypeptide.

In some embodiments, the polypeptide or fragment thereof derived from E. coli comprises an 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 or fragment thereof derived from E. coli comprises an 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 or fragment thereof derived from E. coli comprises an 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 E. coli derived polypeptide or fragment thereof may be derived from an E. coli FimG polypeptide, e.g., having the amino acid sequence of SEQ ID NO:9. In some embodiments, the E. coli derived polypeptide or fragment thereof may be derived from an E. coli FimC polypeptide, e.g., having the amino acid sequence of SEQ ID NO:10.

A. Polypeptides and fragments thereof derived from E. coli FimH

In a preferred embodiment, the polypeptide or fragment thereof is derived from E. coli FimH. In some embodiments, the polypeptide or fragment thereof comprises full-length E. coli FimH. Full-length FimH comprises two domains: an N-terminal lectin domain and a C-terminal pyrin domain, which are connected by a short linker. In some embodiments, the full-length E. coli FimH comprises 279 amino acids, including the full-length mature E. coli FimH protein. In some embodiments, the full-length E. coli FimH comprises 300 amino acids, including the full-length mature E. coli FimH protein and a signal peptide sequence having a length of 21 amino acids. The primary structure of the 300 amino acid-long wild-type FimH is highly conserved across E. coli strains.

An exemplary sequence for 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 responsible for binding to mannosylated uroplakin 1a on the urothelial cell surface. The pilin domain is anchored to the core of the pilus via the donor strand of the subsequent FimG subunit, a process referred to as donor strand complementation.

Starting from the N-terminus, the names of each domain of full-length FimH and exemplary amino acid sequences in parentheses 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 those having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, or 50% 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. Included are variants having 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 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity. Other suitable polypeptides have 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: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 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: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 protein (i) forms part of FimH-FimC; (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: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) is capable of eliciting antibodies in vivo that are immunologically cross-reactive with E. coli FimH.

In some embodiments, the composition comprises 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, where n is 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20 or more). Preferably, the fragment comprises an epitope from those sequences. In some embodiments, the composition comprises a polypeptide having at least 50 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues of any one of the amino acid sequences 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 comprises a polypeptide having at least 50 contiguous 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, the composition comprises 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, the composition comprises a polypeptide having at least 2 ...50 consecutive amino acid residues of any one of the amino acid sequences 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 comprises 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 comprises a polypeptide having at least 90% identity to SEQ ID NO:1. In some embodiments, the composition comprises a polypeptide having at least 95% identity to SEQ ID NO:1. In some embodiments, the composition comprises a polypeptide having at least 99% identity to SEQ ID NO:1. In some embodiments, the composition comprises a polypeptide as set forth in SEQ ID NO:1. In some embodiments, the composition comprises 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 comprises a polypeptide having at least 90% identity to SEQ ID NO:2. In some embodiments, the composition comprises a polypeptide having at least 95% identity to SEQ ID NO:2. In some embodiments, the composition comprises a polypeptide having at least 99% identity to SEQ ID NO:2. In some embodiments, the composition comprises a polypeptide as set forth in SEQ ID NO:2. In some embodiments, the composition comprises 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 comprises a polypeptide having at least 90% identity to SEQ ID NO:3. In some embodiments, the composition comprises a polypeptide having at least 95% identity to SEQ ID NO:3. In some embodiments, the composition comprises a polypeptide having at least 99% identity to SEQ ID NO:3. In some embodiments, the composition comprises a polypeptide as set forth in SEQ ID NO:3. In some embodiments, the composition comprises 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 comprises a polypeptide having at least 90% identity to SEQ ID NO:4. In some embodiments, the composition comprises a polypeptide having at least 95% identity to SEQ ID NO:4. In some embodiments, the composition comprises a polypeptide having at least 99% identity to SEQ ID NO:4. In some embodiments, the composition comprises a polypeptide as set forth in SEQ ID NO:4. In some embodiments, the composition comprises 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 comprises a polypeptide having at least 90% identity to SEQ ID NO:5. In some embodiments, the composition comprises a polypeptide having at least 95% identity to SEQ ID NO:5. In some embodiments, the composition comprises a polypeptide having at least 99% identity to SEQ ID NO:5. In some embodiments, the composition comprises a polypeptide as set forth in SEQ ID NO:5. In some embodiments, the composition comprises 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 comprises a polypeptide having at least 90% identity to SEQ ID NO:6. In some embodiments, the composition comprises a polypeptide having at least 95% identity to SEQ ID NO:6. In some embodiments, the composition comprises a polypeptide having at least 99% identity to SEQ ID NO:6. In some embodiments, the composition comprises a polypeptide as set forth in SEQ ID NO:6. In some embodiments, the composition comprises 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 comprises a polypeptide having at least 90% identity to SEQ ID NO:20. In some embodiments, the composition comprises a polypeptide having at least 95% identity to SEQ ID NO:20. In some embodiments, the composition comprises a polypeptide having at least 99% identity to SEQ ID NO:20. In some embodiments, the composition comprises a polypeptide as set forth in SEQ ID NO:20. In some embodiments, the composition comprises 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 comprises a polypeptide having at least 90% identity to SEQ ID NO:23. In some embodiments, the composition comprises a polypeptide having at least 95% identity to SEQ ID NO:23. In some embodiments, the composition comprises a polypeptide having at least 99% identity to SEQ ID NO:23. In some embodiments, the composition comprises a polypeptide as set forth in SEQ ID NO:23. In some embodiments, the composition comprises 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 comprises a polypeptide having at least 90% identity to SEQ ID NO:24. In some embodiments, the composition comprises a polypeptide having at least 95% identity to SEQ ID NO:24. In some embodiments, the composition comprises a polypeptide having at least 99% identity to SEQ ID NO:24. In some embodiments, the composition comprises a polypeptide as set forth in SEQ ID NO:24. In some embodiments, the composition comprises 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 comprises a polypeptide having at least 90% identity to SEQ ID NO:26. In some embodiments, the composition comprises a polypeptide having at least 95% identity to SEQ ID NO:26. In some embodiments, the composition comprises a polypeptide having at least 99% identity to SEQ ID NO:26. In some embodiments, the composition comprises a polypeptide as set forth in SEQ ID NO:26. In some embodiments, the composition comprises 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 comprises a polypeptide having at least 90% identity to SEQ ID NO:27. In some embodiments, the composition comprises a polypeptide having at least 95% identity to SEQ ID NO:27. In some embodiments, the composition comprises a polypeptide having at least 99% identity to SEQ ID NO:27. In some embodiments, the composition comprises a polypeptide as set forth in SEQ ID NO:27. In some embodiments, the composition comprises 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 comprises a polypeptide having at least 90% identity to SEQ ID NO:28. In some embodiments, the composition comprises a polypeptide having at least 95% identity to SEQ ID NO:28. In some embodiments, the composition comprises a polypeptide having at least 99% identity to SEQ ID NO:28. In some embodiments, the composition comprises a polypeptide as set forth in SEQ ID NO:28. In some embodiments, the composition comprises 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 comprises a polypeptide having at least 90% identity to SEQ ID NO:29. In some embodiments, the composition comprises a polypeptide having at least 95% identity to SEQ ID NO:29. In some embodiments, the composition comprises a polypeptide having at least 99% identity to SEQ ID NO:29. In some embodiments, the composition comprises a polypeptide as set forth in SEQ ID NO:29. In some embodiments, the composition comprises 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 comprises a polypeptide having at least 90% identity to SEQ ID NO: 110. In some embodiments, the composition comprises a polypeptide having at least 95% identity to SEQ ID NO: 110. In some embodiments, the composition comprises a polypeptide having at least 99% identity to SEQ ID NO: 110. In some embodiments, the composition comprises a polypeptide as set forth in SEQ ID NO: 110. In some embodiments, the composition has a affinity for at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, or 80% of SEQ ID NO: 111.
In some embodiments, the composition comprises a polypeptide having at least 90% identity to SEQ ID NO:111. In some embodiments, the composition comprises a polypeptide having at least 95% identity to SEQ ID NO:111. In some embodiments, the composition comprises a polypeptide having at least 99% identity to SEQ ID NO:111. In some embodiments, the composition comprises a polypeptide as set forth in SEQ ID NO:111. In some embodiments, the composition comprises 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 comprises a polypeptide having at least 90% identity to SEQ ID NO: 112. In some embodiments, the composition comprises a polypeptide having at least 95% identity to SEQ ID NO: 112. In some embodiments, the composition comprises a polypeptide having at least 99% identity to SEQ ID NO: 112. In some embodiments, the composition comprises a polypeptide as set forth in SEQ ID NO: 112. In some embodiments, the composition comprises 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 comprises a polypeptide having at least 90% identity to SEQ ID NO: 113. In some embodiments, the composition comprises a polypeptide having at least 95% identity to SEQ ID NO: 113. In some embodiments, the composition comprises a polypeptide having at least 99% identity to SEQ ID NO: 113. In some embodiments, the composition comprises a polypeptide as set forth in SEQ ID NO: 113.

Another example of a suitable polypeptide and fragment thereof derived from E. coli FimH as described herein lacks the wild-type N-terminal signal sequence and is shown as SEQ ID NO:2, which 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 that set forth in SEQ ID NO:1.

In some embodiments, the glycosylation site in the E. coli derived polypeptide or fragment thereof is removed by a mutation in the sequence of the E. coli derived polypeptide or fragment thereof. For example, in some embodiments, the Asn residue at position 7 of the mature E. coli FimH polypeptide (e.g., according to the numbering of SEQ ID NO:2) may be mutated, preferably by substitution. In some embodiments, the Asn residue at position 7 of the lectin domain of the E. coli FimH polypeptide (e.g., according to the numbering of SEQ ID NO:3) may be mutated, preferably by substitution. In some embodiments, the residue substitution is selected from any one of Ser, Asp, Thr, and Gln.

In some embodiments, the Thr residue at position 10 of the mature E. coli FimH polypeptide (e.g., according to the numbering of SEQ ID NO:2) may be mutated, preferably by substitution. In some embodiments, the Thr residue at position 7 of the lectin domain of the E. coli FimH polypeptide (e.g., according to the numbering of SEQ ID NO:3) may be mutated, preferably by substitution. In some embodiments, the residue substitution is selected from any one of Ser, Asp, and Gln.

In some embodiments, the Asn residue at position N235 (e.g., according to the numbering of SEQ ID NO:2) of the mature E. coli FimH polypeptide may be mutated, preferably by substitution. In some embodiments, the Asn residue at position N228 (e.g., according to the numbering of SEQ ID NO:2) of the mature E. coli FimH polypeptide may be mutated, preferably by substitution. In some embodiments, the residue substitution is selected from any one of Ser, Asp, Thr, and Gln.

In some embodiments, the Asn residue at position 70 of the mature E. coli FimH polypeptide (e.g., according to the numbering of SEQ ID NO:2) may be mutated, preferably by substitution. In some embodiments, the Asn residue at position 70 of the lectin domain of the E. coli FimH polypeptide (e.g., according to the numbering of SEQ ID NO:3) may be mutated, preferably by substitution. In some embodiments, the residue substitution is selected from any one of Ser, Asp, Thr, and Gln.

In some embodiments, the Ser residue at position 72 of the mature E. coli FimH polypeptide (e.g., according to the numbering of SEQ ID NO:2) may be mutated, preferably by substitution. In some embodiments, the Ser residue at position 72 of the lectin domain of the E. coli FimH polypeptide (e.g., according to the numbering of SEQ ID NO:3) may be mutated, preferably by substitution. In some embodiments, the residue substitution is selected from any one of Asp, Thr, and Gln.

The term "fragment" as used herein refers to a polypeptide and is defined as any distinct portion of a given polypeptide that is unique or characteristic of that polypeptide. The term as used herein also refers to any distinct 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 that is retained is at least 10% of the activity of the full-length polypeptide. In certain embodiments, the fraction of activity that is 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 that is 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 that is retained is 100% or more of the activity of the full-length polypeptide. In some embodiments, the fragment comprises 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. Complexes of FimH, FimC, and Fragments Thereof In some embodiments, the polypeptide or fragment thereof derived from E. coli FimH is present as a complex with a polypeptide or fragment thereof derived from E. coli FimC. In a preferred embodiment, the polypeptide or fragment thereof derived from E. coli FimH and the polypeptide or fragment thereof derived from E. coli FimC are present in a complex, preferably in a 1:1 ratio in the complex. Without being bound by theory or mechanism, full-length FimH may be stabilized in an active conformation by the periplasmic chaperone FimC, thereby allowing full-length FimH protein to be purified. Thus, in some embodiments, the polypeptide or fragment thereof comprises full-length FimH and full-length FimC.

In some embodiments, the polypeptide or fragment thereof comprises a fragment of FimH and a fragment of FimC. In some embodiments, the polypeptide or fragment thereof comprises full length FimH and a fragment of FimC. An exemplary sequence of E. coli FimC is set forth in SEQ ID NO: 10. In some embodiments, the polypeptide or fragment thereof derived from E. coli comprises a complex-forming fragment of FimH.

A complex-forming fragment of FimH can be any portion or part of the FimH protein that retains the ability to form a complex with FimC or a fragment thereof. Suitable complex-forming fragments of FimH can be obtained or determined by standard assays known in the art, such as co-immunoprecipitation assays, cross-linking, or co-localization by fluorescent staining. SDS-PAGE or Western blots can also be used (e.g., by showing that the FimH fragment and FimC or a fragment thereof are in a complex as evidenced by gel electrophoresis). In certain embodiments, the complex-forming fragment of FimH (i) forms part of a 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) is capable of eliciting antibodies that are immunologically cross-reactive with E. coli FimH in vivo.

In some embodiments, the E. coli derived polypeptide or fragment thereof comprises a full-length FimH that is not complexed with FimC. In further embodiments, the polypeptide or fragment thereof comprises a fragment of FimH that is not complexed with FimC. In some embodiments, the E. coli derived polypeptide or fragment thereof FimC comprises SEQ ID NO: 10. In some embodiments, the complexes may be expressed from the same plasmid, preferably under the control of separate promoters for each polypeptide or fragment thereof.

In some embodiments, a polypeptide or fragment thereof derived from E. coli FimH binds to a polypeptide or fragment thereof derived from E. coli FimC that may be engineered into the structure of the polypeptide or fragment thereof derived from E. coli FimH. The portion of the FimC molecule that is bound to FimH in the complex is referred to as the "donor strand," and the mechanism of formation of the native FimH structure using the strand from FimC that is bound to FimH in the FimCH complex is known as "donor strand complementation."

In some embodiments, a polypeptide or fragment thereof derived from E. coli FimH may be expressed with an appropriate donor strand complemented version of FimH, where the amino acid sequence of FimC that interacts with FimH in the FimCH complex is itself engineered at the C-terminus of FimH to provide a native conformation that does not require the presence of the remainder of the FimC molecule. In some embodiments, a polypeptide or fragment thereof derived from E. coli FimH may be expressed in the form of a complex that includes isolated domains thereof, such as a lectin binding domain and a piling domain, which may be covalently or non-covalently linked to one another. For example, in some embodiments, the binding segment may include an amino acid sequence or other oligomeric structure, including a single polymeric structure.

The methods and compositions of the present invention may include a complex as described herein that is co-expressed or formed in combination with the polypeptides or fragments thereof derived from E. coli.

C. Lectin Domain, Pyrin Domain, and Variants Thereof The conformation and ligand-binding properties of the lectin domain of FimH may be under allosteric control of the pyrin domain of FimH. Under static conditions, the interaction of the two domains of full-length FimH stabilizes the lectin domain in a low affinity state for monomannose (e.g., _Kd ≈ 300 μM) characterized by a shallow binding pocket. Binding to a mannoside ligand may induce a conformational change that results in a moderate affinity state, in which the lectin and pyrin domains remain in close contact. However, upon shear stress, the lectin and pyrin domains may separate, thereby inducing a high affinity state (e.g., _Kd < 1.2 μM).

Due to the absence of negative allosteric control provided by the pyrin domain, the isolated lectin domain of FimH is locked in a high affinity state (e.g., _Kd < 1.2 μM). Isolated recombinant lectin domain locked in a high affinity state. However, locking the adhesin in a low affinity conformation (e.g., _Kd ≈ 300 μM) induces the production of adhesin-inhibiting antibodies. Therefore, there is interest in stabilizing the lectin domain in a low affinity state.

In some embodiments, the polypeptide or fragment thereof derived from E. coli comprises a lectin domain of E. coli FimH. Exemplary sequences of the 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 E. coli FimH comprises a cysteine substitution. In a preferred embodiment, the lectin domain of E. coli FimH comprises a cysteine substitution within the first 50 amino acid residues of the lectin domain. In some embodiments, the lectin domain may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cysteine substitutions. Preferably, the lectin domain comprises two cysteine substitutions. See, e.g., pSB02158 and pSB02198.

Other suitable polypeptides and fragments thereof derived from E. coli FimH include FimH lectin domain variants having various degrees of identity to the sequence set forth in 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 set forth in SEQ ID NO:3. In some embodiments, the composition comprises 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 comprises a polypeptide having at least 90% identity to SEQ ID NO:3. In some embodiments, the composition comprises a polypeptide having at least 95% identity to SEQ ID NO:3. In some embodiments, the composition comprises a polypeptide having at least 99% identity to SEQ ID NO:3. In some embodiments, the composition comprises a polypeptide as set forth in SEQ ID NO:3. In some embodiments, the polypeptide or fragment thereof derived from E. coli comprises the pilin domain of E. coli FimH. Other suitable polypeptides and fragments thereof derived from E. coli FimH include FimH pilin domain variants having 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 set forth in SEQ ID NO:7. In some embodiments, the composition comprises 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 comprises a polypeptide having at least 90% identity to SEQ ID NO:7. In some embodiments, the composition comprises a polypeptide having at least 95% identity to SEQ ID NO:7. In some embodiments, the composition comprises a polypeptide having at least 99% identity to SEQ ID NO:7. In some embodiments, the composition comprises a polypeptide as set forth in SEQ ID NO:7. Other suitable polypeptides and fragments thereof derived from E. coli FimH include FimH lectin domain variants having various degrees of identity to the sequence set forth in 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 set forth in SEQ ID NO:8. In some embodiments, the composition comprises 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 comprises a polypeptide having at least 90% identity to SEQ ID NO:8. In some embodiments, the composition comprises a polypeptide having at least 95% identity to SEQ ID NO:8. In some embodiments, the composition comprises a polypeptide having at least 99% identity to SEQ ID NO:8. In some embodiments, the composition comprises a polypeptide as set forth in SEQ ID NO:8. In some embodiments, the polypeptide or fragment thereof derived from E. coli comprises the pilin domain of E. coli FimH. Other suitable polypeptides and fragments thereof derived from E. coli FimH include FimH pilin domain variants having 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 set forth in SEQ ID NO:24. In some embodiments, the composition comprises 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 comprises a polypeptide having at least 90% identity to SEQ ID NO:24. In some embodiments, the composition comprises a polypeptide having at least 95% identity to SEQ ID NO:24. In some embodiments, the composition comprises a polypeptide having at least 99% identity to SEQ ID NO:24. In some embodiments, the composition comprises a polypeptide as set forth in SEQ ID NO:24. Other suitable polypeptides and fragments thereof derived from E. coli FimH include FimH lectin domain variants having various degrees of identity to the sequence set forth in 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 set forth in SEQ ID NO:26. In some embodiments, the composition comprises 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 comprises a polypeptide having at least 90% identity to SEQ ID NO:26. In some embodiments, the composition comprises a polypeptide having at least 95% identity to SEQ ID NO:26. In some embodiments, the composition comprises a polypeptide having at least 99% identity to SEQ ID NO:26. In some embodiments, the composition comprises a polypeptide as set forth in SEQ ID NO:26. Other suitable polypeptides and fragments thereof derived from E. coli FimH include FimH lectin domain variants having various degrees of identity to the sequence set forth in 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 set forth in SEQ ID NO:110. In some embodiments, the composition comprises 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 comprises a polypeptide having at least 90% identity to SEQ ID NO: 110. In some embodiments, the composition comprises a polypeptide having at least 95% identity to SEQ ID NO: 110. In some embodiments, the composition comprises a polypeptide having at least 99% identity to SEQ ID NO: 110. In some embodiments, the composition comprises a polypeptide as set forth in SEQ ID NO: 110. Other suitable polypeptides and fragments thereof derived from E. coli FimH include FimH lectin domain variants having various degrees of identity to the sequence set forth in 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 set forth in SEQ ID NO:111. In some embodiments, the composition comprises 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 comprises a polypeptide having at least 90% identity to SEQ ID NO:111. In some embodiments, the composition comprises a polypeptide having at least 95% identity to SEQ ID NO:111. In some embodiments, the composition comprises a polypeptide having at least 99% identity to SEQ ID NO:111. In some embodiments, the composition comprises a polypeptide as set forth in SEQ ID NO:111.

In some embodiments, the composition comprises 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, where n is 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20 or more). Preferably, the fragment comprises an epitope from the sequence. In some embodiments, the composition comprises 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 any one of the amino acid sequences 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 comprises a polypeptide having at least 50 contiguous 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 comprises a polypeptide having at least 100 contiguous 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 comprises a polypeptide having at least 150 contiguous 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 comprises a polypeptide having at least 250 contiguous amino acid residues of any one of the amino acid sequences 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 the lectin domain of E. coli FimH or a homolog or variant thereof can be predicted based on a pairwise alignment of the sequence with 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 FimH with SEQ ID NO:1 and identifying sequences that align with 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 the host cell to produce a mature FimH polypeptide. Thus, the FimH expressed by the host cell may not contain an N-terminal signal sequence. In a preferred embodiment, the E. coli derived polypeptide or fragment thereof may be encoded by a nucleotide sequence that does not contain a coding sequence for the wild-type N-terminal signal sequence.

In some embodiments, the E. coli derived polypeptide or fragment thereof comprises a FimH-FimC complex-forming fragment of FimH, an N-terminal signal sequence (e.g., residues 1-21 of SEQ ID NO:1), or a combination thereof. The FimH complex-forming fragment can be any part or portion of the FimH protein that retains the ability to form a complex with FimC.

In some embodiments, the E. coli derived polypeptide or fragment thereof may lack between 1 and 21 amino acid residues at the N-terminus and/or C-terminus of the full-length FimH polypeptide (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 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). In some embodiments, the E. coli derived polypeptide or fragment thereof lacks 1-21 residues at the N-terminus of the full-length FimH polypeptide. In some embodiments, the polypeptide or fragment thereof derived from E. coli lacks 1 to 10 residues at the N-terminus of a full-length FimH polypeptide. In some embodiments, the polypeptide or fragment thereof derived from E. coli lacks 5 to 15 residues at the N-terminus of a full-length FimH polypeptide. In some embodiments, the polypeptide or fragment thereof derived from E. coli lacks 5 to 10 residues at the N-terminus of a full-length FimH polypeptide. In some embodiments, the polypeptide or fragment thereof derived from E. coli lacks 1 to 21 residues at the C-terminus of a full-length FimH polypeptide. In some embodiments, the polypeptide or fragment thereof derived from E. coli lacks 1 to 10 residues at the C-terminus of a full-length FimH polypeptide. In some embodiments, the polypeptide or fragment thereof derived from E. coli lacks 5 to 15 residues at the C-terminus of a full-length FimH polypeptide. In some embodiments, the polypeptide or fragment thereof derived from E. coli lacks 5 to 10 residues at the C-terminus of the full-length FimH polypeptide.

II. Nucleic Acid In one aspect, a nucleic acid encoding a polypeptide or a fragment thereof derived from E. coli is disclosed. One or more nucleic acid constructs encoding a polypeptide or a fragment thereof derived from E. coli can be used for genome integration and subsequent expression of the polypeptide or a fragment thereof derived from E. coli. For example, a single nucleic acid construct encoding a polypeptide or a fragment thereof derived from E. coli can be introduced into a host cell. Alternatively, the coding sequence for a polypeptide or a fragment thereof derived from E. coli can be carried by two or more nucleic acid constructs, which are then introduced into the host cell simultaneously or sequentially.

For example, in one exemplary embodiment, a single nucleic acid construct encodes the lectin domain and the pyrin domain of E. coli FimH. In another exemplary embodiment, one nucleic acid construct encodes the lectin domain of E. coli FimH and a second nucleic acid construct encodes the pyrin domain. In some embodiments, genomic integration is achieved.

The nucleic acid construct may comprise genomic DNA, including 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 expression of an exogenous polypeptide in the 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 comprises a signal sequence encoding a peptide that directs secretion of the E. coli derived polypeptide or fragment thereof. In some embodiments, the nucleic acid comprises the native signal sequence of the E. coli derived polypeptide FimH. In some embodiments, in which the E. coli derived polypeptide or fragment thereof comprises an endogenous signal sequence, the nucleic acid sequence encoding the signal sequence can be codon optimized to increase the level of expression of the protein in the 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 in length. In some embodiments, the signal sequence is 20 amino acids in length. In some embodiments, the signal sequence is 21 amino acids in length.

In some embodiments, when a polypeptide or fragment thereof includes a signal sequence, an endogenous signal sequence naturally associated with the polypeptide can be replaced with a signal sequence that is not associated with the wild-type polypeptide in order to improve the level of expression of the polypeptide or fragment thereof in cultured cells. Thus, in some embodiments, the nucleic acid does not include the native signal sequence of a polypeptide or fragment thereof derived from E. coli. In some embodiments, the nucleic acid does not include the native signal sequence of a polypeptide derived from E. coli FimH. In some embodiments, a polypeptide or fragment thereof derived from E. coli can be expressed with a heterologous peptide, preferably a signal sequence, or other peptide having a specific cleavage site at the N-terminus of a mature protein or polypeptide derived from E. coli or a fragment thereof. For example, a polypeptide or fragment thereof derived from E. coli FimH can be expressed with a heterologous peptide, preferably a signal sequence (e.g., an IgK signal sequence), or other peptide having a specific cleavage site at the N-terminus of a mature E. coli FimH protein. In a preferred embodiment, specific cleavage at the N-terminus of the mature E. coli FimH protein occurs immediately prior to the first 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 a 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 of SEQ ID NO: 18. In some embodiments, the nucleic acid encodes the amino acid sequence of SEQ ID NO: 19. In some embodiments, the nucleic acid encodes the amino acid sequence of SEQ ID NO: 22. In preferred embodiments, the signal sequence is a mouse IgK signal sequence.

Suitable mammalian expression vectors for producing polypeptides or fragments thereof from E. coli are known in the art and may be commercially available, such as the pSecTag2 expression vector from Invitrogen™. An exemplary mouse Ig kappa signal peptide sequence comprises the sequence ETDTLLLWVLLLWVPGSTG (SEQ ID NO:54). In some embodiments, the vector comprises the pBudCE4.1 mammalian expression vector from Thermo Fisher. Additional exemplary suitable vectors include the pcDNA™ 3.1 mammalian expression vector (Thermo Fisher).

In some embodiments, the signal sequence does not include a hemagglutinin signal sequence.

In some embodiments, the nucleic acid comprises a native signal sequence of a polypeptide or fragment thereof derived from E. coli. In some embodiments, the signal sequence is not an IgK signal sequence. In some embodiments, the signal sequence comprises a hemagglutinin signal sequence.

In one aspect, a vector is disclosed herein that includes a coding sequence for a polypeptide or fragment thereof derived from E. coli. Exemplary vectors include plasmids capable of autonomous replication or replication in mammalian cells. Typical expression vectors include suitable promoters, enhancers, and terminators that are useful for regulating expression of the coding sequence in the expression construct. The vector may include a selection marker for obtaining a phenotypic trait (such as conferring resistance to antibiotics such as ampicillin or neomycin) for selection of transformed host cells.

Suitable promoters are known in the art. Exemplary promoters include, for example, CMV promoter, adenovirus, EF1a, GAPDH metallothionine promoter, SV40 early promoter, SV40 late promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, and the like. The promoter may be a constitutive or inducible promoter. One or more vectors may be used (e.g., one vector encoding all of the subunits or domains or fragments thereof, or multiple vectors collectively encoding the subunits or domains or fragments thereof).

Internal ribosome entry sites (IRES) and 2A peptide sequences can also be used. IRES and 2A peptides provide an alternative approach for co-expression of multiple sequences. IRES is a nucleotide sequence that allows translation initiation in the middle of a messenger RNA (mRNA) sequence as part of the larger process of protein synthesis. Normally, in eukaryotes, translation can only be initiated at the 5' end of an mRNA molecule. IRES elements allow the expression of multiple genes in one transcript. IRES-based polycistronic vectors that express multiple proteins from one transcript can reduce leakage of non-expressing clones from selection. 2A peptides allow translation of multiple proteins in one open reading frame into a polyprotein that is subsequently cleaved into individual proteins via a ribosome-skipping mechanism. 2A peptides can provide a more balanced expression of multiple protein products. Exemplary IRES sequences include, for example, EV71 IRES, EMCV IRES, HCV IRES. With respect to genomic integration, integration can be site-specific or random. Site-specific recombination can be achieved by introducing a homologous sequence into the nucleic acid construct described herein. Such a homologous sequence substantially matches an endogenous sequence at a specific target site in the host genome. Alternatively, random integration can be used. Sometimes, protein expression levels can vary depending on the integration site. Therefore, it can be desirable to select multiple clones according to the expression level of the recombinant protein to identify clones that achieve the desired expression level.

Exemplary nucleic acid constructs are further described in the figures, such as any one of Figures 2A-2T.

In one aspect, the nucleic acid sequence encodes an 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:111, SEQ ID NO:112, and SEQ ID NO:113.

III. Host Cell In one aspect, the present invention relates to a cell in which a sequence encoding a polypeptide or a fragment thereof derived from E. coli is expressed in a mammalian host cell. In one embodiment, the polypeptide or a fragment thereof derived from E. coli is transiently expressed in the host cell. In another embodiment, the polypeptide or a fragment thereof derived from E. coli is stably integrated into the genome of the host cell, which expresses the polypeptide or a fragment thereof derived from E. coli when cultured under suitable conditions. In a preferred embodiment, the polynucleotide sequence is expressed with high efficiency and genome stability.

Suitable mammalian host cells are known in the art. Preferably, the host cells are suitable for producing proteins on an industrial manufacturing scale. Exemplary mammalian host cells include any one of the following and their derivatives: 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 (a mouse myeloma cell line), and C127 cells (a non-tumorigenic 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), mouse hybridoma (Sp2/0), mouse thymoma (EL4), Chinese hamster ovary (CHO) and CHO cell derivatives, mouse embryonic (NIH/3T3, 3T3 Li), rat cardiac muscle (H9c2), mouse myoblasts (C2C12), and mouse kidney (miMCD-3). Further examples of mammalian cell lines include NS0/1, Sp2/0, Hep G2, PER. C6, COS-7, TM4, CV1, VERO-76, MDCK, BRL 3A, W138, MMT 060562, TR1, MRC5, and FS4.

Any cell capable of cell culture can be used in accordance with the present invention. In some embodiments, the cell is a mammalian cell. Non-limiting examples of mammalian cells that can be used in accordance with the present invention include BALB/c mouse myeloma line (NS0/1, ECACC No: 85110503); human retinoblastoma cells (PER.C6, Cru cells, Leiden, The Netherlands); SV40 transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney line (293 cells 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); dog 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 hepatocellular carcinoma line (Hep G2). In some preferred embodiments, the cells are CHO cells. In some preferred embodiments, the cells are GS cells.

In addition, any number of commercially available and non-commercially available hybridoma cell lines can be used in accordance with the present invention. The term "hybridoma" as used herein refers to a cell or progeny resulting from the fusion of an immortalized cell and an antibody-producing cell. The hybridoma so obtained is an immortalized cell that produces an antibody. The individual cells used to make the hybridoma can be from any mammalian source, including, but not limited to, rat, pig, rabbit, sheep, pig, goat, and human. In some embodiments, the hybridoma is a trioma cell line that arises when the progeny of a heterohybrid myeloma fusion, the product of a fusion between a human cell and a mouse myeloma cell line, is subsequently fused with a plasma cell. In some embodiments, the hybridoma is any immortalized hybrid cell line that produces an antibody, such as, for example, a quadroma (see, e.g., Milstein et al., Nature, 537:3053, 1983). One skilled in the art will know that hybridoma cell lines may have different nutritional requirements and/or require different culture conditions for optimal growth, and will be able to modify conditions as necessary.

In some embodiments, the cell comprises a first gene of interest, where 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 or fragment thereof derived from E. coli), a dependent 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 separate recombination target sites (RTS) in a site-specific integration (SSI) mammalian cell, where the two RTSs are chromosomally integrated within the NL1 locus or the NL2 locus. See, for example, U.S. Patent Application No. 20200002727 for a description of the NL1, NL2, NL3, NL4, NL5, and NL6 loci. In some embodiments, the first gene of interest is located within the NL1 locus. In some embodiments, the cell includes a second gene of interest, where the second gene of interest is chromosomally integrated. In some embodiments, the second gene of interest includes a reporter gene, a selection gene, a gene of therapeutic interest (such as a polypeptide or fragment thereof derived from E. coli), a dependent gene, or a combination thereof. In some embodiments, the gene of therapeutic interest includes a gene encoding a DtE protein. In some embodiments, the second gene of interest is located between two RTSs. 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 or fragment thereof from E. coli), a dependent 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 RTSs. 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 separate 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 in three separate loci. In some embodiments, at least one of the first gene of interest, the second gene of interest, and the third gene of interest is in 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 in 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 disclosure provides a mammalian cell comprising at least four distinct RTSs, wherein the cell comprises: (a) at least two distinct RTSs chromosomally integrated within the NL1 locus or the NL2 locus; (b) a first gene of interest integrated between the at least two RTSs of (a), the first gene of interest comprising a reporter gene, a gene encoding a DtE protein, a dependent gene, or a combination thereof; and (c) a second gene of interest integrated within a second chromosomal locus distinct from the locus of (a), the second gene of interest comprising a reporter gene, a gene encoding a DtE protein (such as a polypeptide or fragment thereof derived from E. coli), a dependent gene, or a combination thereof. In some embodiments, the present disclosure provides a mammalian cell comprising at least four distinct RTSs, wherein the cell comprises: (a) at least two distinct RTSs chromosomally integrated within the Fer1L4 locus; (b) at least two distinct RTSs chromosomally integrated within the NL1 locus or the NL2 locus; (c) a first gene of interest chromosomally integrated within the Fer1L4 locus, the first gene of interest comprising a reporter gene, a gene encoding a DtE protein, a dependent gene, or a combination thereof; and (d) a second gene of interest chromosomally integrated within the NL1 locus or the NL2 locus of (b), the second gene of interest comprising a reporter gene, a gene encoding a DtE protein (such as a polypeptide or fragment thereof derived from E. coli), a dependent gene, or a combination thereof.

In some embodiments, the present disclosure provides a mammalian cell comprising at least six distinct RTSs, wherein the cell comprises: (a) at least two distinct RTSs and a first gene of interest that are chromosomally integrated within the Fer1L4 locus; (b) at least two distinct RTSs and a second gene of interest that are chromosomally integrated within the NL1 locus; and (c) at least two distinct RTSs and a third gene of interest that 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 linking 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 linking of amino acid sequences in such a manner that a functional protein is produced. In some embodiments, a gene of interest is operably linked to a promoter, where the gene of interest is chromosomally integrated into the host cell. In some embodiments, a gene of interest is operably linked to a heterologous promoter; where the gene of interest is chromosomally integrated into the host cell. In some embodiments, a dependent gene is operably linked to a promoter, where the dependent gene is chromosomally integrated into the host cell genome. In some embodiments, a dependent gene is operably linked to a heterologous promoter; where the dependent gene is chromosomally integrated into the host cell genome. In some embodiments, a gene encoding a DtE protein is operably linked to a promoter, where the gene encoding a DtE protein is chromosomally integrated into the host cell genome. In some embodiments, the gene encoding the DtE protein is operably linked to a heterologous promoter, where the gene encoding the DtE protein is chromosomally integrated into the host cell genome. In some embodiments, the recombinase gene is operably linked to a promoter, where the recombinase gene is chromosomally integrated into the host cell. In some embodiments, the recombinase gene is operably linked to a promoter, where the recombinase gene is chromosomally integrated into the host cell genome. In some embodiments, the recombinase gene is operably linked to a heterologous promoter, where 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, where 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 integration of a nucleic acid sequence into a 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, the chromosomally integrated nucleic acid sequence is stable. In some embodiments, the chromosomally integrated nucleic acid sequence is not located on a plasmid or vector. In some embodiments, the chromosomally integrated nucleic acid sequence is not excised. In some embodiments, the chromosomal integration is mediated by 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 culture. 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 anchorage-dependent cells (e.g., epithelial cells, fibroblasts).

In some embodiments, the expression level or activity of the E. coli derived polypeptide 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 compared to expression of the E. coli derived polypeptide or fragment thereof in a bacterial cell, such as an E. coli host cell. In some embodiments, the expression level of the E. coli derived polypeptide or fragment thereof is increased by at least 2-fold compared to expression of the E. coli derived polypeptide or fragment thereof in an E. coli host cell. In some embodiments, the expression level of the E. coli derived polypeptide or fragment thereof is increased by at least 50-fold compared to expression of the E. coli derived polypeptide or fragment thereof in an E. coli host cell. In some embodiments, the expression level of the E. coli derived polypeptide or fragment thereof is increased by at least 100-fold compared to the expression of the E. coli derived polypeptide or fragment thereof in an E. coli host cell.

The host cells described herein are suitable for large scale cultivation. For example, the cell culture may be 10L, 30L, 50L, 100L, 150L, 200L, 300L, 500L, 1000L, 2000L, 3000L, 4000L, 5000L, 10,000L or more. In some embodiments, the cell culture size is 10L-5000L, 10L-10,000L, 10L-20,000L, 10L-50,000L, 40L-50,000L, 100L-50,000L, 500L-50,000L, 1000L-50,000L, 2000L-50,000L, 3000L-50,000L, 4000L-50,000L, 5 ... 000L to 50,000L, 4500L to 50,000L, 1000L to 10,000L, 1000L to 20,000L, 1000L to 25,000L, 1000L to 30,000L, 15L to 2000L, 40L to 1000L, 100L to 500L, 200L to 400L, or any integer range therebetween. Media components for cell culture are known in the art and may include, for example, buffers, amino acid contents, vitamin contents, salt contents, inorganic salt contents, serum contents, carbon source contents, lipid contents, nucleic acid contents, hormone contents, trace element contents, ammonia contents, cofactor contents, indicator contents, small molecule contents, hydrolysate contents, and enzyme regulator contents.

The terms "culture medium", "cell culture medium" and "culture medium" as used herein refer to a solution containing nutrients that support the growth of mammalian cells. Typically, such solutions provide the essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by cells for minimal growth and/or survival. Such solutions may contain supplemental components that enhance growth and/or survival beyond the minimal proportions, including, but not limited to, hormones and/or other growth factors, specific ions (such as sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds that are usually present at very low final concentrations), inorganic compounds (e.g., iron) that are present at high final concentrations, amino acids, lipids, and/or glucose or other energy sources. In some embodiments, the medium is advantageously formulated to an optimal pH and salt concentration for cell survival and growth. In some embodiments, the medium is a feed medium that is added after the initiation of cell culture.

In some embodiments, the cells can be grown in one of a variety of chemically defined media, in which the components of the media are known and controlled. In some embodiments, the cells can be grown in complex media, in which not all of the components of the media are known and/or controlled. Chemically defined growth media for culturing mammalian cells have been widely developed and published over the last several decades. All components of the defined media are well characterized, and such defined media do not contain complex additives such as serum or hydrolysates. Early media formulations were developed to allow cell growth and survival with little or no concerns regarding protein production. More recently, media formulations have been developed for the express purpose of supporting the culture of highly proliferative recombinant protein producing cells. Such media are preferred for use in the methods of the invention. Such media generally contain large amounts of nutrients, particularly amino acids, that support the growth and/or maintenance of cells at high density. If necessary, these media can be modified by one of skill in the art for use in the methods of the invention. For example, one of skill in the art can reduce the amount of phenylalanine, tyrosine, tryptophan, and/or methionine in these media for use as basal or feed media in the methods disclosed herein.

Not all components of a complex medium are well characterized, and such complex media may contain simple and/or complex carbon sources, simple and/or complex nitrogen sources, and additives such as serum, among others. In some embodiments, complex media suitable for the present invention contain additives such as hydrolysates in addition to other components of defined media as described herein. In some embodiments, defined media typically contain approximately 50 chemical entities at known concentrations in water. Many of them also contain one or more well-characterized proteins such as insulin, IGF-1, transferrin or BSA, while others do not require a protein component and are therefore referred to as protein-free defined media. Typical chemical components of media fall into five broad categories: amino acids, vitamins, inorganic salts, trace elements, and a miscellaneous category that cannot be succinctly classified.

The 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 beyond a minimal ratio, including, but not limited to, hormones and/or other growth factors, specific ions (such as sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds present at very low final concentrations), amino acids, lipids, and/or glucose or other energy sources. In some embodiments, the supplementary components may be added to the initial cell culture. In some embodiments, the supplementary components may be added after the cell culture is initiated. Typically, trace elements refer to various inorganic salts that are included at micromolar or lower levels. For example, commonly included trace elements are zinc, selenium, copper, etc. In some embodiments, iron (ferrous or ferric salts) may be included as a trace element in the initial cell culture medium at micromolar concentrations. Among the trace elements, manganese is also often included as a divalent cation ( _MnCl2 or _MnSO4 ) in the nanomolar to micromolar range. Many less common trace elements are usually added in nanomolar concentrations.

In some embodiments, the medium used in the methods of the invention is a medium suitable for supporting ^high cell densities in cell culture, such as, for example, 1×10 ⁶ cells/mL, 5×10 ⁶ cells/mL, 1×10 ⁷ cells/mL, 5×10 ⁷ cells/mL, 1×10 ^{8 cells/mL or 5×10 8} cells/mL. In some embodiments, the cell culture is a mammalian cell fed-batch culture, preferably a CHO cell fed-batch culture.

In some embodiments, the cell culture medium comprises phenylalanine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises tyrosine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises tryptophan at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises methionine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises leucine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises serine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises threonine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises glycine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises two of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises phenylalanine and tyrosine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises phenylalanine and tryptophan at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises phenylalanine and methionine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises tyrosine and tryptophan at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises tyrosine and methionine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises tryptophan and methionine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises three of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine and glycine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises phenylalanine, tyrosine and tryptophan at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises phenylalanine, tyrosine and methionine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises phenylalanine, tryptophan and methionine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises tyrosine, tryptophan and methionine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises four of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine and glycine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises phenylalanine, tyrosine, tryptophan and methionine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises five of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine and glycine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises six of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises seven of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine and glycine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM or between 0.5 and 1 mM. In some embodiments, the cell culture medium further comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or thirteen of glycine, valine, leucine, isoleucine, proline, serine, threonine, lysine, arginine, histidine, aspartate, glutamate and asparagine at a concentration of 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 15 mM, preferably greater than 2 mM. In some embodiments, the cell culture medium further comprises at least five of glycine, valine, leucine, isoleucine, proline, serine, threonine, lysine, arginine, histidine, aspartate, glutamate and asparagine at concentrations of 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 15 mM, preferably greater than 2 mM. In some embodiments, the cell culture medium further comprises glycine, valine, leucine, isoleucine, proline, serine, threonine, lysine, arginine, histidine, aspartate, glutamate and asparagine at concentrations of 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 15 mM, preferably greater than 2 mM. In some embodiments, the cell culture medium further comprises at least one, two, three, four, five, six, seven, eight, or nine of valine, isoleucine, proline, lysine, arginine, histidine, aspartate, glutamate, and asparagine at a concentration of 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 15 mM, preferably greater than 2 mM. In some embodiments, the cell culture medium further comprises at least five of valine, isoleucine, proline, lysine, arginine, histidine, aspartate, glutamate, and asparagine at a concentration of 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 15 mM, preferably greater than 2 mM. In some embodiments, the cell culture medium further comprises valine, isoleucine, proline, lysine, arginine, histidine, aspartate, glutamate and asparagine at a concentration of 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 15 mM, preferably 2 mM. In some embodiments, the cell culture medium comprises serine at a concentration of 3 mM, 5 mM, 7 mM, 10 mM, 15 mM or 20 mM, preferably more than 10 mM. In some embodiments, the cell culture medium comprises valine at a concentration of 3 mM, 5 mM, 7 mM, 10 mM, 15 mM or 20 mM, preferably more than 10 mM. In some embodiments, the cell culture medium comprises cysteine at a concentration of 3 mM, 5 mM, 7 mM, 10 mM, 15 mM or 20 mM, preferably more than 10 mM. In some embodiments, the cell culture medium comprises isoleucine at a concentration of 3 mM, 5 mM, 7 mM, 10 mM, 15 mM or 20 mM, preferably greater than 10 mM. In some embodiments, the cell culture medium comprises leucine at a concentration of 3 mM, 5 mM, 7 mM, 10 mM, 15 mM or 20 mM, preferably greater than 10 mM. 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 as a basal medium in a method as disclosed herein. In some embodiments, the above cell culture medium is used as a feed medium in a method as disclosed herein.

IV. Methods of Production In one aspect, the present invention includes a method of producing an E. coli derived polypeptide or fragment thereof. The method includes culturing mammalian cells under suitable conditions, thereby expressing the E. coli derived polypeptide or fragment thereof. The method may further include collecting the E. coli derived polypeptide or fragment thereof from the culture. The process may further include purifying the E. coli derived polypeptide or fragment thereof.

In some embodiments, the method produces a yield of the polypeptide or fragment thereof of between 0.1 g/L and 0.5 g/L.

In some embodiments, the cells can be grown in batch or fed-batch culture, where the culture is stopped after sufficient expression of the polypeptide, and the expressed polypeptide is then harvested and optionally purified. In some embodiments, the cells can be grown in perfusion culture, where the culture is not stopped, but rather new nutrients and other components are periodically or continuously added to the culture, while the expressed polypeptide is periodically or continuously harvested.

In some embodiments, the cells can be grown in small scale reaction vessels ranging in volume from a few milliliters to a few liters. In some embodiments, the cells can be grown in large scale commercial bioreactors ranging in volume from approximately at 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 therebetween.

The temperature of the cell culture will be selected primarily based on the range of temperatures at which the cell culture is still viable and high levels of polypeptide are 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 of protein or polypeptide at approximately 37°C. In general, most mammalian cells can grow well and/or produce high levels of protein or polypeptide within the range of about 25°C to 42°C, but the methods taught by the present disclosure are not limited to these temperatures. Certain mammalian cells can grow well and/or produce high levels of protein or polypeptide within the range of about 35°C to 40°C. In certain embodiments, the cell culture is grown one or more times during the cell culture process 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.

The terms "culture" and "cell culture," as used herein, refer to a population of cells suspended in a medium under conditions suitable for the survival and/or growth of the population of cells. As will be apparent to one of ordinary skill in the art, in some embodiments, these terms, as used herein, refer to a combination comprising a population of cells and the medium in which the population is suspended. In some embodiments, the cells of the cell culture comprise mammalian cells.

The present invention can be used with any cell culture method that is applicable to a desired process, such as the production of a recombinant protein (e.g., an antibody). As a non-limiting example, cells can be grown in batch or fed-batch culture, where the culture is stopped after sufficient expression of the recombinant protein (e.g., an antibody) and the expressed protein (e.g., an antibody) is then harvested. Alternatively, as another non-limiting example, cells can be grown in batch-refeed, where the culture is not stopped and new nutrients and other components are added to the culture periodically or continuously while the expressed recombinant protein (e.g., an antibody) is harvested periodically or continuously. Other suitable methods (e.g., spin tube culture) 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 supplied one or more times after the start of the culturing process. Such supplied components typically include nutritional components for the cells that are depleted during the culturing process. Fed-batch cultures are typically stopped at multiple time points to harvest and optionally purify the cells and/or components in the medium. In some embodiments, fed-batch cultures include a basal medium supplemented with a feed medium.

The cells can be grown in any convenient volume selected by the practitioner. For example, the cells can be grown in small scale reaction vessels ranging in volume from a few milliliters to a few liters. Alternatively, the cells can 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, 15,000, 20,000, or 25,000 liters or more, or any volume therebetween.

The temperature of the cell culture will be selected primarily based on the range of temperatures at which the cell culture is still viable and at which high levels of the desired product (e.g., recombinant protein) are produced. In general, most mammalian cells can grow well and/or produce the desired product (e.g., recombinant protein) within a range of about 25°C to 42°C, although the methods taught by the present disclosure are not limited to these temperatures. Certain mammalian cells can grow well and/or produce the desired product (e.g., recombinant protein or antibody) within a range of about 35°C to 40°C. In certain embodiments, the cell culture is grown one or more times during the cell culture process 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. A person of ordinary skill in the art would be able to select a suitable temperature or temperatures to grow the cells at depending on the particular needs of the cells and the particular production requirements of the practitioner. The cells can be grown for any amount of time depending on the needs of the practitioner and the requirements of the cells themselves. In some embodiments, the cells are grown at 37°C. In some embodiments, the cells are grown at 36.5°C.

In some embodiments, the cells can be grown for a greater or lesser amount of time during the initial growth phase (or growth phase), depending on the needs of the practitioner and the requirements of the cells themselves. In some embodiments, the cells are grown for a sufficient period of time to achieve a predefined cell density. In some embodiments, the cells are grown for a sufficient period of time to achieve a cell density that is a given percentage of the maximum cell density that the cells would eventually reach if grown undisturbed. For example, the cells can be grown for a sufficient period of time to achieve a desired viable cell density of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99 percent of the maximum 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 embodiments, the cells are grown for a defined period of time. For example, the cells are grown for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days or more, preferably 4-10 days, 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. In some cases, the cells can be grown for a month or more. The practitioner of the invention will be able to select the duration of the initial growth phase depending on the protein production requirements and the requirements of the cells themselves.

The cell culture can be agitated or shaken during the initial cultivation phase to increase oxygenation and distribution of nutrients to the cells. In the present invention, one skilled in the art will appreciate that it may be advantageous 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, a second set of culture conditions can be applied to shift at least one of the culture conditions such that a metabolic shift occurs in the culture. The metabolic shift can be achieved, for example, by changing the temperature, pH, osmolality, or chemical induction 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, a temperature shift is not the only mechanism by which a suitable 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 invention based on protein production requirements and the requirements of the cells themselves.

When the temperature of the culture is shifted, the temperature shift can be relatively gradual. For example, the temperature change can take hours or days to complete. Alternatively, the temperature shift can be relatively sudden. For example, the temperature change can be completed in less than a few hours. Given suitable production and control equipment, such as is standard in large-scale commercial production of polypeptides or proteins, the temperature change can be completed in less than an hour.

In some embodiments, once the cell culture conditions have been shifted as discussed above, the cell culture is maintained through a subsequent growth phase under a second set of culture conditions that are conducive to the survival and viability of the cell culture and suitable for expression of the desired polypeptide or protein at commercially relevant levels.

As discussed above, the culture can be shifted by shifting one or more of several culture conditions, including, but not limited to, temperature, pH, osmolality, and sodium butyrate levels. In some embodiments, the temperature of the culture is shifted. In this embodiment, the culture is maintained during the subsequent growth phase at a temperature or temperature range that is lower than the temperature or temperature range of the initial growth phase. As discussed above, multiple separate temperature shifts can be used to increase cell density or viability, or to increase recombinant protein expression.

In some embodiments, the cells can be maintained in the subsequent production phase until a desired cell density or production titer is achieved. In another embodiment of the invention, the cells are grown during the subsequent production phase for a predetermined period of time. 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 can be grown for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days or more. In some cases, the cells can be grown for a month or more. The practitioner of the invention will be able to select the duration of the subsequent production phase depending on the polypeptide or protein production requirements and the needs of the cells themselves.

The cell culture can be agitated or shaken during the subsequent production phase to increase oxygenation and distribution of nutrients to the cells. In the present invention, one skilled in the art will appreciate that it may be advantageous 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 includes a growth phase and a production phase.

In some embodiments, step (ii) of any of the methods disclosed herein is applied throughout the cell culture method. In some embodiments, step (ii) of any of the methods disclosed herein is applied as 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 portion 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" may refer to maintaining the concentration of amino acids or metabolites below C1 or C2 during the entire culture process (until harvest), or during a portion of the culture process, such as, for example, the growth phase, a portion of the growth phase, or until a predetermined cell density is obtained.

In some embodiments of any of the above methods, cell growth and/or productivity is increased compared to a control culture, where the control culture is identical except that it does not include step (ii).

In some embodiments of any of the above-described methods, the method of the invention is a method for improving cell proliferation. In some embodiments, the method of the invention is a method for improving cell proliferation at high cell densities in high density cell culture.

High cell density, as used herein, refers to a cell density greater than 1× ¹⁰ cells/mL, 5× ¹⁰ cells/mL, 1× ¹⁰ cells/mL, 5× ¹⁰ cells/mL, 1×10 cells/mL ^or 5× ¹⁰ cells/mL, preferably greater than 1× ¹⁰ cells/mL, more preferably greater than 5× ¹⁰ cells/mL.

In some embodiments, the method of the invention is a method for improving cell proliferation in cell culture, wherein the cell density is greater ^{than 1x106} cells/mL, 5x106 cells/mL, ^1x107 cells/mL, ^5x107 cells/mL, ^1x108 cells/ ^mL or ^5x108 cells/mL. In some embodiments, the method of the invention is a method for improving cell proliferation in cell culture, wherein the maximum cell density is greater than ^1x106 cells/mL, ^5x106 cells/mL, ^1x107 cells/mL, ^5x107 cells/mL, ^1x108 cells/mL or ^5x108 cells/mL.

In some embodiments, cell proliferation is determined by viable cell density (VCD), maximum viable cell density, or integrated viable cell density (IVCC). In some embodiments, cell proliferation 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 culture medium. Viable cell density can be measured by any method known to those of skill in the art. 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 cell culture. The term "cell viability" as used herein refers to the likelihood that cells in culture will survive under a given set of culture conditions or experimental variations. A person of ordinary skill in the art will recognize that any of a number of methods for determining cell viability are encompassed by the present invention. For example, to determine cell viability, a dye (e.g., trypan blue) can be used that does not pass through the membranes of viable cells but can pass through the disrupted membranes of dead or dying cells.

The term "integrated viable cell count (IVCC)" as used herein refers to the area under the viable cell density (VCD) curve. IVCC can be calculated using the following formula: IVCCt ₊₁ = IVCCt + ( _VCDt + _{VCDt +1} ) x (Δt)/2, where Δt is the time difference between time points t and t+1. _IVCCt=0 can be assumed to be negligible. _VCDt and VCDt ₊₁ are the viable cell densities at time points t and t+1.

The term "titer" as used herein refers to, for example, the total amount of recombinantly expressed protein produced by a cell culture in a given amount of medium volume. Titer is typically expressed in grams of protein per liter of medium.

In some embodiments, cell proliferation is increased by at least 5%, 10%, 15%, 20% or 25% compared to a control culture. In some embodiments, cell proliferation is increased by at least 10% compared to a control culture. In some embodiments, cell proliferation is increased by at least 20% compared to a control culture.

In some embodiments, the production rate is determined by titer and/or volumetric production rate.

The term "titer" as used herein refers to, for example, the total amount of recombinantly expressed protein produced by a cell culture in a given amount of medium volume. Titer is typically expressed in grams of protein per liter of medium.

In some embodiments, the production rate is determined by titer. In some embodiments, the production rate is increased by at least 5%, 10%, 15%, 20% or 25% compared to a control culture. In some embodiments, the production rate is increased by at least 10% compared to a control culture. In some embodiments, the production rate is increased by at least 20% compared to a control culture.

In some embodiments, the maximum cell density of the cell culture is greater than ^1x106 cells/mL, ^5x106 cells/mL, ^1x107 cells/mL, ^5x107 cells/mL, ^1x108 cells/mL, or ^5x108 cells/mL. In some embodiments, the maximum cell density of the cell culture is greater than 5x106 cells/mL ^. In some embodiments, the maximum cell density of the cell culture is greater than ^1x108 cells/mL.

V. Purification In some embodiments, the method for purifying an E. coli derived polypeptide or fragment thereof comprises isolating and/or purifying the E. coli derived polypeptide or fragment thereof. In some embodiments, the expressed E. coli derived polypeptide or fragment thereof is secreted into the medium, so that cells and other solids can be removed by centrifugation and/or filtration.

The E. coli derived polypeptide or fragment thereof produced according to the methods described herein can be collected from the host cells and purified using any suitable method known to those of skill in the art. 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. A suitable purification scheme may include two or more of these or other suitable methods. In some embodiments, one or more of the E. coli derived polypeptide or fragment thereof may include a "tag" that facilitates purification, such as an epitope tag or a HIS tag, a Strep tag, etc. Such tagged polypeptides can be conveniently purified, for example, from conditioned medium by chelation or affinity chromatography. Optionally, the tag sequence can be cleaved after purification.

In some embodiments, the E. coli derived polypeptide or fragment thereof may include a tag for affinity purification. Affinity purification tags are known in the art. Examples include, for example, His tags (which bind metal ions), antibodies, maltose binding protein (MBP) (which binds amylose), glutathione-S-transferase (GST) (which binds glutathione), FLAG tags, and Strep tags (which bind streptavidin or derivatives thereof).

In a preferred embodiment, the polypeptide or fragment thereof derived from E. coli does not contain a purification tag.

In some embodiments, the yield of the polypeptide or fragment thereof derived from E. coli 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., at least about 10 L, at least about 20 L, at least about 30 L, at least about 40 L, at least about 50 L, at least about 60 L, at least about 70 L, at least about 80 L, at least about 90 L, at least about 100 L, at least about 150 L, at least about 200 L, at least about 250 L, at least about 300 L, at least about 400 L, at least about 500 L, at least about 600 L, at least about 700 L, at least about 800 L, at least about 900 L, at least about 1000 L, at least about 2000 L, at least about 3000 L, at least about 4000 L, The volume is at least about 5000 L, at least about 6000 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 present invention includes compositions comprising a polypeptide or fragment thereof derived from E. coli. In some embodiments, the compositions elicit an immune response including antibodies that can confer immunity against pathogenic species of E. coli.

In some embodiments, the composition comprises a polypeptide or fragment thereof derived from E. coli as the only antigen. In some embodiments, the composition does not comprise a conjugate.

In some embodiments, the composition comprises a polypeptide or fragment thereof derived from E. coli and an additional antigen. In some embodiments, the composition comprises a polypeptide or fragment thereof derived from E. coli and an additional E. coli antigen. In some embodiments, the composition comprises a polypeptide or fragment thereof derived from E. coli and a glycoconjugate from E. coli.

In some embodiments, the polypeptide or fragment thereof is derived from E. coli FimH.

In some embodiments, the composition comprises a polypeptide or fragment thereof derived from E. coli FimC.

In some embodiments, the composition comprises a polypeptide or fragment thereof derived from E. coli FimH; and a polypeptide or fragment thereof derived from E. coli FimC.

In one aspect, the invention includes a composition comprising a polypeptide or fragment thereof derived from E. coli FimH; and a saccharide, wherein the saccharide is a polypeptide of Formula O1 (e.g., Formula O1A, Formula O1B, and Formula O1C), Formula O2, Formula O3, Formula O4 (e.g., Formula O4:K52 and Formula O4:K6), Formula O5 (e.g., Formula O5ab and Formula O5ac (strain 180/C3)), Formula O6 (e.g., Formula O6:K2;K13;K15 and Formula O6:K54), Formula O7, Formula O8, Formula O9, Formula O10, Formula O11, Formula O12, Formula O13, Formula O14, Formula O15, Formula O16, Formula O17, Formula O18 (e.g., Formula O18A, Formula O18ac, Formula O18A1, Formula O18B, Formula O18C, Formula O18D, Formula O18E, Formula O18F, Formula O18G, Formula O18H, Formula O18I, Formula O18I, Formula O18J, Formula O18J, Formula O18J, Formula O18K ... , and Formula O18B1), Formula O19, Formula O20, Formula O21, Formula O22, Formula O23 (e.g., Formula O23A), Formula O24, Formula O25 (e.g., Formula O25a and Formula O25b), Formula O26, Formula O27, Formula O28, Formula O29, Formula O30, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37, Formula O38, Formula O39, formula O40, formula O41, formula O42, formula O43, formula O44, formula O45 (e.g., formula O45 and formula O45rel), formula O46, formula O48, formula O49, formula O50, formula O51, formula O52, formula O53, formula O54, formula O55, formula O56, formula O57, formula O58, formula O59, formula O60, formula O61, formula O62, formula 62D ₁ , Formula O63, Formula O64, Formula O65, Formula O66, Formula O68, Formula O69, Formula O70, Formula O71, Formula O73 (e.g., Formula O73 (strain 73-1)), Formula O74, Formula O75, Formula O76, Formula O77, Formula O78, Formula O79, Formula O80, Formula O81, Formula O82, Formula O83, Formula O84, Formula O85, Formula O86, Formula O87, Formula O88, Formula O89, Formula O90, Formula O91, Formula O92, Formula O93, Formula O95, Formula O96, Formula O97, Formula O98, Formula O99 , formula O100, formula O101, formula O102, formula O103, formula O104, formula O105, formula O106, formula O107, formula O108, formula O109, formula O110, formula O111, formula O112, formula O113, formula O114, formula O1 15, Formula O116, Formula O117, Formula O118, Formula O119, Formula O120, Formula O121, Formula O123, Formula O124, Formula O125, Formula O126, Formula O127, Formula O128, Formula O129, Formula O130, Formula O131, Formula O132, formula O133, formula O134, formula O135, formula O136, formula O137, formula O138, formula O139, formula O140, formula O141, formula O142, formula O143, formula O144, formula O145, formula O146, formula O14 7. Formula O148, Formula O149, Formula O150, Formula O151, Formula O152, Formula O153, Formula O154, Formula O155, Formula O156, Formula O157, Formula O158, Formula O159, Formula O160, Formula O161, Formula O162, Formula O 163, formula O164, formula O165, formula O166, formula O167, formula O168, formula O169, formula O170, formula O171, formula O172, formula O173, formula O174, formula O175, formula O176, formula O177, formula O178, formula O179, formula O180, formula O181, formula O182, formula O183, formula O184, formula O185, formula O186, and formula O187 (wherein n is an integer from 1 to 100).

In some embodiments, the composition comprises any one of the sugars disclosed herein. In preferred embodiments, the composition comprises any one of the conjugates disclosed herein.

In some embodiments, the composition comprises at least one glycoconjugate from E. coli serotype O25, preferably serotype O25b. In one embodiment, the composition comprises at least one glycoconjugate from E. coli serotype O1, preferably serotype O1a. In one embodiment, the composition comprises at least one glycoconjugate from E. coli serotype O2. In one embodiment, the composition comprises at least one glycoconjugate from E. coli serotype O6.

In one embodiment, the composition comprises at least one glycoconjugate selected from any one of the following E. coli serotypes O25, O1, O2, and O6, preferably O25b, O1a, O2, and O6. In one embodiment, the composition comprises at least two glycoconjugates selected from any one of the following E. coli serotypes O25, O1, O2, and O6, preferably O25b, O1a, O2, and O6. In one embodiment, the composition comprises at least three glycoconjugates selected from any one of the following E. coli serotypes O25, O1, O2, and O6, preferably O25b, O1a, O2, and O6. In one embodiment, the composition comprises glycoconjugates from each of the following E. coli serotypes: O25, O1, O2, and O6, preferably O25b, O1a, O2, and O6.

In one preferred embodiment, the glycoconjugates of any of the above compositions are individually conjugated to CRM _197. In another preferred embodiment, the glycoconjugates of any of the above compositions are individually conjugated to a SCP.

Thus, in some embodiments, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen derived from at least one E. coli serotype. In a preferred embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen derived from more than one E. coli serotype. For example, the composition may comprise O antigens from 12 different serotypes (12v) from two different E. coli serotypes (or "v", valency). In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen derived from three different serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen derived from four different E. coli serotypes. In one embodiment, the composition comprises O antigens from five different E. coli serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen derived from six different E. coli serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen derived from seven different E. coli serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen derived from eight different E. coli serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen derived from nine different E. coli serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen derived from ten different E. coli serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen derived from eleven different E. coli serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen from 12 different serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen from 13 different serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen from 14 different serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen from 15 different serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen from 16 different serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen from 17 different serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen from 18 different serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen from 19 different serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen from 20 different serotypes.

Preferably, the number of E. coli saccharides may range from one serotype (or "v", valency) to 26 different serotypes (26v). In one embodiment, one serotype is present. In one embodiment, two different serotypes are present. In one embodiment, three different serotypes are present. In one embodiment, four different serotypes are present. In one embodiment, five different serotypes are present. In one embodiment, six different serotypes are present. In one embodiment, seven different serotypes are present. In one embodiment, eight different serotypes are present. In one embodiment, nine different serotypes are present. In one embodiment, ten different serotypes are present. In one embodiment, eleven different serotypes are present. In one embodiment, twelve different serotypes are present. In one embodiment, thirteen different serotypes are present. In one embodiment, fourteen different serotypes are present. In one embodiment, fifteen different serotypes are present. In one embodiment, sixteen different serotypes are present. In one embodiment, eighteen different serotypes are present. 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 one embodiment, there are 25 different serotypes. In one embodiment, there are 26 different serotypes. The saccharide is conjugated to the carrier protein to form the glycoconjugate described herein.

In one aspect, the composition comprises a glycoconjugate comprising a polypeptide or fragment thereof derived from E. coli; and an O antigen from at least one E. coli serogroup, the O antigen being conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen from more than one E. coli serotype, each O antigen being conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen from two different E. coli serotypes, each O antigen being conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen from three different E. coli serotypes, each O antigen being conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from four different E. coli serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from five different E. coli serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from six different E. coli serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from seven different E. coli serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from eight different E. coli serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from nine different E. coli serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from ten different E. coli serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises an O antigen from a polypeptide or fragment thereof derived from E. coli; and eleven different E. coli serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from 12 different serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from 13 different serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from 14 different serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from 15 different serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from 16 different serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from 17 different serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from 18 different serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from 19 different serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from 20 different serotypes, each of which is conjugated to a carrier protein.

In another aspect, the composition comprises an O polysaccharide derived from at least one E. coli serotype. In a preferred embodiment, the composition comprises an O polysaccharide derived from more than one E. coli serotype. For example, the composition may comprise an O polysaccharide derived from two different E. coli serotypes to 12 different E. coli serotypes. In one embodiment, the composition comprises an O polysaccharide derived from three different E. coli serotypes. In one embodiment, the composition comprises an O polysaccharide derived from four different E. coli serotypes. In one embodiment, the composition comprises an O polysaccharide derived from five different E. coli serotypes. In one embodiment, the composition comprises an O polysaccharide derived from six different E. coli serotypes. In one embodiment, the composition comprises an O polysaccharide derived from seven different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides from 8 different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides from 9 different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides from 10 different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides from 11 different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides from 12 different serotypes. In one embodiment, the composition comprises O polysaccharides from 13 different serotypes. In one embodiment, the composition comprises O polysaccharides from 14 different serotypes. In one embodiment, the composition comprises O polysaccharides from 15 different serotypes. In one embodiment, the composition comprises O polysaccharides from 16 different serotypes. In one embodiment, the composition comprises O polysaccharides from 17 different serotypes. In one embodiment, the composition comprises O polysaccharides from 18 different serotypes. In one embodiment, the composition comprises O polysaccharides from 19 different serotypes. In one embodiment, the composition comprises O polysaccharides from 20 different serotypes.

In a preferred embodiment, the composition comprises an O polysaccharide from at least one E. coli serotype, the O polysaccharide being conjugated to a carrier protein. In a preferred embodiment, the composition comprises an O polysaccharide from more than one E. coli serotype, the respective O polysaccharide being conjugated to a carrier protein. For example, the composition may comprise an O polysaccharide from two different E. coli serotypes to 12 different E. coli serotypes, the respective O polysaccharide being conjugated to a carrier protein. In one embodiment, the composition comprises an O polysaccharide from three different E. coli serotypes, the respective O polysaccharide being conjugated to a carrier protein. In one embodiment, the composition comprises an O polysaccharide from four different E. coli serotypes, the respective O polysaccharide being conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from five different E. coli serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from six different E. coli serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from seven different E. coli serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from eight different E. coli serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from nine different E. coli serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from ten different E. coli serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from 11 different E. coli serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from 12 different serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from 13 different serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from 14 different serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from 15 different serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from 16 different serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from 17 different serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from 18 different serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from 19 different serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from 20 different serotypes, each of which is conjugated to a carrier protein.

In a most preferred embodiment, the composition comprises O polysaccharides from at least one E. coli serotype, where the O polysaccharide is conjugated to a carrier protein and comprises an O antigen and a core saccharide. In a preferred embodiment, the composition comprises O polysaccharides from more than one E. coli serotype, where each O polysaccharide is conjugated to a carrier protein and comprises an O antigen and a core saccharide. For example, the composition may comprise O polysaccharides from 12 different E. coli serotypes from two different E. coli serotypes, where each O polysaccharide is conjugated to a carrier protein and comprises an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from three different E. coli serotypes, where each O polysaccharide is conjugated to a carrier protein and comprises an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from four different E. coli serotypes, each of which is conjugated to a carrier protein and comprises an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from five different E. coli serotypes, each of which is conjugated to a carrier protein and comprises an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from six different E. coli serotypes, each of which is conjugated to a carrier protein and comprises an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from seven different E. coli serotypes, each of which is conjugated to a carrier protein and comprises an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from 8 different E. coli serotypes, each of which is conjugated to a carrier protein and comprises an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from 9 different E. coli serotypes, each of which is conjugated to a carrier protein and comprises an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from 10 different E. coli serotypes, each of which is conjugated to a carrier protein and comprises an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from 11 different E. coli serotypes, each of which is conjugated to a carrier protein and comprises an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from 12 different serotypes, each of which is conjugated to a carrier protein and comprises an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from 13 different serotypes, each of which is conjugated to a carrier protein and comprises an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from 14 different serotypes, each of which is conjugated to a carrier protein and comprises an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from 15 different serotypes, each of which is conjugated to a carrier protein and comprises an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from 16 different serotypes, each of which is conjugated to a carrier protein and comprises an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from 17 different serotypes, each of which is conjugated to a carrier protein and comprises an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from 18 different serotypes, each of which is conjugated to a carrier protein and comprises an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from 19 different serotypes, each of which is conjugated to a carrier protein and comprises an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from 20 different serotypes, each of which is conjugated to a carrier protein and comprises an O antigen and a core saccharide. In a preferred embodiment, the carrier protein is CRM ₁₉₇ .

In another preferred embodiment, the composition comprises an O polysaccharide conjugated to CRM 197, the O polysaccharide comprising formula O25a, where n is at least 40, and a core saccharide. In a preferred embodiment, the composition further comprises an O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharide comprising formula O25b, where n is at least 40, and a core saccharide. In another embodiment, the composition further comprises an O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharide comprising formula O1a, where n is at least 40, and a core saccharide. In another embodiment, the composition further comprises an O polysaccharide conjugated _{to CRM 197 ,} the O polysaccharide comprising formula O2, where n is at least 40, and a core saccharide. In another embodiment, the composition further comprises an O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharide comprising formula O6, where n is at least 40, and a core saccharide.

In another embodiment, the composition further comprises an O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharide comprising formula O17, where n is at least 40, and a core sugar. In another embodiment, the composition further comprises an O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharide comprising formula O15, where n is at least 40, and a core sugar. In another embodiment, the composition further comprises an O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharide comprising formula O18A, where n is at least 40, and a core sugar. In another embodiment, the composition further comprises an O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharide comprising formula O75, where n is at least 40, and a core sugar. In another embodiment, the composition further comprises an O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharide comprising formula O4, where n is at least 40, and a core sugar. In another embodiment, the composition further comprises an O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharide comprising formula O16, where n is at least 40, and a core sugar. In another embodiment, the composition further comprises an O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharide comprising formula O13, where n is at least 40, and a core sugar. In another embodiment, the composition further comprises an O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharide comprising formula O7, where n is at least 40, and a core sugar.

In another embodiment, the composition further comprises an O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharide comprising formula O8, where n is at least 40, and a core saccharide. In another embodiment, the O polysaccharide comprises formula O8, where n is 1-20, preferably 2-5, more preferably 3. Formula O8 is shown, for example, in FIG. 10B. In another embodiment, the composition further comprises an O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharide comprising formula O9, where n is at least 40, and a core saccharide. In another embodiment, the O polysaccharide comprises formula O9, where n is 1-20, preferably 4-8, more preferably 5. Formula O9 is shown, for example, in FIG. 10B. In another embodiment, the O polysaccharide comprises formula O9a, where n is 1-20, preferably 4-8, more preferably 5. Formula O9a is shown, for example, in FIG. 10B.

In some embodiments, the O polysaccharide is selected from any one of formulas O20ab, O20ac, O52, O97, and O101, where n is 1-20, preferably 4-8, and more preferably 5. See, e.g., FIG. 10B.

As noted above, the composition may comprise any combination of a polypeptide or fragment thereof derived from E. coli; and a conjugated O polysaccharide (antigen). In one exemplary embodiment, the composition comprises a polysaccharide comprising formula O25b, a polysaccharide comprising formula O1A, a polysaccharide comprising formula O2, and a polysaccharide comprising formula O6. More specifically, such compositions comprise: (i) an O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharide comprising formula O25b, where n is at least 40, and a core saccharide; (ii) an O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharide comprising formula O1a, where n is at least 40, and a core saccharide; (iii) an O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharide comprising formula O2, where n is at least 40, and a core saccharide; and (iv) an O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharide comprising formula O6, where n is at least 40, and a core saccharide.

In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and at least one O polysaccharide derived from any E. coli serotype that is not O25a. For example, in one embodiment, the composition does not include a saccharide comprising the formula O25a. Such a composition may include, for example, an O polysaccharide comprising the formula O25b, an O polysaccharide comprising the formula O1A, an O polysaccharide comprising the formula O2, and an O polysaccharide comprising the formula O6.

In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O polysaccharides from two different E. coli serotypes, each O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharides comprising an O antigen and a core saccharide. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O polysaccharides from three different E. coli serotypes, each O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharides comprising an O antigen and a core saccharide. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O polysaccharides from four different E. coli serotypes, each O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharides comprising an O antigen and a core saccharide. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O polysaccharides from five different E. coli serotypes, each O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharides comprising an O antigen and a core saccharide. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O polysaccharides from six different E. coli serotypes, each O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharides comprising an O antigen and a core saccharide. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O polysaccharides from seven different E. coli serotypes, each O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharides comprising an O antigen and a core saccharide. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O polysaccharides from eight different E. coli serotypes, each O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharides comprising an O antigen and a core saccharide. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O polysaccharides from nine different E. coli serotypes, each O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharides comprising an O antigen and a core saccharide. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O polysaccharides from ten different E. coli serotypes, each O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharides comprising an O antigen and a core saccharide. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O polysaccharides from 11 different E. coli serotypes, each O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharides comprising an O antigen and a core saccharide. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O polysaccharides from 12 different serotypes, each O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharides comprising an O antigen and a core saccharide. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O polysaccharides from 13 different serotypes, each O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharides comprising an O antigen and a core saccharide. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O polysaccharides from 14 different serotypes, each O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharides comprising an O antigen and a core saccharide. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O polysaccharides from 15 different serotypes, each O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharides comprising an O antigen and a core saccharide. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O polysaccharides from 16 different serotypes, each O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharides comprising an O antigen and a core saccharide. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O polysaccharides from 17 different serotypes, each O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharides comprising an O antigen and a core saccharide. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O polysaccharides from 18 different serotypes, each O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharides comprising an O antigen and a core saccharide. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O polysaccharides from 19 different serotypes, each O polysaccharide conjugated to CRM ₁₉₇ , the O polysaccharides comprising an O antigen and a core saccharide. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O polysaccharides from 20 different serotypes, each O polysaccharide conjugated to CRM ₁₉₇ , wherein the O polysaccharide comprises an O antigen and a core saccharide.

In one aspect, the invention relates to a composition comprising a polypeptide or fragment thereof derived from E. coli; and a conjugate comprising a saccharide covalently attached to a carrier protein comprising formula O25b, where n is 15±2. In one aspect, the invention relates to a composition comprising a polypeptide or fragment thereof derived from E. coli; and a conjugate comprising a saccharide covalently attached to a carrier protein comprising formula O25b, where n is 17±2. In one aspect, the invention relates to a composition comprising a polypeptide or fragment thereof derived from E. coli; and a conjugate comprising a saccharide covalently attached to a carrier protein comprising formula O25b, where n is 55±2. In another aspect, the invention relates to a composition comprising a polypeptide or fragment thereof derived from E. coli; and a conjugate comprising a saccharide covalently attached to a carrier protein comprising formula O25b, where n is 51±2. In one embodiment, the saccharide further comprises an E. coli R1 core saccharide moiety. In another embodiment, the saccharide further comprises an E. coli K12 core saccharide moiety. In another embodiment, the saccharide further comprises a KDO moiety. Preferably, the carrier protein is CRM _197. In one embodiment, the conjugate is prepared by single end linkage 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 via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. Preferably, the composition further comprises a pharma- ceutically acceptable diluent.

In one embodiment, the immunogenic composition elicits IgG antibodies in humans, which are capable of binding to E. coli serotype O25B 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 an ELISA assay. Thus, a comparison of OPA activity of pre- and post-immunization sera with the immunogenic composition of the invention can be performed and compared for their response to serotype O25B to assess the possibility of increased responders. In one embodiment, the immunogenic composition elicits IgG antibodies in humans, which are capable of killing E. coli serotype O25B as determined by an in vitro opsonophagocytosis assay. In one embodiment, the immunogenic composition elicits functional antibodies in humans, said antibodies capable of killing E. coli serotype O25B, as determined by an in vitro opsonophagocytosis assay. In one embodiment, the immunogenic composition of the invention elicits a higher number of responders (i.e., in vitro opsonophagocytosis assays) to E. coli serotype O25B compared to a pre-immune population.
In one embodiment, the immunogenic composition elicits a titer of at least 1:8 against E. coli serotype O25B in at least 50% of subjects as determined by an 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 O25B in at least 60%, 70%, 80%, or at least 90% of subjects as determined by an 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 O25B in at least 60%, 70%, 80%, or at least 90% of subjects as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention increases the proportion of responders (i.e., individuals with sera with a titer of at least 1:8 against E. coli serotype O25B) compared to a pre-immune population.
In one embodiment, the immunogenic composition of the invention significantly increases the proportion of human subjects with sera having a titer of at least 1:8 as determined by OPA. In one embodiment, the immunogenic composition of the invention significantly increases the OPA titer of human subjects against E. coli serotype O25B as compared to a pre-immune population.

In one aspect, the invention relates to a composition comprising a conjugate comprising a polypeptide or fragment thereof derived from E. coli; and a saccharide covalently attached to a carrier protein, the saccharide comprising formula O1a, where n is 39±2. In another aspect, the invention relates to a composition comprising a conjugate comprising a polypeptide or fragment thereof derived from E. coli; and a saccharide covalently attached to a carrier protein, the saccharide comprising formula O1a, where n is 13±2. In one embodiment, the saccharide further comprises an E. coli R1 core saccharide moiety. In one embodiment, the saccharide further comprises a KDO moiety. Preferably, the carrier protein is CRM _197. In one embodiment, the conjugate is prepared by single end bond 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 via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. Preferably, the composition further comprises a pharma- ceutically acceptable diluent.

In one embodiment, the immunogenic composition elicits IgG antibodies in humans, which are capable of binding to E. coli serotype O1A 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 an ELISA assay. Thus, a comparison of OPA activity of pre- and post-immunization sera with the immunogenic composition of the invention can be performed and compared for their response to serotype O1A to assess the possibility of increased responders. In one embodiment, the immunogenic composition elicits IgG antibodies in humans, which are capable of killing E. coli serotype O1A as determined by an in vitro opsonophagocytosis assay. In one embodiment, the immunogenic composition elicits functional antibodies in humans, said antibodies capable of killing E. coli serotype O1A, as determined by an in vitro opsonophagocytosis assay. In one embodiment, the immunogenic composition of the invention elicits a higher number of responders (i.e., in vitro opsonophagocytosis assays) to E. coli serotype O1A compared to a pre-immune population.
In one embodiment, the immunogenic composition elicits a titer of at least 1:8 against E. coli serotype O1A in at least 50% of subjects as determined by an 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 O1A in at least 60%, 70%, 80%, or at least 90% of subjects as determined by an 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 O1A in at least 60%, 70%, 80%, or at least 90% of subjects as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention increases the proportion of responders (i.e., individuals with sera with a titer of at least 1:8 against E. coli serotype O1A) compared to a pre-immune population.
In one embodiment, the immunogenic composition of the invention significantly increases the proportion of human subjects with sera having a titer of at least 1:8 as determined by OPA. In one embodiment, the immunogenic composition of the invention significantly increases the OPA titer of human subjects against E. coli serotype O1A as compared to a pre-immune population.

In one aspect, the invention relates to a composition comprising a polypeptide or fragment thereof derived from E. coli; and a conjugate comprising a saccharide covalently attached to a carrier protein, the saccharide comprising formula O2, where n is 43±2. In another aspect, the invention relates to a composition comprising a polypeptide or fragment thereof derived from E. coli; and a conjugate comprising a saccharide covalently attached to a carrier protein, the saccharide comprising formula O2, where n is 47±2. In another aspect, the invention relates to a composition comprising a conjugate comprising a saccharide covalently attached to a carrier protein, the saccharide comprising formula O2, where n is 17±2. In another aspect, the invention relates to a composition comprising a conjugate comprising a saccharide covalently attached to a carrier protein, the saccharide comprising formula O2, where n is 18±2. In one embodiment, the saccharide further comprises an E. coli R1 core saccharide moiety. In another embodiment, the saccharide further comprises an E. coli R4 core saccharide moiety. In another embodiment, the saccharide further comprises a KDO moiety. Preferably, the carrier protein is CRM _197. In one embodiment, the conjugate is prepared by single end linkage conjugation. In one embodiment, the conjugate is prepared by reductive amination chemistry, preferably in DMSO buffer. In one embodiment, the sugar is conjugated to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. Preferably, the composition further comprises a pharma- ceutically acceptable diluent.

In one embodiment, the immunogenic composition elicits IgG antibodies in humans, which are capable of binding to E. coli serotype O2 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 an ELISA assay. Thus, a comparison of OPA activity of pre- and post-immunization sera with the immunogenic composition of the invention can be performed and compared for their response to serotype O2 to assess the possibility of increased responders. In one embodiment, the immunogenic composition elicits IgG antibodies in humans, which are capable of killing E. coli serotype O2 as determined by an in vitro opsonophagocytosis assay. In one embodiment, the immunogenic composition elicits functional antibodies in humans, said antibodies capable of killing E. coli serotype O2, as determined by an in vitro opsonophagocytosis assay. In one embodiment, the immunogenic composition of the invention elicits a greater number of responders (i.e., in vitro opsonophagocytosis assays) to E. coli serotype O2 compared to a pre-immune population.
In one embodiment, the immunogenic composition elicits a titer of at least 1:8 against E. coli serotype O2 in at least 50% of subjects as determined by an 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 O2 in at least 60%, 70%, 80%, or at least 90% of subjects as determined by an 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 O2 in at least 60%, 70%, 80%, or at least 90% of subjects as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention increases the proportion of responders (i.e., individuals with sera with a titer of at least 1:8 against E. coli serotype O2) compared to a pre-immune population.
In one embodiment, the immunogenic composition of the invention significantly increases the proportion of human subjects with sera having a titer of at least 1:8 as determined by OPA. In one embodiment, the immunogenic composition of the invention significantly increases the OPA titer of human subjects against E. coli serotype O2 as compared to a pre-immune population.

In one aspect, the invention relates to a composition comprising a polypeptide or fragment thereof derived from E. coli; and a conjugate comprising a saccharide covalently attached to a carrier protein comprising formula O6, where n is 42±2. In another aspect, the invention relates to a composition comprising a polypeptide or fragment thereof derived from E. coli; and a conjugate comprising a saccharide covalently attached to a carrier protein comprising formula O6, where n is 50±2. In another aspect, the invention relates to a composition comprising a conjugate comprising a saccharide covalently attached to a carrier protein comprising formula O6, where n is 17±2. In another aspect, the invention relates to a composition comprising a conjugate comprising a saccharide covalently attached to a carrier protein comprising formula O6, where n is 18±2. In one embodiment, the saccharide further comprises an E. coli R1 core saccharide moiety. In one embodiment, the saccharide further comprises a KDO moiety. Preferably, the carrier protein is CRM ₁₉₇ . In one embodiment, the conjugate is prepared by single end linkage conjugation. In one embodiment, the conjugate is prepared by reductive amination chemistry, preferably in DMSO buffer. In one embodiment, the sugar is conjugated to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. Preferably, the composition further comprises a pharma- ceutically acceptable diluent.

In one embodiment, the immunogenic composition elicits IgG antibodies in humans, which are capable of binding to E. coli serotype O6 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 an ELISA assay. Thus, a comparison of OPA activity of pre- and post-immunization sera with the immunogenic composition of the invention can be performed and compared for their response to serotype O6 to assess the possibility of increased responders. In one embodiment, the immunogenic composition elicits IgG antibodies in humans, which are capable of killing E. coli serotype O6 as determined by an in vitro opsonophagocytosis assay. In one embodiment, the immunogenic composition elicits functional antibodies in humans, said antibodies capable of killing E. coli serotype O6, as determined by an in vitro opsonophagocytosis assay. In one embodiment, the immunogenic composition of the invention elicits a greater number of responders (i.e., in vitro opsonophagocytosis assays) to E. coli serotype O6 compared to a pre-immune population.
In one embodiment, the immunogenic composition elicits a titer of at least 1:8 against E. coli serotype O6 in at least 50% of subjects as determined by an 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 O6 in at least 60%, 70%, 80%, or at least 90% of subjects as determined by an 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 O6 in at least 60%, 70%, 80%, or at least 90% of subjects as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention increases the proportion of responders (i.e., individuals with sera with a titer of at least 1:8 against E. coli serotype O6) compared to a pre-immune population.
In one embodiment, the immunogenic composition of the invention significantly increases the proportion of human subjects with sera having a titer of at least 1:8 as determined by OPA. In one embodiment, the immunogenic composition of the invention significantly increases the OPA titer of human subjects against E. coli serotype O6 as compared to a pre-immune population.

In one aspect, a composition comprises a conjugate comprising a polypeptide derived from E. coli or a fragment thereof; and a sugar covalently attached to a carrier protein, wherein the sugar is selected from the group consisting of Formula O1 (e.g., Formula O1A, Formula O1B, and Formula O1C), Formula O2, Formula O3, Formula O4 (e.g., Formula O4:K52 and Formula O4:K6), Formula O5 (e.g., Formula O5ab and Formula O5ac (strain 180/C3)), Formula O6 (e.g., Formula O6:K2;K13;K15 and Formula O6:K54), Formula O7, Formula O8, Formula O9, Formula O10, Formula O11, Formula O12, Formula O13, Formula O14, Formula O15, Formula O16, Formula O17, Formula O18 (e.g., Formula O18A, Formula O18ac, Formula O18), A1, O18B, and O18B1), O19, O20, O21, O22, O23 (e.g., O23A), O24, O25 (e.g., O25a and O25b), O26, O27, O28, O29, O30, O32, O33, O34, O35, O36, O37, O 38, formula O39, formula O40, formula O41, formula O42, formula O43, formula O44, formula O45 (e.g., formula O45 and formula O45rel), formula O46, formula O48, formula O49, formula O50, formula O51, formula O52, formula O53, formula O54, formula O55, formula O56, formula O57, formula O58, formula O59, formula O60, formula O61, formula O62, formula 62D ₁ , formula O63, formula O64, formula O65, formula O66, formula O68, formula O69, formula O70, formula O71, formula O73 (for example, formula O73 (stock 73-1)), formula O74, formula O75, formula O76, formula O77, formula O78, formula O79, formula O 80, formula O81, formula O82, formula O83, formula O84, formula O85, formula O86, formula O87, formula O88, formula O89, formula O90, formula O91, formula O92, formula O93, formula O95, formula O96, formula O97, formula O98, formula O99, formula O 100, Formula O101, Formula O102, Formula O103, Formula O104, Formula O105, Formula O106, Formula O107, Formula O108, Formula O109, Formula O110, Formula O111, Formula O112, Formula O113, Formula O114, Formula O115, Formula O116, formula O117, formula O118, formula O119, formula O120, formula O121, formula O123, formula O124, formula O125, formula O126, formula O127, formula O128, formula O129, formula O130, formula O131, formula O132, formula O133, formula O134, formula O135, formula O136, formula O137, formula O138, formula O139, formula O140, formula O141, formula O142, formula O143, formula O144, formula O145, formula O146, formula O147, formula O148, Formula O149, Formula O150, Formula O151, Formula O152, Formula O153, Formula O154, Formula O155, Formula O156, Formula O157, Formula O158, Formula O159, Formula O160, Formula O161, Formula O162, Formula O163, Formula O164 , Formula O165, Formula O166, Formula O167, Formula O168, Formula O169, Formula O170, Formula O171, Formula O172, Formula O173, Formula O174, Formula O175, Formula O176, Formula O177, Formula O178, Formula O179, Formula O180, Formula O181, Formula O182, Formula O183, Formula O184, Formula O185, Formula O186, and Formula O187, where n is an integer from 1 to 100, preferably 31 to 90. In one embodiment, the saccharide further comprises an E. coli R1 core saccharide moiety. In one embodiment, the saccharide further comprises an E. coli R2 core saccharide moiety. In one embodiment, the saccharide further comprises an E. coli R3 core saccharide moiety. In another embodiment, the saccharide further comprises an E. coli R4 core saccharide moiety. In one embodiment, the saccharide further comprises an E. coli K12 core saccharide moiety. In another embodiment, the saccharide further comprises a KDO moiety. Preferably, the carrier protein is CRM _197. In one embodiment, the conjugate is prepared by single end linkage 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 via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. Preferably, the composition further comprises a pharma- ceutically acceptable diluent. In one embodiment, the composition further comprises at least 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, up to a maximum of 30 additional conjugates, each conjugate comprising a sugar covalently attached to a carrier protein, wherein the sugar comprises a structure selected from any one of the above formulas.

A. Sugars In one embodiment, sugars are produced by expression (not necessarily overexpression) of a different Wzz protein (eg, WzzB) to control the size of the sugar.

As used herein, the term "sugar" refers to a single sugar moiety or monosaccharide unit and to combinations of two or more single sugar moieties or monosaccharide units covalently linked to form disaccharides, oligosaccharides, and polysaccharides. Sugars may be linear or branched.

In one embodiment, the sugar is produced in a recombinant Gram-negative bacterium. In one embodiment, the sugar is produced in a recombinant E. coli cell. In one embodiment, the sugar is produced in a recombinant Salmonella cell. Exemplary bacteria include E. coli O25K5H1, E. coli BD559, E. coli GAR2831, E. coli GAR865, E. coli GAR868, E. coli GAR869, E. coli GAR872, E. coli GAR878, E. coli GAR896, E. coli GAR1902, E. coli O25a ETC NR-5, E. coli O157:H7:K-, Salmonella enterica Examples of suitable pathogens include E. enterica serovar Typhimurium strain LT2, E. coli GAR2401, Salmonella enterica serovar Enteritidis CVD1943, Salmonella enterica serovar Typhimurium strain CVD1925, Salmonella enterica serovar Paratyphi A CVD1902, and Shigella flexneri CVD1208S. In one embodiment, the bacterium is not E. coli GAR2401. This genetic approach to sugar production allows for efficient production of O polysaccharide and O antigen molecules as vaccine components.

The term "wzz protein" as used herein refers to chain length determinant polypeptides such as, for example, wzzB, wzz, wzz _SF , wzz _ST , fepE, wzz _fepE , wzz1 and wzz2. GenBank accession numbers for 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 gene sequences are U39305 and U39306, respectively. Additional GenBank accession numbers for exemplary wzz gene sequences are: NP_459581 for Salmonella enterica subsp. enterica serovar Typhimurium strain LT2 FepE; AIG66859 for E. coli O157:H7 strain EDL933 FepE; NP_461024 for Salmonella enterica subsp. enterica serovar Typhimurium strain LT2 WzzB; NP_461024 for E. coli K-12 substrain MG1655 For WzzB it is NP_416531, and for E. coli K-12 substrain MG1655 FepE it is NP_415119. In a preferred embodiment, the wzz family protein is any one of wzzB, wzz, wzz _SF , wzz _ST , fepE, wzz _fepE , wzz1 and wzz2, most preferably wzzB, more preferably fepE.

Exemplary wzzB sequences include those set forth in SEQ ID NOs: 30-34. Exemplary FepE sequences include those set forth in SEQ ID NOs: 35-39.

In some embodiments, modified sugars (modified relative to the corresponding wild-type sugars) can be produced in Gram-negative bacteria by expressing (not necessarily overexpressing) a wzz family protein (e.g., fepE) from a Gram-negative bacterium and/or by switching off (i.e., suppressing, deleting, removing) a second wzz gene (e.g., wzzB) to generate high molecular weight sugars, such as lipopolysaccharides, containing intermediate or long O-antigen chains. For example, modified sugars can be produced by expressing (not necessarily overexpressing) wzz2 and switching off wzz1. Or, in another method, modified sugars can be produced by expressing (not necessarily overexpressing) wzzfepE and switching off wzzB. In another embodiment, modified sugars can be produced by expressing (not necessarily overexpressing) wzzB but switching off wzzfepE. In another embodiment, modified sugars can 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 sugar is produced by expressing a wzz family protein having an amino acid sequence having 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 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. 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, and SEQ ID NO:34. In some embodiments, the sugar is produced by expressing a protein having an amino acid sequence having at least 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the 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 produce high molecular weight saccharides containing intermediate or long O antigen chains with at least one, two, three, four, or five increased repeat units compared to the corresponding wild-type O polysaccharide. In one aspect, the invention relates to saccharides produced by gram-negative bacteria expressing (but not necessarily overexpressing) a wzz family protein (e.g., wzzB) in culture to produce high molecular weight saccharides containing intermediate or long O antigen chains with at least one, two, three, four, or five increased repeat units compared to the corresponding wild-type O antigen from the gram-negative bacterium. See the description of O polysaccharides and O antigens below for further exemplary saccharides with an increased number of repeat units compared to the corresponding wild-type saccharide. Desirable chain lengths are those that provide improved or maximal immunogenicity in the context of a given vaccine construct.

In another embodiment, the saccharide comprises any one of the formulas selected from Table 1, wherein the number of repeating units in the saccharide, n, is 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, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 109, 109, 110, 111, 112 , 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 repeating units. Preferably, the saccharide comprises 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 repeating units compared to the corresponding wild-type O polysaccharide. See, for example, Table 24. Methods for determining saccharide length are known in the art. Such methods include nuclear magnetic resonance, mass spectrometry, and size exclusion chromatography, as described in Example 13.

In a preferred embodiment, the invention relates to sugars produced in recombinant E. coli host cells in which the endogenous wzz O-antigen length regulator gene (e.g., wzzB) has been deleted and replaced with a (second) wzz gene (e.g., Salmonella fepE) from a gram-negative bacterium heterologous to the recombinant E. coli host cell, producing high molecular weight sugars, such as lipopolysaccharides, containing intermediate or long O-antigen chains. In some embodiments, the recombinant E. coli host cell contains a wzz gene from Salmonella, preferably Salmonella enterica.

In one embodiment, the host cell comprises a heterologous gene for a wzz family protein as a stably maintained plasmid vector. In another embodiment, the host cell comprises a heterologous gene for a wzz family protein as an integrated gene in the chromosomal DNA of the host cell. Methods for stable expression of plasmid vectors in E. coli host cells and for integration of heterologous genes into the chromosome of E. coli host cells are known in the art. In one embodiment, the host cell comprises a heterologous gene for an O antigen as a stably maintained plasmid vector. In another embodiment, the host cell comprises a heterologous gene for an O antigen as an integrated gene in the chromosomal DNA of the host cell. Methods for stable expression of plasmid vectors in E. coli and Salmonella host cells are known in the art. Methods for integration of heterologous genes into the chromosome of E. coli and Salmonella host cells are known in the art.

In one aspect, the recombinant host cells are cultured in a medium that includes a carbon source. Carbon sources for culturing E. coli are known in the art. Exemplary carbon sources include, but are not limited to, sugar alcohols, polyols, aldol sugars, or keto sugars, such as 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, such as mannitol, inositol, sorbose, glycerol, sorbitol, lactose, and arabinose, as a carbon source. All carbon sources may be added to the medium before the start of the culture or may be added in portions or continuously during the culture.

Exemplary culture media for recombinant host cells contain elements selected _{from any one of KH2PO4, K2HPO4 , ( NH4 ) 2SO4 , sodium} citrate _{, Na2SO4} , _aspartic acid, _glucose , _MgSO4 , _{FeSO4-7H2O , Na2MoO4-2H2O} , _H3BO3 , _CoCl2-6H2O , _CuCl2-2H2O , _{MnCl2-4H2O , ZnCl2 , and CaCl2-2H2O .} Preferably, the medium contains _KH2PO4 , _K2HPO4 , ( _{NH4 ) 2SO4 ,} sodium _citrate , _Na2SO4 , _aspartic acid, _{glucose , MgSO4} , _{FeSO4-7H2O , Na2MoO4-2H2O} , _H3BO3 , _CoCl2-6H2O , _{CuCl2-2H2O , MnCl2-4H2O , ZnCl2 and CaCl2-2H2O .}

As used herein, a medium may be solid or liquid, synthetic (i.e., artificial) or natural, and may contain sufficient nutrients for the culture of recombinant host cells. Preferably, the medium is a liquid medium.

In some embodiments, the medium may further comprise suitable inorganic salts. In some embodiments, the medium may further comprise micronutrients. In some embodiments, the medium may further comprise growth factors. In some embodiments, the medium may further comprise an additional carbon source. In some embodiments, the medium may further comprise suitable inorganic salts, micronutrients, growth factors, and additional carbon sources. Suitable inorganic salts, micronutrients, growth factors, and supplemental carbon sources for culturing E. coli are known in the art.

In some embodiments, the medium may optionally contain additional components such as peptone, N-Z amine, soy enzymatic hydrolysate, additional yeast extract, malt extract, supplemental carbon source and various vitamins. In some embodiments, the medium does not optionally contain such additional components such as peptone, N-Z amine, soy enzymatic hydrolysate, additional yeast extract, malt extract, supplemental carbon source and various vitamins.

Illustrative examples of suitable supplemental carbon sources include, but are not limited to, glucose, fructose, mannitol, starch or other carbohydrates such as starch hydrolysates, cellulose hydrolysates, 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 comprises a nitrogen source. Suitable nitrogen sources 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; nitrates or nitrites, and other nitrogen-containing materials, including amino acids, meat extracts, peptones, fish meal, fish hydrolysates, corn infusions, casein hydrolysates, soybean meal hydrolysates, yeast extracts, dry yeast, ethanol-yeast distillates, soybean flour, cottonseed flour, and the like, in pure or unrefined preparations.

In some embodiments, the medium includes inorganic salts. 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 phosphate.

In some embodiments, the medium comprises suitable growth factors. Illustrative examples of suitable micronutrients, growth factors, etc. include, but are not limited to, coenzyme A, pantothenic acid, pyridoxine-HCl, biotin, thiamine, riboflavin, flavin mononucleotide, flavin adenine dinucleotide, DL-6,8-thioctic acid, folic acid, vitamin B ₁₂ , 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, etc., either as pure or partially purified compounds or present in natural sources. The amounts can be empirically determined by one of skill in the art according to methods and techniques known in the art.

In another embodiment, the modified sugars described herein (as compared to the corresponding wild-type sugars) are synthetically produced, e.g., in vitro. Synthetic production or synthesis of sugars can facilitate avoidance of costly and time-intensive production processes. In one embodiment, sugars are synthesized, e.g., by using a sequential glycosylation strategy or a combination of a sequential glycosylation strategy and a [3+2] block synthesis strategy from a suitably protected monosaccharide intermediate. For example, thiol glycosides and glycosyl trichloroacetimidate derivatives can be used as glycosyl donors in glycosylation. In one embodiment, sugars synthesized in vitro have the same structure as sugars produced by recombinant means, such as by engineering the wzz family proteins described above.

The sugars produced (by recombinant or synthetic means) can be, for example, from the following E. coli serotypes: O1 (e.g., O1A, O1B, and O1C), O2, O3, O4 (e.g., O4:K52 and O4:K6), O5 (e.g., O5ab and O5ac (strain 180/C3)), O6 (e.g., O6:K2; K13; K15 and O6:K54), O7, O8, O9, O10, O11, O12, O13, O14, O15, O16, O17, O18 (e.g., O18A, O18ac, O18A1, O18B, O18C, O18D, O18E, O18F, O18F, O18G, O18H ... , and O18B1), O19, O20, O21, O22, O23 (e.g., O23A), O24, O25 (e.g., O25a and O25b), O26, O27, O28, O29, O30, O32, O33, O34, O35, O36, O37, O38, O39, O40, O41, O42, O43, O44, O45 (e.g., O45 and O45rel), O46, O48, O49, O50, O51, O52, O53, O54, O55, O56, O57, O58, O59, O60, O61, O62, 62D ₁ , O63, O64, O65, O66, O68, O69, O70, O71, O73 (e.g., O73 (73-1 strain)), O74, O75, O76, O77, O78, O79, O80, O81, O82, O83, O84, O85, O86, O87, O88, O89, O90, O91, O92, O93, O95, O96, O97, O98, O99, O100, O101, O102, O103, O104, O105, O106, O107, O108, O109, O110, 0111, O112, O113, O114, O 115, O116, O117, O118, O119, O120, O121, O123, O124, O125, O126, O127, O128, O129, O130, O13 1, O132, O133, O134, O135, O136, O137, O138, O139, O140, O141, O142, O143, O144, O145, O146 , O147, O148, O149, O150, O151, O152, O153, O154, O155, O156, O157, O158, O159, O160, O161, O The present invention includes structures derived from any E. coli serotype, including any one of O162, O163, O164, O165, O166, O167, O168, O169, O170, O171, O172, O173, O174, O175, O176, O177, O178, O179, O180, O181, O182, O183, O184, O185, O186, and O187.

Individual polysaccharides are typically purified (enriched with respect to the amount of polysaccharide-protein conjugates) by methods known in the art, such as, for example, dialysis, concentration operations, diafiltration operations, tangential flow filtration, sedimentation, elution, centrifugation, sedimentation, ultrafiltration, depth filtration, and/or column chromatography (ion exchange chromatography, multimode ion exchange chromatography, DEAE, and hydrophobic interaction chromatography). Preferably, the polysaccharides are purified by methods that include tangential flow filtration.

The purified polysaccharides can be activated (e.g., chemically activated) to render them capable of reacting (e.g., directly to a carrier protein or via a linker such as an eTEC spacer) and then incorporated into the glycoconjugates of the invention, as further described herein.

In one preferred embodiment, the saccharide of the invention is derived from an E. coli serotype that is serotype O25a. In another preferred embodiment, the serotype is O25b. In another preferred embodiment, the serotype is O1A. In another preferred embodiment, the serotype is O2. In another preferred embodiment, the serotype is O6. In another preferred embodiment, the serotype is O17. In another preferred embodiment, the serotype is O15. In another preferred embodiment, the serotype is O18A. In another preferred embodiment, the serotype is O75. In another preferred embodiment, the serotype is O4. In another preferred embodiment, the serotype is O16. In another preferred embodiment, the serotype is O13. In another preferred embodiment, the serotype is O7. In another preferred embodiment, the serotype is O8. In another preferred embodiment, the serotype is O9.

As used herein, reference to any of the above serotypes refers to a serotype that includes a repeating unit structure (O unit as described below) that is known in the art and unique to the corresponding serotype. For example, the term "O25a" serotype (also known in the art as serotype "O25") refers to a serotype that includes the formula O25 shown in Table 1. As another example, the term "O25b" serotype refers to a serotype that includes the formula O25b shown in Table 1.

As used herein, serotypes are generally referred to herein unless otherwise specified, e.g., the term "O18" generally refers to include O18A, O18ac, O18A1, O18B, and O18B1.

As used herein, the term "O1" generally refers to the inclusion of species of formulae that include the generic name "O1" in the formula name set forth in Table 1, such as any one of formulae O1A, O1A1, O1B, and O1C, respectively, as set forth in Table 1. Thus, "O1 serotype" generally refers to serotypes that include any one of formulae O1A, O1A1, O1B, and O1C.

As used herein, the term "O6" generally refers to species of formulae that include the generic name "O6" in the formula name set forth in Table 1, such as any one of the formulae O6:K2; K13; K15; and O6:K54, respectively, as set forth in Table 1. Thus, "O6 serotype" generally refers to serotypes that include any one of the formulae O6:K2; K13; K15; and O6:K54.

Other examples of terms generally referring to species of formulae that include a generic term in the formula name listed in Table 1 include "O4", "O5", "O18", and "O45".

As used herein, the term "O2" refers to the formula O2 shown in Table 1. The term "O2 O antigen" refers to a saccharide that includes the formula O2 shown in Table 1.

As used herein, references to O antigens from the above serotypes refer to saccharides that include the formula labeled with the corresponding serotype name. For example, the term "O25B O antigen" refers to a saccharide that includes the formula O25B as shown in Table 1.

As another example, the term "O1 O antigen" generally refers to saccharides that include formulas that include the term "O1," such as formula O1A, formula O1A1, formula O1B, and formula O1C, each of which is shown in Table 1.

As another example, the term "O6 O antigen" generally refers to saccharides that include formulas that include the term "O6," such as formula O6:K2; formula O6:K13; formula O6:K15, and formula O6:K54, each of which is shown in Table 1.

B. O Polysaccharides As used herein, the term "O polysaccharide" refers to any structure that contains an O antigen, provided that the structure does not contain whole cells or lipid A. For example, in one embodiment, the O polysaccharide comprises lipopolysaccharide that does not have lipid A attached. The step of removing lipid A is known in the art and includes, for example, heat treatment with the addition of acid. An exemplary process includes treatment with 1% acetic acid at 100° C. for 90 minutes. This process is combined with a process to isolate the removed lipid A. An exemplary process for isolating lipid A includes ultracentrifugation.

In one embodiment, O polysaccharide refers to a structure consisting of O antigen, in which case O polysaccharide is synonymous with the term O antigen. In one preferred embodiment, O polysaccharide refers to a structure comprising repeating units of O antigen, without the core saccharide. Thus, in one embodiment, O polysaccharide does not comprise an E. coli R1 core portion. In another embodiment, O polysaccharide does not comprise an E. coli R2 core portion. In another embodiment, O polysaccharide does not comprise an E. coli R3 core portion. In another embodiment, O polysaccharide does not comprise an E. coli R4 core portion. In another embodiment, O polysaccharide does not comprise an E. coli K12 core portion. In another preferred embodiment, O polysaccharide refers to a structure comprising an O antigen and a core saccharide. In another embodiment, O polysaccharide refers to a structure comprising an O antigen, a core saccharide, and a KDO portion.

Methods for purifying O polysaccharide, including the core oligosaccharide, from LPS are known in the art. For example, after purification of LPS, the purified LPS can be hydrolyzed by heating in 1% (v/v) acetic acid at 100° C. for 90 minutes, followed by ultracentrifugation at 142,000×g for 5 hours at 4° C. The supernatant containing the O polysaccharide is lyophilized and stored at 4° C. In certain embodiments, deletion of capsule synthesis genes to allow simple purification of O polysaccharide is described.

The O polysaccharide can be isolated by methods including, but not limited to, mild acid hydrolysis to remove lipid A from the LPS. Other embodiments may include the use of hydrazine as an agent for the preparation of the O polysaccharide. Preparation of the LPS can be accomplished by methods known in the art.

In certain embodiments, O polysaccharides purified from wild-type, modified, or attenuated Gram-negative bacterial strains expressing (but not necessarily overexpressing) a Wzz protein (e.g., wzzB) are provided for use in conjugate vaccines. In preferred embodiments, O polysaccharide chains are purified from Gram-negative bacterial strains expressing (but not necessarily overexpressing) a wzz protein for use as a vaccine antigen as a conjugate or complexed vaccine.

In one embodiment, the O polysaccharide is 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, The O polysaccharide has a molecular weight that is increased by 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 times. In a preferred embodiment, the O polysaccharide has a molecular weight that is increased by at least 1 fold and at most 5 fold compared to the corresponding wild-type O polysaccharide. In another embodiment, the O polysaccharide has a molecular weight that is increased by at least two-fold and at most four-fold compared to the corresponding wild-type O polysaccharide. The increase in molecular weight of the O polysaccharide compared to the corresponding wild-type O polysaccharide is preferably associated with an increase in the number of O antigen repeat units. In one embodiment, the increase in molecular weight of the O polysaccharide is due to a wzz family protein.

In one embodiment, the O polysaccharide has about 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, 5 The O polysaccharide of the present invention has an increased molecular weight of 0, 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. In one embodiment, the O polysaccharide of the present invention has an increased molecular weight of at least 1 kDa and at most 200 kDa compared to the corresponding wild-type O polysaccharide. In one embodiment, the molecular weight is increased by at least 5 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 12 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 15 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 18 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 20 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 21 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 22 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 30 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 1 kDa and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 5 kDa and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 12 kDa and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 15 kDa and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 20 kDa and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 1 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 5 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 12 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 15 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 18 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 20 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 30 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 90 kDa. In one embodiment, the molecular weight is increased by at least 12 kDa and at most 85 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 70 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 60 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 50 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 49 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 48 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 47 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 46 kDa. In one embodiment, the molecular weight is increased by at least 20 kDa and at most 45 kDa. In one embodiment, the molecular weight is increased by at least 20 kDa and at most 44 kDa. In one embodiment, the molecular weight is increased by at least 20 kDa and at most 43 kDa. In one embodiment, the molecular weight is increased by at least 20 kDa and at most 42 kDa. In one embodiment, the molecular weight is increased by at least 20 kDa and at most 41 kDa. Such an increase in the molecular weight of the O polysaccharide compared to the corresponding wild-type O polysaccharide is preferably associated with an increase in the number of O antigen repeat units. In one embodiment, the increase in the molecular weight of the O polysaccharide is due to a wzz family protein. See, for example, Table 21.

In another embodiment, the O polysaccharide comprises any one of the formulas selected from Table 1, wherein the number of repeating units n in the O polysaccharide is 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 repeating units. Preferably, the saccharide comprises 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 repeating units compared to the corresponding wild-type O polysaccharide. See, e.g., Table 21.

C. O Antigens O antigens are part of the lipopolysaccharide (LPS) in the outer membrane of gram-negative bacteria. O antigens are found on the cell surface and are variable cellular components. The variability of O antigens provides the basis for serotyping of gram-negative bacteria. The current E. coli serotyping scheme includes O polysaccharides 1-181.

O antigens contain oligosaccharide repeating units (O units), whose wild-type structures usually contain 2-8 residues from a wide range of sugars. An exemplary E. coli O antigen O unit is shown in Table 1; see also Figures 9A-9C and 10A-10B.

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 an embodiment, the saccharide may comprise a structure selected from any one of formula O1a, formula O2, formula O6, formula O8, formula O9a, formula O9, formula O20ab, formula O20ac, formula O25b, formula O52, formula O97, and formula O101. In a further embodiment, the saccharide may comprise a structure selected from any one of formula O1a, formula O2, formula O6, and formula O25b.

In one embodiment, the saccharide of the present invention may be an oligosaccharide. Oligosaccharides have a small number of repeating units (typically 5-15 repeating units) and are typically derived synthetically or by hydrolysis of a polysaccharide. In such an embodiment, the saccharide may comprise a structure selected from any one of formulas O1a, O2, O6, O8, O9a, O9, O20ab, O20ac, O25b, O52, O97, and O101. In a further embodiment, the saccharide may comprise a structure selected from any one of formulas O1a, O2, O6, and O25b.

Preferably, all saccharides of the invention and in the immunogenic compositions of the invention are polysaccharides. High molecular weight polysaccharides are capable of inducing certain antibody immune responses due to epitopes present on their antigenic surface. Isolation and purification of high molecular weight polysaccharides is preferably contemplated for use in the conjugates, compositions and methods of the invention.

In some embodiments, the number of repeating O units in each individual O-antigen polymer (and thus the length and molecular weight of the polymer chain) depends on the wzz chain length regulator, an inner membrane protein. Different wzz proteins result in different ranges of mode lengths (4 to over 100 repeat units). The term "mode length" refers to the number of repeating O units. Gram-negative bacteria often have two different Wzz proteins that result in two different OAg mode chain lengths, one long and one short. Expression (not necessarily overexpression) of a wzz family protein (e.g., wzzB) in Gram-negative bacteria can allow for manipulation of O-antigen length to shift or bias bacterial production of a certain length range of O-antigens and enhance production of high yields of high molecular weight lipopolysaccharide. In one embodiment, a "short" mode length as used herein refers to a small number of repeating O units, e.g., 1 to 20. In one embodiment, a "long" mode length as used herein refers to a number of repeating O units greater than 20, up to 40. In one embodiment, a "very long" mode length as used herein refers to greater than 40 repeating O units.

In one embodiment, the sugar produced has an increase of at least 10 repeat units, 15 repeat units, 20 repeat units, 25 repeat units, 30 repeat units, 35 repeat units, 40 repeat units, 45 repeat units, 50 repeat units, 55 repeat units, 60 repeat units, 65 repeat units, 70 repeat units, 75 repeat units, 80 repeat units, 85 repeat units, 90 repeat units, 95 repeat units, or 100 repeat units compared to the corresponding wild-type O polysaccharide.

In another embodiment, the saccharide of the present invention has a 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 increases in repeat units. Preferably, the saccharide comprises 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 repeating units compared to the corresponding wild-type O polysaccharide. See, for example, Table 21. Methods for determining the length of the saccharide are known in the art. Such methods include nuclear magnetic resonance, mass spectrometry, and size exclusion chromatography, as described in Example 13.

Methods for determining the number of repeating units in a saccharide are also known in the art. For example, the number of repeating units (or "n" in the formula) can be calculated by dividing the molecular weight of the polysaccharide (not including the molecular weight of the core sugar or KDO residue) by the molecular weight of the repeating unit (i.e., the molecular weight of the corresponding structure in the formula shown in Table 1, which can theoretically be calculated as the sum of the molecular weights of each monosaccharide in the formula). The molecular weight of each monosaccharide in the formula is known in the art. For example, the molecular weight of the repeating unit of formula O25b is about 862 Da. For example, the molecular weight of the repeating unit of formula O1a is about 845 Da. For example, the molecular weight of the repeating unit of formula O2 is about 829 Da. For example, the molecular weight of the repeating unit of formula O6 is about 893 Da. When determining the number of repeating units in a conjugate, the molecular weight of the carrier protein and the protein:polysaccharide ratio are taken into account. As defined herein, "n" refers to the number of repeating units in a polysaccharide molecule (shown in parentheses in Table 1). As is known in the art, in biopolymers, the repeating structures may incorporate regions of imperfect repeats, e.g., missing branches. Furthermore, it is known in the art that polysaccharides isolated and purified from natural sources, such as bacteria, may be heterogeneous in size and branching. In such cases, n may represent the average or median value for n of the molecules in the population.

In one embodiment, the O polysaccharide has an increase of at least one repeating unit of the O antigen compared to the corresponding wild-type O polysaccharide. The repeating units of the O antigen are shown in Table 1. In one embodiment, the O polysaccharide has an increase of at least one repeating unit of the O antigen compared to the corresponding wild-type O polysaccharide. The repeating units of the O antigen are shown in Table 1. In one embodiment, the O polysaccharide has an increase of at least one repeating unit of the O antigen compared to the corresponding wild-type O polysaccharide. 2, 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 repeating units. Preferably, the saccharide has a total of at least 3 and at most 80 repeating units. In another embodiment, the O polysaccharide has a nucleotide sequence similar to that of the corresponding wild-type O polysaccharide, but is more similar to that of the corresponding wild-type O polysaccharide, and is 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 unit increases.

In one embodiment, the saccharide is an O antigen formula (e.g., a formula such as those shown in Table 1 (see also Figures 9A-9C and Figures 10A-10B)) in which n is 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 and O antigens that are integers of at most 200, 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 can be combined with any maximum value 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 comprises an O antigen, where 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 O 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 O antigen formulas is at least 10 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 20 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 25 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 30 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 40 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 50 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 30 and at most 90. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 85. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 70. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 60. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 50. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 49. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 48. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 47. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 46. In one embodiment, n in any one of the O antigen formulas is at least 36 and at most 45. In one embodiment, n in any one of the O antigen formulas is at least 37 and at most 44. In one embodiment, n in any one of the O antigen formulas is at least 38 and at most 43. In one embodiment, n in any one of the O antigen formulas is at least 39 and at most 42. In one embodiment, n in any one of the O antigen formulas is at least 39 and at most 41.

For example, in one embodiment, n in the sugar 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 and 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 and 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 sugar structure can be determined by methods and means known in the 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 prior to conjugation has a molecular weight of 5 kDa to 400 kDa. In other such embodiments, the saccharide has a molecular weight of 10 kDa to 400 kDa; 5 kDa to 400 kDa; 5 kDa to 300 kDa; 5 kDa to 200 kDa; 5 kDa to 150 kDa; 10 kDa to 100 kDa; 10 kDa to 75 kDa; 10 kDa to 60 kDa; 10 kDa to 40 kDa; 10 kDa to 100 kDa; 10 kDa to 20 kDa. 00 kDa; 15 kDa to 150 kDa; 12 kDa to 120 kDa; 12 kDa to 75 kDa; 12 kDa to 50 kDa; 12 to 60 kDa; 35 kDa to 75 kDa; 40 kDa to 60 kDa; 35 kDa to 60 kDa; 20 kDa to 60 kDa; 12 kDa to 20 kDa; or 20 kDa to 50 kDa. In further embodiments, the polysaccharide has a molecular weight of 7 kDa to 15 kDa; 8 kDa to 16 kDa; 9 kDa to 25 kDa; 10 kDa to 100 kDa; 10 kDa to 60 kDa; 10 kDa to 70 kDa; 10 kDa to 160 kDa; 15 kDa to 600 kDa; 20 kDa to 1000 kDa; 20 kDa to 600 kDa; 20 kDa to 400 kDa; 30 kDa to 1,000 kDa; 30 kDa to 60 kDa; 30 kDa to 50 kDa or 5 kDa to 60 kDa. Any whole integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

As used herein, the term "molecular weight" of a polysaccharide or carrier protein-polysaccharide conjugate refers to the molecular weight calculated by size exclusion chromatography (SEC) in combination with a multi-angle laser light scattering detector (MALLS).

Polysaccharides may become slightly reduced in size during normal purification procedures. Additionally, as described herein, polysaccharides may be subjected to sizing techniques prior to conjugation. Mechanical or chemical sizing may be used. Chemical hydrolysis may be performed using acetic acid. Mechanical sizing may be performed using high pressure homogenization shear. The molecular weight ranges above refer to purified polysaccharides prior to conjugation (e.g., prior to activation).

D. Core Oligosaccharide The core oligosaccharide is located between lipid A and the O-antigen outer region in wild-type E. coli LPS. More specifically, the core oligosaccharide is the portion of the polysaccharide that contains the linkage between the O-antigen and lipid A in wild-type E. coli. This linkage involves a ketosidic bond between the hemiketal function of the innermost 3-deoxy-d-manno-octa-2-ulosonic acid (KDO) residue and the hydroxyl group of the GlcNAc residue of lipid A. The core oligosaccharide region shows a high degree of similarity among wild-type E. coli strains. It usually contains 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. KDO residues have the following formula: KDO:

を含む。

Includes.

The outer region of the core oligosaccharide shows greater 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 the general structure of the carbohydrate backbone of the outer core oligosaccharides of the five known chemotypes. HepII is the last residue of the inner core oligosaccharide. The outer core oligosaccharides all share a structural theme, with a (hexose) ₃ carbohydrate backbone and two side chain residues, although the order of the hexoses in the backbone and the nature, position, and linkage of the side chain residues can all vary. The structures of the R1 and R4 outer core oligosaccharides are very similar, differing only in a single β-linked residue.

The core oligosaccharide of wild-type E. coli is classified in the art into five different chemotypes based on the structure of the distal oligosaccharide: E. coli R1, E. coli R2, E. coli R3, E. coli R4, and E. coli K12.

In a preferred embodiment, the compositions described herein comprise a saccharide conjugate comprising a core oligosaccharide in which the O polysaccharide is bound to an O antigen. In one embodiment, the composition induces an immune response against at least 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 comprise a saccharide conjugate in which the O polysaccharide does not comprise a core oligosaccharide bound to an O antigen. In one embodiment, such a composition induces an immune response against at least one of the core E. coli chemotypes: E. coli R1, E. coli R2, E. coli R3, E. coli R4, and E. coli K12, even though the saccharide conjugate with the O polysaccharide does not comprise a core oligosaccharide.

E. coli serotypes can be characterized according to one of five chemotypes. Table 2 lists exemplary serotypes characterized according to chemotype. The bolded serotypes represent the serotypes most commonly associated with the indicated core chemotype. Thus, in a preferred embodiment, the composition induces an immune response against at least one of the core E. coli chemotypes: E. coli R1, E. coli R2, E. coli R3, E. coli R4, and E. coli K12, including an immune response against any one of the respective corresponding E. coli serotypes.

In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having an R1 chemotype, e.g., selected from saccharides having formula O25a, formula O6, formula O2, formula O1, formula O75, formula O4, formula O16, formula O8, formula O18, formula O9, formula O13, formula O20, formula O21, formula O91, and formula O163, where n is 1-100. In some embodiments, the saccharide in the composition further comprises an E. coli R1 core moiety, e.g., as shown in FIG. 24.

In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having an R1 chemotype, e.g., selected from saccharides having formula O25a, formula O6, formula O2, formula O1, formula O75, formula O4, formula O16, formula O18, formula O13, formula O20, formula O21, formula O91, and formula O163, where n is 1-100, preferably 31-100, preferably 31-90, more preferably 35-90, and most preferably 35-65. In some embodiments, the saccharide in the composition further comprises an E. coli R1 core moiety in the saccharide.

In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having an R2 chemotype, e.g., selected from saccharides having formula O21, formula O44, formula O11, formula O89, formula O162, and formula O9, where n is 1-100, preferably 31-100, preferably 31-90, more preferably 35-90, and most preferably 35-65. In some embodiments, the saccharide in the composition further comprises an E. coli R2 core moiety, e.g., as shown in FIG. 24.

In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having an R3 chemotype, e.g., selected from saccharides having formula O25b, O15, O153, O21, O17, O11, O159, O22, O86, and O93, where n is 1-100, preferably 31-100, preferably 31-90, more preferably 35-90, and most preferably 35-65. In some embodiments, the saccharide in the composition further comprises an E. coli R3 core moiety, e.g., as shown in FIG. 24.

In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having an R4 chemotype, e.g., selected from saccharides having formula O2, formula O1, formula O86, formula O7, formula O102, formula O160, and formula O166, where n is 1-100, preferably 31-100, preferably 31-90, more preferably 35-90, and most preferably 35-65. In some embodiments, the saccharide in the composition further comprises an E. coli R4 core moiety, e.g., as shown in FIG. 24.

In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having a K-12 chemotype (e.g., selected from a saccharide having the formula O25b and a saccharide having the formula O16, where n is 1-100, preferably 31-100, preferably 31-90, more preferably 35-90, most preferably 35-65). In some embodiments, the saccharide in the composition further comprises an E. coli K-12 core portion, e.g., as shown in FIG. 24.

In some embodiments, the saccharide comprises a core saccharide. Thus, in one embodiment, the O polysaccharide further comprises an E. coli R1 core portion. In another embodiment, the O polysaccharide further comprises an E. coli R2 core portion. In another embodiment, the O polysaccharide further comprises an E. coli R3 core portion. In another embodiment, the O polysaccharide further comprises an E. coli R4 core portion. In another embodiment, the O polysaccharide further comprises an E. coli K12 core portion.

In some embodiments, the saccharide does not include a core saccharide. Thus, in one embodiment, the O polysaccharide does not include an E. coli R1 core portion. In another embodiment, the O polysaccharide does not include an E. coli R2 core portion. In another embodiment, the O polysaccharide does not include an E. coli R3 core portion. In another embodiment, the O polysaccharide does not include an E. coli R4 core portion. In another embodiment, the O polysaccharide does not include an E. coli K12 core portion.

E. Conjugated O Antigens Chemical conjugation of O antigens, or preferably O polysaccharides, to protein carriers can improve the immunogenicity of O antigens or O polysaccharides. However, variability in polymer size is a real challenge for production. In commercial use, the size of the sugar can affect compatibility with various conjugation synthesis strategies, uniformity of the product, and immunogenicity of the conjugate. Controlling the expression of Wzz family protein chain length regulators by engineering the O antigen synthesis pathway allows the production of O antigen chains of desired length in various Gram-negative bacterial strains, including E. coli.

In one embodiment, the purified sugars are chemically activated to produce activated sugars capable of reacting with a carrier protein. Once activated, each sugar is separately conjugated to a carrier protein to form a conjugate, i.e., a glycoconjugate. As used herein, the term "glycoconjugate" refers to a sugar covalently attached to a carrier protein. In one embodiment, the sugar is directly attached to the carrier protein. In another embodiment, the sugar is attached to the carrier protein via a spacer/linker.

The conjugates can be prepared by a scheme in which a carrier is attached to the O antigen at one or more sites along the O antigen, or by a scheme in which at least one residue of the core oligosaccharide is activated.

In one embodiment, each sugar is conjugated to the same carrier protein.

If the protein carrier is the same for two or more sugars in the composition, the sugars can be conjugated to the same molecule of carrier protein (e.g., a carrier molecule to which two or more different sugars are conjugated).

In a preferred embodiment, the sugars are each individually conjugated to different molecules of the protein carrier (each molecule of the protein carrier has only one type of sugar conjugated to it). In said embodiment, the sugars are said to be individually conjugated to the carrier protein.

Chemical activation of the saccharide and subsequent conjugation to the carrier protein can be accomplished by the activation and conjugation methods disclosed herein. After conjugation of the polysaccharide to the carrier protein, the saccharide conjugate is purified (enriched with respect to the amount of polysaccharide-protein conjugate) by various techniques. These techniques include concentration/diafiltration operations, precipitation/elution, column chromatography, and depth filtration. After the individual saccharide conjugates are purified, they are mixed to formulate the immunogenic compositions of the invention.

Activation. The present invention further relates to activated polysaccharides produced from any of the embodiments described herein, where the polysaccharide is activated with a chemical reagent that generates reactive groups for conjugation to a linker or carrier protein. In some embodiments, the saccharides of the present invention are activated prior to conjugation to a carrier protein. In some embodiments, the degree of activation does not substantially 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 affect the degree of conjugation as measured by the number of modified lysine residues in a carrier protein, such as CRM ₁₉₇ (determined by amino acid analysis). For example, in some embodiments, the degree of activation does not significantly increase the number of modified lysine residues in a carrier protein (determined by amino acid analysis) by 3-fold compared to the number of modified lysine residues in a carrier protein of a conjugate with a reference polysaccharide at the same activation degree. 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 reduce the optimal saccharide/protein ratio.

In some embodiments, the activated sugar has a percentage of activation in which the number of moles of thiol per sugar repeat unit of the activated sugar is 1-100%, such as 2-80%, 2-50%, 3-30%, and 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%, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, 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 can be combined with any maximum value to define a range.

In one embodiment, the polysaccharide is activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester, and the activated polysaccharide is then coupled to amino groups on a carrier protein, preferably CRM ₁₉₇ or tetanus toxoid, either directly or through a spacer (linker) group.

For example, the spacer may be cystamine or cysteamine to obtain a thiolated polysaccharide that can be coupled to the carrier by a thioether bond obtained after reaction with a maleimide-activated carrier protein (e.g., using N-[γ-maleimidobutyryloxy]succinimide ester (GMBS)) or a haloacetylated carrier protein (e.g., 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]propionate (SBAP)). In one embodiment, a cyanate ester (which may be generated by CDAP chemistry) is coupled with hexanediamine or adipic dihydrazide (ADH) and the amino-derivatized sugar is conjugated to a carrier protein (e.g., CRM ₁₉₇ ) via carboxyl groups on the protein carrier using carbodiimide (e.g., EDAC or EDC) chemistry.

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 that can be formed by reaction of a free hydroxyl group of a sugar with CDI, followed by reaction with the protein to form a carbamate bond. 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 of the CDI carbamate intermediate to an amino group on the protein (CDI chemistry).

Molecular weight. In some embodiments, the sugar conjugate comprises a sugar having a molecular weight of 10 kDa to 2,000 kDa. In other embodiments, the sugar has a molecular weight of 50 kDa to 1,000 kDa. In other embodiments, the sugar has a molecular weight of 70 kDa to 900 kDa. In other embodiments, the sugar has a molecular weight of 100 kDa to 800 kDa. In other embodiments, the sugar has a molecular weight of 200 kDa to 600 kDa. In further embodiments, the saccharide is between 100 kDa and 1000 kDa; 100 kDa and 900 kDa; 100 kDa and 800 kDa; 100 kDa and 700 kDa; 100 kDa and 600 kDa; 100 kDa and 500 kDa; 100 kDa and 400 kDa; 100 kDa and 300 kDa; 150 kDa and 1,000 kDa; 150 kDa and 900 kDa; 150 kDa and 8 00kDa; 150kDa to 700kDa; 150kDa to 600kDa; 150kDa to 500kDa; 150kDa to 400kDa; 150kDa to 300kDa; 200kDa to 1,000kDa; 200kDa to 900kDa; 200kDa to 800kDa; 200kDa to 700kDa; 200kDa to 600kDa; 200kDa to 500kDa; 200k Da ~ 400kDa; 200kDa ~ 300kDa; 250kDa ~ 1,000kDa; 250kDa ~ 900kDa; 250kDa ~ 800kDa; 250kDa ~ 700kDa; 2 50kDa - 600kDa; 250kDa - 500kDa; 250kDa - 400kDa; 250kDa - 350kDa; 300kDa - 1,000kDa; 300kDa - 900kDa a; 300 kDa to 800 kDa; 300 kDa to 700 kDa; 300 kDa to 600 kDa; 300 kDa to 500 kDa; 300 kDa to 400 kDa; 400 kDa to 1,000 kDa; 400 kDa to 900 kDa; 400 kDa to 800 kDa; 400 kDa to 700 kDa; 400 kDa to 600 kDa; 500 kDa to 600 kDa. In one embodiment, sugar conjugates having such molecular weights are produced by single-end conjugation. In another embodiment, sugar conjugates having such molecular weights are produced by reductive amination chemistry (RAC) prepared in an aqueous buffer. Any whole integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

In some embodiments, the sugar conjugates of the present invention have a molecular weight of 400 kDa to 15,000 kDa; 500 kDa to 10,000 kDa; 2,000 kDa to 10,000 kDa; 3,000 kDa to 8,000 kDa; or 3,000 kDa to 5,000 kDa. In other embodiments, the sugar conjugates have a molecular weight of 500 kDa to 10,000 kDa. In other embodiments, the sugar conjugates have a molecular weight of 1,000 kDa to 8,000 kDa. In yet other embodiments, the sugar conjugates have a molecular weight of 2,000 kDa to 8,000 kDa or 3,000 kDa to 7,000 kDa. In further embodiments, the glycoconjugates of the invention are of a molecular weight of 200 kDa to 20,000 kDa; 200 kDa to 15,000 kDa; 200 kDa to 10,000 kDa; 200 kDa to 7,500 kDa; 200 kDa to 5,000 kDa; 200 kDa to 3,000 kDa; 200 kDa to 1,000 kDa; 500 kDa to 20,000 kDa; 500 kDa to 15,000 kDa; 500 kDa to 12,500 kDa; 500 kDa to 10,000 kDa. Da; 500kDa to 7,500kDa; 500kDa to 6,000kDa; 500kDa to 5,000kDa; 500kDa to 4,000kDa; 500kDa to 3,000kDa; 500kDa to 2,000kDa; 500 kDa ~ 1,500kDa; 500kDa ~ 1,000kDa; 750kDa ~ 20,000kDa; 750kDa ~ 15,000kDa; 750kDa ~ 12,500kDa; 750kDa ~ 10,000kDa; 750kDa ~7,500kDa;750kDa~6,000kDa;750kDa~5,000kDa;750kDa~4,000kDa;750kDa~3,000kDa;750kDa~2,000kDa;750kDa~1,500 kDa; 1,000kDa to 15,000kDa; 1,000kDa to 12,500kDa; 1,000kDa to 10,000kDa; 1,000kDa to 7,500kDa; 1,000kDa to 6,000kDa; 1,000k Da to 5,000 kDa; 1,000 kDa to 4,000 kDa; 1,000 kDa to 2,500 kDa; 2,000 kDa to 15,000 kDa; 2,000 kDa to 12,500 kDa; 2,000 kDa to 10,000 kDa; 2,000 kDa to 7,500 kDa; 2,000 kDa to 6,000 kDa; 2,000 kDa to 5,000 kDa; 2,000 kDa to 4,000 kDa; or 2,000 kDa to 3,000 kDa. In one embodiment, glycoconjugates having such molecular weights are produced by eTEC conjugation as described herein. In another embodiment, glycoconjugates having such molecular weights are produced by reductive amination chemistry (RAC). In another embodiment, glycoconjugates having such molecular weights are produced by reductive amination chemistry (RAC) prepared in DMSO.

In further embodiments, the glycoconjugates of the present invention have a molecular weight of 1,000 kDa to 20,000 kDa; 1,000 kDa to 15,000 kDa; 2,000 kDa to 10,000 kDa; 2000 kDa to 7,500 kDa; 2,000 kDa to 5,000 kDa; 3,000 kDa to 20,000 kDa; 3,000 kDa to 15,000 kDa; 3,000 kDa to 12,500 kDa; 4,000 kDa to 10,000 kDa; 4,000 kDa to 7,500 kDa; 4,000 kDa to 6,000 kDa; or 5,000 kDa to 7,000 kDa. In one embodiment, glycoconjugates having such molecular weights are produced by reductive amination chemistry (RAC). In another embodiment, glycoconjugates having such molecular weights are produced by reductive amination chemistry (RAC) prepared in DMSO. In another embodiment, glycoconjugates having such molecular weights are produced by eTEC conjugation as described herein.

In further embodiments, the glycoconjugates of the invention have a molecular weight of 5,000 kDa to 20,000 kDa; 5,000 kDa to 15,000 kDa; 5,000 kDa to 10,000 kDa; 5,000 kDa to 7,500 kDa; 6,000 kDa to 20,000 kDa; 6,000 kDa to 15,000 kDa; 6,000 kDa to 12,500 kDa; 6,000 kDa to 10,000 kDa or 6,000 kDa to 7,500 kDa.

The molecular weight of the glycoconjugates can be measured by SEC-MALLS. Any whole integer within any of the above ranges is contemplated as an embodiment of the present disclosure. The glycoconjugates of the present invention can also be characterized by the sugar to carrier protein ratio (w/w). In some embodiments, the ratio of polysaccharide to carrier protein (w/w) in the glycoconjugate is 0.5 to 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 0.5-2.0, 0.5-1.5, 0.8-1.2, 0.5-1.0, 1.0-1.5, or 1.0-2.0. In further embodiments, the saccharide to carrier protein ratio (w/w) is 0.8-1.2. In preferred embodiments, the polysaccharide to carrier protein ratio in the conjugate is 0.9-1.1. In some such embodiments, the carrier protein is CRM ₁₉₇ .

Glycoconjugates can also be characterized by their molecular size distribution (K _d ). Size exclusion chromatography media (CL-4B) can be used to determine the relative molecular size distribution of the conjugates. Size exclusion chromatography (SEC) is used in a gravity-fed column to profile the molecular size distribution of the conjugates. Large molecules that are excluded from small pores in the media elute more quickly than small molecules. A fraction collector is used to collect the column eluate. Fractions are tested colorimetrically by sugar assay. For the determination of K _d , the column is calibrated to establish the fraction at which molecules are completely excluded (V ₀ ), (K _d =0), and the fractions that show maximum retention (V _i ), (K _d =1). The fraction at which a particular sample characteristic is reached (V _e ) is related to K _d by the formula K _d =(V _e -V ₀ )/(V _i -V ₀ ).

Free saccharides. The saccharide conjugates and immunogenic compositions of the present invention may include free saccharides that are not covalently conjugated to a carrier protein but are nevertheless present in the saccharide conjugate composition. The free saccharides may be non-covalently associated with the saccharide conjugate (i.e., non-covalently bound, adsorbed, or entrapped within or with it). In a preferred embodiment, the saccharide conjugate comprises at most 50%, 45%, 40%, 35%, 30%, 25%, 20%, or 15% free polysaccharides relative to the total amount of polysaccharides. In a preferred embodiment, the saccharide conjugate comprises less than about 25% free polysaccharides relative to the total amount of polysaccharides. In a preferred embodiment, the saccharide conjugate comprises at most about 20% free polysaccharides relative to the total amount of polysaccharides. In a preferred embodiment, the saccharide conjugate comprises at most about 15% free polysaccharides relative to the total amount of polysaccharides. In another preferred embodiment, the saccharide conjugate 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% free polysaccharide relative to the total amount of polysaccharide. In a preferred embodiment, the saccharide conjugate comprises less than about 8% free polysaccharide relative to the total amount of polysaccharide. In a preferred embodiment, the saccharide conjugate comprises at most about 6% free polysaccharide relative to the total amount of polysaccharide. In a preferred embodiment, the saccharide conjugate comprises at most about 5% free polysaccharide relative 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 bonds. In other embodiments, the conjugate comprises at least one covalent bond between the carrier protein and the saccharide every 5-10 saccharide repeating units; every 2-7 saccharide repeating units; every 3-8 saccharide repeating units; every 4-9 saccharide repeating units; every 6-11 saccharide repeating units; every 7-12 saccharide repeating units; every 8-13 saccharide repeating units; every 9-14 saccharide repeating units; every 10-15 saccharide repeating units; every 2-6 saccharide repeating units; every 3-7 saccharide repeating units; every 4-8 saccharide repeating units; every 6-10 saccharide repeating units; every 7-11 saccharide repeating units; every 8-12 saccharide repeating units; every 9-13 saccharide repeating units; every 10-14 saccharide repeating units; every 10-20 saccharide repeating units; every 4-25 saccharide repeating units, or every 2-25 saccharide repeating units. In a frequent embodiment, the carrier protein is CRM ₁₉₇ . In another embodiment, at least one bond between the carrier protein and the saccharide is present 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 integer within any of the above ranges is contemplated as an embodiment of the present 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 the extent of conjugated lysine (degree of conjugation). Evidence for lysine modification of the carrier protein due to covalent attachment to the polysaccharide can be obtained by amino acid analysis using routine methods known to those skilled 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 material. In preferred embodiments, the degree of conjugation of the glycoconjugates of the invention is 2-15, 2-13, 2-10, 2-8, 2-6, 2-5, 2-4, 3-15, 3-13, 3-10, 3-8, 3-6, 3-5, 3-4, 5-15, 5-10, 8-15, 8-12, 10-15 or 10-12. In one embodiment, the degree of conjugation of the glycoconjugates of the invention is about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14 or about 15. In a preferred embodiment, the degree of conjugation of the glycoconjugates of the invention is 4 to 7. In some such embodiments, the carrier protein is CRM ₁₉₇ .

The frequency of attachment of glycans to lysines on the carrier protein is another parameter for characterizing the glycoconjugates of the invention. For example, in some embodiments, at least one covalent bond between the carrier protein and the polysaccharide occurs for every 4 saccharide repeat units of the polysaccharide. In another embodiment, a covalent bond between the carrier protein and the polysaccharide occurs at least once for every 10 saccharide repeat units of the polysaccharide. In another embodiment, a covalent bond between the carrier protein and the polysaccharide occurs at least once for every 15 saccharide repeat units of the polysaccharide. In another embodiment, a covalent bond between the carrier protein and the polysaccharide occurs at least once for every 25 saccharide repeat units of the polysaccharide. O-acetylation. In some embodiments, the saccharides of the invention are O-acetylated. In some embodiments, the sugar conjugate comprises a sugar having a degree of O-acetylation of 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 75-100%, 80-100%, 90-100%, 50-90%, 60-90%, 70-90%, or 80-90%. In other embodiments, the degree of O-acetylation is 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, or about 100%. Percent O-acetylation refers to the percentage of a given sugar relative to 100% (each repeat unit is fully acetylated relative to its acetylated structure).

In some embodiments, the sugar conjugate is prepared by reductive amination. In some embodiments, the sugar conjugate is a single end-linked conjugated sugar, where the sugar is covalently attached directly to the carrier protein. In some embodiments, the sugar conjugate is covalently attached to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer.

Reductive amination. In one embodiment, the sugar is conjugated to the carrier protein by reductive amination (e.g., as described in U.S. Patent Application Publication Nos. 2006/0228380, 2007/0231340, 2007/0184071 and 2007/0184072, WO 2006/110381, WO 2008/079653, and WO 2008/143709).

Reductive amination involves (1) oxidation of the sugar, and (2) reduction of the activated sugar and carrier protein to form a conjugate. Prior to oxidation, the sugar may be hydrolyzed. Mechanical or chemical hydrolysis may be used. Chemical hydrolysis may be performed using acetic acid.

The oxidation step may include reaction with periodate. As used herein, the term "periodate" refers to both periodate and periodic acid. The term also includes both metaperiodate (IO ₄ ⁻ ) and orthoperiodate (IO ₆ ⁵⁻ ) and various salts of periodic acid (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 (NaIO ₄ ). 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 a piperidine-N-oxy or pyrrolidine-N-oxy compound, in the presence of an oxidizing agent to selectively oxidize the primary hydroxyl. In the reaction, the actual oxidizing agent is an N-oxoammonium salt in the catalytic cycle. In one aspect, the stable nitroxyl or nitroxide radical compound is a piperidine-N-oxy or pyrrolidine-N-oxy compound. In one aspect, the stable nitroxyl or nitroxide radical compound bears a TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) or PROXYL (2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety. In one aspect, the stable nitroxyl radical compound is TEMPO or a derivative thereof. In one aspect, the oxidizing agent is a molecule bearing an N-halo moiety. In one embodiment, the oxidizing agent is selected from any one of N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione, dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione, diiodoisocyanuric acid, and 1,3,5-triiodo-1,3,5-triazinane-2,4,6-trione. Preferably, the oxidizing agent is N-chlorosuccinimide.

After the sugar oxidation step, the sugar is said to be activated and is referred to hereinafter as "activated". The activated sugar and carrier protein can be lyophilized (freeze-dried) independently (separate lyophilization) or together (co-lyophilization). In one embodiment, the activated sugar and carrier protein are co-lyophilized. In another embodiment, the activated polysaccharide and carrier protein are lyophilized independently.

In one embodiment, lyophilization is performed 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 in the conjugation process is the reduction of the activated sugar and carrier protein with a reducing agent (so-called reductive amination) to form the conjugate. Suitable reducing agents include cyanoborohydrides such as sodium cyanoborohydride, sodium triacetoxyborohydride or sodium or zinc borohydride in the presence of a Bronsted or Lewis acid, 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 some embodiments, the reduction reaction is carried out in an 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 pH 6.0-8.5, 7.0-8.0, or 7.0-7.5), and in other embodiments, the reaction is carried out in an aprotic solvent. In some embodiments, the reduction reaction is carried out in DMSO (dimethyl sulfoxide) or DMF (dimethylformamide) solvent. DMSO or DMF solvent can be used to reconstitute the lyophilized activated sugar and carrier protein.

At the end of the reduction reaction, there may be unreacted aldehyde groups remaining in the conjugate, which can be capped using a suitable capping agent. In one embodiment, the capping agent is sodium borohydride (NaBH ₄ ). After conjugation (reduction reaction and, if necessary, capping), the glycoconjugate can be purified (enriched in terms of the amount of polysaccharide-protein conjugate) by various techniques known to those skilled in the art. These techniques include dialysis, concentration/diafiltration operations, tangential flow filtration sedimentation/elution, column chromatography (DEAE or hydrophobic interaction chromatography), and depth filtration. The glycoconjugate can be purified by diafiltration and/or ion exchange chromatography and/or size exclusion chromatography. In an embodiment, the glycoconjugate is purified by diafiltration or ion exchange chromatography or size exclusion chromatography. In one embodiment, the glycoconjugate is sterile filtered.

In a preferred embodiment, a glycoconjugate derived from an E. coli serotype selected from any one of O25B, O1, O2, and O6 is prepared by reductive amination. In a preferred embodiment, a glycoconjugate derived from an E. coli serotype selected from any one of O25B, O1, O2, and O6 is prepared by reductive amination.

In one aspect, the present invention relates to a carrier protein, e.g.

［式中、ｎは、１以上の任意の整数である］
によって表される、式Ｏ２５Ｂの糖に連結されているＣＲＭ_１９７を含むコンジュゲートに関する。好ましい実施形態では、ｎは、少なくとも３１、３２、３３、３４、３５、３６、３７、３８、３９、４０、および最大でも２００、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、または５０の整数である。任意の最小値と任意の最大値とを組み合わせて、範囲を定義することができる。例示的な範囲としては、例えば、少なくとも１～最大でも１０００；少なくとも１０～最大でも５００；および少なくとも２０～最大でも８０が挙げられる。１つの好ましい実施形態では、ｎは少なくとも３１～最大でも９０、より好ましくは４０～９０、最も好ましくは６０～８５である。

[In the formula, n is any integer equal to or greater than 1]
In a preferred embodiment, n is an integer of at least 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, and at most 200, 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 can be combined with any maximum value to define a range. Exemplary ranges include, for example, at least 1 and at most 1000; at least 10 and at most 500; and at least 20 and at most 80. In one preferred embodiment, n is at least 31 and at most 90, more preferably 40-90, and most preferably 60-85.

In another aspect, the invention relates to a conjugate comprising a carrier protein, e.g., CRM 197, linked to a saccharide having any one of the following structures shown in Table 1 (see also Figures 9A-9C and Figures _10A -10B), where n is an integer greater than or equal to 1:

Without being bound by theory or mechanism, it is believed that in some embodiments, a stable conjugate requires a level of antigen modification that balances preservation of the structural integrity of the antigen's key immunogenic epitopes.

Activation and Formation of Aldehydes. In some embodiments, the sugars of the present invention are activated, resulting in the formation of aldehydes. In such embodiments in which the sugars are activated, the percentage (%) of activation (or degree of oxidation (DO)) (see, e.g., Example 31) refers to the moles of sugar repeating units per mole of aldehyde of the activated polysaccharide. For example, in some embodiments, the sugars are activated by periodate oxidation of vicinal diols on the repeating units of the polysaccharide, resulting in the formation of aldehydes. Varying the molar equivalent (meq) of sodium periodate to the sugar repeating units and the temperature during oxidation results in varying levels of degree of oxidation (DO).

Sugar and aldehyde concentrations are typically determined by colorimetric assays. An alternative reagent is a TEMPO (2,2,6,6-tetramethylpiperidine 1-oxyl radical)-N-chlorosuccinimide (NCS) mixture, which results in the formation of aldehydes from primary alcohol groups.

In some embodiments, the activated sugar has a degree of oxidation that is 1-100, such as, for example, 2-80, 2-50, 3-30, and 4-25 moles of sugar repeating units per mole of aldehyde of the activated sugar. 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, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, or 90 or more, 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 can be combined to define a range. The value of the degree of oxidation can be expressed as a percentage (%) of activation. For example, in one embodiment, a DO value of 10 refers to 1 activated saccharide repeat unit out of a total of 10 saccharide repeat units in the activated saccharide, in which case a DO value of 10 can be expressed as 10% activation.

In some embodiments, the conjugates prepared by reductive amination chemistry include a carrier protein and a saccharide, the saccharide being selected from the group consisting of Formula O1 (e.g., Formula O1A, Formula O1B, and Formula O1C), Formula O2, Formula O3, Formula O4 (e.g., Formula O4:K52 and Formula O4:K6), Formula O5 (e.g., Formula O5ab and Formula O5ac (strain 180/C3)), Formula O6 (e.g., Formula O6:K2;K13;K15 and Formula O6:K54), Formula O7, Formula O8, Formula O9, Formula O10, Formula O11, Formula O12, Formula O13, Formula O14, Formula O15, Formula O16, Formula O17, Formula O18 (e.g., Formula O18A, Formula O18ac, Formula O18A1, Formula O18B, and Formula O18C), Formula O18B1), Formula O19, Formula O20, Formula O21, Formula O22, Formula O23 (e.g., Formula O23A), Formula O24, Formula O25 (e.g., Formula O25a and Formula O25b), Formula O26, Formula O27, Formula O28, Formula O29, Formula O30, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37, Formula O38, Formula O3 9, Formula O40, Formula O41, Formula O42, Formula O43, Formula O44, Formula O45 (e.g., Formula O45 and Formula O45rel), Formula O46, Formula O48, Formula O49, Formula O50, Formula O51, Formula O52, Formula O53, Formula O54, Formula O55, Formula O56, Formula O57, Formula O58, Formula O59, Formula O60, Formula O61, Formula O62, Formula O62D ₁ , formula O63, formula O64, formula O65, formula O66, formula O68, formula O69, formula O70, formula O71, formula O73 (e.g., formula O73 (strain 73-1)), formula O74, formula O75, formula O76, formula O77, formula O78, formula O79, formula O80, formula O81, formula O82, formula O83, formula O84, formula O85, formula O86, formula O87, formula O88, formula O89, formula O90, formula O91, formula O92, formula O93, formula O95, formula O96, formula O97, formula O98, Formula O99, Formula O100, Formula O101, Formula O102, Formula O103, Formula O104, Formula O105, Formula O106, Formula O107, Formula O108, Formula O109, Formula O110, Formula O111, Formula O112, Formula O113, Formula O 114, Formula O115, Formula O116, Formula O117, Formula O118, Formula O119, Formula O120, Formula O121, Formula O123, Formula O124, Formula O125, Formula O126, Formula O127, Formula O128, Formula O129, Formula O13 0, Formula O131, Formula O132, Formula O133, Formula O134, Formula O135, Formula O136, Formula O137, Formula O138, Formula O139, Formula O140, Formula O141, Formula O142, Formula O143, Formula O144, Formula O145 , formula O146, formula O147, formula O148, formula O149, formula O150, formula O151, formula O152, formula O153, formula O154, formula O155, formula O156, formula O157, formula O158, formula O159, formula O160, formula Formula O161, Formula O162, Formula O163, Formula O164, Formula O165, Formula O166, Formula O167, Formula O168, Formula O169, Formula O170, Formula O171, Formula O172, Formula O173, Formula O174, Formula O175, Formula O176, Formula O177, Formula O178, Formula O179, Formula O180, Formula O181, Formula O182, Formula O183, Formula O184, Formula O185, Formula O186, and Formula O187. In some embodiments, the sugar in the conjugate comprises a formula where n is an integer between 1 and 1000, between 5 and 1000, preferably between 31 and 100, more preferably between 35 and 90, and most preferably between 35 and 65.

Single-end linked conjugates. In some embodiments, the conjugates are single-end linked conjugated saccharides in which the saccharide is covalently linked to the carrier protein at one end of the saccharide. In some embodiments, the single-end linked conjugated polysaccharide has a terminal saccharide. For example, a conjugate is single-end linked when one end of the polysaccharide (the terminal sugar residue) is covalently linked to the carrier protein. In some embodiments, a conjugate is single-end linked when the terminal sugar residue of the polysaccharide is covalently linked to the carrier protein via a linker. Such linkers may include, for example, a cystamine linker (A1), a 3,3'-dithiobis(propanoic acid 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 sugar is conjugated to the carrier protein via a 3-deoxy-d-manno-octa-2-ulosonic acid (KDO) residue to form a single-end linked conjugate. See, e.g., 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 of a protein (e.g., a carrier protein) and an antigen, e.g., an O antigen (e.g., O25B), prepared in a host cell background, where the host cell machinery binds the antigen to the protein (e.g., N-linkage). Glycoconjugates include bioconjugates prepared by means that do not require preparation of the conjugate in a host cell, e.g., conjugation by chemical linkage of the protein with the sugar, as well as saccharide antigen (e.g., oligosaccharide and polysaccharide)-protein conjugates.

Thiol-activated sugars. In some embodiments, the sugars of the present invention are thiol-activated. In such embodiments in which the sugar is thiol-activated, the percentage (%) of activation refers to the moles of thiol per sugar repeat unit of the activated polysaccharide. Sugar and thiol concentrations are typically determined by Ellman's assay for quantification of sulfhydryls. For example, in some embodiments, the sugar comprises 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 sugar is covalently attached to the carrier protein via a bivalent heterobifunctional linker (also referred to herein as a "spacer"). The linker preferably provides a thioether bond between the sugar and the carrier protein, resulting in a sugar conjugate referred to herein as a "thioether sugar conjugate." 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 comprises a carrier protein and a saccharide, wherein the saccharide is selected from the group consisting of Formula O1 (e.g., Formula O1A, Formula O1B, and Formula O1C), Formula O2, Formula O3, Formula O4 (e.g., Formula O4:K52 and Formula O4:K6), Formula O5 (e.g., Formula O5ab and Formula O5ac (strain 180/C3)), Formula O6 (e.g., Formula O6:K2;K13;K15 and Formula O6:K54), Formula O7, Formula O8, Formula O9, Formula O10, Formula O11, Formula O12, Formula O13, Formula O14, Formula O15, Formula O16, Formula O17, Formula O18 (e.g., Formula O18A, Formula O18ac, Formula O18A1, Formula O18B, and Formula O18B1), ), Formula O19, Formula O20, Formula O21, Formula O22, Formula O23 (e.g., Formula O23A), Formula O24, Formula O25 (e.g., Formula O25a and Formula O25b), Formula O26, Formula O27, Formula O28, Formula O29, Formula O30, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37, Formula O38, Formula O39, Formula O40, formula O41, formula O42, formula O43, formula O44, formula O45 (e.g., formula O45 and formula O45rel), formula O46, formula O48, formula O49, formula O50, formula O51, formula O52, formula O53, formula O54, formula O55, formula O56, formula O57, formula O58, formula O59, formula O60, formula O61, formula O62, formula 62D ₁ , formula O63, formula O64, formula O65, formula O66, formula O68, formula O69, formula O70, formula O71, formula O73 (e.g., formula O73 (strain 73-1)), formula O74, formula O75, formula O76, formula O77, formula O78, formula O79, formula O80, formula O81, formula O82, formula O83, formula O84, formula O85, formula O86, formula O87, formula O88, formula O89, formula O90, formula O91, formula O92, formula O93, formula O95, formula O96, formula O97, formula O98, Formula O99, Formula O100, Formula O101, Formula O102, Formula O103, Formula O104, Formula O105, Formula O106, Formula O107, Formula O108, Formula O109, Formula O110, Formula O111, Formula O112, Formula O113, Formula O 114, Formula O115, Formula O116, Formula O117, Formula O118, Formula O119, Formula O120, Formula O121, Formula O123, Formula O124, Formula O125, Formula O126, Formula O127, Formula O128, Formula O129, Formula O13 0, Formula O131, Formula O132, Formula O133, Formula O134, Formula O135, Formula O136, Formula O137, Formula O138, Formula O139, Formula O140, Formula O141, Formula O142, Formula O143, Formula O144, Formula O145 , formula O146, formula O147, formula O148, formula O149, formula O150, formula O151, formula O152, formula O153, formula O154, formula O155, formula O156, formula O157, formula O158, formula O159, formula O160, formula Formula O161, Formula O162, Formula O163, Formula O164, Formula O165, Formula O166, Formula O167, Formula O168, Formula O169, Formula O170, Formula O171, Formula O172, Formula O173, Formula O174, Formula O175, Formula O176, Formula O177, Formula O178, Formula O179, Formula O180, Formula O181, Formula O182, Formula O183, Formula O184, Formula O185, Formula O186, and Formula O187. In some embodiments, the sugar in the conjugate comprises a formula where n is an integer between 1 and 1000, between 5 and 1000, preferably between 31 and 100, more preferably between 35 and 90, and most preferably between 35 and 65.

For example, in one embodiment, the single-end linked conjugate includes a carrier protein and a sugar having a structure selected from formula O8, formula O9a, formula O9, formula O20ab, formula O20ac, formula O52, formula O97, and formula O101, where n is an integer from 1 to 10.

F. eTEC Conjugates In one aspect, the invention generally relates to glycoconjugates comprising saccharides derived from Escherichia coli (E. coli) as described above covalently conjugated to a carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer (e.g., as described in U.S. Pat. No. 9,517,274 and International Patent Publication WO2014027302, which are incorporated by reference in their entireties), immunogenic compositions comprising such glycoconjugates, and methods for the preparation and use of such glycoconjugates and immunogenic compositions. The glycoconjugates comprise a saccharide covalently conjugated to a carrier protein via one or more eTEC spacers, where the saccharide is covalently conjugated to the eTEC spacer by a carbamate bond and the carrier protein is covalently conjugated to the eTEC spacer by an amide bond. The eTEC spacer contains seven linear atoms (ie, --C(O)NH( _CH.sub.2 ) _{.sub.2SCH.sub.2C} (O)-- ₎ and provides stable thioether and amide bonds between the sugar and the carrier protein.

The eTEC-binding sugar conjugate of the present invention is represented by the general formula (I):

（式中、ｅＴＥＣスペーサーを含む原子は中央の囲みに含まれる）
によって表すことができる。

(wherein the atoms comprising the eTEC spacer are included in the central box)
It can be expressed as:

In the sugar conjugate of the present invention, the sugar may be a polysaccharide or an oligosaccharide.

The carrier protein to be incorporated in the glycoconjugates of the present invention is selected from the group of carrier proteins generally suitable for such purposes, as further described herein or as known to those of skill in the art. In a particular embodiment, the carrier protein is CRM ₁₉₇ .

In another aspect, the present invention provides a method for making a sugar conjugate comprising a sugar as described herein conjugated to a carrier protein via an eTEC spacer, the method comprising the steps of: a) reacting the sugar with a carbonic acid derivative in an organic solvent to produce an activated sugar; b) reacting the activated sugar with cystamine or cysteamine, or a salt thereof, to produce a thiolated sugar; c) reacting the thiolated sugar with a reducing agent to produce an activated thiolated sugar comprising one or more free sulfhydryl residues; d) reacting the activated thiolated sugar with one or more α-haloacetate derivatives to produce an activated thiolated sugar comprising one or more free sulfhydryl residues; and an activated carrier protein containing an amide group to produce a thiolated sugar-carrier protein conjugate; and e) reacting the thiolated sugar-carrier protein conjugate with (i) a first capping reagent capable of capping unconjugated α-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 sugar, thereby producing an eTEC-linked sugar conjugate.

In a common embodiment, the carbonate derivative is 1,1'-carbonyl-di-(1,2,4-triazole) (CDT) or 1,1'-carbonyldiimidazole (CDI). Preferably, the carbonate derivative is CDT and the organic solvent is a polar aprotic solvent such as dimethylsulfoxide (DMSO). In a preferred embodiment, the thiolated sugar is produced by reaction of an activated sugar with a bifunctional symmetric thioalkylamine reagent, cystamine or a salt thereof. Alternatively, the thiolated sugar can be formed by reaction of an activated sugar with cysteamine or a salt thereof. The eTEC-linked sugar conjugates produced by the methods of the present invention can be represented by the general formula (I):

In a common embodiment, the first capping reagent is N-acetyl-L-cysteine, which reacts with unconjugated α-haloacetamide groups on lysine residues of the carrier protein to form S-carboxymethylcysteine (CMC) residues that are covalently attached to the activated lysine residues via a thioether bond.

In other embodiments, the second capping reagent is iodoacetamide (IAA), which reacts with the unconjugated free sulfhydryl groups of the activated thiolated sugar to provide a capped thioacetamide. Step e) often involves capping with both the first and second capping reagents. In certain embodiments, step e) involves 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, after reaction with the first and/or second capping reagent, reaction with a reducing agent, e.g., DTT, TCEP, or mercaptoethanol.

The eTEC-linked sugar conjugates and immunogenic compositions of the invention may contain free sulfhydryl residues. In some examples, the activated thiolated sugars formed by the methods provided herein contain multiple free sulfhydryl residues, some of which may not undergo covalent conjugation to the carrier protein during the conjugation step. Such remaining free sulfhydryl residues are capped by reaction with an thiol-reactive capping reagent, e.g., iodoacetamide (IAA), to cap the potentially reactive functional groups. Other thiol-reactive capping reagents, e.g., maleimide-containing reagents, and the like, are also contemplated.

Furthermore, the eTEC-binding glycoconjugates and immunogenic compositions of the present invention may contain residual unconjugated carrier protein and may contain activated carrier protein that has been modified during the capping process step.

In some embodiments, step d) further comprises providing an activated carrier protein that includes one or more α-haloacetamide groups prior to reacting the activated thiolated sugar with the activated carrier protein. In a common embodiment, the activated carrier protein includes one or more α-bromoacetamide groups.

In another aspect, the present invention provides an eTEC-linked glycoconjugate comprising a sugar as described herein conjugated to a carrier protein via an eTEC spacer produced according to any of the methods disclosed herein.

In some embodiments, the carrier protein is CRM ₁₉₇ , and the covalent bond between CRM ₁₉₇ and the polysaccharide via the eTEC spacer occurs at least once per 4, 10, 15, or 25 saccharide repeat units of the polysaccharide.

For each of the aspects of the invention, and in particular for certain embodiments of the methods and compositions described herein, the eTEC-binding sugar conjugate comprises a sugar described herein, such as a sugar 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 the immunogenic composition comprises an eTEC-binding saccharide conjugate comprising a saccharide described herein. In some embodiments, the saccharide is derived from E. coli.

In some embodiments, the eTEC-binding glycoconjugate comprises a carrier protein and a glycoconjugate of Formula O1 (e.g., Formula O1A, Formula O1B, and Formula O1C), Formula O2, Formula O3, Formula O4 (e.g., Formula O4:K52 and Formula O4:K6), Formula O5 (e.g., Formula O5ab and Formula O5ac (strain 180/C3)), Formula O6 (e.g., Formula O6:K2;K13;K15 and Formula O6:K54), Formula O7, Formula O8, Formula O9, Formula O10, Formula O11, Formula O12, Formula O13, Formula O14, Formula O15, Formula O16, Formula O17, Formula O18 (e.g., Formula O18A, Formula O18ac, Formula O18A1, Formula O18B, and Formula O18B1), Formula O19, Formula O20, Formula O21, Formula O22, Formula O23, Formula O24, Formula O25, Formula O26, Formula O27, Formula O28, Formula O29, Formula O30, Formula O31, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37, Formula O38, Formula O39, Formula O40, Formula O41, Formula O42, Formula O43, Formula O44, Formula O45, Formula O46, Formula O47, Formula O48, Formula O49, Formula O50, Formula O51, Formula O52, Formula O53, Formula O54, Formula O55, Formula O56, Formula O57, Formula O58, Formula O59, Formula O60, Formula O61, Formula O62, Formula O63, Formula O64, Formula O65, Formula O66, Formula O67, Formula O68, Formula O69, Formula O70, Formula O71, Formula O72, Formula O 19, Formula O20, Formula O21, Formula O22, Formula O23 (e.g., Formula O23A), Formula O24, Formula O25 (e.g., Formula O25a and Formula O25b), Formula O26, Formula O27, Formula O28, Formula O29, Formula O30, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37, Formula O38, Formula O39, Formula O4 0, formula O41, formula O42, formula O43, formula O44, formula O45 (e.g., formula O45 and formula O45rel), formula O46, formula O48, formula O49, formula O50, formula O51, formula O52, formula O53, formula O54, formula O55, formula O56, formula O57, formula O58, formula O59, formula O60, formula O61, formula O62, formula O62D ₁ , Formula O63, Formula O64, Formula O65, Formula O66, Formula O68, Formula O69, Formula O70, Formula O71, Formula O73 (e.g., Formula O73 (strain 73-1)), Formula O74, Formula O75, Formula O76, Formula O77, Formula O78, Formula O79, Formula O80, Formula O81, Formula O82, Formula O83, Formula O84, Formula O85, Formula O86, Formula O87, Formula O88, Formula O89, Formula O90, Formula O91, Formula O92, Formula O93, Formula O95, Formula O96, Formula O97, Formula O98, Formula O99, formula O100, formula O101, formula O102, formula O103, formula O104, formula O105, formula O106, formula O107, formula O108, formula O109, formula O110, formula O111, formula O112, formula O113, formula O11 4, Formula O115, Formula O116, Formula O117, Formula O118, Formula O119, Formula O120, Formula O121, Formula O123, Formula O124, Formula O125, Formula O126, Formula O127, Formula O128, Formula O129, Formula O130, Formula O131, Formula O132, Formula O133, Formula O134, Formula O135, Formula O136, Formula O137, Formula O138, Formula O139, Formula O140, Formula O141, Formula O142, Formula O143, Formula O144, Formula O145, Formula O 146, Formula O147, Formula O148, Formula O149, Formula O150, Formula O151, Formula O152, Formula O153, Formula O154, Formula O155, Formula O156, Formula O157, Formula O158, Formula O159, Formula O160, Formula O16 and a sugar comprising a structure selected from any one of formulas O162, O163, O164, O165, O166, O167, O168, O169, O170, O171, O172, O173, O174, O175, O176, O177, O178, O179, O180, O181, O182, O183, O184, O185, O186, and O187. In some embodiments, the sugar in the conjugate comprises a formula where n is an integer between 1 and 1000, between 5 and 1000, preferably between 31 and 100, more preferably between 35 and 90, and most preferably between 35 and 65.

The number of lysine residues in the carrier protein that become conjugated to a saccharide can be characterized as the range of conjugated lysines. For example, in some embodiments of the immunogenic composition, CRM ₁₉₇ may contain 4-16 of the 39 lysine residues covalently attached to a saccharide. Another way to express this parameter is that about 10% to about 41% of the CRM ₁₉₇ lysines are covalently attached to a saccharide. In other embodiments, CRM ₁₉₇ may contain 2-20 of the 39 lysine residues covalently attached to a saccharide. Another way to express this parameter is that about 5% to about 50% of the CRM ₁₉₇ lysines are covalently attached to a saccharide.

In frequent embodiments, the carrier protein is CRM ₁₉₇ and the covalent bond between CRM ₁₉₇ and the polysaccharide via the eTEC spacer occurs at least once per 4, 10, 15 or 25 saccharide repeat units of the polysaccharide.

In other embodiments, the conjugate comprises at least one covalent bond between the carrier protein and the saccharide every 5-10 saccharide repeat units; every 2-7 saccharide repeat units; every 3-8 saccharide repeat units; every 4-9 saccharide repeat units; every 6-11 saccharide repeat units; every 7-12 saccharide repeat units; every 8-13 saccharide repeat units; every 9-14 saccharide repeat units; every 10-15 saccharide repeat units; every 2-6 saccharide repeat units; every 3-7 saccharide repeat units; every 4-8 saccharide repeat units; every 6-10 saccharide repeat units; every 7-11 saccharide repeat units; every 8-12 saccharide repeat units; every 9-13 saccharide repeat units; every 10-14 saccharide repeat units; every 10-20 saccharide repeat units; or every 4-25 saccharide repeat units.

In another embodiment, at least one bond between the carrier protein and the saccharide is present 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 glycoconjugates of the present invention is a carrier protein to which the sugar is conjugated. The terms "protein carrier" or "carrier protein" or "carrier" can be used interchangeably herein. The carrier protein should be suitable for standard conjugation procedures.

One component of the conjugate is a carrier protein to which the O polysaccharide is conjugated. In one embodiment, the conjugate comprises a carrier protein conjugated to the core oligosaccharide of the O polysaccharide (see FIG. 24). In one embodiment, the conjugate comprises 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. The carrier protein should be suitable for standard conjugation procedures.

In a preferred embodiment, the carrier protein of the conjugate 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, C. difficile toxins A or B, and PsaA. In an embodiment, the carrier protein of the conjugate of the invention is DT (diphtheria toxoid). In another embodiment, the carrier protein of the conjugate of the invention is TT (tetanus toxoid). In another embodiment, the carrier protein of the conjugate of the invention is PD (Haemophilus influenzae protein D - see, e.g., EP 0594610B). In some embodiments, the carrier protein comprises poly(L-lysine) (PLL).

In a preferred embodiment, the saccharide is conjugated to CRM ₁₉₇ protein, which is a non-toxic form of diphtheria toxin but is immunologically indistinguishable from diphtheria toxin. CRM ₁₉₇ is produced by C. difficile infected with the non-toxigenic phage β197tox ^- , _which was created by nitrosoguanidine mutagenesis of toxigenic corynebacterium beta. CRM ₁₉₇ protein has the same molecular weight as diphtheria toxin but differs from it by a single base change (guanine to adenine) in the structural gene. This single base change causes an amino acid substitution (glutamic acid to glycine) in the mature protein, eliminating the toxicity of diphtheria toxin. CRM ₁₉₇ protein is a safe and effective T-cell dependent carrier for saccharides.

Thus, in some embodiments, the conjugates of the invention comprise CRM ₁₉₇ as a carrier protein, with a sugar covalently attached to CRM ₁₉₇ .

In a preferred embodiment, the carrier protein of the glycoconjugate is DT (diphtheria toxin), TT (tetanus toxoid) or fragment C of TT, CRM197 (a non-toxic but antigenically identical variant of diphtheria toxin), other DT variants (CRM176, CRM228, CRM45 (Uchida et al., J. Biol. Chem. 218:3838-3844, 1973), CRM9, CRM45, CRM102, CRM103 or CRM107; and Nicholls and Youle in Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker, et al., J. Biol. Chem. 218:3838-3844, 1973). and other mutations described by U.S. Patent Nos. 4,709,017 and 4,950,740; deletions or mutations of at least one or more of the residues Lys516, Lys526, Phe530 and/or Lys534 and other mutations disclosed in U.S. Patent Nos. 5,917,017 or 6,455,673; or fragments disclosed in U.S. Patent No. 5,843,711), which are detoxified in some manner. ply, e.g., dPLY-GMBS (WO04081515, PCT/EP2005/010258) or dPLY-formol, PhtX, including PhtA, PhtB, PhtD, PhtE (the sequences of PhtA, PhtB, PhtD or PhtE are disclosed in WO00/37105 or WO00/39299) and fusions of Pht proteins, e.g., PhtDE fusions, PhtBE fusions, PhtA-E (WO01/98334, WO03/54007, WO2009/000826), pneumococcal pneumolysin (Kuo et al. (1995) Infect. lmmun 63:2706-13), OMPC (meningococcal outer membrane protein, usually extracted from N. meningitidis serogroup B - EP0372501), PorB (from N. meningitidis), PD (Haemophilus influenzae influenza) protein D; see, e.g., EP 0594610B), or immunologically functional equivalents thereof, synthetic peptides (EP 0378881, EP 0427347), heat shock proteins (WO 93/17712, WO 94/03208), pertussis proteins (WO 98/58668, EP 0471177), cytokines, lymphokines, growth factors or hormones (WO 91/01146), artificial proteins comprising multiple human CD4+ T cell epitopes derived from various pathogen-derived antigens (Falugi et al. (2001) Eur J Immunol 31:3816-3824), e.g., N19 protein (Baraldoi et al. (2004) Infect Immunol 31:3816-3824), 72:4884-7), pneumococcal surface protein PspA (WO 02/091998), iron uptake protein (WO 01/72337), Clostridium difficile toxin A or B (WO 00/61761), transferrin binding protein, pneumococcal adhesion protein (PsaA), recombinant Pseudomonas aeruginosa exotoxin A, particularly non-toxic mutants thereof, such as exotoxin A carrying 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) can also be used as carrier proteins. Other suitable carrier proteins include inactivated bacterial toxins such as cholera toxoid (e.g., as described in International Patent Application WO 2004/083251), E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa.

In some embodiments, the carrier protein is, 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 (EPA) of P. aeruginosa, maltose binding protein (MBP), flagellin, detoxified hemolysin A of S. aureus, clumping factor A, clumping factor B, cholera toxin B subunit (CTB), Streptococcus pneumoniae, or the like. The carrier protein is selected from any one of C. pneumoniae pneumolysin and detoxified variants thereof, C. jejuni AcrA, C. jejuni native glycoprotein, 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 glycoconjugate 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, C. difficile toxin A or B, and PsaA. In an embodiment, the carrier protein of the glycoconjugate of the invention is DT (diphtheria toxoid). In another embodiment, the carrier protein of the glycoconjugate 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, for example, EP 0594610B).

In a preferred embodiment, the capsular saccharide of the invention is conjugated to CRM ₁₉₇ protein, _which is a non-toxic form of diphtheria toxin, but is immunologically indistinguishable from diphtheria toxin. CRM ₁₉₇ is produced by C. diphtheriae infected with the non-toxigenic phage β197tox-, which was created by nitrosoguanidine mutagenesis of toxigenic corynephrage beta (Uchida, T. et al., 1971, Nature New Biology 233:8-11). The CRM ₁₉₇ protein has the same molecular weight as diphtheria toxin, but differs from it by a single base change in the structural gene (guanine to adenine). This single base change causes an amino acid substitution in the mature protein (glutamic acid to glycine), eliminating the toxicity of diphtheria toxin. CRM ₁₉₇ protein is a safe and effective T cell dependent carrier for sugars. Further details regarding CRM ₁₉₇ and its production can be found, for example, in U.S. Patent No. 5,614,382.

Thus, in a frequent embodiment, the glycoconjugates of the invention comprise CRM ₁₉₇ as a carrier protein, to _which the capsular polysaccharide is covalently attached.

In a further embodiment, the carrier protein of the glycoconjugate is SCP (streptococcal C5a peptidase). All human isolates of β-hemolytic streptococci produce a highly conserved cell wall protein, SCP (streptococcal C5a peptidase), that specifically inactivates C5a. The scp gene encodes a polypeptide containing 1,134-1,181 amino acids (Brown et al., PNAS, 2005, vol. 102, no. 51, pp. 18391-18396). The first 31 residues are the export signal presequence, which are removed during passage through the cell membrane. The next 68 residues act as a prosequence and need to be removed to produce active SCP. The next 10 residues can be removed without losing protease activity. At the other end, starting from Lys-1034, there are four consecutive 17-residue motifs, followed by a cell sorting and cell wall binding signal. This signal combination consists of a 20-residue hydrophilic sequence containing the LPTTND sequence, a 17-residue hydrophobic sequence, and a short basic carboxyl terminus.

SCPs can be divided into domains (see FIG. 1B in Brown et al., PNAS, 2005, vol. 102, no. 51, pp. 18391-18396). These domains include the pre/pro domain (containing the export signal pre sequence (typically the first 31 residues) and the pro sequence (typically the next 68 residues)), the protease domain (split into two parts (protease part 1 is typically residues 89-333/334, protease domain part 2 is typically residues 467/468-583/584), the protease associated domain (PA domain) (typically the first 31 residues) and the protease associated domain (PA domain) (typically the first 31 residues). typically residues 333/334-467/468), three fibronectin type III (Fn) domains (Fn1, typically residues 583/584-712/713; Fn2, typically residues 712/713-928/929/930; Fn3, typically residues 929/930-1029/1030/1031), and a cell wall anchor domain (typically residues 1029/1030/1031 to the C-terminus).

In one embodiment, the carrier protein of the glycoconjugate of the invention is GBS-derived SCP (SCPB). An example of SCPB is provided in SEQ ID NO: 3 of WO 97/26008. See also SEQ ID NO: 3 of WO 00/34487.

In another embodiment, the carrier protein of the glycoconjugate of the invention is a GAS-derived SCP (SCPA). Examples of SCPA can be found in SEQ ID NO: 1 and SEQ ID NO: 2 of WO 97/26008. See also SEQ ID NO: 1, 2 and 23 of WO 00/34487.

In a further embodiment, the carrier protein of the glycoconjugate of the present invention is an SCP as set forth in SEQ ID NO: 150 or 151 of WO2014/136064.

H. Dosage of the Composition Dosage regimens can be adjusted to obtain the optimum desired response. For example, a single dose of the polypeptide or fragment thereof derived from E. coli can be administered, or multiple divided doses can be administered over time, or the dose can be correspondingly reduced or increased as dictated by the exigencies of the situation. It should be noted that dosage values can vary with the type and severity of the condition to be alleviated and can include single or multiple doses. Furthermore, it should be understood that for any particular subject, specific dosing regimens should be adjusted over time according to the individual's needs and the professional judgment of the person administering or supervising the administration of the composition, and that the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions. It will be understood that the determination of suitable dosing and regimens for administration of therapeutic proteins is well known in the relevant art and is within the skill of the art once provided with the teachings disclosed herein.

In some embodiments, the amount of E. coli derived polypeptide or fragment thereof in the composition may range from about 10 μg to about 300 μg of each protein antigen. In some embodiments, the amount of E. coli derived polypeptide or fragment thereof in the composition may range from about 20 μg to about 200 μg of each protein antigen.

The amount of glycoconjugate in each dose is selected as an amount that will induce an immunoprotective response without significant adverse side effects in a typical vaccine. Such an amount will vary depending on which particular immunogen is used and how it is delivered.

The amount of a particular saccharide conjugate in an immunogenic composition can be calculated based on the total polysaccharide for that conjugate (conjugated and unconjugated). For example, a saccharide conjugate containing 20% free polysaccharide will have about 80 μg of conjugated polysaccharide and about 20 μg of unconjugated polysaccharide in a 100 μg polysaccharide dose. The amount of saccharide conjugate may vary depending on the E. coli serotype. The saccharide concentration can be determined by uronic acid assay.

The "immunogenic amounts" of different polysaccharide components in the immunogenic composition may vary and may each comprise about 1.0 μg, about 2.0 μg, about 3.0 μg, about 4.0 μg, about 5.0 μg, about 6.0 μg, about 7.0 μg, about 8.0 μg, about 9.0 μg, about 10.0 μg, about 15.0 μg, about 20.0 μg, about 30.0 μg, about 40.0 μg, about 50.0 μg, about 60.0 μg, about 70.0 μg, about 80.0 μg, about 90.0 μg, or about 100.0 μg of any particular polysaccharide antigen. Generally, each dose will contain 0.1 μg to 100 μg, particularly 0.5 μg to 20 μg, more particularly 1 μg to 10 μg, and even more particularly 2 μg to 5 μg of polysaccharide for a given serotype. Any whole integer within any of the above ranges is contemplated as an embodiment of the present disclosure. In one embodiment, each dose will contain 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 15 μg, or 20 μg of polysaccharide for a given serotype.

Amount of carrier protein. Generally, each dose will comprise from 5 μg to 150 μg of carrier protein, particularly from 10 μg to 100 μg of carrier protein, more particularly from 15 μg to 100 μg of carrier protein, more particularly from 25 μg to 75 μg of carrier protein, more particularly from 30 μg to 70 μg of carrier protein, more particularly from 30 μg to 60 μg of carrier protein, more particularly from 30 μg to 50 μg of carrier protein, and even more particularly from 40 μg to 60 μg of carrier protein. In one embodiment, the carrier protein is CRM ₁₉₇ . In one embodiment, each dose is about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29 μg, about 30 μg, about 31 μg, about 32 μg, about 33 μg, about 34 μg, about 35 μg, about 36 μg, about 37 μg, about 38 μg, about 39 μg, about 40 μg, about 41 μg, about 42 μg, about 43 μg, about 44 μg, about 45 μg, about 46 μg, about 47 μg, about 48 μg, about 49 μg, about 50 μg, about 51 μg, about 52 μg, about 53 μg, about 54 μg, about 55 μg, about 56 μg, about 57 μg, about 58 μg, about 59 μg, about 60 μg, about 61 μg, about 62 μg, about 63 μg, about 64 μg, about 65 μg, about 66 μg, about 67 μg, about 68 μg, about 69 μg, about 70 μg, about 71 μg, about 72 μg, about 73 μg, about 74 μg, about 75 μg, about 76 μg, about 77 μg, about 78 μg, about 79 μg, about 80 μg, about 81 μg, about 82 μg, about 83 μg, about 84 μg, about 85 μg, about 86 μg, about 87 μg, about 88 μg, about 89 μg, about 90 μg, about 91 μg, about 92 μg, about 93 μg, about 94 μg, about 95 μg, about 96 μg, The carrier protein may comprise about 0 μg, about 51 μg, about 52 μg, about 53 μg, about 54 μg, about 55 μg, about 56 μg, about 57 μg, about 58 μg, about 59 μg, about 60 μg, about 61 μg, about 62 μg, about 63 μg, about 64 μg, about 65 μg, about 66 μg, about 67 μg, 68 μg, about 69 μg, about 70 μg, about 71 μg, about 72 μg, about 73 μg, about 74 μg or about 75 μg of carrier protein. In one embodiment, the carrier protein is CRM _197. In another embodiment, the carrier protein is SCP.

I. Adjuvants 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. An antigen may act primarily as a delivery system, primarily as an immune modulator, or have strong characteristics 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 some embodiments, the immunogenic compositions disclosed herein include an aluminum salt (alum) (e.g., aluminum phosphate, aluminum sulfate, or aluminum hydroxide) as an adjuvant. In a preferred embodiment, the immunogenic compositions disclosed herein include aluminum phosphate or aluminum hydroxide as an adjuvant. In some embodiments, the immunogenic compositions disclosed herein include 0.1 mg/mL to 1 mg/mL or 0.2 mg/mL to 0.3 mg/mL elemental aluminum in the form of aluminum phosphate. In some embodiments, the immunogenic compositions disclosed herein include about 0.25 mg/mL elemental aluminum in the form of aluminum phosphate.

Examples of known suitable immunomodulatory adjuvants that can be used in humans include, but are not limited to, saponin extract from Aquila bark (QS21, QuilA), TLR4 agonists such as MPLA (monophosphoryl lipid A), 3DMPL (3-O-deacylated MPL) or GLA-AQ, LT/CT mutants, cytokines such as various interleukins (e.g., IL-2, IL-12) or GM-CSF, AS01, etc.

Examples of known suitable immunomodulatory adjuvants that exhibit both delivery and immunomodulatory properties that can be used in humans include, but are not limited to, ISCOMS (see, e.g., Sjolander et al. (1998) J. Leukocyte Biol. 64:713; WO90/03184, WO96/11711, WO00/48630, WO98/36772, WO00/41720, WO2006/134423 and WO2007/026190) or GLA-EM, which is a combination of a TLR4 agonist and an oil-in-water emulsion.

For veterinary applications, such as animal testing, those skilled in the art can easily use, but are not limited to, Freund's complete 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), or other suitable adjuvant. 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP19835A, 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, can be used.

Further exemplary adjuvants for enhancing the efficacy of the immunogenic compositions disclosed herein include, but are not limited to, (1) for example, (a) SAF containing 10% squalane, 0.4% Tween 80, 5% Pluronic® blocking polymer L121, and thr-MDP microfluidized to a submicrometer emulsion or vortexed to generate a larger particle size emulsion, and (b) RIBI™ Adjuvant System (RAS) (Ribi™) containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components such as monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (DETOX™). (2) oil-in-water emulsion formulations, such as those available from Immunochem, Hamilton, Mont., with or without muramyl peptides (see below) or other specific immune stimulants such as bacterial cell wall components; (3) glycerol-based formulations, such as those available from Immunochem, Hamilton, Mont., with or without muramyl peptides (see below) or other specific immune stimulants such as bacterial cell wall components; (4) glycerol-based formulations, such as those available from Immunochem, Hamilton, Mont., with or without muramyl peptides (see below) or other specific immune stimulants such as bacterial cell wall components; (5) glycerol-based formulations, such as those available from Immunochem, Hamilton, Mont., with or without muramyl peptides (see below) or other specific immune stimulants such as bacterial cell wall components; (6) glycerol-based formulations, such as those available from Immunochem, Hamilton, Mont., with or without muramyl peptides (see below) or other specific immune stimulants such as bacterial cell wall components; (3) saponin adjuvants such as saponin, such as sucrose, glycerol, tert-butyl phosphate (IL-1), tert-butyl phosphate (IL-2), tert-butyl phosphate (IL-3), tert-butyl phosphate (IL-4), tert-butyl phosphate (IL-5), tert-butyl phosphate (IL-6), tert-butyl phosphate (IL-7), tert-butyl phosphate (IL-8), tert-butyl phosphate (IL-9), tert-butyl phosphate (IL-10), tert-butyl phosphate (IL-11), tert-butyl phosphate (IL-12), tert-butyl phosphate (IL-13), tert-butyl phosphate (IL-14), tert-butyl phosphate (IL-15), tert-butyl phosphate (IL-16), tert-butyl phosphate (IL-17), tert-butyl phosphate (IL-18), tert-butyl phosphate (IL-19), tert-butyl phosphate (IL-20), tert-butyl phosphate (IL-21), tert-butyl phosphate (IL-22), tert-butyl phosphate (IL-23), tert-butyl phosphate (IL-24), tert-butyl phosphate (IL-25), tert-butyl phosphate (IL-26), tert-butyl phosphate (IL-27), tert-butyl phosphate (IL-28), tert-butyl phosphate (IL-29), tert-butyl phosphate (IL-30), tert-butyl phosphate (IL-31), tert-butyl phosphate (IL-32), tert-butyl phosphate (IL-33), tert-butyl phosphate (IL-34), tert-butyl phosphate (IL-35), tert-butyl phosphate (IL-36), tert-butyl phosphate (IL-37), tert-butyl phosphate (IL (5) cytokines such as interferons (e.g., gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF); (6) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions (e.g., EP 0835318, EP 0735898, E (7) polyoxyethylene ethers or polyoxyethylene esters (see, e.g., WO 99/52549); (8) polyoxyethylene sorbitan ester surfactants in combination with octoxynol (see, e.g., WO 01/21207) or polyoxyethylene alkyl ether or ester surfactants in combination with at least one additional non-ionic surfactant such as octoxynol (see, e.g., WO 01/21152); (9) saponins and and immunostimulatory oligonucleotides (e.g., CpG oligonucleotides) (e.g., WO 00/62800); (10) particles of immunostimulants and metal salts (see, e.g., WO 00/23105); (11) saponin and oil-in-water emulsions (e.g., WO 99/11241); (12) saponin (e.g., QS21) + 3dMPL + IM2 (optionally + sterol) (e.g., WO 98/57659); (13) other substances that act as immunostimulants to enhance the efficacy of the composition. Examples of muramyl peptides include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25 acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), and N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE).

In another embodiment, the adjuvant is a liposomal QS21 formulation as described in Example 35. In another embodiment, the adjuvant is a liposomal MPLA formulation as described in Example 35. In a further embodiment, the adjuvant is a liposomal MPLA/QS21 formulation as described in Example 35.

In an embodiment of the invention, the immunogenic compositions disclosed herein include a CpG oligonucleotide as an adjuvant. As used herein, CpG oligonucleotide refers to an immunostimulatory CpG oligodeoxynucleotide (CpG ODN), and thus the terms are used interchangeably unless otherwise indicated. An immunostimulatory CpG oligodeoxynucleotide contains one or more immunostimulatory CpG motifs that are unmethylated cytosine-guanine dinucleotides, optionally within the context of certain preferred bases. The methylation state of a 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 that contains a 5' unmethylated cytosine linked by a phosphate bond to a 3' guanine and activates the immune system by binding to Toll-like receptor 9 (TLR-9). In another embodiment, the immunostimulatory oligonucleotide may contain one or more methylated CpG dinucleotides that activate the immune system through TLR9, but not as potently as if the CpG motifs were unmethylated. The CpG immunostimulatory oligonucleotide may contain one or more palindromes, which in turn may encompass the CpG dinucleotide. CpG oligonucleotides are described in several issued patents, published patent applications, and other publications, including U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; and 6,339,068.

In one embodiment of the present invention, the immunogenic composition disclosed herein comprises any of the CpG oligonucleotides described on page 3, line 22 to page 12, line 36 of WO2010/125480.

Various classes of CpG immunostimulatory oligonucleotides have been identified. These are referred to as the A, B, C and P classes and are described in more detail in WO 2010/125480 at page 3, line 22 to page 12, line 36. The methods of the invention encompass the use of these various classes of CpG immunostimulatory oligonucleotides.

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

In some embodiments, antigens are fused or conjugated to the exterior of the nanostructure to stimulate the generation of an adaptive immune response against the presented epitope. In some embodiments, the immunogenic complexes further include adjuvants or other immunomodulatory compounds attached to the exterior and/or encapsulated within the cage to help tailor the type of immune response generated with each pathogen.

In some embodiments, the nanostructure comprises a single assembly that includes multiple identical first nanostructure-associated polypeptides.

In an alternative embodiment, the nanostructure comprises a plurality of assemblies comprising a plurality of identical first nanostructure-associated polypeptides, and a plurality of second assemblies, each of the second assemblies comprising a plurality of identical second nanostructure-associated polypeptides.

A variety of nanostructure platforms can be used in generating the immunogenic compositions described herein. In some embodiments, the nanostructures used are formed by multiple copies of a single subunit. In some embodiments, the nanostructures used are formed by multiple copies of multiple different subunits.

The nanostructures are typically ball-like in shape and/or have rotational symmetry (e.g., 3- and 5-fold axes), for example, with the icosahedral structures exemplified herein.

In some embodiments, the antigen is presented on a self-assembling nanoparticle, such as a self-assembling nanostructure derived from ferritin (FR), E2p, Qβ, and I3-01. E2p is a redesigned variant of dihydrolipoyl acyltransferase from Bacillus stearothermophilus. I3-01 is an engineered protein that can self-assemble into ultrastable nanoparticles. The sequences of the subunits of these proteins are known in the art. In a first aspect, disclosed herein are nanostructure-related polypeptides that include an amino acid sequence that is at least 75% identical over its length and at least one identified interface position to an 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 nanostructures. The nanostructure-related polypeptides were designed such that they can self-assemble in pairs to form nanostructures, such as icosahedral nanostructures.

In some embodiments, the nanostructure comprises: (a) a plurality of first assemblies, each of which comprises a plurality of identical first nanostructure-associated polypeptides, the first nanostructure-associated polypeptides comprising an amino acid sequence of a nanostructure-associated polypeptide selected from the group consisting of SEQ ID NOs: 59-92; and (b) a plurality of second assemblies, each of which comprises a plurality of identical second nanostructure-associated polypeptides, the second nanostructure-associated polypeptides comprising an amino acid sequence of a nanostructure-associated polypeptide selected from the group consisting of SEQ ID NOs: 59-92, and the second nanostructure-associated polypeptides are different from the first nanostructure-associated polypeptides, wherein the plurality of first assemblies non-covalently interact with the plurality of second assemblies to form the nanostructure.

The nanostructure includes symmetrically repeated non-natural non-covalent polypeptide-polypeptide interfaces that orient the first and second assemblies into a nanostructure, such as one with icosahedral symmetry.

SEQ ID NOs:59-92 provide amino acid sequences of exemplary nanostructure-associated polypeptides. The number of interface residues in the exemplary nanostructure-associated polypeptides of SEQ ID NOs:59-92 ranges from 4 to 13 residues. In various embodiments, the nanostructure-associated polypeptide comprises 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 to an amino acid sequence of a nanostructure-associated polypeptide selected from the group consisting of SEQ ID NOs:59-92, and is identical at at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 identified interface positions (depending on the number of interface residues in a given nanostructure-associated polypeptide). In other embodiments, the nanostructure-associated polypeptide comprises 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 at least 20%, 25%, 33%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 100% identical at the identified interface positions to the amino acid sequence of a nanostructure-associated polypeptide selected from the group consisting of SEQ ID NOs: 59-92. In further embodiments, the nanostructure-associated polypeptide comprises a nanostructure-associated polypeptide having an amino acid sequence of a nanostructure-associated polypeptide selected from the group consisting of SEQ ID NOs: 59-98.

In one non-limiting embodiment, nanostructure-associated polypeptides can be modified to facilitate covalent attachment to a "cargo" of interest. In one non-limiting example, nanostructure-associated polypeptides can be modified, such as by introducing various cysteine residues at defined positions, to facilitate attachment to one or more antigens of interest, such that the nanostructure of the nanostructure-associated polypeptide provides a scaffold to provide multiple antigens for delivery as a vaccine, resulting in an improved immune response.

In some embodiments, some or all of the naturally occurring cysteine residues present in the nanostructure-associated polypeptides, but not intended to be used for conjugation, can be mutated to other amino acids to facilitate conjugation at predefined positions. In another non-limiting embodiment, the nanostructure-associated polypeptides can be modified by conjugation (covalent or non-covalent) with moieties that help facilitate "endosomal escape". In applications involving delivery of a molecule of interest to a target cell, such as targeted delivery, a critical step can be escape from endosomes (membrane-bound organelles), which are the entry point for the delivery vehicle into the cell. Endosomes mature into lysosomes to degrade their contents. Thus, unless the delivery vehicle somehow "escapes" from the endosome before the endosome becomes a lysosome, the delivery vehicle will be degraded and will not be able to perform its function. There are various lipids or organic polymers that disrupt endosomes, allowing escape into the cytosol. Thus, in this embodiment, the nanostructure-associated polypeptides can be modified, for example, by introducing cysteine residues that allow for chemical conjugation of such lipids or organic polymers to the monomers or resulting assembly surfaces. In another non-limiting example, the nanostructure-associated polypeptides can be modified, for example, by introducing cysteine residues that allow for chemical conjugation of fluorophores or other imaging agents that allow visualization of the nanostructures in vitro or in vivo.

Surface amino acid residues on nanostructure-associated polypeptides can be mutated to improve the stability or solubility of protein subunits or assembled nanostructures. As will be appreciated by those of skill in the art, if a nanostructure-associated polypeptide has significant sequence homology to an existing protein family, multiple sequence alignments of other proteins from that family can be used to guide the selection of amino acid mutations at non-conserved positions that may increase protein stability and/or solubility, a process referred to as consensus protein design (9).

To impart an overall positive or negative charge to the protein surface, the surface amino acid residues on the nanostructure-associated polypeptides can be mutated to positively charged (Arg, Lys) or negatively charged (Asp, Glu) amino acids. In one non-limiting embodiment, the surface amino acid residues on the nanostructure-associated polypeptides can be mutated to impart a high net charge to the interior surface of the self-assembling nanostructure. Such nanostructures can then be used to package or encapsulate cargo molecules with an opposite net charge through electrostatic interactions between the nanostructure interior surface and the cargo molecule. In one non-limiting embodiment, the surface amino acid residues on the nanostructure-associated polypeptides can be mutated primarily to arginine or lysine residues to impart a net positive charge to the interior surface of the self-assembling nanostructure. A solution containing the nanostructure-associated polypeptide can then be mixed in the presence of a nucleic acid cargo molecule, such as dsDNA, ssDNA, dsRNA, ssRNA, cDNA, miRNA., siRNA, shRNA, piRNA, or other nucleic acid, to encapsulate the nucleic acid inside the self-assembling nanostructure. Such nanostructures 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 include 60 copies of a first nanostructure-associated polypeptide and 60 copies of a second nanostructure-associated polypeptide. In one such embodiment, the number of identical first nanostructure-associated polypeptides in each first assembly is different from the number of identical second nanostructure-associated polypeptides in each second assembly. For example, in one embodiment, the nanostructure includes 12 first assemblies and 20 second assemblies; in this embodiment, each first assembly may include, for example, 5 copies of an identical first nanostructure-associated polypeptide and each second assembly may include, for example, 3 copies of an identical second nanostructure-associated polypeptide. In another embodiment, the nanostructure includes 12 first assemblies and 30 second assemblies; in this embodiment, each first assembly may include, for example, 5 copies of an identical first nanostructure-associated polypeptide and each second assembly may include, for example, 2 copies of an identical second nanostructure-associated polypeptide. In a further embodiment, the nanostructure comprises 20 first assemblies and 30 second assemblies; in this embodiment, each first assembly may comprise, for example, three copies of the same first nanostructure-associated polypeptide, and each second assembly may comprise, for example, two copies of the same second nanostructure-associated polypeptide. All of these embodiments may form a synthetic nanomaterial with icosahedral symmetry.

VIII. Combinations with saccharides and/or polypeptides derived from Klebsiella pneumoniae or fragments thereof Klebsiella 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 death in at-risk vulnerable populations. O-antigen serotypes are highly prevalent among strains causing invasive disease globally, making derived O-antigen glycoconjugates 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 O1 (and d-Gal-III variants), O2 (and d-Gal-III variants), O2ac, O3, O4, O5, O7, O8, and O12. In a preferred embodiment, any of the compositions disclosed herein may further comprise a polypeptide derived from K. pneumoniae selected from a polypeptide or immunogenic fragment thereof derived from K. pneumoniae type I pilus protein; or a polypeptide or immunogenic fragment thereof derived from K. pneumoniae type III pilus protein; or a combination thereof.

As is known in the art, K. pneumoniae O1 and O2 O antigens and their corresponding v1 and v2 subtypes are polymeric galactans that differ in the structure of their repeating units. K. pneumoniae O1 and O2 antigens contain homopolymeric galactose units (or galactans). K. pneumoniae O1 and O2 antigens each contain D-galactan I units (sometimes referred to as O2a repeating units), while the O1 antigen differs in that the O1 antigen has a D-galactan II cap structure. D-galactan III (d-Gal-III) is a variant of D-galactan I. The structures of the base galactans I and III that define the two different serotype O2 subtypes, O2v1 and O2v2; and the structures of the derived chimeras resulting from capping with galactan II to give subtypes O1v1 and O1v2 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 Klebsiella pneumoniae O1 comprises a repeating unit of [→3)-β-D-Galf-(1→3)-α-D-Galp-(1→]. In some embodiments, the saccharide derived from Klebsiella pneumoniae O1 comprises a repeating unit of [→3)-α-D-Galp-(1→3)-β-D-Galp-(1→]. In some embodiments, the saccharide derived from Klebsiella pneumoniae O1 comprises a repeating unit of [→3)-α-D-Galp-(1→3)-β-D-Galp-(1→]. The resulting saccharide comprises a repeating unit of [→3)-β-D-Galf-(1→3)-α-D-Galp-(1→] and a repeating unit of [→3)-α-D-Galp-(1→3)-β-D-Galp-(1→]. In some embodiments, the saccharide derived from K. pneumoniae O1 comprises a repeating unit of →3)-β-D-Galf-(1→3)-[α-D-Galp-(1→4)]-α-D-Galp-(1→] (referred to as a D-Gal-III repeating unit). O. 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 O2 comprises a repeating unit of [→3)-α- _D -Galp-(1→3)-β- _D -Galf-(1→], which may be a component of the K. pneumoniae serovar O2a antigen. In some embodiments, the saccharide derived from K. pneumoniae O2 comprises a repeating unit of [→3)-β- _D -GlcpNAc-(1→5)-β- _D -Galf-(1→] (which may be an element of the K. pneumoniae serovar O2c antigen). In some embodiments, the saccharide derived from K. pneumoniae O2 comprises a modification of the O2a repeating unit with the side chain addition of a (1→4)-linked Galp residue (which may be an element of the K. pneumoniae O2afg antigen). In some embodiments, the saccharide derived from K. pneumoniae O2 comprises a modification of the O2a repeating unit with the side chain addition of a (1→2)-linked Galp residue (which may be an element of the K. pneumoniae O2aeh antigen). (Whitfield C. (1992) J. et al. Bacteriol. 174, 4913-4919).

Without being bound by mechanism or theory, it has been disclosed in the art that the O-antigen polysaccharide structures of K. pneumoniae serotypes O3 and O5 are identical to those of E. coli serotypes O9a (formula O9a) and O8 (formula O8), respectively.

In some embodiments, the saccharide derived from Klebsiella pneumoniae O4 comprises a repeating unit of [→4)-α-D-Galp-(1→2)-β-D-Ribf-(1→)]. In some embodiments, the saccharide from K. pneumoniae O7 comprises a repeat unit of [→2-a-L-Rhap-(1→2)-β-D-Ribf-(1→3)-α-L-Rhap-(1→3)-α-L-Rhap-(1→]. In some embodiments, the saccharide from K. pneumoniae O8 serotype comprises the same repeat unit structure as K. pneumoniae O2a, but is non-stoichiometrically O-acetylated. In some embodiments, the saccharide from K. pneumoniae O12 serotype comprises a repeat unit of [α-Rhap-(1→3)-β-GlcpNAc] disaccharide repeat unit.

In one aspect, the invention includes a composition comprising a polypeptide or fragment thereof derived from E. coli FimH; and at least one saccharide that is or is derived from at least one K. pneumoniae serotype selected from O1 (and d-Gal-III variants), O2 (and d-Gal-III variants), O2ac, O3, O4, O5, O7, O8, and O12. In some embodiments, the composition comprises saccharides from or derived from one or more of serotypes O1, O2, O3, and O5, or a combination thereof. In some embodiments, the composition comprises saccharides from or derived from each of serotypes O1, O2, O3, and O5.

In another aspect, the invention provides a method for the preparation of a saccharide comprising at least one saccharide that is or is derived from at least one K. pneumoniae serotype selected from O1 (and d-Gal-III variants), O2 (and d-Gal-III variants), O2ac, O3, O4, O5, O7, O8, and O12; and a saccharide of formula O1 (e.g., formula O1A, formula O1B, and formula O1C), formula O2, formula O3, formula O4 (e.g., formula O4:K52 and formula O4:K6), formula O5 (e.g., formula O5ab and formula O5ac (strain 180/C3)), formula O6 (e.g., formula O6:K2;K13;K15 and formula O6:K54), formula O7, formula O8, formula O9, formula O10, formula O11, formula O12, formula O13, formula O14, formula O15, formula O16, formula O17, formula O18, formula O19, formula O20, formula O21, formula O22, formula O23, formula O24, formula O25, formula O26, formula O27, formula O28, formula O29, formula O30, formula O31, formula O32, formula O33, formula O34, formula O35, formula O36, formula O37, formula O38, formula O39, formula O40, formula O41, formula O42, formula O43, formula O44, formula O45, formula O46, formula O47, formula O48, formula O49, formula O50, formula O51, formula O52, formula O53, formula O54, formula O55, formula O56, formula O57, formula O58, formula O59, formula O61, formula O62, formula O63, formula O64, formula O65, formula O66 5, Formula O16, Formula O17, Formula O18 (e.g., Formula O18A, Formula O18ac, Formula O18A1, Formula O18B, and Formula O18B1), Formula O19, Formula O20, Formula O21, Formula O22, Formula O23 (e.g., Formula O23A), Formula O24, Formula O25 (e.g., Formula O25a and Formula O25b), Formula O26, Formula O27, Formula O28, Formula O29, Formula O30, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37, Formula O38, Formula O39, Formula O40, Formula O41, Formula O42, Formula O43, Formula O44, Formula O45, Formula O46, Formula O47, Formula O48, Formula O49, Formula O50, Formula O51, Formula O52, Formula O53, Formula O54, Formula O55, Formula O56, Formula O57, Formula O58, Formula O59, Formula O60, Formula O61, Formula O62, Formula O63, Formula O64, Formula O65, Formula O66, Formula O67, Formula O68, Formula O69, Formula O70, Formula O71, Formula O72, Formula O73, Formula O74, Formula O75, Formula O76, Formula O77, Formula O78, Formula O79, Formula O80, Formula O81, Formula O82, Formula O83, Formula O84, Formula O85, Formula O86, Formula O87, Formula O88, Formula O89, Formula O90, Formula O91, Formula O92, Formula O93, Formula O94, Formula O95, Formula O96, Formula O97, Formula O98, Formula O99, Formula O100, Formula O101, Formula O102, Formula O103, Formula O1 4, formula O35, formula O36, formula O37, formula O38, formula O39, formula O40, formula O41, formula O42, formula O43, formula O44, formula O45 (e.g., formula O45 and formula O45rel), formula O46, formula O48, formula O49, formula O50, formula O51, formula O52, formula O53, formula O54, formula O55, formula O56, formula O57, formula O58, formula O59, formula O60, formula O61, formula O62, formula 62D ₁ , Formula O63, Formula O64, Formula O65, Formula O66, Formula O68, Formula O69, Formula O70, Formula O71, Formula O73 (e.g., Formula O73 (strain 73-1)), Formula O74, Formula O75, Formula O76, Formula O77, Formula O78, Formula O79, Formula O80, Formula O81, Formula O82, Formula O83, Formula O84, Formula O85, Formula O86, Formula O87, Formula O88, Formula O89, Formula O90, Formula O91, Formula O92, Formula O93, Formula O95, Formula O96, Formula O97, Formula O98, Formula O99, Formula O100, formula O101, formula O102, formula O103, formula O104, formula O105, formula O106, formula O107, formula O108, formula O109, formula O110, formula O111, formula O112, formula O113, formula O114, formula O115 , formula O116, formula O117, formula O118, formula O119, formula O120, formula O121, formula O123, formula O124, formula O125, formula O126, formula O127, formula O128, formula O129, formula O130, formula O131, formula O13 2. Formula O133, Formula O134, Formula O135, Formula O136, Formula O137, Formula O138, Formula O139, Formula O140, Formula O141, Formula O142, Formula O143, Formula O144, Formula O145, Formula O146, Formula O147, Formula O 148, formula O149, formula O150, formula O151, formula O152, formula O153, formula O154, formula O155, formula O156, formula O157, formula O158, formula O159, formula O160, formula O161, formula O162, formula O163, formula The compositions include compositions that include saccharides derived from an E. coli O antigen having a structure selected from any one of: O164, O165, O166, O167, O168, O169, O170, O171, O172, O173, O174, O175, O176, O177, O178, O179, O180, O181, O182, O183, O184, O185, O186, and O187, where n is an integer between 1 and 100. In some embodiments, the compositions include saccharides from or derived from one or more of K. pneumoniae serotypes O1, O2, O3, and O5, or a combination thereof. In some embodiments, the composition comprises saccharides from or derived from each of K. pneumoniae serotypes O1, O2, O3, and O5. In some embodiments, the composition comprises saccharides from an E. coli O antigen having formula O9, and does not include saccharides from K. pneumoniae serotype O3. In some embodiments, the composition comprises saccharides from an E. coli O antigen having formula O8, and does not include saccharides from K. pneumoniae serotype O5.

In another aspect, the invention relates to a polypeptide or fragment thereof derived from E. coli FimH; and at least one K. pneumoniae serotype selected from O1 (and d-Gal-III variants), O2 (and d-Gal-III variants), O2ac, O3, O4, O5, O7, O8, and O12, or or at least one saccharide derived therefrom; and a saccharide of formula O1 (e.g., formula O1A, formula O1B, and formula O1C), formula O2, formula O3, formula O4 (e.g., formula O4:K52 and formula O4:K6), formula O5 (e.g., formula O5ab and formula O5ac (strain 180/C3)), formula O6 (e.g., formula O6:K2;K13;K15 and formula O6:K54), formula O7, formula O8, formula O9, formula O10, formula O11 , Formula O12, Formula O13, Formula O14, Formula O15, Formula O16, Formula O17, Formula O18 (e.g., Formula O18A, Formula O18ac, Formula O18A1, Formula O18B, and Formula O18B1), Formula O19, Formula O20, Formula O21, Formula O22, Formula O23 (e.g., Formula O23A), Formula O24, Formula O25 (e.g., Formula O25a and Formula O25b), Formula O26, Formula O27, Formula O28, Formula O29, Formula O30, Formula O31, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37, Formula O38, Formula O39, Formula O40, Formula O41, Formula O42, Formula O43, Formula O44, Formula O45, Formula O46, Formula O47, Formula O48, Formula O49, Formula O50, Formula O51, Formula O52, Formula O53, Formula O54, Formula O55, Formula O56, Formula O57, Formula O58, Formula O59, Formula O60, Formula O61, Formula O62, Formula O63, Formula O64, Formula O65, Formula O66, Formula O67, Formula O68, Formula O69, Formula O70, Formula O71, Formula O72, Formula O73, Formula O74, Formula O75, Formula O76, Formula O77, Formula O78, Formula O79, Formula O80, Formula O81, Formula O82, Formula O83, Formula O84, Formula O85, Formula O86, Formula O87, Formula O88, Formula O89, Formula O90, Formula O91, Formula O92, Formula O93, Formula O94, Formula O95, Formula O96, Formula O97, Formula O98, Formula O99, Formula O1 2, formula O33, formula O34, formula O35, formula O36, formula O37, formula O38, formula O39, formula O40, formula O41, formula O42, formula O43, formula O44, formula O45 (e.g., formula O45 and formula O45rel), formula O46, formula O48, formula O49, formula O50, formula O51, formula O52, formula O53, formula O54, formula O55, formula O56, formula O57, formula O58, formula O59, formula O60, formula O61, formula O62, formula 62D ₁ , Formula O63, Formula O64, Formula O65, Formula O66, Formula O68, Formula O69, Formula O70, Formula O71, Formula O73 (e.g., Formula O73 (strain 73-1)), Formula O74, Formula O75, Formula O76, Formula O77, Formula O78, Formula O79, Formula O80, Formula O81, Formula O82, Formula O83, Formula O84, Formula O85, Formula O86, Formula O87, Formula O88, Formula O89, Formula O90, Formula O91, Formula O92, Formula O93, Formula O95, Formula O96, Formula O97, Formula O98, Formula O99, Formula O100 , Formula O101, Formula O102, Formula O103, Formula O104, Formula O105, Formula O106, Formula O107, Formula O108, Formula O109, Formula O110, Formula O111, Formula O112, Formula O113, Formula O114, Formula O115, Formula O116, Formula O117, Formula O118, Formula O119, Formula O120, Formula O121, Formula O123, Formula O124, Formula O125, Formula O126, Formula O127, Formula O128, Formula O129, Formula O130, Formula O131, Formula O132, Formula O133, Formula O134, formula O135, formula O136, formula O137, formula O138, formula O139, formula O140, formula O141, formula O142, formula O143, formula O144, formula O145, formula O146, formula O147, formula O148, formula O149, formula O150, formula O151, formula O152, formula O153, formula O154, formula O155, formula O156, formula O157, formula O158, formula O159, formula O160, formula O161, formula O162, formula O163, formula O164, formula O165, formula O The present invention relates to compositions comprising a saccharide having a structure selected from any one of Formula O166, Formula O167, Formula O168, Formula O169, Formula O170, Formula O171, Formula O172, Formula O173, Formula O174, Formula O175, Formula O176, Formula O177, Formula O178, Formula O179, Formula O180, Formula O181, Formula O182, Formula O183, Formula O184, Formula O185, Formula O186, and Formula O187, where n is an integer between 1 and 100, preferably between 31 and 90. In some embodiments, the composition comprises a saccharide derived from an E. coli O antigen having formula O9 and does not include a saccharide derived from K. pneumoniae serovar O3. In some embodiments, the composition comprises a saccharide derived from an E. coli O antigen having the formula O8 and does not include a saccharide derived from K. pneumoniae serotype O5.

In some embodiments, the composition comprises at least one saccharide derived from any one of K. pneumoniae types selected from the group consisting of O1, O2, O3, and O5.

In some embodiments, the composition comprises at least one saccharide derived from K. pneumoniae type O1. In one aspect of this embodiment, the K. pneumoniae O antigen is selected from subtype v1 (O1v1) or subtype v2 (O1v2). In one aspect of this embodiment, the K. pneumoniae O antigen is selected from subtype v1 (O1v1) and subtype v2 (O1v2). In some embodiments, the composition comprises at least one saccharide derived from K. pneumoniae type O2. In one aspect of this embodiment, the K. pneumoniae O antigen is selected from subtype v1 (O2v1) or subtype v2 (O2v2). In one aspect of this embodiment, the K. pneumoniae O antigen is selected from subtype v1 (O2v1) and subtype v2 (O2v2). In another aspect, the K. pneumoniae O antigen is selected from the group consisting of a) serotype O1 subtype v1 (O1v1), b) serotype O1 subtype v2 (O1v2), c) serotype O2 subtype v1 (O2v1), and d) serotype O2 subtype v2 (O2v2). In one aspect of this embodiment, the K. pneumoniae O antigen is subtype v1 (O1v1). In one aspect of this embodiment, the K. pneumoniae O antigen is subtype v2 (O1v2). In one aspect of this embodiment, the K. pneumoniae O antigen is subtype v1 (O2v1). In one aspect of this embodiment, the K. pneumoniae O antigen is subtype v2 (O2v2). In another aspect of this embodiment, the composition comprises one, two, three or four K. pneumoniae O antigens selected from the group consisting of a) serotype O1 subtype v1 (O1v1), b) serotype O1 subtype v2 (O1v2), c) serotype O2 subtype v1 (O2v1), and d) serotype O2 subtype v2 (O2v2). In some embodiments, the composition comprises 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 O1, O2, O3, and O5, and a second saccharide is derived from any one of K. pneumoniae types selected from the group consisting of O1 (and d-Gal-III variants), O2 (and d-Gal-III variants), O2ac, O3, O4, O5, O7, O8, and O12. For example, in some embodiments, the composition comprises at least one saccharide derived from K. pneumoniae type O1 and at least one saccharide derived from K. pneumoniae type O2. In a preferred embodiment, the saccharide derived from K. pneumoniae is conjugated to a carrier protein; the saccharide derived from E. coli is conjugated to a carrier protein.

In another aspect, the invention includes a composition comprising a polypeptide or fragment thereof derived from E. coli FimH; and at least one saccharide derived from any one of K. pneumoniae types selected from the group consisting of O1, O2, O3, and O5.

In another aspect, the invention provides a method for the preparation of a saccharide comprising at least one saccharide derived from any one of K. pneumoniae types selected from the group consisting of O1, O2, O3, and O5; and a saccharide of formula O1 (e.g., formula O1A, formula O1B, and formula O1C), formula O2, formula O3, formula O4 (e.g., formula O4:K52 and formula O4:K6), formula O5 (e.g., formula O5ab and formula O5ac (strain 180/C3)), formula O6 (e.g., formula O6:K2;K13;K15 and formula O6:K54), formula O7, formula O8, formula O9, formula O10, formula O11, formula O12, formula O13, formula O14, formula O15, formula O16, formula O17, formula O18 (e.g., formula O18A, formula O18a, formula O18b, formula O18c, formula O18d, formula O18e, formula O18f ... c, Formula O18A1, Formula O18B, and Formula O18B1), Formula O19, Formula O20, Formula O21, Formula O22, Formula O23 (e.g., Formula O23A), Formula O24, Formula O25 (e.g., Formula O25a and Formula O25b), Formula O26, Formula O27, Formula O28, Formula O29, Formula O30, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37 , formula O38, formula O39, formula O40, formula O41, formula O42, formula O43, formula O44, formula O45 (e.g., formula O45 and formula O45rel), formula O46, formula O48, formula O49, formula O50, formula O51, formula O52, formula O53, formula O54, formula O55, formula O56, formula O57, formula O58, formula O59, formula O60, formula O61, formula O62, formula 62D ₁ , Formula O63, Formula O64, Formula O65, Formula O66, Formula O68, Formula O69, Formula O70, Formula O71, Formula O73 (e.g., Formula O73 (strain 73-1)), Formula O74, Formula O75, Formula O76, Formula O77, Formula O78, Formula O79, Formula O80, Formula O81, Formula O82, Formula O83, Formula O84, Formula O85, Formula O86, Formula O87, Formula O88, Formula O89, Formula O90, Formula O91, Formula O92, Formula O93, Formula O95, Formula O96, Formula O97, Formula O98, Formula O99, formula O100, formula O101, formula O102, formula O103, formula O104, formula O105, formula O106, formula O107, formula O108, formula O109, formula O110, formula O111, formula O112, formula O113, formula O11 4. Formula O115, Formula O116, Formula O117, Formula O118, Formula O119, Formula O120, Formula O121, Formula O123, Formula O124, Formula O125, Formula O126, Formula O127, Formula O128, Formula O129, Formula O130, Formula O131, formula O132, formula O133, formula O134, formula O135, formula O136, formula O137, formula O138, formula O139, formula O140, formula O141, formula O142, formula O143, formula O144, formula O145, formula O1 46, Formula O147, Formula O148, Formula O149, Formula O150, Formula O151, Formula O152, Formula O153, Formula O154, Formula O155, Formula O156, Formula O157, Formula O158, Formula O159, Formula O160, Formula O161 , Formula O162, Formula O163, Formula O164, Formula O165, Formula O166, Formula O167, Formula O168, Formula O169, Formula O170, Formula O171, Formula O172, Formula O173, Formula O174, Formula O175, Formula O176, Formula O177, Formula O178, Formula O179, Formula O180, Formula O181, Formula O182, Formula O183, Formula O184, Formula O185, Formula O186, and Formula O187. In some embodiments, the composition comprises a saccharide derived from an E. coli O antigen having formula O9, and does not include a saccharide derived from K. pneumoniae serovar O3. In some embodiments, the composition comprises a saccharide derived from an E. coli O antigen having the formula O8 and does not include a saccharide derived from K. pneumoniae serotype O5.

In some embodiments, the composition comprises at least one saccharide derived from K. pneumoniae type O1 and at least one saccharide derived from E. coli having a structure selected from the group consisting of formula O8 and formula O9. In another embodiment, the composition comprises at least one saccharide derived from K. pneumoniae type O2 and at least one saccharide derived from E. coli having a structure selected from the group consisting of formula O8 and formula O9. In another embodiment, the composition comprises at least one saccharide derived from K. pneumoniae type O1; at least one saccharide derived from K. pneumoniae type O2; and at least one saccharide derived from E. coli having a structure selected from the group consisting of formula O8 and formula O9.

In one embodiment, the invention provides a method of inducing an immune response against K. pneumoniae in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition comprising at least one glycoconjugate derived from E. coli serotype O8 or O9, wherein the immunogenic composition does not comprise a glycoconjugate derived from K. pneumoniae serotype O5 or O3. In one aspect, the composition comprises a saccharide derived from an E. coli O antigen having formula O8 and does not comprise a saccharide derived from K. pneumoniae serotype O5. In another aspect, the composition comprises a saccharide derived from an E. coli O antigen having formula O9 and does not comprise a saccharide derived from K. pneumoniae serotype O3.

In another embodiment, the invention provides a method of inducing an immune response against E. coli in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition comprising at least one glycoconjugate derived from K. pneumoniae serotype O5 or O3, or a variant thereof, wherein the immunogenic composition does not comprise a glycoconjugate derived from E. coli serotype O8 or O9. In one aspect, the composition comprises saccharides derived from K. pneumoniae serotype O5 and does not comprise a saccharide derived from an E. coli O antigen having formula O8. In another aspect, the composition comprises saccharides derived from K. pneumoniae serotype O3 and does not comprise a saccharide derived from an E. coli O antigen having formula O9.

In some embodiments, the composition comprises at least one saccharide that is or is derived from at least one K. pneumoniae serotype selected from O1 (and d-Gal-III variants), O2 (and d-Gal-III variants), O2ac, O3, O4, O5, O7, O8, and O12; and at least one saccharide derived from E. coli having a structure selected from the group consisting of formula O8 and formula O9. In some embodiments, the composition comprises at least one saccharide that is or is derived from at least one K. pneumoniae serotype selected from O1 (and d-Gal-III variants), O2 (and d-Gal-III variants), O2ac, O3, O4, O5, O7, O8, and O12; and at least one saccharide derived from E. coli having a structure selected from the group consisting of formula O1A, formula O1B, formula O2, formula O6, and formula O25B.

In some embodiments, the composition further comprises a polypeptide derived from K. pneumoniae selected from a polypeptide derived from K. pneumoniae type I pilus protein or an immunogenic fragment thereof; or a polypeptide derived from K. pneumoniae type III pilus 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 for the use of a composition described herein, a nucleic acid encoding an E. coli FimH polypeptide or a vector for expressing same, or a composition comprising the polypeptide or nucleic acid as a medicament or in the manufacture of a medicament, for raising an immune response against E. coli or K. pneumoniae or for preventing E. coli or K. pneumoniae infection in a subject.

In another aspect, the disclosure provides a method of eliciting an immune response against E. coli or 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 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, e.g., a vaccine, comprising an E. coli or K. pneumoniae composition described herein.

As used herein, "subject" means a mammal, preferably a human. In certain embodiments, the human is a child, e.g., an infant. In certain other embodiments, the human is a woman, particularly a pregnant woman. The compositions of the present invention can be administered to a subject with or without an adjuvant. The effective amount administered to the subject is an amount sufficient to elicit an immune response in the subject against E. coli or K. pneumoniae antigens. Subjects that can be selected for treatment include those at risk of developing an E. coli or K. pneumoniae infection due to exposure or potential exposure to E. coli or K. pneumoniae. Because humans can become infected with E. coli or K. pneumoniae by age 2, entire birth cohorts are included as appropriate populations for immunization. This can be done, for example, by initiating immunization regimens in pregnant women (or women of childbearing age) to protect infants still in utero by passive transfer of antibodies any time from birth to 6 months of age, from 6 months to 5 years of age, and in subjects over 50 years of age.

Administration of 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, transdermal, mucosal, or oral administration.

The total dose of the composition provided to a subject during a single administration may vary, as would be known to one of skill in the art.

It is also possible to provide one or more booster doses of one or more vaccine compositions. If a booster vaccination is performed, typically such a booster vaccination will be administered to the same subject one week to ten years, preferably two weeks to six months, after the subject is first administered the composition (in such a case referred to as a "primary vaccination"). In an alternative booster vaccination regimen, the subject may be administered a different vector, e.g., one or more adenoviruses, or other vectors, e.g., modified vaccinia virus Ankara (MVA), or DNA, or protein, after the primary vaccination. For example, the subject may be administered the recombinant viral vector as a primary vaccination and then boosted with the composition described herein.

In certain embodiments, the administration includes an initial administration and at least one booster administration. In certain other embodiments, the administration is provided annually. In yet other embodiments, the administration is provided annually along with an influenza vaccine.

The vaccines provided by the present disclosure can be used with one or more other vaccines. For example, in adults, they can be used with influenza vaccines, Prevnar, tetanus vaccines, diphtheria vaccines, and whooping cough vaccines. For use in children, the vaccines provided by the present disclosure can be used with any other vaccines indicated for pediatric patients.

In order to better understand the present invention, the following examples are set forth. These examples are merely illustrative and should not be construed as limiting the scope of the invention in any manner. The following examples are intended to illustrate some embodiments of the present invention.

Example 1
The bacterial pilus adhesins FimH and FmlH enable Escherichia coli to exploit distinct urinary tract microenvironments by recognition of specific host cell glycoproteins. FimH binds to mannosylated uroplakin receptors in urothelial cells, while FmlH binds to galactose or N-acetylgalactosamine O-glycans on epithelial surface proteins in the kidney and inflamed bladder. FimH fimbriae also play a role in colonization of the intestine by enterotoxigenic E. coli (ETEC) and multidrug-resistant invasive E. coli through binding to highly mannosylated proteins on the intestinal epithelium.

Full-length FimH is composed of two domains: an N-terminal lectin domain and a C-terminal pyrin domain, which are connected by a short linker. The lectin domain of FimH contains the carbohydrate recognition domain responsible for binding to mannosylated uroplakin 1a on the urothelial cell surface. The pyrin domain is anchored to the core of the pilus via the donor strand of the subsequent FimG subunit, a process called donor strand complementation.

The conformation and ligand-binding properties of the lectin domain of FimH are under the allosteric control of the pyrin domain of FimH. Under static conditions, the interaction of the two domains of full-length FimH stabilizes the lectin domain in a low affinity state for monomannose (e.g., _Kd ≈ 300 μM) characterized by a shallow binding pocket. Binding to a mannoside ligand induces a conformational change that leads to a moderate affinity state, in which the lectin and pyrin domains remain in close contact. However, upon shear stress, the lectin and pyrin domains separate, thereby inducing a high affinity state (e.g., _Kd < 1.2 μM).

Due to the absence of negative allosteric control provided by the pyrin domain, the isolated lectin domain of FimH is locked in a high affinity state. Isolated recombinant lectin domains locked in a high affinity state exhibit high stability. However, locking the adhesin in a low binding conformation induces the production of adhesin-inhibiting antibodies. Therefore, there is interest in stabilizing the lectin domain in a low affinity state.

Table 3 describes the FimH constructs used to generate various constructs to address these needs.

All FimH constructs tested were monomeric proteins of the expected molecular weight.

ＦｉｍＣ－ＦｉｍＨ複合体の予想分子量は、５３．１ｋＤａである；
ＦｉｍＣの予想分子量は、２４ｋＤａである。

The predicted molecular weight of the FimC-FimH complex is 53.1 kDa;
The predicted molecular weight of FimC is 24 kDa.

Example 2
Mammalian Expression of FimH Lectin Binding Domain This non-limiting example relates to the production of an E. coli derived polypeptide or fragment thereof in a HEK cell line. The yields were relatively high compared to the expression of an E. coli derived polypeptide or fragment thereof in an E. coli host cell.

To achieve production of FimH variants from mammalian cells, different non-homologous signal sequences were analyzed for secretion of proteins and fragments using the SignalP prediction algorithm. The wild-type FimH leader sequence was also analyzed. Predictions showed that the wild-type FimH leader sequence could function for secretion of FimH variants in mammalian cells, however, the secreted variants were predicted to be cleaved at the W20 residue of full-length wild-type FimH (see SEQ ID NO:1) rather than the F22 residue of full-length wild-type FimH (see SEQ ID NO:1). The hemagglutinin signal sequence was predicted to be non-functional. The mouse IgK signal sequence was predicted to produce the N-terminus of F22 of SEQ ID NO:1, or the F1 residue of the mature protein.

Based on these analyses, DNA was synthesized to recombinantly produce constructs expressing the FimH lectin binding domain containing the wild-type FimH leader. Constructs were also prepared to express the FimH lectin binding domain containing the mIgK signal sequence. To facilitate purification, an affinity purification tag, such as a His tag, was introduced at the C-terminus of the E. coli derived polypeptide or fragment thereof.

The expression plasmid was transfected into HEK host cells, i.e., EXPI293 mammalian cells.

Polypeptides or fragments thereof derived from E. coli were successfully expressed. For example, preferred N-terminal processing using the mIgK signal sequence fused to the maturation start of FimH at F22 was demonstrated by MS for the pSB01892 FimHdscG construct. Processing appears to be correct for the lectin domain construct pSB01878, and mass spectrometry data supports this.

The preferred N-terminal processing (i.e., processing at F22 in SEQ ID NO:1) was not demonstrated in the native FimH leader peptide.

The pSB01877 and pSB01878 constructs are in the pcDNA3.1(+) mammalian expression vector. Cells were diluted and subsequently used in a 20 ml transfection. 1 ug/ml DNA for each construct was used to transfect cells using the Expifectamine protocol in a 125 ml flask. After 72 hours, cell viability was still good so expression could be continued for up to 96 hours. Samples were taken at 72 hours and 10 ul of each was run on an SDS PAGE gel to check for expression.

After 96 hours, conditioned media was harvested and 0.25 ml of Nickel Excel resin was added in batch binding overnight at 4°C with rotation. Elution was in TrisCl pH 8.0, NaCl, imidazole. See Figure 4.

pSB01878 predicts a mass consistent with an N-terminal F22. Glycosylation is present at one or two sites (+1 mass from each N-D deamidation).

Glycosylation mutants were constructed. See, for example, pSB02081, pSB02082, pSB02083, pSB02088, and pSB02089. The glycosylation mutants expressed the polypeptide of interest. See Figure 5 for results.

FimH lectin domain lock mutants were also constructed. See, for example, pSB02158. Expression results of the pSB02158 construct are shown in Figure 6B.

Fluorescence polarization assay using 0.5 pmoles of fluorescein-conjugated aminophenyl-mannopyranoside (APMP). The assay was run at room temperature, 300 RPM, for 64 hours. The results are shown in Figure 6C.

Example 3
Mammalian expression of FimH/C complex, pSB01879 and pSB01880 To produce FimH/C complex, dual expression constructs were prepared with FimC under the EF1 alpha promoter and FimH containing either wild type or mIgK signal peptide. These were cloned into the pBudCE4.1 mammalian expression vector (ThermoFisher) and a C-terminal His tag was added to FimC. FimC variants were designed with the mIgK signal peptide for secretion because this gave a positive prediction to obtain 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 EF1 alpha promoter in vector pBudCE4.1 and the FimH fragment under the CMV promoter in the same vector. The vector pBudCE4.1 is an expression vector from Thermo Fisher with two promoters for expression in mammalian cells. The FimC fragment insert (pSB01881 insert) was subcloned by digesting with NotI and XhoI and subcloning into the pBudCE4.1 vector at the same sites. These were plated on 2xYT Zeocin 50ug/ml plates. Colonies were inoculated into 2xYT with Zeocin 50ug/ml and grown overnight at 37°C to prepare the plasmid. They were checked for inserts by digesting with NotI and XhoI and all colonies had an insert size of about 722bp.

pSB01881 was digested with HindIII and BamHI, and pSB01879 insert and pSB01880 insert DNA were digested with HindIII and BamHI. These fragments were gel isolated, subcloned into pSB01881 vector and plated onto 2xYTzeo 50ug/ml plates. Colonies from each were inoculated into 2xYT zeo 50ug/ml and grown overnight at 37°C, plasmids were prepared and digested with NotI and XhoI to test for FimC inserts and with HindIII and BamHI to test for FimH inserts. All clones had inserts of the predicted size at both cloning sites. pSB01879-1 and pSB01880-1 clones were subsequently used for expression.

The FimH/FimC complex has also been demonstrated to be expressed in EXPI293 cells. Expression can be optimized by switching promoters such as EF1α, CAG, Ub, Tub, or other promoters.

The preferred N-terminal processing (i.e., processing at F22 in SEQ ID NO:1) was not demonstrated in the native FimH leader peptide.

Shown below are exemplary results from SignalP4.1 (DTU Bioinformatics), used for signal peptide prediction. Additional signal peptides are predicted to produce a preferred N-terminus of Phe at position 1 of the mature FimH polypeptide or fragments thereof. Below is just a representative sample set of four common signal sequences.

The following signal peptide sequence was predicted to result in a preferred N-terminus of Phe at position 1 of the mature FimH polypeptide or fragment thereof:

The following signal peptide sequences are not predicted to result in a preferred N-terminus of Phe at position 1 of the mature FimH polypeptide or fragment thereof:

Example 4
Mammalian Expression of Donor Strand Complementary Fusions of FimH and FimG Peptides Several linker lengths were tested, and recombinant expression was set up using these linkers fusing FimH to the N-terminal FimG peptide in both wild-type FimH and the mlgK signal peptide fused to F22 of FimH.

The FimH donor strand-complemented FimG construct has also been shown to have robust expression in EXPI293 cells.

The preferred N-terminal processing (i.e., processing at F22 in SEQ ID NO:1) was not demonstrated in the native FimH leader peptide.

For donor strand complementation constructs, oligonucleotides were designed to produce base constructs containing various linkers and the FimG peptide in pcDNA3.1(+). A unique BstEII site was incorporated into FimH at residues G294 V295 T296 according to the numbering of SEQ ID NO:1. The same BstEII site was incorporated into the linker to produce the base constructs.

The base construct for pSB01882-01895 was constructed. Primers were used to PCR amplify pcDNA3.1(+) with ACCUPRIME PFX DNA polymerase (Thermo Fisher), the PCR product was digested with NdeI (in the CMV promoter) and BamHI, and cloned into pcDNA3.1(+) digested with NdeI and BamHI, and gel isolated to remove the fragment.

Separate transient transfections were performed with pSB01877, 01878, 01879, 01880, 01885, and 01892 together with EXPI293 cells as controls.

Constructs pSB01882 to pSB01895 were used in transient transfection expression studies in EXPI293 cells from Thermo Fisher following the manufacturer's protocol. See Figure 3 showing results after expression in 20mL EXPI293 cells for 72 hours with a 10ul conditioned media load; high levels of expression were observed; FimH/FimC complexes were present after expression from pSB01879 and pSB01880 constructs; 20ml conditioned media was batch bound to Nickel Excel, washed with 40CV, eluted in imidazole.

Additional FimH donor strand complementation constructs were prepared. See, for example, pSB02198, pSB02199, pSB02200, pSB02304, pSB02305, pSB02306, pSB02307, pSB02308 constructs. Expression of the pSB2198 FimH dscG lock mutant construct is shown in FIG. 7. The pSB2198 FimH dscG Lock mutant yielded 12 mg/L from transient expression.

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

Example 5
The molecular weight fragment has a signal peptide that is processed

Example 6
The N-terminal α-amino group of Phe1 (according to the numbering of SEQ ID NO:2) in the FimH mature protein provides important polar recognition for D-mannose Without being bound by theory or mechanism, it has been suggested that precise signal peptide cleavage just before Phe1 (according to the numbering of SEQ ID NO:2) of the FimH mature protein is important for expressing a functional FimH protein. Alteration of the N-terminal α-amino group, such as by adding amino acids at the N-terminus before Phe1 of the FimH protein, may disrupt hydrogen bonding interactions with the O2-, O5-, and O6-atoms of D-mannose, introducing steric repulsion with D-mannose, thereby blocking mannose binding. This is confirmed by our experimental observation that adding an extra Gly residue before Phe1 of SEQ ID NO:2 results in no detectable mannose binding.

After analysis of the crystal structure of FimH bound to D-mannose, the following was observed: the N-terminal α-amino group of Phe1, together with the side chains of Asp54 of FimH according to the numbering of SEQ ID NO:2 and Gln133 of FimH according to the numbering of SEQ ID NO:2, provide an important polar recognition motif for D-mannose, and mutations and alterations of these polar interactions abolish mannose binding.

(Example 7)
The side chain of Phe1 in FimH does not directly interact with D-mannose but rather is buried inside FimH, suggesting that Phe1 may be replaced by other residues, such as aliphatic hydrophobic residues (Ile, Leu, or Val).
Analysis of the crystal structures of FimH in complex with D-mannose and its analogues (e.g., PDB ID: 1QUN) indicates that the side chain of Phe1 (according to the numbering of SEQ ID NO:2) does not directly interact with D-mannose, but rather stabilizes the binding pocket by stacking its aromatic ring with the side chains of Val56, Tyr95, Gln133 and Phe144 (according to the numbering of SEQ ID NO:2).

Alternative N-terminal residues in place of Phe may stabilize the FimH protein, regulate mannose binding, and allow accurate signal peptide cleavage. Such residues have been identified by visual inspection of the crystal structure of FimH or by modeling of the protein using BioLuminate™ [BioLuminate, Schrödinger LLC, New York, 2017], Discovery Studio™ [Discovery Studio Modeling Environment, Dassault Systèmes, San Diego, 2017], MOE™ [Molecular Operating Environment, Chemical Computing Group Inc. , Montreal, 2017], and by more quantitative selection using computational protein design software such as Rosetta™ [Rosetta, University of Washington, Seattle, 2017]. Examples are shown in Figures 9A-9C. The replacement amino acid can be an aliphatic hydrophobic amino acid (e.g., Ile, Leu, and Val). Figure 11 depicts computational mutagenesis scanning of Phe1 with other amino acids, e.g., Ile, Leu, and Val, that have aliphatic hydrophobic side chains that can stabilize the FimH protein and modulate mannose binding.

(Example 8)
Mutation of Asn7, according to numbering of SEQ ID NO:2, in the FimH protein may remove a putative N-glycosylation site and prevent deamidation without affecting mannose, mAb21, or mAb475 binding.
Overexpression of secreted E. coli FimH from mammalian cell systems can result in N-linked glycosylation at residue Asn7 according to the numbering of SEQ ID NO: 2. In addition, residue Asn7 is highly susceptible to deamidation since it is exposed to solvent and is followed by a Gly residue.

Analysis of the crystal structure of FimH in complex with D-mannose and its analogs (e.g., PDB ID: 1QUN) indicates that Asn7 is more than 20 Å away from the mannose-binding site, and mutations at that site should not affect mannose binding. Thus, mutation of Asn7 to other amino acids (e.g., Ser, Asp, and Gln) could effectively remove the putative N-glycosylation site and prevent deamidation.

(Example 9)
E. coli and S. enterica strains Clinical and derivative strains are listed in Table 10. Additional reference strains included O25K5H1, a clinical O25a serotype strain; and S. enterica serotype Typhimurium strain LT2.

We constructed a gene knockout in an E. coli strain that removes the target open reading frame but leaves a short scar sequence.

For simplicity, the hydrolyzed O antigen chains and core sugar are shown next to the O polysaccharide (OPS).

(Example 10)
Oligonucleotide primers for wzzB, fepE and O antigen gene cluster cloning

(Example 11)
Plasmids Plasmid vectors and subclones are listed in Table 12. PCR fragments carrying 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 (FIGS. 12A-12B). This plasmid is based on a pUC replicon. Primers P3 and P4 were used to amplify the E. coli wzzB gene along with its native promoter, which are designed to bind to regions in the proximal and distal genes encoding UDP-glucose-6-dehydrogenase and phosphoribosyl adenine nucleotidyl hydrolase, respectively (annotated in Genbank MG1655 NC_000913.3). A PCR fragment containing the Salmonella fepE gene and promoter was amplified using primers previously described. Similar E. coli fepE primers were designed based on available Genbank genome sequences or in-house generated whole genome data (for GAR2401 and O25K5H1). The low copy number plasmid pBAD33 was used to express O-antigen biosynthesis genes under the control of the arabinose promoter. The plasmid was first modified to facilitate cloning (by 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 O25b biosynthesis operon is illustrated in Figures 12A-12B.

Example 12
O-antigen purification The fermentation broth was treated with acetic acid to a final concentration of 1-2% (final pH of 4.1). Extraction and delipidation of OAg was achieved by heating the acid-treated broth to 100° C. for 2 h. At the end of acid hydrolysis, the batch was cooled to ambient temperature and 14% NH ₄ OH was added to a final pH of 6.1. The neutralized broth was centrifuged and the centrate was collected. CaCl ₂ in sodium phosphate was added to the centrate and the resulting slurry was incubated at room temperature for 30 min. Solids were removed by centrifugation and the centrate was concentrated 12-fold using a 10 kDa membrane before being diafiltered twice against water. The retentate retaining OAg was then purified using a carbon filter. The carbon filtrate was diluted 1:1 (v/v) with 4.0 M ammonium sulfate. The final ammonium sulfate concentration was 2 M. The ammonium sulfate-treated carbon filtrate was further purified using a membrane with 2 M ammonium sulfate as the running buffer. OAg was collected in the effluent. For long OAg, the HIC filtrate was concentrated and then buffer exchanged against water (20 diavolumes) using a 5 kDa membrane. For short (native) OAg polysaccharides, the MWCO was further reduced to enhance yield.

Example 13
Conjugation of O25b long chain O antigen to CRM ₁₉₇ A first set of long chain O25b polysaccharide-CRM ₁₉₇ conjugates was produced using periodate oxidation followed by conjugation using reductive amination chemistry (RAC) (Table 14). The conjugates vary in three activation levels (low, medium and high) by varying the oxidation level. The conjugates were produced by reacting lyophilized activated polysaccharide with lyophilized CRM ₁₉₇ reconstituted in DMSO medium using sodium cyanoborohydride as the reducing agent. The conjugation reaction was carried out for 24 h at 23° C. followed by capping for 3 h using sodium borohydride. After the conjugation quenching step, the conjugates were purified by UF/DF using 100K MWCO regenerated cellulose membranes using 5 mM succinate/0.9% NaCl, pH 6.0. Final filtration of the conjugate was performed using a 0.22 μm membrane.

Unless expressly indicated otherwise, the conjugates disclosed throughout the examples below include a core sugar moiety.

Long Chain O Antigen Expression Provided by Heterologous Polymerase Chain Length Regulators Initial E. coli strain construction focused on the O25 serotype. The goal was to overexpress heterologous wzzB or fepE genes to see if they would provide longer chain lengths in an O25 wzzB knockout strain. First, blood isolates were screened by PCR to identify strains of O25a and O25b subtypes. Strains were then screened for susceptibility to ampicillin. One ampicillin-sensitive O25b isolate, GAR2401, was identified in which a wzzB deletion was introduced. Similarly, a wzzB deletion was made in O25a strain O25K5H1. For genetic complementation of these mutations, the wzzB genes from GAR2401 and O25K5H1 were subcloned into a high copy PCR-BluntII cloning vector and introduced into both strains by electroporation. Additional wzzB genes from E. coli K-12 and S. enterica serovar Typhimurium LT2; as well as the fepE genes from E. coli O25K5H1, GAR2401, O25a ETEC, NR-5, O157:H7:K- and S. enterica serovar Typhimurium LT2 were cloned and introduced.

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 OD ₆₀₀ units). LPS size was estimated by measuring a ladder visible from an internal native E. coli LPS standard and from a subset of samples showing a broad chain length distribution (differing by one repeat unit). On the left side of FIG. 13A, the LPS profile of a plasmid transformant of O25a O25K5HΔwzzB is shown; on the right side, a similar profile of an O25b GAR2401ΔwzzB transformant is shown. Immunoblots of replicate gels probed with O25-specific serum are shown in FIG. 13B.

The results of this experiment show that introduction of the homologous wzzB gene into the E. coli O25aΔwzzB host restores expression of the short O25 LPS (10-20x), similar to Salmonella LT2 wzzB. Introduction of the O25b wzzB gene from GAR2401 does not, suggesting that the WzzB enzyme from this strain is defective. Comparison of the E. coli WzzB amino acid sequence suggests that A210E and P253S substitutions may be responsible. Importantly, Salmonella LT2 fepE and E. coli fepE derived from O25a O25K5H1 confer the ability to express very long (VL) OAg LPS, with Salmonella LT2 fepE producing an OAg that exceeds in size that produced by E. coli fepE.

A similar pattern of expression was observed for GAR2401ΔwzzB transformants: E. coli O25a or K12 strain wzzB restored the ability to produce short LPS. Salmonella LT2 fepE produced the longest LPS, E. coli fepE produced a slightly shorter LPS, while Salmonella LT2 wzzB yielded an intermediate-sized long LPS (L). The ability of other E. coli fepE genes to produce very long LPS was evaluated in separate experiments using transformants of E. coli O25aΔwzzB. The fepE genes from GAR2401, an O25a ETEC strain, and an O157 Shigella toxin-producing strain also conferred the ability to produce very long LPS, although not as long as the LPS produced for Salmonella LT2 fepE (Figure 14).

Once Salmonella LT2 fepE was established in serotype O25a and O25b strains, which generate the longest LPS of the polymerase regulators evaluated, we next sought to determine whether it also produces very long LPS in other E. coli serotypes. Wild-type bacteremia isolates of serotypes O1, O2, O6, O15, and O75 were transformed with the Salmonella fepE plasmid and extracted LPS. The results, shown in FIG. 15, confirm that Salmonella fepE can confer the ability to make very long LPS in other common serotypes associated with blood infections. The results also show that plasmid-based expression of Salmonella fepE appears to override the chain length control normally exhibited by endogenous wzzB in these strains.

Plasmid-based expression of O-antigens in a common E. coli host strain From a bioprocess development perspective, the ability to produce O-antigens of different serotypes in a common E. coli host instead of multiple strains would greatly simplify the production 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 the control of an arabinose-regulated promoter. This plasmid is compatible with (and can co-exist with) the Salmonella LT2 fepE plasmid in E. coli, as it carries a different (p15a) replicon and a different selection marker (chloramphenicol vs. kanamycin). In initial experiments, pBAD33 O25b operon plasmid subclones were co-transfected with the Salmonella LT2 fepE plasmid into GAR2401ΔwzzB and transformants were grown in the presence or absence of 0.2% arabinose. The results, shown in Figures 16A-16B, indicated that very long O-antigen LPS was produced in an arabinose-dependent manner.

Cloned O-antigen gene clusters from other serotypes were similarly evaluated, and the results are shown in Figure 17. Co-expression of Salmonella LT2 fepE with pBAD33-OAg plasmid yielded detectable long-chain LPS corresponding to O1, O2 (for two of the four clones), O16, O21, and O75 serotypes. For unknown reasons, pBAD33-O6 plasmid failed to yield detectable LPS in all four isolates tested. Although expression levels were variable, the results indicate that expression of long-chain O-antigens in a common host is feasible. However, in some cases, further optimization to improve expression, for example by modification of the plasmid promoter sequence, may be necessary.

The profiles of LPS from different serotype O25 E. coli strains with or without the Salmonella LT2 fepE plasmid are shown in Figure 18. Two strains were tested for fermentation, extraction and purification of O antigens: GAR2831 for the production of native short O25b OAg; and GAR2401ΔwzzB/fepE for the production of long O25b OAg. The corresponding short and long forms of LPS shown in the SDS-PAGE gel in Figure 18 are highlighted in red. Polysaccharides were extracted and purified directly from the fermented bacteria using acetic acid. Size exclusion chromatography profiles of purified short and long or very long O25b polysaccharides are shown in Figures 19A-19B. The properties of the two lots of short polysaccharide (from GAR2831) are compared to a single very long polysaccharide preparation (from strain GAR2401ΔwzzB/fepE). The molecular weight of the long O antigen was 3.3 times larger than that of the short O antigen, and the number of repeat units was estimated to be about 65 (very long) and about 20. See Table 13.

The very long O25b O antigen polysaccharide was conjugated to diphtheria toxoid CRM ₁₉₇ using a conventional reductive amination process. Three different lots of the sugar conjugate were prepared with varying degrees of periodate activation: medium (5.5%), low (4.4%) and high (8.3%). The resulting preparations and the unconjugated polysaccharide were shown to be free of endotoxin contamination (Table 14).

A group of four rabbits (New Zealand White females) were vaccinated with 10mcg of the sugar conjugate and 20mcg of QS21 adjuvant, respectively, according to the schedule shown in Figure 20A, and serum was collected (VAC-2017-PRL-EC-0723). It is noteworthy that the 10mcg dose is at the lower end of the range routinely administered to rabbits in the evaluation of bacterial sugar conjugates (20-50mcg being more typical). One group of rabbits was also vaccinated with unconjugated polysaccharide, using the same dose (10mcg polysaccharide + 20mcg QS21 adjuvant) and the same dosing schedule in a separate study (VAC-2017-PRL-GB-0698).

Rabbit antibody responses to the three O25b glycoconjugate preparations were evaluated in a LUMINEX assay in which carboxybeads were coated with methylated human serum albumin pre-bound with unconjugated O25b long polysaccharide. The presence of O25b-specific IgG antibodies in serum samples was detected using a phycoerythrin (PE)-labeled anti-IgG secondary antibody. The profile of the immune response observed in sera collected at week 0 (pre-immunization), week 6 (post-dose 2, PD2), week 8 (post-dose 3, PD3) and week 12 (post-dose 4, PD4) in the best responding rabbits (one from each group of four) is shown in Figures 21A-21C. No significant pre-immunization serum IgG titers were detected in any of the twelve rabbits. In contrast, O25b antigen-specific antibody responses were detected in post-vaccination sera from rabbits in all three groups, with responses in the low activation glycoconjugate group tending to be slightly higher than in the medium or high activation glycoconjugate groups. Maximal responses were observed by the post-dose 3 time point. One rabbit in the low activation group and one rabbit in the high activation group failed to respond to vaccination (non-responders).

To assess the impact of CRM ₁₉₇ carrier protein conjugation on the immunogenicity of long O25b OAg polysaccharide, the presence of antibodies in sera from rabbits vaccinated with unconjugated polysaccharide was compared to sera from rabbits vaccinated with the less active CRM ₁₉₇ glycoconjugate (FIGS. 22A-22F). Unexpectedly, free polysaccharide was not immunogenic and elicited virtually no IgG responses in immune and pre-immune sera (FIG. 22A). In contrast, over a range of serum dilutions (1:100-1:6400), O25b OAg-specific IgG mean fluorescence intensity values (MFI) approximately 10-fold higher than pre-immune serum levels were observed in PD4 sera from three of four rabbits vaccinated with O25b OAg-CRM ₁₉₇ . These results demonstrate the necessity of carrier protein conjugation to generate IgG antibodies against O25b OAg polysaccharide at the 10 mcg dose level.

Bacteria grown on TSA plates were suspended in PBS, adjusted to an OD of _2.0 , and fixed in 4% paraformaldehyde in PBS. After blocking for 1 h in 4% BSA/PBS, bacteria were incubated with serial dilutions of pre-immune and PD3 immune sera in 2% BSA/PBS, and bound IgG was detected with a PE-labeled secondary F(ab) antibody.

The specificity of the O25b antibodies elicited by O25b 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 Figures 23A-23C, pre-immune rabbit antibodies were unable to bind to wild-type serotype O25b isolates GAR2831 and GAR2401 or the K-12 E. coli strain, whereas the matched PD3 antibody stained O25b bacteria in a concentration-dependent manner. The negative control K-12 strain, lacking the ability to express OAg, showed only very weak binding of the PD3 antibody, most likely due to the presence of exposed inner core oligosaccharide epitopes on its surface. Introduction of the Salmonella fepE plasmid into wild-type O25b isolates resulted in significantly enhanced staining, consistent with a higher density of immunogenic epitopes provided by the longer OAg polysaccharide.

Conclusion: The described results show that Salmonella fepE is not only a determinant of very long O-antigen polysaccharides in Salmonella species, but can also confer the ability to make very long OAgs to E. coli strains of different O-antigen serotypes. This property can be exploited to produce O-antigen vaccine polysaccharides with improved properties for bioprocess development by facilitating purification and chemical conjugation to suitable carrier proteins, as well as potentially enhancing immunogenicity through the formation of higher molecular weight conjugates.

(Example 14)
Initial rabbit studies generated IgG responses to the first polyclonal antibody reagent and RAC O25b OAg-CRM _197. Long chain O25b polysaccharide-CRM ₁₉₇ conjugates were produced using periodate oxidation followed by conjugation using reductive amination chemistry (RAC) (Table 14). See also Table 24.

In rabbit study 1 (VAC-2017-PRL-EC-0723) (also described above in Example 13), 5 rabbits/group using 10 μg of L-, M- or H-activated RAC (+QS21) received the composition according to the schedule shown in FIG. 20A. Unconjugated free O25b polysaccharide was observed to be non-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 using L-RAC (AlOH ₃ , QS21, or no adjuvant) received the 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 O25b polysaccharide described in Example 13 and had their sera tested at week 18.

More specifically, a composition comprising 50 μg unconjugated O25b, 100 μg AlOH ₃ adjuvant was administered to rabbit 4-1. A composition comprising 50 μg unconjugated O25b, 100 μg AlOH ₃ adjuvant was administered to rabbit 4-2. A composition comprising 50 μg unconjugated O25b, 50 μg QS-21 adjuvant was administered to rabbit 5-1. A composition comprising 50 μg unconjugated O25b, 50 μg QS-21 adjuvant was administered to rabbit 5-2. A composition comprising 50 μg unconjugated O25b and no adjuvant was administered to rabbit 6-1. A composition comprising 50 μg unconjugated O25b and no adjuvant was administered to rabbit 6-2.

(Example 15)
Rabbit Study with O25b RAC Conjugate: dLIA Serum Dilution Titers O25b dLIA serum dilution titers from rabbit study 2 (VAC-2018-PRL-EC-077) versus the best responding rabbit from study 1 (VAC-2017-PRL-EC-0723). For these experiments, a modified direct binding Luminex assay was performed in which a polylysine conjugate of O25b long chain O antigen was passively adsorbed onto Luminex carboxy beads in place of the methylated serum albumin long chain O antigen mixture previously described. The use of polylysine-O25b conjugate improved the sensitivity of the assay and the quality of the IgG concentration dependent response and allowed the determination of serum dilution by use of curve fitting (4 parameter non-linear equation). O25b IgG titers in serum from the highest titer rabbit from the first study are compared to serum from the second study rabbit in Table 15.

Higher doses in a second rabbit study (50/20 μg vs. 10 μg) did not improve IgG titers.

A two-month break enhances IgG responses (not observed with shorter intervals).

Alum appears to enhance IgG responses in rabbits compared to QS21 or no adjuvant.

An opsonophagocytosis assay (OPA) using baby rabbit complement (BRC) and HL60 cells as a source of neutrophils was established to measure the functional immunogenicity of O-antigen glycoconjugates. Pre-frozen bacterial stocks of E. coli GAR2831 were grown in Luria Broth (LB) medium at 37° C. Cells were pelleted, suspended in PBS supplemented with 20% glycerol at a concentration of 1 OD ₆₀₀ units per ml, and frozen. Pre-titrated and thawed bacteria were diluted to ^0.5x105 CFU/ml in HBSS (Hank's Balanced Salt Solution) containing 1% gelatin, 10μL ( ¹⁰³ CFU) was mixed with 20μL of serially diluted serum in a U-bottom tissue culture microplate, and the mixture was shaken at 700 rpm (BELLCO Shaker) for 30min at 37°C in a 5% _CO2 incubator. 10 μl of 2.5% complement (baby rabbit serum, PEL-FREEZ 31061-3, prediluted in HBG) and 20 μL of HL-60 cells (0.75×10 ⁷ /ml) and 40 μL of HBG were added to a U-bottom tissue culture microplate and the mixture was shaken at 700 rpm (BELLCO Shaker) for 45 min at 37° C. in a 5% CO ₂ incubator. 10 μL of each 100 μL reaction was then transferred to the corresponding well of a pre-wetted MILLIPORE MULTISCREENHTS HV filter plate that had been prepared by adding 100 μL of water, vacuum filtering, and adding 150 μL of 50% LB. The filter plate was vacuum filtered and incubated overnight at 37° C. in a 5% CO ₂ incubator. The following day, colonies were counted after fixing, staining, and destaining with COOMASSIE dye and destaining solutions using an IMMUNOSPOT® analyzer and IMMUNOCAPTURE software. To establish the specificity of OPA activity, immune sera were preincubated with 100 μg/mL purified long chain O25b O antigen before mixing with the other assay components in the OPA reaction. The OPA assay includes control reactions that do not contain HL60 cells or complement to demonstrate the dependence of the 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 were determined (Table 16, Figures 26A-26B). Pre-incubation with unconjugated O25b long chain O-antigen polysaccharide blocked bactericidal activity indicating the specificity of OPA (Figure 19C). Table 16 shows the OPA titers.

Rabbit 2-3 was dosed as follows: Rabbit 2-3 dosing: 10/10/10/10 μg RAC conjugate + QS21, bled after dose 4 (PD). Rabbit 1-2 was dosed as follows: 50/20/20/20 μg RAC conjugate + Al(OH) ₃ , bled on PD4.

(Example 16)
O-antigen O25b IgG levels elicited by unconjugated O25b long chain O-antigen polysaccharide and induced O25b RAC/DMSO long chain O-antigen saccharide conjugate Groups of 10 CD-1 mice were administered 0.2 or 2.0 μg/animal O25b RAC/DMSO long chain O-antigen saccharide conjugate by subcutaneous injection at weeks 0, 5 and 13 and bled at weeks 3 (after dose 1, PD1), 6 (after dose 2, PD2) and 13 (after dose 3, PD3) for immunogenicity studies. Antigen-specific IgG levels were determined by quantitative Luminex assay (see details in Example 15) using an O25b-specific mouse mAb as an internal standard. Baseline IgG levels (dotted line) were determined in pooled serum from 20 randomly selected non-vaccinated mice. Free unconjugated O25b long chain O antigen polysaccharide immunogen did not induce IgG above baseline levels at any time point. In contrast, IgG responses were observed after two doses of the O25b-CRM197 RAC long chain conjugate glycoconjugate: robust and homogenous IgG responses were observed by PD3, with intermediate and more variable IgG levels observed at PD2. GMT IgG values (ng/ml) are shown with 95% CI error bars. See Figures 27A-27C.

(Example 17)
Specificity of O25b Baby Rabbit Complement (BRC) OPA A-B) O25b RAC/DMSO long chain O antigen post-immune sera from rabbits 2-3 and 1-2 (but not matched pre-immune control sera) show bactericidal OPA activity. C) OPA activity of immune sera from rabbits 1-2 was blocked by pre-incubation with 100 μg/mL long chain O antigen O25b polysaccharide. GAR2831 bacterial strain was incubated with serial dilutions of HL60, 2.5% BRC and serum for 1 h at 37° C. and viable bacteria were enumerated by counting microcolonies (CFU) on filter plates. See Figures 26A-26C.

(Example 18)
RAC and eTEC O25b long chain glycoconjugates are more immunogenic than single terminal glycoconjugates BRC OPA assay with carbapenem-resistant fluoroquinolone-resistant MDR strain Atlas187913. Groups of 20 CD-1 mice were vaccinated with 2 μg glycoconjugates according to the same schedule as shown in Figures 28A-28B and OPA responses were determined after dose 2 (PD2) (Figure 28A) and dose 3 (PD3) (Figure 28B). Bars indicate GMT with 95% CI. Percentage of responders above unvaccinated baseline is shown. Log-transformed data from different groups were evaluated to assess whether differences were statistically significant using unpaired t-tests with Welch's correction (Graphpad Prism). Results are summarized in Table 17. See Figures 28A-28B. In mice vaccinated with 2 μg of eTEC O1a long-chain glycoconjugate, OPA titers against O1a at PD2 and PD3 (data not shown) were observed to be higher than OPA titers against O25b at PD2 and PD3, respectively, as shown in Table 17.

(Example 19)
OPA immunogenicity of eTEC chemistry can be improved by modifying the level of polysaccharide activation BRC OPA assay with carbapenem-resistant fluoroquinolone-resistant MDR strain Atlas187913. Groups of 20 CD-1 mice were vaccinated with 0.2 μg or 2 μg of the indicated long-chain O25b eTEC glycoconjugates and OPA responses were determined at PD2. Pooled log-transformed data from the 4% and 17% activation groups were evaluated to determine statistically significant differences in OPA responses using an unpaired t-test with Welch's correction (Graphpad Prism). GMTs and percentage of responders for individual groups are summarized in Table 18. See Figure 29.

(Example 20)
Challenge studies show that long chain E. coli O25b eTEC conjugates elicit protection after three doses Groups of 20 CD-1 mice immunized with a 2 μg dose according to the indicated schedule were challenged IP with 1×10 ⁹ bacteria of GAR2831 strain. Survival was monitored over a 6 day period thereafter. Groups of mice vaccinated with activated eTEC glycoconjugates at levels of 4%, 10% or 17% were protected from lethal infection, whereas unvaccinated control mice or mice vaccinated with 2 μg of unconjugated O25b long chain polysaccharide were not. See Figures 30A-30B.

(Example 21)
Process for the preparation of eTEC-linked sugar conjugates Sugar activation and thiolation with cystamine dihydrochloride. Sugars are reconstituted in anhydrous dimethylsulfoxide (DMSO). The water content of the solution is determined by Karl Fischer (KF) analysis and adjusted to reach a water content of 0.1-1.0%, typically 0.5%.

To initiate activation, a solution of 1,1'-carbonyl-di-1,2,4-triazole (CDT) or 1,1'-carbonyldiimidazole (CDI) is freshly prepared in DMSO at a concentration of 100 mg/mL. The sugar 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 35°C. Water was added to quench residual CDI/CDT in the activation reaction solution. The amount of water to be added is determined and calculations are performed to ensure that the final water content is 2-3% of the total water content. The reaction is allowed to proceed for 0.5 hours at rt. Cystamine dihydrochloride is freshly prepared in anhydrous DMSO at a concentration of 50 mg/mL. The activated sugar is reacted with 1-2 molar equivalents of cystamine dihydrochloride. Alternatively, the activated sugar is reacted with 1-2 molar equivalents of cystamine hydrochloride. The thiolation reaction is allowed to proceed at rt for 5-20 hours to produce thiolated sugars. The thiolation level is determined by the amount of CDT/CDI added.

Reduction and purification of activated thiolated sugars. To the reaction mixture of thiolated sugars, 3-6 molar equivalents of a solution of tris(2-carboxyethyl)phosphine (TCEP) are added and allowed to proceed at rt for 3-5 hours. The reaction mixture is then diluted 5-10-fold by addition to pre-chilled 10 mM sodium dihydrogen phosphate and filtered through a 5 μm filter. Diafiltration of the thiolated sugar is performed against 30-40 diavolumes of pre-chilled 10 mM sodium dihydrogen phosphate. An aliquot of the retentate of activated thiolated sugar is removed and sugar concentration and thiol content are determined (Ellman assay).

Activation and purification of bromoacetylated carrier protein. Free amino groups of the carrier protein are bromoacetylated by reaction with a bromoacetylating agent such as bromoacetic acid N-hydroxysuccinimide ester (BAANS), bromoacetyl bromide, or another suitable reagent.

Carrier protein (in 0.1 M sodium phosphate, pH 8.0±0.2) is first held at pH 7 and 8±3°C prior to activation. To the protein solution is added N-hydroxysuccinimide ester of bromoacetic acid (BAANS) as a dimethylsulfoxide (DMSO) stock solution (20 mg/mL) at a ratio of 0.25-0.5 BAANS:protein (w/w). The reaction is mixed gently for 30-60 minutes at 5±3°C. The resulting bromoacetylated (activated) protein is purified, for example, by UF/DF using a 10 kDa MWCO membrane using 10 mM phosphate (pH 7.0) buffer. After purification, the protein concentration of the bromoacetylated carrier protein is estimated by Lowry's protein assay.

The degree of activation is determined by a total bromide assay by ion-exchange liquid chromatography (ion chromatography) with suppressed conductivity detection. Bound bromide on activated bromoacetylated proteins is cleaved from the proteins in the assay sample preparation and quantified together with any free bromide that may be present. Remaining covalently bound bromide on the protein is liberated by converting it 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% NaCl pH 7 (PBS) and then brought to 0.1 M NaHCO ₃ pH 7.0 using a 1 M stock solution. BAANS was added at a CRM ₁₉₇ :BAANS ratio of 1:0.35 (w:w) using a BAANS stock solution in DMSO at 20 mg/mL. The reaction mixture was incubated at 3° C.-11° C. for 30 min-1 hr and then purified by UF/DF using a 10K MWCO membrane and 10 mM sodium phosphate/0.9% NaCl, pH 7.0. The purified activated CRM ₁₉₇ was assayed by Lowry assay to determine protein concentration and then diluted to 5 mg/mL in PBS. Sucrose was added as a cryoprotectant to a concentration of 5% wt/vol, and the activated protein was frozen and stored at −25° C. until required for conjugation.

Bromoacetylation of lysine residues in CRM ₁₉₇ was highly consistent, resulting in activation of 15-25 lysines from the 39 available lysines. The reaction produced high yields of activated protein.

Conjugation of activated thiolated sugars to bromoacetylated carrier proteins. The bromoacetylated carrier protein and activated thiolated sugars are then added. The sugar/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 for 20±4 hours at 5° C.

Capping of remaining reactive functional groups. Unreacted bromoacetylated residues on the carrier protein are quenched by reaction with 2 molar equivalents of N-acetyl-L-cysteine as a capping reagent at 5°C for 3-5 hours. Remaining free sulfhydryl groups are capped with 4 molar equivalents of iodoacetamide (IAA) at 5°C for 20-24 hours.

Purification of eTEC-bound glycoconjugates. The conjugation reaction (post IAA capping) mixture is filtered through a 0.45 μm filter. Ultrafiltration/diafiltration of the glycoconjugate is performed against 5 mM succinate-0.9% saline, pH 6.0. The glycoconjugate retentate is then filtered through a 0.2 μm filter. An aliquot of the glycoconjugate is removed for assay. The remaining glycoconjugate is stored at 5° C. See Tables 21, 22, 23, 24, and 25.

(Example 22)
Preparation of E. coli-O25B ETEC conjugate Activation process - Activation of E. coli-O25b lipopolysaccharide. Lyophilized E. coli-O25b polysaccharide was reconstituted in anhydrous dimethyl sulfoxide (DMSO). The water content of the lyophilized O25b/DMSO solution was determined by Karl Fischer (KF) analysis. The water content was adjusted by adding WFI to the O25b/DMSO solution to achieve a water content of 0.5%.

To initiate activation, 1,1'-carbonyldiimidazole (CDI) was freshly prepared as 100 mg/mL in DMSO solution. Prior to the thiolation step, E. coli-O25b polysaccharide was activated with various amounts of CDI. CDI activation was carried out at rt or 35°C for 1-3 h. Water was added to quench residual CDI in the activation reaction solution. The amount of water to be added was determined and calculations were performed to ensure that the final water content was 2-3% of the total water content. The reaction was allowed to proceed for 0.5 h at rt.

Thiolation of activated E. coli-O25b polysaccharide. Cystamine dihydrochloride was freshly prepared in anhydrous DMSO, and 1-2 molar equivalents of cystamine dihydrochloride were added to the reaction solution of activated polysaccharide. The reaction was allowed to proceed for 20±4 hours at rt.

Reduction and purification of activated thiolated E. coli-O25b polysaccharide. A solution of 3-6 molar equivalents of tris(2-carboxyethyl)phosphine (TCEP) was added to the thiolated sugar reaction mixture and allowed to proceed at rt for 3-5 hours. The reaction mixture was then diluted 5-10 fold by addition to pre-chilled 10 mM sodium dihydrogen phosphate and filtered through a 5 μm filter. Diafiltration of the thiolated sugar was performed against 40 diavolumes of pre-chilled 10 mM sodium dihydrogen phosphate using a 5K MWCO ultrafiltration membrane cassette. The thiolated O25b polysaccharide retentate was removed for both sugar concentration and thiol (Ellman) assays. A flow diagram of the activation process is provided in FIG. 32A.

Conjugation process - conjugation of thiolated E. coli-O25b polysaccharide to bromoacetylated CRM _197. CRM 197 carrier protein was separately activated by bromoacetylation as described in Example 21 and then reacted with activated E. coli-O25b polysaccharide for the conjugation reaction. Bromoacetylated CRM _{197 and} thiolated O25b polysaccharide were mixed together in a reaction vessel. The sugar/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 for 20±4 hours at 5°C.

Capping of reactive groups on bromoacetylated CRM ₁₉₇ and thiolated E. coli-O25b polysaccharide. Unreacted bromoacetylated residues on CRM ₁₉₇ protein were capped by reaction with 2 molar equivalents of N-acetyl-L-cysteine at 5° C. for 3-5 hours, followed by capping of remaining free sulfhydryl groups on the thiolated O25b-polysaccharide with 4 molar equivalents of iodoacetamide (IAA) at 5° C. for 20-24 hours.

Purification of eTEC-bound E. coli-O25b glycoconjugates. The conjugation solution was filtered through 0.45 μm or 5 μm filters. Diafiltration of the O25b glycoconjugates was performed using a 100K MWCO ultrafiltration membrane cassette. Diafiltration was performed against 5 mM succinate-0.9% saline, pH 6.0. The E. coli-O25b glycoconjugate 100K retentate was then filtered through a 0.22 μm filter and stored at 5°C.

A flow diagram of the conjugation process is provided in Figure 32B.

Results Reaction parameters and characterization data for several batches of E. coli-O25b eTEC glycoconjugates are shown in Table 19. CDI activation-thiolation with cystamine dihydrochloride produced glycoconjugates with sugar yields of 41-92% and less than 5% to 14% free sugar. See also Tables 21, 22, 23, 24, and 25.

(Example 23)
Procedure for preparation of E. coli O-antigen polysaccharide-CRM ₁₉₇ eTEC conjugate (applies to O-antigens from E. coli serotypes O25b, O1a, O2, and O6)
Polysaccharide Activation. E. coli O antigen polysaccharide is reconstituted in anhydrous dimethylsulfoxide (DMSO). To initiate activation, various amounts of 1,1'-carbonyldiimidazole (CDI) (1-10 molar equivalents) are added to the polysaccharide solution and the reaction is allowed to proceed for 1-5 hours at rt or 35°C. Water (2-3%, v/v) is then added to quench residual CDI in the activation reaction solution. The reaction is allowed to proceed for 0.5 hours at rt before adding 1-2 molar equivalents of cystamine dihydrochloride. The reaction is allowed to proceed for 5-20 hours at rt before being treated with 3-6 molar equivalents of tris(2-carboxyethyl)phosphine (TCEP) to produce thiolated saccharides. The thiolation level is determined by the amount of CDI added.

The reaction mixture is then diluted 5-10 fold by addition to pre-chilled 10 mM sodium dihydrogen phosphate and filtered through a 5 μm filter. Diafiltration of the thiolated sugar is performed against 30-40 diavolumes of pre-chilled 10 mM sodium dihydrogen phosphate. An aliquot of the retentate of activated thiolated sugar is removed and sugar concentration and thiol content are determined (Ellman assay).

Activation of carrier protein (CRM ₁₉₇ ). CRM ₁₉₇ (in 0.1 M sodium phosphate, pH 8.0±0.2) is initially held at pH ≈8 and 8±3° C. prior to activation. To the protein solution is added N-hydroxysuccinimide ester of bromoacetic acid (BAANS) as a dimethylsulfoxide (DMSO) stock solution (20 mg/mL) at a ratio of 0.25-0.5 BAANS:protein (w/w). The reaction is mixed gently for 30-60 minutes at 5±3° C. The resulting bromoacetylated (activated) protein is purified, for example, by UF/DF using a 10 kDa MWCO membrane using 10 mM phosphate (pH 7.0) buffer. After purification, the protein concentration of the bromoacetylated carrier protein is estimated by Lowry's protein assay.

Conjugation. The activated CRM ₁₉₇ and activated E. coli O antigen polysaccharide are then added to the reactor and mixed. The sugar/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 for 20±4 hours at 5° C. Unreacted bromoacetylated residues on the carrier protein are quenched by reacting with 2 molar equivalents of N-acetyl-L-cysteine as a capping reagent for 3-5 hours at 5° C. Remaining free sulfhydryl groups are capped with 4 molar equivalents of iodoacetamide (IAA) for 20-24 hours at 5° C. The reaction mixture is then purified using UF/DF performed against 5 mM succinate-0.9% saline, pH 6.0. The purified conjugate is then filtered through a 0.2 μm filter. See Tables 21, 22, 23, 24, and 25.

(Example 24)
General Procedure - Conjugation of O-Antigen (from E. coli serotypes O1, O2, O6, 25b) polysaccharides by reductive amination chemistry (RAC) Conjugation in dimethylsulfoxide (RAC/DMSO)
Polysaccharide activation. Polysaccharide oxidation was carried out in 100 mM sodium phosphate buffer (pH 6.0±0.2) by sequential addition of calculated amounts of 500 mM sodium phosphate buffer (pH 6.0) and water for injection (WFI) to obtain a final polysaccharide concentration of 2.0 g/L. If necessary, the reaction pH was adjusted to approximately pH 6.0. After pH adjustment, the reaction temperature was cooled to 4° C. Oxidation was initiated by the addition of approximately 0.09-0.13 molar equivalents of sodium periodate. The oxidation reaction was carried out at 5±3° C. for approximately 20±4 h.

Concentration and diafiltration of the activated polysaccharides were performed using a 5K MWCO ultrafiltration cassette. Diafiltration was performed against 20x diavolume of WFI. The purified activated polysaccharides were then stored at 5±3°C. The purified activated sugars are characterized by, inter alia, (i) sugar concentration by colorimetric assay; (ii) aldehyde concentration by colorimetric assay; (iii) degree of oxidation; and (iv) molecular weight by SEC-MALLS.

Blending of activated polysaccharide with sucrose excipient and lyophilization. The activated polysaccharide was blended with sucrose in a ratio of 25 grams of sucrose per gram of activated polysaccharide. The bottles of blended mixture were then lyophilized. After lyophilization, the bottles containing the lyophilized activated polysaccharide were stored at -20±5°C. The calculated amount of CRM ₁₉₇ protein was shell frozen and lyophilized separately. The lyophilized CRM ₁₉₇ was stored at -20±5°C.

Reconstitution of lyophilized activated polysaccharide and carrier protein. The lyophilized activated polysaccharide was reconstituted in anhydrous dimethylsulfoxide (DMSO). Upon complete dissolution of the polysaccharide, an equal volume of anhydrous DMSO was added to the lyophilized CRM ₁₉₇ for reconstitution.

Conjugation and Capping. The renatured activated polysaccharide was combined with the renatured CRM ₁₉₇ in a reaction vessel and mixed thoroughly to obtain a clear solution before initiating conjugation with sodium cyanoborohydride. The final polysaccharide concentration in the reaction solution was approximately 1 g/L. Conjugation was initiated by adding 0.5-2.0 molar equivalents of sodium cyanoborohydride to the reaction mixture and incubating at 23±2° C. for 20-48 h. The conjugation reaction was terminated by adding 2 molar equivalents of sodium borohydride (NaBH ₄ ) to cap unreacted aldehydes. The capping reaction continued for 3±1 h at 23±2° C.

Conjugate purification. 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 membranes with 100-300K MWCO.

The diluted conjugate solution was passed through a 5 μm filter and diafiltration was performed using 5 mM succinate/0.9% saline (pH 6.0) as the medium. After diafiltration was completed, the conjugate retentate was transferred through a 0.22 μm filter. The conjugate was further diluted with 5 mM succinate/0.9% saline (pH 6) to a target sugar concentration of approximately 0.5 mg/mL. Alternatively, the conjugate is purified by tangential flow filtration using a 100-300K MWCO membrane using 20 mM histidine-0.9% saline (pH 6.5). A final 0.22 μm 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) adapted from E. coli serotypes O25B, O1A, O2, and O6. Activation and diafiltration of the polysaccharides were carried out in the same manner as for the DMSO-based conjugation.

The filtered activated saccharide was mixed with CRM ₁₉₇ at a polysaccharide to protein mass ratio ranging from 0.4 to 2 w/w, depending on the serotype. This input ratio was chosen to control the polysaccharide to CRM ₁₉₇ ratio in the resulting conjugate.

The combined mixture was then lyophilized. During conjugation, the polysaccharide and protein mixture was dissolved in 0.1 M sodium phosphate buffer at polysaccharide concentrations ranging from 5 to 25 g/L depending on the serotype, and the pH was adjusted to 6.0 to 8.0 depending on the serotype. Conjugation was initiated by adding 0.5 to 2.0 molar equivalents of sodium cyanoborohydride to the reaction mixture and incubating at 23±2° C. for 20 to 48 h. The conjugation reaction was terminated by adding 1 to 2 molar equivalents of sodium borohydride (NaBH ₄ ) to cap unreacted aldehydes.

Alternatively, the filtered activated saccharide and the calculated amount of CRM ₁₉₇ protein can be shell frozen and lyophilized separately, then mixed upon dissolution in 0.1 M sodium phosphate buffer before conjugation proceeds as described above.

(Example 26)
Procedure for the preparation of E. coli O-antigen polysaccharide-CRM ₁₉₇ single-ended conjugate Lipopolysaccharide (LPS), a common component of the outer membrane of gram-negative bacteria, comprises lipid A, a core region, and an O-antigen (also called O-specific polysaccharide or O-polysaccharide). The O-antigen repeating units of different serotypes differ in their composition, structure and serological characteristics. The O-antigen used in the present invention is linked to a core domain that contains a saccharide unit called 2-keto-3-deoxyoctanoic acid (KDO) at its chain end. It differs from some conjugation methods that are based on random activation of the polysaccharide chain (e.g., activation with sodium periodate, or carbodiimide). The present invention discloses a conjugation process that includes selective activation of KDO with a disulfide amine linker upon unmasking of the thiol functional group, which is then conjugated to the bromo-activated CRM ₁₉₇ protein as depicted in Figure 31 (preparation of single-ended conjugate).

Conjugation based on cystamine linker (A1). O-antigen polysaccharide and cystamine (50-250 molar equivalents of KDO) were mixed in phosphate buffer and the pH was adjusted to 6.0-7.0. To the mixture, sodium cyanoborohydride (NaCNBH3) (5-30 molar equivalents of KDO) was added and the mixture was stirred at 37°C for 48-72 h. Upon cooling to room temperature and diluting with an equal volume of phosphate buffer, the mixture was treated with tris(2-carboxyethyl)phosphine (TCEP) (1.2 molar equivalents of cystamine were added). The mixture was then purified by diafiltration using a 5 KDa MWCO membrane against 10 mM sodium dihydrogen phosphate solution to provide thiol containing O-antigen polysaccharide. The thiol content can be determined by Ellman assay.

Conjugation was then allowed to proceed by mixing the above thiol-activated O-antigen polysaccharide with the bromo-activated CRM ₁₉₇ protein in a ratio of 0.5-2.0. The pH of the reaction mixture was adjusted to 8.0-10.0 with 1M NaOH solution. The conjugation reaction was allowed to proceed for 24±4 hours at 5° C. Unreacted bromo residues on the carrier protein were quenched by reacting with 2 molar equivalents of N-acetyl-L-cysteine for 3-5 hours at 5° C. The remaining free sulfhydryl groups were then capped after adding 3 molar equivalents of iodoacetamide (associated with the addition of N-acetyl-L-cysteine). The capping reaction was allowed to proceed for an additional 3-5 hours at 5° C. and the pH of both capping steps was maintained at 8.0-10.0 by the addition of 1M NaOH. The resulting conjugates were obtained after UF/DF using a 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'-dithiobis(propanoic acid dihydrazide) linker (A4) O antigen polysaccharide and 3,3'-dithiobis(propanoic acid dihydrazide) (5-50 molar equivalents of KDO) were mixed in acetate buffer and the pH was adjusted to 4.5-5.5. To the mixture, sodium cyanoborohydride (NaCNBH ₃ ) (5-30 molar equivalents of KDO) was added and the mixture was stirred at 23-37°C for 24-72 h. The mixture was then treated with tris(2-carboxyethyl)phosphine (TCEP) (1.2 molar equivalents of 3,3'-dithiobis(propanoic acid dihydrazide) linker was added). The mixture was then purified by diafiltration using a 5 KDa MWCO membrane against 10 mM sodium dihydrogen phosphate solution to provide the thiol containing O antigen polysaccharide. Thiol content can be determined by Ellman assay.

Conjugation was then allowed to proceed by mixing the above thiol-activated O-antigen polysaccharide with the bromo-activated CRM ₁₉₇ protein in a ratio of 0.5-2.0. The pH of the reaction mixture was adjusted to 8.0-10.0 with 1M NaOH solution. The conjugation reaction was allowed to proceed for 24±4 hours at 5° C. Unreacted bromo residues on the carrier protein were quenched by reacting with 2 molar equivalents of N-acetyl-L-cysteine for 3-5 hours at 5° C. The remaining free sulfhydryl groups were then capped after adding 3 molar equivalents of iodoacetamide (associated with the addition of N-acetyl-L-cysteine). The capping reaction was allowed to proceed for an additional 3-5 hours at 5° C. and the pH of both capping steps was maintained at 8.0-10.0 by the addition of 1M NaOH. The resulting conjugate was obtained after UF/DF using a 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) O-antigen polysaccharide and 2,2'-dithio-N,N'-bis(ethane-2,1-diyl)bis(2-(aminooxy)acetamide) (5-50 molar equivalents of KDO) were mixed in acetate buffer and the pH was adjusted to 4.5-5.5. The mixture was then stirred at 23-37°C for 24-72 h, after which sodium cyanoborohydride ( _NaCNBH3 ) (5-30 molar equivalents of KDO) was added and the mixture was stirred for an additional 3-24 h. The mixture was then treated with tris(2-carboxyethyl)phosphine (TCEP) (1.2 molar equivalents of linker were added). The mixture was then purified by diafiltration using a 5 KDa MWCO membrane against 10 mM sodium dihydrogen phosphate solution to provide thiol containing O-antigen polysaccharides. The thiol content can be determined by Ellman assay.

Conjugation was then allowed to proceed by mixing the above thiol-activated O-antigen polysaccharide with the bromo-activated CRM ₁₉₇ protein in a ratio of 0.5-2.0. The pH of the reaction mixture was adjusted to 8.0-10.0 with 1M NaOH solution. The conjugation reaction was allowed to proceed for 24±4 hours at 5° C. Unreacted bromo residues on the carrier protein were quenched by reacting with 2 molar equivalents of N-acetyl-L-cysteine for 3-5 hours at 5° C. The remaining free sulfhydryl groups were then capped after adding 3 molar equivalents of iodoacetamide (associated with the addition of N-acetyl-L-cysteine). The capping reaction was allowed to proceed for an additional 3-5 hours at 5° C. and the pH of both capping steps was maintained at 8.0-10.0 by the addition of 1M NaOH. The resulting conjugate was obtained after UF/DF using a 30 KDa MWCO membrane against 5 mM succinate-0.9% saline, pH 6.0.

(Example 29A)
Preparation of Bromo-activated CRM ₁₉₇ CRM ₁₉₇ is prepared in 0.1 M sodium phosphate solution, pH 8.0±0.2, and cooled to 5±3° C. To the protein solution is added N-hydroxysuccinimide ester of bromoacetic acid (BAANS) as a dimethylsulfoxide (DMSO) stock solution (20 mg/mL) at a ratio of 0.25-0.5 BAANS:protein (w/w). The reaction is mixed gently for 30-60 minutes at 5±3° C. The resulting bromoacetylated (activated) protein is purified, for example, by UF/DF using a 10 kDa MWCO membrane using 10 mM phosphate (pH 7.0) buffer. After purification, the protein concentration of the bromoacetylated carrier protein is estimated by Lowry's protein assay.

(Example 29B)
Preparation of E. coli O-Ag-TT Conjugate. Fifty mg of lyophilized E. coli serotype O25b long chain polysaccharide, lot number 709766-30 (approximately 6.92 mg/mL, MW: approximately 39 kDa) was used for tetanus toxoid (TT) conjugation.

E. coli serotype O1a long chain polysaccharide 710958-142-3 (approximately 6.3 mg/mL, MW: approximately 44.3 kDa) (50 mg, 7.94 mL) was freeze-dried.

E. coli serotype O6 long chain polysaccharide 710758-121-1 (approximately 16.8 mg/mL, MW: approximately 44 kDa) (50 mg, 2.98 mL) was freeze-dried.

Each of the above lyophilized polysaccharides was dissolved in WFI to approximately 5-10 mg/mL and 0.5 mL (100 mg of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) in 1 mL acetonitrile) was added and stirred at RT. Triethylamine (TEA) 0.2 M (2 mL) 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 (2 x 50 mL) using a filter tube. It was then diluted with HEPES and saline to a final HEPES concentration of approximately 0.25 M.

TT was prepared as above and the pH of the reaction was adjusted to approximately 9.1-9.2. The reaction mixture was stirred at RT.

After 20-24 h, the reaction was quenched with glycine (0.5 mL). It was then concentrated using a MWCO regenerated cellulose membrane and diafiltered against saline. Filtered and analyzed. See Table 26.

(Example 30)
Further Results of O-Antigen Fermentation, Purification, and Conjugation The exemplary process described below is generally applicable to all E. coli serotypes. The production of each polysaccharide involved batch production fermentation followed by chemical inactivation before downstream purification.

Strains and storage. The strain used for biosynthesis of the short chain O-antigen was a clinical wild-type strain of E. coli. The long chain O-antigen was produced using a derivative of the short chain producer that was engineered by the Wanner-Datsenko method to have a deletion of the native wzzb gene and complemented by the "long" elongation function fepE from Salmonella. The fepE function was expressed from its native promoter on a high copy colE1-based "topo" vector or a low copy derivative of the colE1-based vector pET30a, in which the T7 promoter region had been deleted.

Cell banks were prepared by growing cells in animal-free LB or minimal medium to an OD of at least _3.0 . The broth was then diluted in fresh medium and mixed with 80% glycerol to obtain a final concentration of 20% glycerol with an OD of _2.0 /mL.

Media used for seed culture and fermentation. The seed and fermentation media used share the _{following formulations: KH2PO4, K2HPO4, (NH4)2SO4 , sodium citrate , Na2SO4 , aspartic acid} , _glucose , _{MgSO4 , FeSO4-7H2O} , _{Na2MoO4-2H2O , H3BO3} , _CoCl2-6H2O , _{CuCl2-2H2O , MnCl2-4H2O , ZnCl2 , and CaCl2-2H2O .}

Seed and fermentation conditions. Seed was inoculated at 0.1% from a single seed vial. Seed flasks were incubated at 37° C. for 16-18 hours to typically achieve 10-20 OD ₆₀₀ /mL.

Fermentation was carried out in a 10 L stainless steel stationary steam sterilized fermenter.

Inoculation of the fermentation medium was typically 1:1000 from a seed of 10 OD _600. The batch phase, during which growth proceeds on 10 g/L batch glucose, typically lasts 8 hours. Upon glucose exhaustion, there is a sudden rise in dissolved oxygen, at which point glucose is fed to the fermentation. Fermentation then typically proceeds for 16-18 hours, yielding a harvest of >120 OD ₆₀₀ /mL.

Initial evaluation of short/long chain O-antigen production for serotypes O1a, O2, O6 and O25b. Wild-type strains for O1a, O2, O6 and O25b were fermented in batch mode in supplemented minimal medium to OD ₆₀₀ =15-20. Upon glucose exhaustion resulting in a sudden decrease in oxygen consumption, a growth-limiting glucose feed was applied from a glucose solution for 16-18 hours. A cell density of 124-145 OD ₆₀₀ units/mL was reached. The pH of the harvest broth was then adjusted to approximately 3.8 and heated to 95°C for 2 hours. The hydrolyzed broth was then cooled to 25°C, brought to pH 6.0 and centrifuged to remove solids. The resulting supernatant was then applied to a SEC-HPLC column for O-antigen quantification. Productivities ranging from 2240-4180 mg/L were obtained. The molecular weight of the purified short chain O-antigen from these batches was found to range from 10-15 kDa. It was also found that SEC chromatography of the O2 and O6 hydrolysates revealed distinct and separate contaminating polysaccharides that were not evident in the O1a and O25b hydrolysates.

Long chain O1a, O2, O6 and O25b O antigens were accessed by fermentation of the respective wzzb deleted strains carrying a heterologous complementary fepE gene on a high copy kanamycin selectable topoplasmid. Fermentations were performed with kanamycin selection but for short chains. Final cell densities observed at OD ₆₀₀ /mL of 124-177 were associated with O antigen productivities of 3500-9850 mg/L. Complementation-based synthesis of long chain O antigens was at least as productive as in the parental short chain strains, and in some cases more productive. The molecular weights of purified O antigen polysaccharides were 33-49 kDa, or approximately three times the size of the corresponding short chains.

Long-chain hydrolysates for O2 and O6 show evidence of a contaminating polysaccharide peak, observed as a shoulder on the major O-antigen peak in the case of long-chain antigens; O1 and O25b were found to show no evidence of production of contaminating polysaccharides, as is more readily seen for the short-chain parent.

Growth rate inhibition was found to be associated with the presence of a toporeplicon lacking fepE. Furthermore, the Δwzzb mutation itself had no deleterious effect on growth rate, indicating that the growth rate perturbation was transmitted by the plasmid vector.

Evaluation of strains for production of O11, O13, O16, O21 and O75 O antigens. Several wild-type strains of serotypes O11, O13, O16, O21 and O75 were evaluated for their tendency to produce undesirable polysaccharides in fermentation by SEC-HPLC. Strains for O11, O13, O16, O21 and O75 were selected for the absence of contaminating polysaccharides and for their ability to produce more than 1000 mg/L O antigens and for displaying antibiotic susceptibility profiles that allow Wanner-Datsenko recombination for the introduction of the Δwzzb trait.

Chloramphenicol-selectable versions of topo-fepE and pET-fepE were constructed that allow the introduction of fepE into O11, O13, O16, O21 and O75 wzzb strains, which are generally known to be kanamycin resistant. The resulting topo-fepE and pET-fepE-bearing strains were fermented with chloramphenicol selection and supernatants from acid hydrolyzed broths were evaluated by SEC-HPLC. Both high copy (topo) and low copy (pET) fepE constructs directed synthesis of O antigen with productivity comparable to the parent wild type for each. No expression of potentially interfering polysaccharides was observed.

Evaluation of growth rates for wzzb plasmid-bearing strains showed that O11, O13, and O21 were retarded by the presence of topo-fepE, but not by pET-fepE; O16 and O75 strains showed acceptable growth rates independent of replicon choice.

The purification process for the polysaccharides included acid hydrolysis to liberate the O-antigens. A crude suspension of serotype-specific E. coli cultures in fermentation reactors was directly treated with acetic acid to a final pH of 3.5±0.5, and the acidified broth was heated to a temperature of 95±5° C. for at least 1 h. This treatment cleaves the labile bond between the KDO at the proximal end of the oligosaccharides and lipid A, thus liberating the O-Ag chains. The acidified broth containing the liberated O-Ag was cooled to 20±10° C. and then neutralized to pH 7±1.0 using NH ₄ OH. The process further included several centrifugation, filtration, and concentration/diafiltration operational steps.

(Example 31)
Conjugation (RAC/DMSO) to tested O antigens (O4, O11, O21, O75)

(Example 32)
Prepared PLL conjugates

(Example 33)
Stable Mammalian Cell Expression of E. coli Polypeptides The SSI (site-specific integration) stable expression system was used to generate stable CHO clones expressing the FimH GSD or FimH LD.

The host CHO cells are an engineered cell line from a CHOK1SV GS-KO background (see, e.g., U.S. Patent Application 20200002727 for a description of the CHOK1SV GS-KO host cell line). Briefly, a landing pad containing a green fluorescent protein (GFP) gene surrounded by two FRT sites was targeted to a transcriptional hotspot within the genome of the host cell. The GFP gene can be exchanged for a GS gene and a gene of interest, also surrounded by FRT sites, from an LVEC vector that is co-expressed with flippase recombinase (FLPe). This system not only advantageously has a growth and productivity profile comparable to random integration, but also exhibits genotypic and phenotypic stability for at least up to 100 generations.

As referred to herein, the word "FRT site" refers to a nucleotide sequence at which the product of the yeast 2 μm plasmid flippase (FLP) gene, FLP recombinase, can catalyze site-specific recombination. A variety of non-identical FRT sites are known in the art. The sequences of the various FRT sites are similar in that they all contain identical 13 base pair inverted repeats adjacent to an 8 base pair asymmetric core region where recombination occurs. It is the asymmetric core region that is responsible for the orientation of the site and the variation among the various FRT sites. Illustrative (non-limiting) examples of these include the naturally occurring FRT (F), as well as several mutant or variant FRT sites such as FRT F1 and FRT F2.

As referred to herein, the term "landing pad" refers to a nucleic acid sequence that comprises a first recombination target site that is chromosomally integrated into a host cell. In some embodiments, the landing site comprises two or more recombination target sites that are 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, the landing pads are integrated into up to 1, 2, 3, 4, 5, 6, 7, or 8 distinct chromosomal loci. In some embodiments, the landing pads are integrated into up to 1, 2, or 3 distinct chromosomal loci. In some embodiments, the landing pads are integrated into up to 4 distinct chromosomal loci.

LVEC expression vectors and FLPe expression vectors for FimH GSD or FimH LD were co-transfected into SSI host cells by electroporation using either BioRad Gene Pulser Xcell or Amaxa 4D-Nucleofector. Cells were then cultured in glutamine-free medium to select for cells with the GS gene integrated into the landing pad site. Cells usually recover in 2-3 weeks. Single cell cloning was then performed in 96-well plates, either by FACS or limiting dilution. Titers from wells containing cells were ranked to narrow down to the top 48 clones. A second round of fed-batch screening was performed in 24-deep well plates to narrow down the clones to the top 12. A third round of fed-batch screening was performed in Ambr15 to narrow down the clones to the top 3. Ambr250 experiments were used to identify the best clones. For the top clones, after their identification, master cell banks and working cell banks were generated.

(Example 34)
Cell Line Development and Production Reactor Expression of FimH-DSG WT and FimH _LD WT Proteins The examples described herein describe exemplary production of both FimH-DSG WT and FimH _LD WT proteins from stable CHO cell lines in which the coding sequences for each protein have been stably integrated into the CHO genome.

In a production bioreactor environment, the selected stable CHO cell lines were able to produce target protein at approximately 1 gram per liter of culture for FimH-DSG WT and 250 milligrams per liter of culture for FimH _LD WT. The seed train in the production reactor was continuously scaled up from a vial thaw of the working cell bank and expanded by three passage cycles in shake flasks using an inoculation viable cell density of 0.3×10^6 cells/ml in shake flasks to obtain sufficient cells for the production reactor. The cells were grown at 36.5° C., 5% CO ₂ for 3-4 days.

The production reactor was seeded from the final shake flask, targeting an inoculum cell density of 1x10^6 cells/ml. The production reactor was grown for 7 days at 36.5°C, targeting 5-10% _CO2 saturation, using a pH of 7.05 (+/- 0.15). pH is controlled with sodium/potassium bicarbonate for base control and _CO2 sparge for acid control. Dissolved oxygen is controlled at a set point of 40% using pure oxygen and via sparge. Temperature was adjusted to 31°C on day 7. The reactor was fed on day 1 using a feeding strategy that adds feed in relation to viable cell density, which is achieved by using a feed factor of 0.75 to ensure that feed components do not run off during operation. Feed is then added continuously over the course of a day to obtain the desired volume of feed.

The production reactor was harvested on day 13 and the harvest culture was centrifuged and 0.22 μm filtered prior to downstream processing.

(Example 35)
Immunogenicity of FimH and O antigen combinations in mice E. coli FimH lectin binding domain (FimH _LD ) and full-length (FimH-DSG or FimCH) variants were evaluated in mouse immunogenicity studies assessing the ability of elicited antibodies to neutralize binding of piliated E. coli to mannosylated ligands. Wild-type FimH antigen secreted and purified in high yields from mammalian cells was as immunogenic in mice as the analogous native antigen purified in low yields from the E. coli periplasm. By comparison, the full-length FimH antigen, containing both the lectin and pilin domains, was significantly more immunogenic than the FimH _LD . The use of adjuvant formulations containing QS21 was necessary to generate robust and functional immune responses against these fimbrial antigens. Follow-up studies investigated the immunogenicity of FimH in combination with tetravalent O-antigen glycoconjugate mixtures and different adjuvants. After the second dose, mice vaccinated with the liposomal QS21 formulation consistently induced higher functional responses to the combined antigens in both FimH neutralization assays and O-antigen-specific OPA assays compared to groups that received no adjuvant or liposomal monophosphoryl lipid A (MPLA).

Animal immunogenicity. CD-1 mice aged 6-8 weeks were obtained from Charles River and groups of 10 or 20 animals were administered 0.1 mL of test antigen or buffer control subcutaneously (SC). Polyclonal anti-FimH rabbit control serum was prepared by immunizing rabbits with 3 doses of E. coli FimH _LD antigen with 100 μg of complete Freund's adjuvant (at wk0) followed by 100 μg of antigen in incomplete Freund's adjuvant (at wk4 and 8) (Covance). Each vaccination was administered with 0.5 mL containing 50 μg of antigen at two sites (SC-dorsal).

Adjuvant formulations. The diluent for unadjuvanted antigens was 10 mM phosphate buffer, pH 6.1. For _AlPO4 , 50 μg (100 μL of 0.5 mg/ml suspension) was given per mouse dose. The default dose of Quillaja Saponaria-21 (QS21) used in preclinical trials was 20 μg per mouse from a stock containing 5.1 mg/mL QS-21, 5 mM succinate, 60 mM NaCl, 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, with a liposome size of 71 nm as determined by dynamic light scattering, was used at 5 μg per dose from a stock containing 15 mM phosphate buffer, pH 6.1, 4 mg/mL DOPC, 1 mg/mL cholesterol, 0.2 mg/mL MPLA (Lot 00714551-0018-2XLipoMPL). Liposomal MPLA/QS21 formulations, with a particle size of 75 nm for MPLA-QS21 liposomes as determined by dynamic light scattering, were used at 5 μg each per dose from stocks containing 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).

Expression plasmids and derived FimH pilus antigens used in immunogenicity studies. The following expression plasmids were used to generate recombinant proteins for mouse experiments:
> pSB02083 - FimH _LD (mIgK signal pept, N28S, N91S), processed protein sequence:
FACKTASGTAIPIGGGSANVYVNLAPCVNVNVGQNCVVDLSTQIFCHNDYPETITDYVTLQRGSAYGGVLSSFSGTVKYSGS SYPFPTSETPRVYNSRTDKPWPVALYLTPVSSAGGGVAIKAGSLIAVLILRQTNNYNSDDFQFVWNIYANNNDVVVPTGG CDVSARDVTVTLPDYPGSVPIPLTVYCAKSQNLGYYLSGTTADAGNSIFTNTASFSPAQGVGVQLTRQGTIIPANNTVSLGAVGTSAVSLGLTANYAARTGGQVTAGNVQSIIGVTFVYQGGSSGGGADVTITVNGKVVAKGGHHHHHHHH (SEQ ID NO: 110);
> pSB02158 - FimH _LD -LM (mIgK signal pept, N28S N91S V48C L55C), processed protein sequence:
FACKTASGTAIPIGGGSANVYVNLAPCVNVGQNCVVDLSTQIFCHNDYPETITDYVTLQRGSAYGGVLSSFSGTVKYSGSSYPFPTTSETPRVVYNSRTDKPWPVALYLTPVSSAGGVAIKAGSLIA VLILRQTNNYNSDDFQFVWNIYANNDVVVPTGGHHHHHHHH (SEQ ID NO: 111);
> pSB02307 - FimH-DSG (mIgK signal pept, N28S N91S N249Q 7aa linker FimG A1..K14), processed protein sequence:
FACKTASGTAIPIGGGSAN VYVNLAPVVNVGQNLVVDLSTQIFCHNDYPETITDYVTLQRGSAYGGVLSSFSGTVKYSGS SYPFPTSETPRVYNSRTDKPWPVALYLTPVSSAGGGVAIKAGSLIAVLILRQTNNYNSDDFQFVWNIYANNNDVVVPTGG CDVSARDVTVTLPDYPGSVPIPLTVYCAKSQNLGYYLSGTTADAGNSIFTNTASFSPAQGVGVQLTRQGTIIPANNTVSLGAVGTSAVSLGLTANYAARTGGQVTAGNVQSIIGVTFVYQGGSSGGGADVTITVNGKVVAKGGHHHHHHHH (SEQ ID NO: 112);
> pSB02198 - FimH-DSG-LM (mIgK signal pept, N28S N91S 249Q V48C L55C 7aa linker FimG A1..K14), processed protein sequence:
FACKTASGTAIPIGGGSANVYVNLAPVVNVGQNLVVDLSTQIFCHNDYPETITDYVTLQRGSAYGGVLSSFSGTVKYSGSSYPFPTTSETPRVVYNSRTDKPWPVALYLTPVSSAGGVAIKAGSLIA VLILRQTNNYNSDDFQFVWNIYANNDVVVPTGGHHHHHHHH (SEQ ID NO: 113)

The amino acid numbering is based on the full-length amino acid sequence of FimH from strain J96, although academic publications use numbering based on the N-terminal phenylalanine (position 21) exposed after signal peptide processing. Other mutations remove glycosylation site asparagine residues in the lectin domain (N28S and N91S) and in the pyrin domain (N249Q) of the full-length FimH-DSG construct. The donor chain G peptide added to the C-terminus stabilizes FimH. The 7 amino acid Ringer length was optimized for mammalian expression as described in the examples above.

FimH whole cell neutralization assay. A FimH neutralization assay was developed to measure the inhibition by serum antibodies of binding of fimbriated E. coli to mannoside ligands. The ligands were yeast mannan immobilized on microplates or bladder 5637 cells expressing the mannosylated uroplakin receptor UPIa.

For the yeast mannan assay, black microtiter 96-well plates (Maxisorb, Nunc) were coated with 20 μg/ml yeast mannan (Sigma-Aldrich) in PBS buffer. Wells were blocked with 1% bovine serum albumin (BSA, Sigma-Aldrich) in PBS for 20 min. 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/l sodium bicarbonate (Sigma) and 0.3 g/l l-glutamine, grown at 37°C with 5% _CO2 , and used between passages 10 and 24. Surface expression of the uroplakin 1a receptor was confirmed by immunofluorescence staining with a polyclonal antibody (Novus #NBP214694). E. coli serotype O25bUTI 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 using rabbit immune serum against the FimH _LD antigen. Specificity of bacterial binding to mannan or bladder cells was established by inclusion of the negative control compound methyl α-D-mannopyranoside (Sigma), which reduces binding by more than 95% at the 50 mM level. Eight two-fold serial dilutions of test sera starting at 1:100 (in PBS, 0.1% BSA) were added to immobilized yeast mannan or 5637 cell monolayers after co-incubation with ^1x107 E. coli at 37°C for 1 h. Serially diluted anti-FimH _LD rabbit serum was used as an internal control on each plate. Plates were incubated at 37°C for 1 h before washing off unbound bacteria. Bound E. coli was stained with 3 μg/ml O25b-specific mAb conjugated to Alexafluor 488 for 45 min at RT. The mAb was reconstituted from the variable light and heavy chain sequences of the O25b antibody 3E9-11 (Nagy E, Nagy G, Szijarto V, et al. Antibodies to multi-drug resistant Escherichia coli: Arsanis Biosciences GmbH, Austria. 2014: 76pp). Human pTT5 IgG expression plasmid was used as the cloning vector. Fluorescence intensity of individual wells was read on a ClarioStar Plus instrument. A sigmoidal dose-response variable slope curve fit (Graphpad Prism) was used to interpolate _IC50 inhibition values. Titers are the reciprocal of the serum dilution at which half-maximal inhibition is observed. Vaccine antigen responders were defined as neutralization titers greater than 80% inhibition at a starting serum dilution of 1:100. Additionally, serum dilution titrations of binding activity must meet the variable slope sigmoidal curve fit parameters ( ^R2 >0.95, interpolate Log IC50 values or induce experimental repeats at wider dilutions until resolution). Statistical significance (p-values) of differences in responses between groups was determined using unpaired t-tests 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 with bacterial detection by elicited anti-O-Ag IgG. In this case, detection is based on photon emission catalyzed by firefly luciferase after release of ATP from permeabilized visible bacteria. The use of this reagent simplified detection by eliminating washing steps, automated liquid handling and facilitated the implementation of a more efficient 384-well assay format (Bravo, Agilent). The miniaturized assay reduced the reaction volume from 100 μL to 30 μL and the number of bacteria required from 1×10 ⁷ to 1×10 ⁶ per well. After dilution 1:1 in PBS, Bactiter Glo was added to all wells and, after mixing, read on a ClarioStar reader in luminometer mode. Serotype O6:K2 E. coli UTI strain CFT073 (ATCC® 700928™) was selected for use in the semi-automated 384-well assay because of its increased sensitivity compared to the O25b UTI strain PFEEC0547. Both strains produced similar results in bridging studies comparing individual serum titers in a subset of mouse vaccinated groups (data not shown).

E. coli O-Ag Mouse IgG Direct Luminex Immunoassay (dLIA). Long chain E. coli O-Ag polysaccharides of serotypes O25b, O1a, O2 and O6 produced in-house by eBPD (Pfizer) were conjugated to poly-L-lysine and then to Luminex bead microspheres using EDC/NHS. The use of beads with different spectral addresses for each O antigen allowed for 4-fold multiplexing. Beads were incubated with serially diluted individual mouse sera or control mAbs with shaking for 18 h at 4°C. After washing, bound serotype-specific IgG was detected with a PE-conjugated goat anti-mouse IgG mouse secondary antibody (90 min incubation at RT). Microplates were read on a FlexMap 3D instrument (Biorad). Serotype-specific mouse IgG mAbs (generated in-house) with similar binding properties were used as internal standards to quantify IgG levels. Plots of the standard curves for each mAb yielded overlapping linear slope profiles over 10 ³ serum dilutions (log luminescence vs. log serum dilution). A lower limit of quantification (LLOQ) for the 4-plex IgG dLIA of 0.15259 μg/mL was calculated from the standard curve bias and was the same for each of the four antigens.

E. coli Opsonophagocytosis Assay (OPA). Clinical E. coli invasive blood isolates were obtained from the Pfizer-sponsored Antimicrobial Testing Leadership and Surveillance (ATLAS) database maintained by the International Health Management Associates (IHMA) clinical laboratory. Strains were genotypically characterized by WGS using the Illumina Miseq platform, including in silico serotyping for prediction of O antigen and K capsule type. Serotype O1a, O2, O6 and O25b strains expressing both O and K antigens when grown in Dulbecco's modified Eagle medium (DMEM) under cell banking conditions, and multidrug resistant uncapsulated strains of O6 and O25b serotypes were used as test strains. O antigen expression was confirmed by flow cytometry using antigen-specific monoclonal or polyclonal antibodies generated in-house. K1 antigen expression was confirmed using K1 capsule mAb mAb13D9-151 (obtained from NRC, Canada). K2 antigen expression was detected using K2 typing serum (Statens Serum Institute). Expression of K5 heparosan capsule was confirmed by increased surface exposure of endogenous O antigens upon treatment with heparinase. The following E. coli clinical strains were developed for the OPA assay:
O1a:K1 (PFEEC0435, ST-95),
O2:K1- (PFEEC0146, ST-95, ticarcillin/clavulanic acid®),
O6:K- (PFEEC0412, ST-127, ticarcillin/clavulanic acid (registered trademark), ESBL);
O6:K2 (PFEEC0150, ST-127),
O25b:K- (PFEEC0068, ST-131, Imipenem (registered trademark), Fluoroquinolone (registered trademark)),
O25b:K5 (PFEEC0066, ST-131, Fluoroquinolone®).

Pre-frozen bacterial stocks were grown in DMEM to an OD ₆₀₀ of 0.5-1.0 and glycerol was added to a final concentration of 20% before freezing. Thawed bacteria before titration were diluted to 1x10 ⁵ CFU/ml in OPA buffer (Hank's Balanced Salt Solution (Life Technologies), 0.1% gelatin, 1 mM MgCl ₂ , 2.5 mM CaCl ₂ ) and 20 μL (10 ³ CFU) of bacterial suspension was opsonized with 10 μL of serially diluted serum over 30 min at RT in 384-well tissue culture microplates. Conditions such as the need for an opsonization step, the level of baby rabbit complement, the length of assay incubation, and the ratio of HL60 to bacteria were slightly varied and optimized according to the assay strain used. Subsequently, 10 μl of 2-6% complement (baby rabbit serum, Pel-Freez) and 10 μL of HL-60 cells (100-200:1 ratio) were added to each well and the mixture was shaken at 2000 rpm for 45-60 min at 37° C. in a 5% CO ₂ incubator. 10 μL of each 50 μL reaction was transferred to corresponding wells of a pre-wetted 384-well Millipore MultiScreen HTS HV filter plate containing 50 μL of water. After vacuum filtration of the liquid, 50 μL of 50% DMEM was applied, filtered, and the plate was incubated overnight at 37° C. in a sealed Ziploc bag. Colonies were counted the following day 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 μg/mL of homologous serotype purified O antigen before the opsonization step. The OPA assay included control reactions without HL60 cells or complement to demonstrate the dependence of the 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 study. The first study was designed to evaluate the immunogenicity of recombinant FimH _LD antigen expressed and purified from E. coli periplasm at different dose levels and with different adjuvants. Purification of FimH _LD 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). The second study compared the activity of E. coli FimH _LD with that of mammalian-expressed variants of the FimH _LD and full-length FimH-DsG. Finally, the third study investigated the immunogenicity of combinations of FimH-Dsg with a mixture of tetravalent long-chain O-antigen glycoconjugates and different adjuvant formulations.

Immunogenicity of the FimH antigen. The induction of functional anti-FimH antibodies induced by various FimH constructs was measured using 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:e1004857) 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) by E. coli. The functional potency of the antigen variants was quantified by determining the serum neutralization titers that prevent binding of fimbriated bacteria to the yeast mannoside ligand. The titer is defined as the reciprocal of the serum dilution at which 50% of the bacteria bind to the assay microplate in an 8-point 2-fold titration of vaccinated animal sera. Results of initial testing of FimH _LD antigen purified from E. coli are shown in Figures 36A and 36B and Table 34. The vaccination schedule is shown in Figure 36A and individual mouse responses are plotted in Figure 36B.

Mouse immunogenicity experiment 1 - Comparison of different E. coli periplasmically produced FimH constructs. After vaccination with three 3 μg doses of FimH _LD antigen, only 25% of mice in the non-adjuvanted group or the group adjuvanted with 50 μg _AlPO4 produced neutralizing antibody titers in the yeast mannan assay. In contrast, 61% of mice in the group adjuvanted with 20 μg QS21/PS80 produced neutralizing titers. In this case, the geometric mean titer (GMT) was 5-fold higher than the other groups (p value less than 0.05). At the 30 μg FimH _LD dose level with QS21/PS80, a further threshold increase in GMT and a 78% rate of sero-responder animals was observed. In this adjuvant context, the presence of disulfide mutations, designed and predicted to enhance functional immunogenicity by locking the FimH _LD 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 domain. A 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 to the wild-type FimHLD construct. Approximately 3-fold lower GMTs were observed for the lock mutant compared to the wild-type, approaching statistical significance (p=0.09). A key observation from this study is that the E. coli FimH _LD antigen requires the potent QS21 adjuvant formulation to elicit robust levels of neutralizing antibodies in mice.

Periplasmic immunogenicity compared to mammalian-produced FimH constructs. In a second study, the immunogenicity of FimH _LD and full-length FimH protein expressed in E. coli was compared to similar variants expressed in Expi293 mammalian cells. Full-length FimH expressed in E. coli complexed with its periplasmic chaperone FimC was used as a benchmark. Neutralization data comparing the relative levels of functional antibody induction by E. coli against mammalian-expressed antigens with or without the lock mutation after three vaccinations are shown in Figures 37A and 37B and Table 35. Antigens were administered at 10 μg each, along with 20 μg QS21/PS80, according to the schedule shown in Figure 37B. Mice in the E. coli-expressed FimH _LD group yielded a neutralization assay GMT of 300 and a 55% responder rate, which was not statistically different from the similar mammalian-expressed FimH _LD group, which showed a GMT of 194 and a 45% responder rate. Mammalian full-length FimH-DSG was significantly more immunogenic than its FimH _LD counterpart, exhibiting a two-fold higher GMT (529 vs. 194) and improved serum responder rate (75% vs. 45%). As in the first study, the presence of the lock mutation did not confer an advantage in terms of generating a functional neutralizing response in the context of the FimH _LD (E. coli or 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 selected for further investigation. We speculate that the presence of the FimH pilin domain in this construct complexed with the C-terminal FimG donor chain peptide may provide additional functional epitopes and/or enhanced stability.

Immunogenicity of co-formulated mammalian-produced FimH-DSG and tetravalent O-antigen glycoconjugates in mice. The third mouse study was designed to address the impact of the combination of O-antigen and adjuvant formulations on FimH-DSG immunogenicity. CRM ₁₉₇ long chain O-antigen glycoconjugates of the four most common serotypes O25b, O1a, O2 and O6 were mixed and administered at a dose of 2 μg each as a baseline control group (FIG. 38). Other vaccine groups evaluated the impact on immunogenicity of three adjuvants, including 10 μg FimH-DSG, either individually or in combination with the tetravalent O-antigen. The adjuvants were 20 μg QS21/PS80 per default dose formulation (used in FimH studies 1 and 2); a liposomal synthetic MPLA formulation (5 μg per dose); and a liposomal synthetic MPLA/QS21 formulation (5 μg each per dose). Sera were tested in functional FimH neutralization and O-antigen-specific opsonophagocytosis (OPA) assays at post-dose 2 (PD2) and post-dose 3 (PD3) time points.

The results of the yeast mannan FimH binding neutralization assay at PD2 and PD3 are shown in Figures 39A and 39B, along with the GMTs and responder rates summarized in Table 36.

Because antibody responses to individual FimH and monovalent serotype O25b antigens were submaximal after two doses in previous experiments, it was expected that the PD2 time point would best distinguish the effects of vaccine antigens and adjuvant compositions. Indeed, by PD3, responder rates and FimH neutralization GMTs were not significantly different between any of the groups (p>0.05). At PD2, the liposomal adjuvant formulation had no significant effect on the group containing FimH-DSG alone. In contrast, for groups vaccinated with the FimH-DSG/O antigen combination, significantly lower FimH neutralization GMTs were observed for the no adjuvant group, or the liposomal MPLA group, compared with groups of mice vaccinated with either the liposomal MPLA/QS21 or the FimH-DSG alone group. PD1 sera were also tested, but none of the mice showed neutralizing titers.

Pooled sera from each vaccine group were tested at PD2 and PD3 time points using a bladder cell binding neutralization assay. Bladder 5637 cells constitutively express the mannosylated uroplakin receptor UPIa, the 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:e1000415-e; Katnik-Prastowska I, Lis J, Matejuk A. Glycosylation of uroplakins. Implications for bladder infection. physiopathology. Glycoconjugate journal 2014;31:623-36). The results shown in Figures 40A and 40B and Table 37 were consistent with the findings of the yeast mannan neutralization assay using the individual sera. Again, the FimH-DSG/O antigen combination formulated with liposomal MPLA or without adjuvant produced a significantly weaker neutralization response than the liposomal MPLA/QS21 group, which responded similarly to groups of mice vaccinated with QS21/PS80 or liposomal MPLA/QS21 and FimH-DSG without the O antigen. The only slight difference between the mannan and bladder neutralization assay data was a trend toward weaker responses at the end for the liposomal MPLA formulation for groups vaccinated with either FimH-DSG alone or the FimH-DSG/O antigen combination.

The levels of O antigen-specific total IgG produced for 4-plex dLIA following vaccination of four groups of mice with tetravalent O antigen alone or tetravalent O antigen combined with FimH-DSG and liposomal adjuvant are shown in Figure 41 and Table 38. Serotype O25b-specific IgG responses after two doses were significantly lower than those elicited by serotype O1a, O2 and O6 O antigens, consistent with results from 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, Seroyen J, et al. Immunogenicity and safety of a tetravalent E. coli O-antigen bioconjugate vaccine in animal models. Vaccine 2016;34:4152-60). The lowest levels of anti-O Ag IgG across the O25b groups at PD2 were observed for the unadjuvanted FimH-DSG and O antigen combination, with only a single titer exceeding 5-fold the baseline IgG responder threshold; in comparison, the unadjuvanted O antigen only group produced a higher responder rate of 56% (5/9), but the difference in GMT between the two groups (0.79 vs. 0.24 μg/mL IgG) 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). Responders are defined as mice with sera that produced a complete dilution-dependent killing response that met the variable slope curve fit criteria. Despite higher responder rates and titers between groups than for serotype O25b, IgG titers for serotype O1a with the FimH-DSG and O antigen combination were also significantly improved by liposomal MPLA/QS21 by 5.9-fold and 4.6-fold at PD2 and PD3, respectively. The effect of MPLA/QS21 on O2 and O6 O antigens was less pronounced than for O1a and O25b, due to smaller overall differences in GMTs between vaccinated groups.

Functional bactericidal antibodies induced by individual O antigens of the tetravalent composition were evaluated in OPA assays using invasive clinical strains. Selection of OPA strains was based on a combination of the following factors: expression of both O antigen and K capsule under in vitro growth conditions and/or multidrug resistance; and the ability to identify comparable baby rabbit serum complements capable of facilitating bactericidal killing in the presence of neutrophil-like HL60 cells and O antigen-specific antibodies. Although E. coli O antigens are thought to be the primary determinants for preventing nonspecific killing by serum complement, the 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 O 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 O(-) and K(-) mutants of a uropathogenic Escherichia coli O75: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).

The results of the OPA assay for the four encapsulated O1a, O2, O6, and O25b strains and the two multidrug-resistant nonencapsulated O6 and O25b strains are shown in Figure 42 and Table 39. With the exception of the O6:K-strain assay, which showed a relatively high baseline titer of 174 for prevaccination sera, sera from unvaccinated mice for all other strains produced titers that were at the assay limit of detection (LOD) or starting serum dilution of 100. Individual OPA responses to tetravalent O antigens are comparable to IgG binding antibody titers (Figure 41, Table 38). For example, a significant positive impact of the liposomal MPLA/QS21 formulation on the immunogenicity of the FimH-DSG tetravalent O antigen combination was confirmed in the OPA for encapsulated serotype O1a, O2, and O6 strains, reporting responder rates of 70% or more after two vaccine doses. In contrast, only after the third dose does the O25b:K5 OPA strain report titers that reach this level of functional activity with the MPLA/QS21 formulation. Compared to their capsulated O6:K1 and O25b:K5 counterparts, the acapsulated O6 and O25b strains produced higher OPA titers and responder rates, especially among non-adjuvanted groups of mice vaccinated with O antigen alone or the FimH-DSG/tetravalent O antigen combination. This higher sensitivity is consistent with the absence of the corresponding K2 or K5 capsules, which have previously been shown to confer resistance to phagocytosis (Burns SM, Hull SI, Loss of resistance to ingestion and phagocytic killing by O(-) and K(-) mutants of a uropathogenic Escherichia coli O75: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).

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 infection. Such a formulation may be advantageous for the prevention of recurrent UTI infections and sepsis in elderly patients. The results confirm the benefit of FimH-DSG and the QS21 adjuvant to enhance functional immune responses against the long-chain O25b O-antigen, which are individually less immunogenic than the analogous serotype O1a, O2, and O6 O-antigen glycoconjugates in mice.

(Example 36)
Antibodies elicited by CRM197 conjugates of E. coli serotype O8 and O9 O antigens exhibit cross-protective bactericidal activity against K. pneumoniae serotype O5 and O3 invasive isolates.
This example demonstrates that antibodies elicited by short single-ended native CRM ₁₉₇ polymannan conjugates of E. coli serotype O8 and O9 O antigens are bactericidal and cross-protective against Klebsiella O5 and O3 strains expressing equivalent or related O antigens.

E. coli and K. pneumoniae share a common polymannan O antigen that is synthesized by enzymes encoded by highly homologous biosynthetic gene clusters. The E. coli O8 and O9 O antigen polysaccharides are linear mannose homopolymers whose repeating units differ in the number of monosaccharide linkages and residues. Their counterparts in K. pneumoniae are the serotype O5 and O3 O antigens. Biosynthesis of E. coli and K. pneumoniae polymannan O antigens involves a mechanism of O unit translocation and chain synthesis that is distinct from other E. coli O antigens. In this case, chain elongation is regulated by a biosynthetic WbdA-WbdD complex (King JD, Berry S, et al. Proceedings of the National Academy of Sciences 2014;111:6407-12) separate from the Wzx/Wzy-dependent pathway in which chain length is controlled by the WzzB or FepE enzymes. As a result, native polymannan O antigens can only be produced in their short chain form, which requires different bioprocessing methods for purification and carrier protein conjugation than the engineered long chain E. coli O antigens. The same mechanisms and limitations apply to the predominant K. pneumoniae serotype O1 and O2 O antigens, which are polygalactans composed of galactose residues.

The structural relationships between these O antigens and their subtypes are shown in Figure 33. The E. coli O8 and K. pneumoniae O5 O antigens are identical (Vinogradov E et al., J Biol Chem 2002;277:25070-81). The E. coli O9 and K. pneumoniae O3 O antigens share common tetrameric O9a/O3a and pentameric O9/O3 repeat unit subtypes, whereas the trimeric O3b subtype is found only in K. pneumoniae. These subtypes can be identified serologically and genotypically (Guachalla LM, et al. Scientific Reports 2017;7:6635). The serotype O3a locus is distinguished by a single point mutation in wbdA (C80R). A similar point mutation in the E. coli O9 wbdA enzyme (C55R) converts the O9 polysaccharide to O9a (Kido N, Kobayashi H. Journal of bacteriology 2000;182:2567-73). The O3b subtype has sufficient nucleotide variation in the sequence of the WbdD enzyme to require a separate reference sequence. The KaptiveWeb algorithm (Wick RR et al., J Clin Microbiol 2018;56), implemented in Pfizer's BigSdb whole genome sequencing (WGS) pipeline, designates O3 loci as either O3/O3a (covered by the same reference sequence) or O3b.

Materials and Methods a. Production of E. coli serotypes O8 and O9 CRM ₁₉₇ immune serum in rabbits For the execution of the study at Covance, two groups of 4 female New Zealand White rabbits each were used. Animals were administered 10 μg/animal serotype O8 or O9 CRM ₁₉₇ conjugate per dose with CFA/IFA as adjuvant. Native O8 and O9 O antigens were conjugated using single-end chemistry. 1 mL doses of 10 μg antigen each were split between two subcutaneous vaccination sites. Vaccinations were given at weeks 0, 6, and 14, with blood taken at weeks 7 and 15, corresponding to post-dose 2 (PD2) and post-dose 3 (PD3) time points.

b. Bacterial strains E. coli and K. pneumoniae clinical isolates were obtained from the Pfizer-sponsored Antimicrobial Testing Leadership and Surveillance (ATLAS) collection 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 multilocus 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). O-antigen serotypes were predicted using embedded in silico serotyping algorithms for E. coli and K. pneumoniae (Wick RR et al., J Clin Microbiol 2018;56; Joensen KG et al., J Clin Microbiol 2015;53:2410-26).

c. E. coli O8 and O9 CRM ₁₉₇ Conjugates Serotype O8 and O9a 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 chain native E. coli O8 and O9 O antigens with a disulfide amine linker. Once the thiol functional group is exposed, it is then conjugated to the bromoactivated CRM ₁₉₇ protein as described in Example 26 herein.

d. Bactericidal Assays Pre-frozen E. coli and K. pneumoniae stocks were prepared by growing strains to an _OD600 between 0.5 and 1.0 in DMEM or LB medium, and glycerol was added to a final concentration of 20% before freezing. Specific assay conditions varied according to the optimized conditions for each strain. Pre-titrated thawed bacteria were diluted to ^1x105 CFU/ml in OPA buffer (Hank's Balanced Salt Solution (Life Technologies) and 0.1% gelatin), and 20 μL ( ¹⁰³ CFU) of the bacterial suspension was opsonized with 20 μL of serially diluted sera for 30 min at room temperature in tissue culture microplates. Subsequently, 10 μl of complement (Baby Rabbit Serum or IgG/IgM depleted human serum, Pel-Freez) and 20 μL of HL-60 cells (at a ratio of 100-200:1) were added to each well with OPA buffer to a final volume of 100 μL. The reaction mixture was shaken for 60 minutes at 37° C. in a 5% CO ₂ incubator. In some cases, bacteria were combined directly with complement and HL60 without a prior opsonization step and shaken for 60 minutes at 37° C. under 5% CO _2. After incubation, 10 μL of each reaction was transferred to the corresponding well of a pre-wetted Millipore MultiScreen HTS HV filter plate containing 100 μL of water. After vacuum filtration of the liquid, 100 μL of 50% bacterial growth medium was applied, filtered, and the plates were incubated overnight at 37° C. in a sealed Ziploc® bag. The next day, microcolonies were counted using an ImmunoSpot® analyzer and ImmunoCapture software after staining with Coomassie dye. For the E. coli serotype O9 assay, OPA was miniaturized in a 384-well format for a 50 μL reaction volume. To demonstrate 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 that did not contain HL60 cells or complement to demonstrate that any observed killing was dependent on these components. In the Klebsiella serotype O5 assay, where the presence of HL60 had no effect, serum bactericidal responses were performed in the absence of effector cells.

Results a. Selection of E. coli and K. pneumoniae strains 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 using O-antigen specific rabbit antisera. Empirical screening of serum complement was then performed to identify individual matched lots across a range of concentrations that provided a suitable balance of low levels of non-specific killing combined with high infectivity in the presence of immune serum. Additional assay optimization parameters included adjustment of the ratio of HL60 effector cells to bacteria, shaker speed, the presence or absence of a plate sealer and the inclusion of an opsonization pre-incubation step.

b. E. coli O8 and K. pneumoniae O5 O-antigen immune serum cross-protection and specificity E. coli O8 strain EC0305 and K. pneumoniae O5 strain KP0121 were selected for assay development. Both are blood isolates. EC0305 is resistant to cephalosporins and tetracyclines, and KP0121 is resistant to ampicillin. An OPA assay was developed for E. coli O8 strain EC0305 with conditions including 3.0% BRC, a bacteria to HL60 ratio of 1:100, and a single-step 60-minute OPA incubation reaction. Since the bactericidal activity of K. pneumoniae O5 strain KP0121 in the presence of immune serum 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 a source of complement. The results of the bactericidal assays using these E. coli O8 and K. pneumoniae O5 strains are shown in Figures 34A-34B. With E. coli serotype O8 OPA, rabbit immune serum generated after administration of two doses of O8-CRM ₁₉₇ conjugate showed effective O-antigen specific killing that was blocked by pre-adsorption of immune serum with free O8 O-antigen polysaccharide. Complete killing was observed at serum dilutions below 1:1000. Matched pre-immune serum from the same rabbit was inactive. The same rabbit sera were evaluated in K. pneumoniae O5 SBA and found to be similarly bactericidal at a serum dilution of 1:2000. Killing was blocked by free O8 O antigen and was absent in pre-immune serum. In this case, serum matrix prozone blocked SBA activity at serum dilutions below 1:1000.

c. E. coli O9 and K. pneumoniae O3 OPA O antigen immune serum cross-protection and specificity E. coli O9a strain EC0611 and K. pneumoniae O3b strain KP0009 were selected for assay development. EC0611 is resistant to ampicillin while KP0009 is resistant to cephalosporins, fluoroquinolones, and tetracyclines. Both are UTI isolates from kidney and bladder infections, respectively. The O9a O antigen used to generate the CRM ₁₉₇ conjugate and resulting immune serum has a tetrameric polymannan repeat unit structure and is identical to the O9a EC0611 assay strain O antigen; however, it is structurally non-homologous to the K. pneumoniae O3b O antigen KP0009 assay strain, which is predicted to express a shorter trimeric repeat unit based on the sequence of its wbdD gene (see FIG. 33). OPA results with E. coli O9a and K. pneumoniae O3b strains indicate that the anti-E. coli O9a immune serum is effective against both (FIGS. 35A-35B). Complete killing in OPA was observed at serum dilutions below 1:8,000 for E. coli O9a strains and below 1:1,600 for Klebsiella O3b strains. Specificity was demonstrated by lack of activity with free O9a O antigen and preadsorption of sera with matched preimmune serum.

Conclusions E. coli serotype O8 and O9 polymannan CRM ₁₉₇ conjugates elicit functional antibodies capable of killing not only homologous E. coli clinical strains but also Klebsiella serotype O5 and O3 strains in bactericidal assays. The results confirm that these conjugates elicit antibodies that are cross-protective against isolates of both species expressing structurally related polymannan O antigens.

The following sections describe further embodiments of the present invention:

C1. A polypeptide derived from FimH or a fragment thereof; and a polypeptide of formula O1 (e.g., formula O1A, formula O1B, formula O1C), formula O2, formula O3, formula O4 (e.g., formula O4:K52 and formula O4:K6), formula O5 (e.g., formula O5ab and formula O5ac (strain 180/C3)), formula O6 (e.g., formula O6:K2;K13;K15 and formula O6:K54), formula O7, formula O8, formula O9, formula O10, formula O11, formula O12, formula O13, formula O14, formula O15, formula O16, formula O17, formula O18 (e.g., formula O18A, formula O18ac, formula O18A1, formula O18B and formula O18B1), formula O19, formula O20, formula O21, formula O22, formula O23 (e.g., formula O23A), formula O24, formula O25 (e.g., formula O25a and formula O25b), formula O26, formula O27, formula O28, formula O29, formula O30, formula O32, formula O33, formula O34, formula O35, formula O36, formula O37, formula O38, formula O39, formula O40, formula O41, formula O42, formula O43, formula O44, formula O45, formula O45 (e.g., formula O45 and formula O45rel), formula O46, formula O48, formula O49, formula O50, formula O51, formula O52, formula O53, formula O54, formula O55, formula O56, formula O57, formula O58, formula O59, formula O60, formula O61, formula O62, formula 62D ₁ , formula O63, formula O64, formula O65, formula O66, formula O68, formula O69, formula O70, formula O71, formula O73 (e.g., formula O73 (strain 73-1)), formula O74, formula O75, formula O76, formula O77, formula O78, formula O79, formula O80, formula O81, formula O82, formula O83, formula O84, formula O85, formula O86, formula O87, formula O88, formula O89, formula O90, formula O91, formula O92, formula O93, formula O95, formula O96, formula O97, formula O98, formula O99, formula O100, formula O101, formula O102, formula O103, formula O104, formula O105, formula O106, formula O107, formula O108, formula O109, formula O110, formula O111, formula O112, formula O113, formula O114, formula O115, Formula O116, Formula O117, Formula O118, Formula O119, Formula O120, Formula O121, Formula O123, Formula O124, Formula O125, Formula O126, Formula O127, Formula O128, Formula O129, Formula O130, Formula O131, Formula O132 , formula O133, formula O134, formula O135, formula O136, formula O137, formula O138, formula O139, formula O140, formula O141, formula O142, formula O143, formula O144, formula O145, formula O146, formula O147, formula O1 48, Formula O149, Formula O150, Formula O151, Formula O152, Formula O153, Formula O154, Formula O155, Formula O156, Formula O157, Formula O158, Formula O159, Formula O160, Formula O161, Formula O162, Formula O163, Formula O 164, Formula O165, Formula O166, Formula O167, Formula O168, Formula O169, Formula O170, Formula O171, Formula O172, Formula O173, Formula O174, Formula O175, Formula O176, Formula O177, Formula O178, Formula O179, Formula O180, Formula O181, Formula O182, Formula O183, Formula O184, Formula O185, Formula O186, and Formula O187 (wherein n is an integer from 1 to 100).

C2. The sugar is of formula O1 (e.g., formula O1A, formula O1B, and formula O1C), formula O2, formula O3, formula O4 (e.g., formula O4:K52 and formula O4:K6), formula O5 (e.g., formula O5ab and formula O5ac (strain 180/C3)), formula O6 (e.g., formula O6:K2;K13;K15 and formula O6:K54), formula O7, formula O10, formula O16, formula O17, formula O18 (e.g., formula O18A, formula O18ac, formula O18A1, formula O18B, and formula O18B1), formula O21, formula O23 (e.g., formula O23A), formula O24, formula O25 (e.g., formula O25a and formula O25b), formula O26, formula O28, formula O4 4, formula O45 (e.g., formula O45 and formula O45rel), formula O55, formula O56, formula O58, formula O64, formula O69, formula O73 (e.g., formula O73 (strain 73-1)), formula O75, formula O77, formula O78, formula O86, formula O88, formula O90, formula O98, formula O104, formula O111, formula O113, formula O 114, Formula O119, Formula O121, Formula O124, Formula O125, Formula O126, Formula O127, Formula O128, Formula O136, Formula O138, Formula O141, Formula O142, Formula O143, Formula O147, Formula O149, Formula O152, Formula O157, Formula O158, Formula O159, Formula O164, Formula O173, Formula O62D ₁ , Formula O22, Formula O35, Formula O65, Formula O66, Formula O83, Formula O91, Formula O105, Formula O116, Formula O117, Formula O139, Formula O153, Formula O167, and Formula O172 (wherein n is an integer from 20 to 100).

C3. The sugar is of formula O1 (e.g., formula O1A, formula O1B, and formula O1C), formula O2, formula O3, formula O4 (e.g., formula O4:K52 and formula O4:K6), formula O5 (e.g., formula O5ab and formula O5ac (strain 180/C3)), formula O6 (e.g., formula O6:K2;K13;K15 and formula O6:K54), formula O7, formula O10, formula O16, formula O17, formula O18 (e.g., formula O18A, formula O18ac, formula O18A1, formula O18B, and formula O18B1), formula O21, formula O23 (e.g., formula O23A), formula O24, formula O25 (e.g., formula O25a and formula O25b), formula O26, formula O28, formula O44 , Formula O45 (e.g., Formula O45 and Formula O45rel), Formula O55, Formula O56, Formula O58, Formula O64, Formula O69, Formula O73 (e.g., Formula O73 (strain 73-1)), Formula O75, Formula O77, Formula O78, Formula O86, Formula O88, Formula O90, Formula O98, Formula O104, Formula O111, Formula O113, Formula O1 14, the composition according to item C2, comprising a structure selected from formula O119, formula O121, formula O124, formula O125, formula O126, formula O127, formula O128, formula O136, formula O138, formula O141, formula O142, formula O143, formula O147, formula O149, formula O152, formula O157, formula O158, formula O159, formula O164, formula O173, and formula 62D ₁ (wherein n is an integer from 20 to 100).

C4. The composition according to claim C2, comprising a structure selected from formula O1 (e.g., formula O1A, formula O1B, and formula O1C), formula O2, formula O6 (e.g., formula O6:K2; K13; K15 and formula O6:K54), formula O15, formula O16, formula O21, formula O25 (e.g., formula O25a and formula O25b), and formula O75.

C5. The composition according to claim C2, comprising a structure selected from formula O4, formula O11, formula O21, and formula O75.

C6. The composition according to item C1, which does not contain a structure selected from formula O8, formula O9a, formula O9, formula 20ab, formula O20ac, formula O52, formula O97, and formula O101.

C7. The composition according to item C1, which does not contain a structure selected from formula O12.

C8. The composition according to C4, produced by expressing a wzz family protein in a gram-negative bacterium that produces the sugar.

C9. The composition of claim C8, wherein the wzz family protein is selected from the group consisting of wzzB, wzz, wzz _SF , wzz _ST , fepE, wzz _fepE , wzz1 and wzz2.

C10. The composition according to item C8, wherein the wzz family protein is wzzB.

C11. The composition according to item C8, wherein the wzz family protein is fepE.

C12. The composition according to item C8, wherein the wzz family proteins are wzzB and fepE.

C13. The composition according to item C8, wherein the wzz family protein is derived from Salmonella enterica.

C14. The composition according to item 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 item 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, and SEQ ID NO:34.

C16. The composition according to item C8, wherein the wzz family protein comprises an array 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 claim C1, wherein the sugar is synthetically synthesized.

C18. The composition of any one of claims C1 to C17, further comprising an E. coli R1 moiety.

C19. The composition of any one of claims C1 to C17, further comprising an E. coli R2 moiety.

C20. The composition of any one of claims C1 to C17, further comprising an E. coli R3 moiety.

C21. The composition of any one of claims C1 to C17, further comprising an E. coli R4 moiety.

C22. The composition of any one of claims C1 to C17, further comprising an E. coli K-12 moiety.

C23. The composition according to any one of items C1 to C22, further comprising a 3-deoxy-d-manno-octa-2-ulosonic acid (KDO) moiety.

C24. The composition of any one of claims C1 to C17, further not comprising an E. coli R1 moiety.

C25. The composition of any one of claims C1 to C17, further not comprising an E. coli R2 moiety.

C26. The composition of any one of claims C1 to C17, further not comprising an E. coli R3 moiety.

C27. The composition of any one of claims C1 to C17, further not comprising an E. coli R4 moiety.

C28. The composition of any one of claims C1 to C17, further not comprising an E. coli K-12 moiety.

C29. The composition according to any one of items C1 to C22, further not comprising a 3-deoxy-d-manno-octa-2-ulosonic acid (KDO) moiety.

C30. The composition according to any one of items C1 to C23, which does not contain lipid A.

C31. The composition according to any one of items C1 to C30, wherein the polysaccharide has a molecular weight of 10 kDa to 2,000 kDa, or 50 kDa to 2,000 kDa.

C32. The composition according to any one of items C1 to C31, having an average molecular weight of 20 to 40 kDa.

C33. The composition according to any one of claims C1 to C32, wherein the sugar has an average molecular weight of 40,000 to 60,000 kDa.

C34. The composition according to any one of items C1 to C33, wherein n is an integer from 31 to 90.

C35. A composition comprising a polypeptide or fragment thereof derived from FimH; and a conjugate comprising a sugar covalently attached to a carrier protein, wherein the sugar is derived from E. coli.

C36. A composition comprising a polypeptide derived from FimH or a fragment thereof; and a conjugate comprising a sugar according to any one of items C1 to C34, covalently bound to a carrier protein.

C37. The carrier protein is selected from the group consisting 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, and exotoxin A from Pseudomonas aeruginosa; detoxified exotoxin A (EPA) from P. aeruginosa, maltose binding protein (MBP), detoxified hemolysin A from S. aureus, clumping factor A, clumping factor B, cholera toxin B subunit (CTB), Streptococcus pneumoniae, and the like. A composition comprising a polypeptide derived from FimH or a fragment thereof selected from any one of C. pneumoniae pneumolysin and detoxified variants thereof, C. jejuni AcrA, C. jejuni native glycoprotein and Streptococcal C5a peptidase (SCP); and a conjugate according to any one of items C35 to C36.

C38. The composition of any one of claims C35 to C37, wherein the carrier protein is CRM ₁₉₇ .

C39. The composition according to any one of claims C35 to C37, wherein the carrier protein is tetanus toxoid (TT).

C40. The composition according to any one of items C35 to C37, wherein the carrier protein is poly(L-lysine).

C41. The composition according to any one of items C35 to C39, wherein the conjugate is prepared by reductive amination.

C42. The composition according to any one of claims C35 to C39, prepared by CDAP chemical reaction.

C43. The composition according to any one of items C35 to C39, which is a single-end-linked conjugated sugar.

C44. The composition according to any one of claims C35 to C39, conjugated to a carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer.

C45. The composition of claim C44, wherein the sugar is conjugated to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer, the sugar being covalently attached to the eTEC spacer via a carbamate bond and the carrier protein being covalently attached to the eTEC spacer via an amide bond.

C46. The composition of any one of items C44 to C45, wherein CRM ₁₉₇ comprises 2 to 20, or 4 to 16 lysine residues covalently attached to the polysaccharide via an eTEC spacer.

C47. The composition according to any one of claims C35 to C46, wherein the sugar:carrier protein ratio (w/w) is 0.2 to 4.

C48. The composition according to any one of claims C35 to C46, wherein the sugar to protein ratio is at least 0.5 and at most 2.

C49. The composition according to any one of claims C35 to C46, wherein the sugar to protein ratio is 0.4 to 1.7.

C50. The composition of any one of claims C43 to C49, wherein the sugar is conjugated to the carrier protein via a 3-deoxy-d-manno-octa-2-ulosonic acid (KDO) residue.

C51. A composition comprising a polypeptide derived from FimH or a fragment thereof, in which the sugar comprises a structure selected from formula O8, formula O9a, formula O9, formula O20ab, formula O20ac, formula O52, formula O97, and formula O101 (wherein n is an integer from 1 to 10); and a conjugate comprising the sugar covalently attached to a carrier protein.

C52. A composition comprising a polypeptide derived from FimH or a fragment thereof; and a sugar according to any one of items C1 to C34, and a pharma- ceutically acceptable diluent.

C53. A composition comprising a polypeptide derived from FimH or a fragment thereof; and a conjugate according to any one of items C35 to C51, and a pharma- ceutically acceptable diluent.

C54. The composition according to claim C53, comprising at most about 25% free sugars compared to the total amount of sugars in the composition.

C55. The composition according to any one of claims C52 to C53, further comprising an adjuvant.

C56. The composition of any one of claims C52 to C53, further comprising aluminum.

C57. The composition according to any one of claims C52 to C53, further comprising QS-21.

C58. The composition according to any one of claims C52 to C53, further comprising a CpG oligonucleotide.

C59. The composition according to any one of claims C52 to C53, which does not contain an adjuvant.

C60. A composition comprising a polypeptide or fragment thereof derived from FimH; and a saccharide derived from E. coli conjugated to a carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer, wherein the polysaccharide is covalently attached to the eTEC spacer via a carbamate bond and the carrier protein is covalently attached to the eTEC spacer via an amide bond.

C61. The composition of claim C60, wherein the sugar is an O antigen derived from E. coli.

C62. The composition of claim C60, further comprising a pharma- ceutically acceptable excipient, carrier, or diluent.

C63. The composition of claim C60, wherein the sugar is an O antigen derived from E. coli.

C64. A composition comprising a polypeptide derived from FimH or a fragment thereof; and a saccharide according to any one of items C1 to C17 conjugated to a carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer, in which the polysaccharide is covalently linked to the eTEC spacer via a carbamate bond and the carrier protein is covalently linked to the eTEC spacer via an amide bond.

C65. A composition comprising a polypeptide derived from FimH or a fragment thereof; and (i) a conjugate of E. coli O25B antigen covalently coupled to a carrier protein, (ii) a conjugate of E. coli O1A antigen covalently coupled to a carrier protein, (iii) a conjugate of E. coli O2 antigen covalently coupled to a carrier protein, and (iv) a conjugate of O6 antigen covalently coupled to a carrier protein, wherein the E. coli O25B antigen has a structure of the formula O25B, where n is an integer greater than 30.

C66. The carrier protein is selected from the group consisting 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, and exotoxin A from Pseudomonas aeruginosa; detoxified exotoxin A (EPA) from P. aeruginosa, maltose binding protein (MBP), detoxified hemolysin A from S. aureus, clumping factor A, clumping factor B, cholera toxin B subunit (CTB), Streptococcus pneumoniae, and the like. The composition of claim C65, wherein the antibacterial agent is selected from any one of C. pneumoniae pneumolysin and detoxified variants thereof, C. jejuni AcrA, C. jejuni native glycoprotein and Streptococcal C5a peptidase (SCP).

C67. A composition comprising a polypeptide derived from FimH or a fragment thereof; and (i) a conjugate of E. coli O25B antigen covalently coupled to a carrier protein, (ii) a conjugate of E. coli O4 antigen covalently coupled to a carrier protein, (iii) a conjugate of E. coli O11 antigen covalently coupled to a carrier protein, and (iv) a conjugate of O21 antigen covalently coupled to a carrier protein, wherein the E. coli O25B antigen has a structure of formula O75, where n is an integer greater than 30.

C68. The carrier protein is selected from the group consisting 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, and exotoxin A from Pseudomonas aeruginosa; detoxified exotoxin A (EPA) from P. aeruginosa, maltose binding protein (MBP), detoxified hemolysin A from S. aureus, clumping factor A, clumping factor B, cholera toxin B subunit (CTB), Streptococcus pneumoniae, and the like. The composition of claim C67, wherein the C5a peptidase is selected from any one of C. pneumoniae pneumolysin and detoxified variants thereof, C. jejuni AcrA, C. jejuni native glycoprotein and Streptococcal C5a peptidase (SCP).

C69. A method for making a composition comprising a conjugate comprising a polypeptide derived from FimH or a fragment thereof; and a sugar conjugated to a carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer, comprising the steps of: a) reacting the sugar with 1,1'-carbonyl-di-(1,2,4-triazole) (CDT) or 1,1'-carbonyldiimidazole (CDI) in an organic solvent to produce an activated sugar; b) reacting the activated sugar with cystamine or cysteamine or a salt thereof to produce a thiolated sugar; c) reacting the thiolated sugar with a reducing agent to produce an activated thiolated sugar comprising one or more free sulfhydryl residues; d) reacting the activated thiolated sugar with one or more α-haloacetamides. and e) reacting the thiolated sugar-carrier protein conjugate with (i) a first capping reagent capable of capping unconjugated α-haloacetamide groups of the activated carrier protein; and/or (ii) a second capping reagent capable of capping unconjugated free sulfhydryl residues of the activated carrier protein, thereby producing an eTEC-binding sugar conjugate, the sugar being derived from E. coli; the method further comprising expressing in a recombinant mammalian cell a polynucleotide encoding a polypeptide or fragment thereof derived from FimH, and isolating the polypeptide or fragment thereof.

C70. The method of claim C69, comprising producing a composition according to any one of claims C1 to C34.

C71. The method according to any one of items C69 to C70, wherein the capping step e) comprises reacting the thiolated sugar-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 of any one of items C69 to C71, further comprising blending the sugars by reaction with a triazole or imidazole to provide a blended sugar, wherein prior to step a), the blended sugars are shell frozen, lyophilized, and reconstituted in an organic solvent.

C73. The method of any one of items C69 to C72, further comprising purifying the thiolated polysaccharide produced in step c), the purification step comprising diafiltration.

C74. The method of any one of items C69 to C73, further comprising purifying the eTEC-bound glycoconjugate by diafiltration.

C75. The method according to any one of items C69 to C74, wherein the organic solvent in step a) is a polar aprotic solvent selected from any one of dimethylsulfoxide (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 containing KH2PO4, K2HPO4, (NH4)2SO4 , sodium citrate , Na2SO4 , aspartic} acid, glucose, _{MgSO4 , FeSO4-7H2O} , _Na2MoO4-2H2O , _H3BO3 , _{CoCl2-6H2O , CuCl2-2H2O , MnCl2-4H2O , ZnCl2 , and CaCl2-2H2O .}

C77. A medium according to item C76, used for culturing E. coli.

C78. A method for producing a sugar according to any one of claims C1 to C34, comprising culturing recombinant E. coli in a medium; and producing the sugar by culturing the cells in the medium, whereby the cells produce the sugar.

C79. The method of claim _C78 , wherein the medium comprises _{an element selected from any one of KH2PO4, K2HPO4, (NH4)2SO4, sodium citrate, Na2SO4 , aspartic acid , glucose , MgSO4 , FeSO4-7H2O , Na2MoO4-2H2O , H3BO3 , CoCl2-6H2O , CuCl2-2H2O , MnCl2-4H2O , ZnCl2 ,} and _{CaCl2-2H2O .}

C80. The method according to claim C78, wherein the medium comprises soy hydrolysate.

C81. The method according to claim C78, wherein the medium contains yeast extract.

C82. The method of claim C78, wherein the medium does not further contain soy hydrolysate and yeast extract.

C83. The method of claim C78, wherein the E. coli cell comprises a heterologous wzz family protein selected from any one of wzzB, wzz, wzz _SF , wzz _ST , fepE, wzz _fepE , wzz1, and wzz2.

C84. The method of claim C78, wherein the E. coli cells contain a Salmonella enterica wzz family protein selected from any one of wzzB, wzz, wzz _SF , wzz _ST , fepE, wzz _fepE , wzz1 and wzz2.

C85. The method of claim 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 of claim C78, wherein the culture results in a yield of greater than 120 OD600/mL.

C87. The method of claim C78, further comprising purifying the sugar.

C88. The method of claim C78, wherein the purification step comprises any one of the following: dialysis, a concentration operation, a diafiltration operation, tangential flow filtration, sedimentation, elution, centrifugation, sedimentation, ultrafiltration, depth filtration, and column chromatography (ion exchange chromatography, multimode ion exchange chromatography, DEAE, and hydrophobic interaction chromatography).

C89. A method for inducing an immune response in a mammal, comprising administering to a subject a composition described in any one of items C1 to C68.

C90. The method of claim C89, wherein the immune response includes induction of anti-E. coli O-specific polysaccharide serum antibodies.

C91. The method of claim C89, wherein the immune response includes induction of anti-E. coli IgG antibodies.

C92. The method of claim C89, wherein the immune response includes induction of bactericidal activity against E. coli.

C93. The method of claim C89, wherein the immune response includes induction of opsonophagocytic antibodies against E. coli.

C94. The method of claim C89, wherein the immune response comprises a geometric mean titer (GMT) level of at least 1,000 to 200,000 after the first dose.

C95. The method of claim C89, wherein the composition comprises a saccharide having the formula O25, where n is an integer between 40 and 100, and the immune response comprises a geometric mean titer (GMT) level of at least 1,000 to 200,000 after the first dose.

C96. The method of claim C89, wherein the mammal is at risk for any one of the conditions selected from urinary tract infection, cholecystitis, cholangitis, diarrhea, hemolytic uremic syndrome, neonatal meningitis, urosepsis, intraperitoneal infection, meningitis, complicated pneumonia, wound infection, post-prostate biopsy related infection, neonatal/infant sepsis, neutropenic fever, and other bloodstream infections; pneumonia, bacteremia, and sepsis.

C97. The method of claim C89, wherein the mammal has any one of the conditions selected from urinary tract infection, cholecystitis, cholangitis, diarrhea, hemolytic uremic syndrome, neonatal meningitis, urosepsis, intraperitoneal infection, meningitis, complicated pneumonia, wound infection, post-prostate biopsy related infection, neonatal/infant sepsis, neutropenic fever, and other bloodstream infections; pneumonia, bacteremia, and sepsis.

C98. (i) A method for inducing an immune response in a subject against extraintestinal pathogenic Escherichia coli, (ii) for inducing an immune response in a subject against extraintestinal pathogenic Escherichia coli, or (iii) for inducing the production of opsonophagocytic antibodies in a subject that are specific for extraintestinal pathogenic Escherichia coli, comprising administering to the subject an effective amount of a composition according to any one of items C1 to C68.

C99. The method of claim C98, wherein the subject is at risk of developing a urinary tract infection.

C100. The method of claim C98, wherein the subject is at risk of developing bacteremia.

C101. The method of claim C98, wherein the subject is at risk of developing sepsis.

C102. A composition comprising a polypeptide derived from FimH or a fragment thereof; and (i) a conjugate of E. coli O25B antigen covalently coupled to a carrier protein, (ii) a conjugate of E. coli O1A antigen covalently coupled to a carrier protein, (iii) a conjugate of E. coli O2 antigen covalently coupled to a carrier protein, and (iv) a conjugate of O6 antigen covalently coupled to a carrier protein, wherein the E. coli O25B antigen has a structure of the formula O25B, where n is an integer greater than 30.

C103. The carrier protein is selected from the group consisting of poly(L-lysine), detoxified exotoxin A (EPA) of Pseudomonas aeruginosa, CRM197, maltose binding protein (MBP), diphtheria toxoid, tetanus toxoid, detoxified hemolysin A of Staphylococcus aureus, clumping factor A, clumping factor B, cholera toxin B subunit (CTB), cholera toxin, detoxified variant of cholera toxin, Streptococcus pneumoniae, and Streptococcus pneumoniae. The composition according to item C102, wherein the β-glucosidase is selected from the group consisting of C. pneumoniae pneumolysin and its detoxified variants, C. jejuni AcrA, C. jejuni native glycoprotein and Streptococcal C5a peptidase (SCP).

C104. (i) A method for inducing an immune response in a subject against extraintestinal pathogenic Escherichia coli, (ii) for inducing an immune response in a subject against extraintestinal pathogenic Escherichia coli, or (iii) for inducing the production of opsonophagocytic antibodies in a subject that are specific for extraintestinal pathogenic Escherichia coli, comprising administering to the subject an effective amount of the composition described in item C51.

C105. The method of claim C104, wherein the subject is at risk of developing a urinary tract infection.

C106. The method of claim C104, wherein the subject is at risk of developing bacteremia.

C107. The method of claim C104, wherein the subject is at risk of developing sepsis.

C108. A composition comprising a polypeptide derived from FimH or a fragment thereof; and a saccharide comprising an increase of at least 5 repeating units compared to the corresponding wild-type O polysaccharide of E. coli.

C109. The composition of claim C108, wherein the saccharide comprises the formula O25a and the E. coli is E. coli serotype O25a.

C110. The composition of claim C108, wherein the saccharide comprises the formula O25b and the E. coli is E. coli serotype O25b.

C111. The composition of claim C108, wherein the sugar comprises the formula O2 and the E. coli is E. coli serotype O2.

C112. The composition of claim C108, wherein the sugar comprises the formula O6 and the E. coli is E. coli serotype O6.

C113. The composition of claim C108, wherein the sugar comprises the formula O1 and the E. coli is E. coli serotype O1.

C114. The composition of claim C108, wherein the saccharide comprises the formula O17 and the E. coli is E. coli serotype O17.

C115. The sugar is formula O1, formula O2, formula O3, formula O4, formula O5, formula O6, formula O7, formula O8, formula O9, formula O10, formula O11, formula O12, formula O13, formula O14, formula O15, formula O16, formula O17, formula O18, formula O19, formula O20, formula O21, formula O22, formula O23, formula O24, formula O25, formula O25b, formula O26, Formula O27, Formula O28, Formula O29, Formula O30, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37, Formula O38, Formula O39, Formula O40, Formula O41, Formula O42, Formula O43, Formula O44, Formula O45, Formula O46, Formula O48, Formula O49, Formula O50, Formula O51, Formula O52, Formula O53, Formula O54, Formula O55, Formula O56, Formula O57, Formula O58, Formula O59, Formula O60, Formula O61, Formula O62, Formula O63, Formula O64, Formula O65, Formula O66, Formula O68, Formula O69, Formula O70, Formula O71, Formula O73, Formula O74, Formula O75, Formula O76, Formula O77, Formula O78, Formula O79, Formula O80, Formula O81, Formula O82, Formula O83, Formula O84, Formula O85, Formula O86, Formula O87, Formula O88, Formula O89, Formula O90, Formula O91, Formula O92, Formula O93, Formula O95, Formula O96, Formula O97, Formula O98, Formula O99, Formula O100, Formula O101, Formula O102, Formula O103, Formula O104, Formula O105, Formula O106, Formula O107, Formula O 108, Formula O109, Formula O110, Formula O111, Formula O112, Formula O113, Formula O114, Formula O115, Formula O116, Formula O117, Formula O118, Formula O119, formula O120, formula O121, formula O123, formula O124, formula O125, formula O126, formula O127, formula O128, formula O129, formula O130, Formula O131, Formula O132, Formula O133, Formula O134, Formula O135, Formula O136, Formula O137, Formula O138, Formula O139, Formula O140, Formula O141 , formula O142, formula O143, formula O144, formula O145, formula O146, formula O147, formula O148, formula O149, formula O150, formula O151, formula O15 2. The composition according to item C108, comprising a structure selected from formula O153, formula O154, formula O155, formula O156, formula O157, formula O158, formula O159, formula O160, formula O161, formula O162, formula O163, formula O164, formula O165, formula O166, formula O167, formula O168, formula O169, formula O170, formula O171, formula O172, formula O173, formula O174, formula O175, formula O176, formula O177, formula O178, formula O179, formula O180, formula O181, formula O182, formula O183, formula O184, formula O185, formula O186, and formula O187 (wherein n is an integer from 5 to 1000).

C116. E. coli: O1, O2, O3, O4, O5, O6, O7, O8, O9, O10, O11, O12, O13, O14, O15, O16, O17, O18, O19, O20, O21, O22, O23, O24, O25, O25b, O26, O27, O28, O29, O30, O32, O33, O34, O35, O36, O37, O38, O39, O40, O41, O42, O43, O44, O45, O46, O48, O49, O50, O51, O52, O53, O54 , O55, O56, O57, O58, O59, O60, O61, O62, O63, O64, O65, O66, O68, O69, O70, O71, O73, O74, O75, O76, O77, O78, O79, O80, O81, O82, O83 , O84, O85, O86, O87, O88, O89, O90, O91, O92, O93, O95, O96, O97, O98, O99, O100, O101, O102, O103, O104, O105, O106, O107, O108, O10 9, O110, 0111, O112, O113, O114, O115, O116, O117, O118, O119, O120, O121, O123, O124, O125, O126, O127, O128, O129, O130, O131, O 132, O133, O134, O135, O136, O137, O138, O139, O140, O141, O142, O143, O144, O145, O146, O147, O148, O149, O150, O151, O152, O153 The composition according to item C108, wherein the E. coli serotype is selected from the group consisting of O154, O155, O156, O157, O158, O159, O160, O161, O162, O163, O164, O165, O166, O167, O168, O169, O170, O171, O172, O173, O174, O175, O176, O177, O178, O179, O180, O181, O182, O183, O184, O185, O186, and O187.

C117. The composition of claim C108, wherein the sugar is produced by increasing the repeating units of the O polysaccharide produced by a gram-negative bacterium in culture, comprising overexpressing a wzz family protein in the gram-negative bacterium that produces the sugar.

The composition of claim C117, wherein the overexpressed wzz family protein is selected from the group consisting of wzzB, wzz, wzz _SF , wzz _ST , fepE, wzz _fepE , wzz1 and wzz2.

C119. The composition according to item C117, wherein the overexpressed wzz family protein is wzzB.

C120. The composition according to item C117, wherein the overexpressed wzz family protein is fepE.

C121. The composition according to item C117, wherein the overexpressed wzz family proteins are wzzB and fepE.

C122. The composition according to claim C108, wherein the sugar is synthetically synthesized.

C123. A composition comprising a polypeptide derived from FimH or a fragment thereof; and a conjugate comprising a sugar according to item C108, covalently bound to a carrier protein.

The composition according to claim C123, wherein the carrier protein is CRM ₁₉₇ .

C125. The sugar is formula O1, formula O2, formula O3, formula O4, formula O5, formula O6, formula O7, formula O8, formula O9, formula O10, formula O11, formula O12, formula O13, formula O14, formula O15, formula O16, formula O17, formula O18, formula O19, formula O20, formula O21, formula O22, formula O23, formula O24, formula O25, formula O25b, formula O26, Formula O27, Formula O28, Formula O29, Formula O30, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37, Formula O38, Formula O39, Formula O40, Formula O41, Formula O42, Formula O43, Formula O44, Formula O45, Formula O46, Formula O48, Formula O49, Formula O50, Formula O51, Formula O52, Formula O53, Formula O54, Formula O55, Formula O56, Formula O57, Formula O58, Formula O59, Formula O60, Formula O61, Formula O62, Formula O63, Formula O64, Formula O65, Formula O66, Formula O68, Formula O69, Formula O70, Formula O71, Formula O73, Formula O74, Formula O75, Formula O76, Formula O77, Formula O78, Formula O79, Formula O80, Formula O81, Formula O82, Formula O83, Formula O84, Formula O85, Formula O86, Formula O87, Formula O88, Formula O89, Formula O90, Formula O91, Formula O92, Formula O93, Formula O95, Formula O96, Formula O97, Formula O98, Formula O99, Formula O100, Formula O101, Formula O102, Formula O103, Formula O104, Formula O105, Formula O106, Formula O107, Formula O 108, Formula O109, Formula O110, Formula O111, Formula O112, Formula O113, Formula O114, Formula O115, Formula O116, Formula O117, Formula O118, Formula O119, formula O120, formula O121, formula O123, formula O124, formula O125, formula O126, formula O127, formula O128, formula O129, formula O130, Formula O131, Formula O132, Formula O133, Formula O134, Formula O135, Formula O136, Formula O137, Formula O138, Formula O139, Formula O140, Formula O141 , formula O142, formula O143, formula O144, formula O145, formula O146, formula O147, formula O148, formula O149, formula O150, formula O151, formula O15 2. The composition according to item C123, comprising a structure selected from formula O153, formula O154, formula O155, formula O156, formula O157, formula O158, formula O159, formula O160, formula O161, formula O162, formula O163, formula O164, formula O165, formula O166, formula O167, formula O168, formula O169, formula O170, formula O171, formula O172, formula O173, formula O174, formula O175, formula O176, formula O177, formula O178, formula O179, formula O180, formula O181, formula O182, formula O183, formula O184, formula O185, formula O186, and formula O187 (wherein n is an integer from 5 to 1000).

C126. The composition of claim C123, wherein the saccharide comprises an increase of at least 5 repeating units compared to the corresponding wild-type O polysaccharide.

C127. The composition according to claim C1, further comprising a pharma- ceutically acceptable diluent.

C128. The composition according to claim C127, further comprising an adjuvant.

C129. The composition of claim C127, further comprising aluminum.

C130. The composition described in item C127, further comprising QS-21.

C131. The composition according to item C127, which does not contain an adjuvant.

C132. A method for inducing an immune response in a subject, comprising administering to the subject a composition described in item C127.

C133. The composition according to claim C123, further comprising a pharma- ceutically acceptable diluent.

C134. A method for inducing an immune response in a subject, comprising administering to the subject the composition described in item C133.

C135. The method of claim C132 or C134, wherein the immune response includes induction of anti-E. coli O-specific polysaccharide serum antibodies.

C136. The method according to claim C135, wherein the anti-E. coli O-specific polysaccharide serum antibody is an IgG antibody.

C137. The method according to claim C135, wherein the anti-E. coli O-specific polysaccharide serum antibody is an IgG antibody having bactericidal activity against E. coli.

C138. An immunogenic composition comprising a polypeptide or fragment thereof derived from FimH, in which the polysaccharide is covalently linked to the eTEC spacer via a carbamate bond and the carrier protein is covalently linked to the eTEC spacer via an amide bond; and a saccharide derived from Escherichia coli (E. coli) conjugated to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer.

C139. The immunogenic composition of claim C138, further comprising a pharma- ceutically acceptable excipient, carrier, or diluent.

C140. The immunogenic composition according to claim C138, wherein the saccharide is an O antigen derived from E. coli.

C141. The sugar is formula O1, formula O2, formula O3, formula O4, formula O5, formula O6, formula O7, formula O8, formula O9, formula O10, formula O11, formula O12, formula O13, formula O14, formula O15, formula O16, formula O17, formula O18, formula O19, formula O20, formula O21, formula O22, formula O23, formula O24, formula O25, formula O25b, formula O26, Formula O27, Formula O28, Formula O29, Formula O30, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37, Formula O38, Formula O39, Formula O40, Formula O41, Formula O42, Formula O43, Formula O44, Formula O45, Formula O46, Formula O48, Formula O49, Formula O50, Formula O51, Formula O52, Formula O53, Formula O54, Formula O55, formula O56, formula O57, formula O58, formula O59, formula O60, formula O61, formula O62, formula O63, formula O64, formula O65, formula O66, formula O68, formula O69, formula O70, formula O71, formula O73, formula O74, formula O75, formula O76, formula O77, formula O78, formula O79, formula O80, formula O81, formula O82, formula O83, formula O84, formula O85, formula O86, formula O87, formula O88, formula O89, formula O90, formula O91, formula O92, formula O93, formula O95, formula O96, formula O97, formula O98, formula O99, formula O100, formula O101, formula O102, formula O103, formula O104, formula O105, formula O106, formula O107, formula O10 8, Formula O109, Formula O110, Formula O111, Formula O112, Formula O113, Formula O114, Formula O115, Formula O116, Formula O117, Formula O118, Formula O1 19, Formula O120, Formula O121, Formula O123, Formula O124, Formula O125, Formula O126, Formula O127, Formula O128, Formula O129, Formula O130, Formula O 131, Formula O132, Formula O133, Formula O134, Formula O135, Formula O136, Formula O137, Formula O138, Formula O139, Formula O140, Formula O141, Formula O142, formula O143, formula O144, formula O145, formula O146, formula O147, formula O148, formula O149, formula O150, formula O151, formula O152, formula The immunogenic composition according to item C138, comprising a structure selected from O153, O154, O155, O156, O157, O158, O159, O160, O161, O162, O163, O164, O165, O166, O167, O168, O169, O170, O171, O172, O173, O174, O175, O176, O177, O178, O179, O180, O181, O182, O183, O184, O185, O186, and O187 (wherein n is an integer from 5 to 1000).

C142. The immunogenic composition according to claim C138, wherein the saccharide has a degree of O-acetylation of 75-100%.

C143. The immunogenic composition according to item C138, wherein the carrier protein is CRM197.

C144. The immunogenic composition of claim C143, wherein CRM197 comprises 2 to 20 lysine residues covalently attached to the polysaccharide via an eTEC spacer.

C145. The immunogenic composition of claim C143, wherein CRM197 comprises 4 to 16 lysine residues covalently attached to the polysaccharide via an eTEC spacer.

C146. The immunogenic composition according to claim C138, further comprising an additional antigen.

C147. The immunogenic composition according to item C138, further comprising an adjuvant.

C148. The immunogenic composition according to claim 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 item C138, which does not contain an adjuvant.

C150. An immunogenic composition comprising a polypeptide derived from FimH or a fragment thereof; and a glycoconjugate comprising a sugar derived from E. coli conjugated to a carrier protein, the glycoconjugate being prepared using reductive amination.

C151. The immunogenic composition of claim C150, further comprising a pharma- ceutically acceptable excipient, carrier, or diluent.

C152. The immunogenic composition according to claim C150, wherein the saccharide is an O antigen derived from E. coli.

C153. The sugar is formula O1, formula O2, formula O3, formula O4, formula O5, formula O6, formula O7, formula O8, formula O9, formula O10, formula O11, formula O12, formula O13, formula O14, formula O15, formula O16, formula O17, formula O18, formula O19, formula O20, formula O21, formula O22, formula O23, formula O24, formula O25, formula O25b, formula O26, Formula O27, Formula O28, Formula O29, Formula O30, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37, Formula O38, Formula O39, Formula O40, Formula O41, Formula O42, Formula O43, Formula O44, Formula O45, Formula O46, Formula O48, Formula O49, Formula O50, Formula O51, Formula O52, Formula O53, Formula O54, Formula O55, formula O56, formula O57, formula O58, formula O59, formula O60, formula O61, formula O62, formula O63, formula O64, formula O65, formula O66, formula O68, formula O69, formula O70, formula O71, formula O73, formula O74, formula O75, formula O76, formula O77, formula O78, formula O79, formula O80, formula O81, formula O82, formula O83, formula O84, formula O85, formula O86, formula O87, formula O88, formula O89, formula O90, formula O91, formula O92, formula O93, formula O95, formula O96, formula O97, formula O98, formula O99, formula O100, formula O101, formula O102, formula O103, formula O104, formula O105, formula O106, formula O107, formula O10 8, Formula O109, Formula O110, Formula O111, Formula O112, Formula O113, Formula O114, Formula O115, Formula O116, Formula O117, Formula O118, Formula O1 19, Formula O120, Formula O121, Formula O123, Formula O124, Formula O125, Formula O126, Formula O127, Formula O128, Formula O129, Formula O130, Formula O 131, Formula O132, Formula O133, Formula O134, Formula O135, Formula O136, Formula O137, Formula O138, Formula O139, Formula O140, Formula O141, Formula O142, formula O143, formula O144, formula O145, formula O146, formula O147, formula O148, formula O149, formula O150, formula O151, formula O152, formula The immunogenic composition according to item C150, comprising a structure selected from O153, O154, O155, O156, O157, O158, O159, O160, O161, O162, O163, O164, O165, O166, O167, O168, O169, O170, O171, O172, O173, O174, O175, O176, O177, O178, O179, O180, O181, O182, O183, O184, O185, O186, and O187 (wherein n is an integer from 5 to 1000).

C154. The immunogenic composition according to claim C150, wherein the saccharide has a degree of O-acetylation of 75-100%.

C155. The immunogenic composition according to item C150, wherein the carrier protein is CRM197.

C156. The immunogenic composition according to claim C150, further comprising an additional antigen.

C157. The immunogenic composition according to item C150, further comprising an adjuvant.

C158. The immunogenic composition according to claim 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 item C150, which does not contain an adjuvant.

C160. A method for inducing an immune response in a subject, comprising administering to the subject a composition described in any one of items C138 to C159.

C161. The method of claim C160, wherein the immune response includes induction of anti-E. coli O-specific polysaccharide serum antibodies.

C162. The method according to claim C135, wherein the anti-E. coli O-specific polysaccharide serum antibody is an IgG antibody.

C163. The method according to claim C135, wherein the anti-E. coli O-specific polysaccharide serum antibody is an IgG antibody having bactericidal activity against E. coli.

C164. A polypeptide or fragment thereof derived from FimH; and a polypeptide of formula O1, formula O1A, formula O1B, formula O1C, formula O2, formula O3, formula O4, formula O4:K52, formula O4:K6, formula O5, formula O5ab, formula O5ac, formula O6, formula O6:K2;K13;K15, formula O6:K54, formula O7, formula O8, formula O9, formula O10, formula O11, formula O12, formula O13, formula O14, formula O15, formula O16, formula O17, formula O18, formula O18A, formula O18ac, formula O18A1, formula O18B, formula O18B1, formula O19, formula O20, formula O21, formula O22, Formula O23, Formula O23A, Formula O24, Formula O25, Formula O25a, Formula O25b, Formula O26, Formula O27, Formula O28, Formula O29, Formula O30, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37, Formula O38, Formula O39, Formula O40, Formula O41, Formula O42 , formula O43, formula O44, formula O45, formula O45, formula O45rel, formula O46, formula O48, formula O49, formula O50, formula O51, formula O52, formula O53, formula O54, formula O55, formula O56, formula O57, formula O58, formula O59, formula O60, formula O61, formula O62, formula 62D ₁ , formula O63, formula O64, formula O65, formula O66, formula O68, formula O69, formula O70, formula O71, formula O73, formula O73, formula O74, formula O75, formula O76, formula O77, formula O78, formula O79, formula O80, formula O8 1, Formula O82, Formula O83, Formula O84, Formula O85, Formula O86, Formula O87, Formula O88, Formula O89, Formula O90, Formula O91, Formula O92, Formula O93, Formula O95, Formula O96, Formula O97, Formula O98, Formula O99, Formula O1 00, Formula O101, Formula O102, Formula O103, Formula O104, Formula O105, Formula O106, Formula O107, Formula O108, Formula O109, Formula O110, Formula O111, Formula O112, Formula O113, Formula O114, Formula O 115, Formula O116, Formula O117, Formula O118, Formula O119, Formula O120, Formula O121, Formula O123, Formula O124, Formula O125, Formula O126, Formula O127, Formula O128, Formula O129, Formula O130, Formula O 131, Formula O132, Formula O133, Formula O134, Formula O135, Formula O136, Formula O137, Formula O138, Formula O139, Formula O140, Formula O141, Formula O142, Formula O143, Formula O144, Formula O145, Formula O146, formula O147, formula O148, formula O149, formula O150, formula O151, formula O152, formula O153, formula O154, formula O155, formula O156, formula O157, formula O158, formula O159, formula O160, formula 1. A composition comprising a saccharide comprising a structure selected from any one of formulas O161, O162, O163, O164, O165, O166, O167, O168, O169, O170, O171, O172, O173, O174, O175, O176, O177, O178, O179, O180, O181, O182, O183, O184, O185, O186, O187, wherein n is greater than the number of repeating units in the corresponding wild-type E. coli polysaccharide.

C165. The composition according to item C164, wherein n is an integer from 31 to 100.

C166. The composition of claim C164, wherein the sugar comprises a structure according to any one of formulas O1A, O1B, and O1C, O2, O6, and O25B.

C167. The composition of claim C164, wherein the sugar is produced in a recombinant host cell expressing 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 item C167, wherein the protein comprises any one of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34.

C169. A synthetically synthesized sugar according to item C164.

C170. A composition comprising a conjugate comprising a polypeptide derived from FimH or a fragment thereof; and a carrier protein covalently bound to a saccharide, wherein the saccharide is selected from the group consisting of Formula O1, Formula O1A, Formula O1B, Formula O1C, Formula O2, Formula O3, Formula O4, Formula O4:K52, Formula O4:K6, Formula O5, Formula O5ab, Formula O5ac, Formula O6, Formula O6:K2;K13;K15, Formula O6:K54, Formula O7, Formula O8, Formula O9, Formula O10, Formula O11, Formula O12, Formula O13, Formula O14, Formula O15, Formula O16, Formula O17, Formula O18, Formula O18A, Formula O18ac, Formula O18A1, Formula O18B, Formula O18B1, Formula O19, Formula O20, Formula O21, Formula O22, Formula O23, Formula O23A, Formula O24, Formula O25, Formula O25a, Formula O25b, Formula O26, Formula O2 7, Formula O28, Formula O29, Formula O30, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37, Formula O38, Formula O39, Formula O40, Formula O41, Formula O42, Formula O43, Formula O44, Formula O45, Formula O45, Formula O45rel, Formula O46, Formula O48, Formula O49, Formula O50, Formula O5 1, Formula O52, Formula O53, Formula O54, Formula O55, Formula O56, Formula O57, Formula O58, Formula O59, Formula O60, Formula O61, Formula O62, Formula O62D ₁ , formula O63, formula O64, formula O65, formula O66, formula O68, formula O69, formula O70, formula O71, formula O73, formula O73, formula O74, formula O75, formula O76, formula O77, formula O78, formula O79, formula O80, formula O81, formula O82, formula O83, formula O84, formula O85, formula O86, formula O87, formula O88, formula O89, formula O90, formula O91, formula O92, formula O93, formula O95, formula O96, formula O97, formula O98, formula O99, formula O100, formula O1 01, Formula O102, Formula O103, Formula O104, Formula O105, Formula O106, Formula O107, Formula O108, Formula O109, Formula O110, Formula O111, Formula O112, Formula O113, Formula O114, Formula O115, Formula O116, Formula O117, Formula O118, Formula O119, Formula O120, Formula O121, Formula O123, Formula O124, Formula O125, Formula O126, Formula O127, Formula O128, Formula O129, Formula O130, Formula O131, Formula O132, Formula O1 33, Formula O134, Formula O135, Formula O136, Formula O137, Formula O138, Formula O139, Formula O140, Formula O141, Formula O142, Formula O143, Formula O144, Formula O145, Formula O146, Formula O147, Formula O148 , formula O149, formula O150, formula O151, formula O152, formula O153, formula O154, formula O155, formula O156, formula O157, formula O158, formula O159, formula O160, formula O161, formula O162, formula O163, formula O 164, Formula O165, Formula O166, Formula O167, Formula O168, Formula O169, Formula O170, Formula O171, Formula O172, Formula O173, Formula O174, Formula O175, Formula O176, Formula O177, Formula O178, Formula O179, Formula O180, Formula O181, Formula O182, Formula O183, Formula O184, Formula O185, Formula O186, or Formula O187, wherein n is an integer from 1 to 100.

C171. The composition of claim C170, wherein the sugar comprises any one of formula O25b, formula O1A, formula O2, and formula O6.

C172. The composition of claim C170, wherein the sugar further comprises any one of an E. coli R1 portion, an E. coli R2 portion, an E. coli R3 portion, an E. coli R4 portion, and an E. coli K-12 portion.

C173. The composition of claim C170, wherein the sugar does not further comprise any one of an E. coli R1 portion, an E. coli R2 portion, an E. coli R3 portion, an E. coli R4 portion, and an E. coli K-12 portion. The composition of claim C170, wherein the sugar does not further comprise an E. coli R2 portion.

C174. The composition of claim C170, wherein the sugar further comprises a 3-deoxy-d-manno-octa-2-ulosonic acid (KDO) moiety.

C175. 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, or exotoxin A from Pseudomonas aeruginosa; detoxified exotoxin A (EPA) from P. aeruginosa, maltose binding protein (MBP), detoxified hemolysin A from S. aureus, clumping factor A, clumping factor B, cholera toxin B subunit (CTB), Streptococcus pneumoniae, The composition of item C170, wherein the antibacterial agent is selected from any one of C. pneumoniae pneumolysin and detoxified variants thereof, C. jejuni AcrA, C. jejuni native glycoprotein and Streptococcal C5a peptidase (SCP).

The composition according to claim C170, wherein the carrier protein is CRM ₁₉₇ .

C177. The composition according to claim C170, wherein the carrier protein is a tetanus toxoid.

C178. The composition according to claim C170, wherein the sugar to protein ratio is at least 0.5 and at most 2.

C179. The composition according to claim C170, prepared by reductive amination.

C180. The composition of claim C170, wherein the sugar is conjugated to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer.

C181. The composition of claim C170, wherein the sugar is a single-end-linked conjugated sugar.

C182. The composition according to claim C174, wherein the sugar is conjugated to the carrier protein via a 3-deoxy-d-manno-octa-2-ulosonic acid (KDO) residue.

C183. The composition of claim C170, prepared by CDAP chemical reaction.

C184. A polypeptide derived from FimH or a fragment thereof; and a composition comprising (a) a conjugate comprising a carrier protein covalently bound to a saccharide comprising formula O25b, (b) a conjugate comprising a carrier protein covalently bound to a saccharide comprising formula O1A, (c) a conjugate comprising a carrier protein covalently bound to a saccharide comprising formula O2, (d) a conjugate comprising a carrier protein covalently bound to a saccharide comprising formula O6, (e) a conjugate comprising a carrier protein covalently bound to a saccharide comprising formula O6.

C185. The composition of claim C184, further comprising a conjugate comprising a carrier protein covalently bound to a sugar having a structure selected from any one of formulas O15, O16, O17, O18, and O75, where n is an integer from 31 to 90.

C186. The composition according to claim C184, comprising at most about 25% free sugars compared to the total amount of sugars in the composition.

C187. A method for eliciting an immune response against Escherichia coli in a mammal, comprising administering to the mammal an effective amount of a composition described in any one of items C184 to C186.

C188. The method of claim C187, wherein the immune response comprises opsonophagocytic antibodies against E. coli.

C189. The method of claim C187, wherein the immune response protects the mammal from E. coli infection.

C190. (a) A mammalian cell comprising a first gene of interest encoding a polypeptide or a fragment thereof derived from E. coli, the gene being integrated between at least two recombination target sites (RTS).

C191. The embodiment of item C190, wherein the two RTSs are chromosomally integrated within the NL1 locus or the NL2 locus.

C192. The embodiment of claim C190, wherein the first gene of interest further comprises a reporter gene, a gene encoding a difficult to express protein, an auxiliary gene, or a combination thereof.

C193. The embodiment of claim C190, further comprising a second gene of interest integrated into a second chromosomal locus different from the locus of (a), the second gene of interest comprising a reporter gene, a gene encoding a difficult to express protein, an auxiliary gene, or a combination thereof.

C194. A recombinant mammalian cell comprising a polynucleotide encoding a polypeptide or a fragment thereof derived from E. coli.

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 residues 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 contain a glycine residue immediately preceding the phenylalanine residue at position 1 of the polypeptide.

C200. The recombinant cell according to C195, wherein the polypeptide does not contain an N-glycosylation site at position 7 of the polypeptide.

C201. The recombinant cell according to C199, wherein the polypeptide does not contain 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 contain an N-glycosylation site at position 70 of the polypeptide.

C204. The recombinant cell according to C203, wherein the polypeptide does not contain an Asn residue at position 70 of the polypeptide.

C205. The recombinant cell according to C203, wherein the polypeptide does not contain a Ser residue at position 70 of the polypeptide.

C206. The recombinant cell of C194, 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.

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 sites include positions N235 and 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 of C215, wherein the lectin domain comprises a mass of about 17,022 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 a 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 contain a signal peptide selected from the group consisting of a native FimH leader peptide, an influenza hemagglutinin signal peptide, and a human respiratory syncytial virus A (A2 strain) fusion glycoprotein F0 signal peptide.

C227. The recombinant cell according to C194, wherein the polypeptide comprises a mouse IgK signal peptide sequence.

C228. The recombinant cell according to C194, wherein the polypeptide comprises any one of a signal peptide sequence selected from a human IgG receptor FcRn large subunit p51 signal peptide and a human IL10 protein signal peptide.

C229. The recombinant cell of C195, wherein the polypeptide comprises an arginine to proline mutation 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 higher than the expression level of a 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 the mammalian cell.

C233. The recombinant cell of C194, wherein the polynucleotide sequence is codon-optimized for expression in the cell.

C234. The recombinant cell according to C194, which 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, which is a CHO cell.

C238. The recombinant cell according to C237, wherein the CHO cell is a CHO-K1 cell, a CHO-DUXB11 cell, a CHO-DG44 cell, or a CHO-S cell.

C239. The recombinant cell according to C194, wherein the polypeptide is soluble.

C240. A recombinant cell according to C194, wherein the polypeptide is secreted from the cell.

C241. The recombinant cell of C195, wherein the polypeptide comprises an N28Q substitution according to the numbering of SEQ ID NO:1.

C242. The recombinant cell of C195, wherein the polypeptide comprises an N28D substitution according to the numbering of SEQ ID NO:1.

C243. The recombinant cell of C195, wherein the polypeptide comprises an N28S substitution according to the numbering of SEQ ID NO:1.

C244. The recombinant cell of 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 of C195, wherein the polypeptide comprises the 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 a fragment thereof comprises a substitution selected from any one of V48C and L55C according to the numbering of SEQ ID NO:1.

C247. A culture comprising the recombinant cells described in C194, the culture being at least 5 liters in size.

C248. The culture of C242, wherein the yield of the polypeptide or fragment thereof is at least 0.05 g/L.

C249. The culture of C248, wherein the yield of the polypeptide or fragment thereof is at least 0.10 g/L.

C250. A method for producing a polypeptide or fragment thereof from E. coli, comprising expressing the polypeptide or fragment thereof by culturing a recombinant mammalian cell according to C194 under suitable conditions; and collecting the polypeptide or fragment thereof.

C251. The method of C250, further comprising purifying the polypeptide or fragment thereof.

C252. The method of 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 of C250, wherein the yield of the polypeptide or fragment thereof is at least 0.05 g/L.

C254. The method of 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 described in C255, further comprising a sugar having a structure selected from any one of the formulas in Table 1.

C257. The composition according to C256, wherein the sugar is covalently attached to a carrier protein.

C258. The carrier protein is selected from the group consisting 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 (EPA) from P. aeruginosa, maltose binding protein (MBP), detoxified hemolysin A from S. aureus, clumping factor A, clumping factor B, cholera toxin B subunit (CTB), Streptococcus pneumoniae, The composition of C257, wherein the active ingredient is selected from any one of C. pneumoniae pneumolysin and detoxified variants thereof, C. jejuni AcrA, C. jejuni native glycoprotein 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 of C257, wherein the sugar is covalently attached to the carrier protein by reductive amination.

C263. The composition of C257, wherein the sugar is covalently attached to the carrier protein by CDAP chemistry.

C264. The composition of C257, wherein the sugar is covalently attached to the carrier protein by a single end-linked conjugation.

C265. The composition of C257, wherein the sugar is covalently attached to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer.

C266. A polypeptide comprising 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, and SEQ ID NO:27.

C267. A polypeptide or fragment thereof derived from FimH; and a polypeptide of formula O1, formula O1A, formula O1B, formula O1C, formula O2, formula O3, formula O4, formula O4:K52, formula O4:K6, formula O5, formula O5ab, formula O5ac, formula O6, formula O6:K2;K13;K15, formula O6:K54, formula O7, formula O8, formula O9, formula O10, formula O11, formula O12, formula O13, formula O14, formula O15, formula O16, formula O17, formula O18, formula O18A, formula O18ac, formula O18A1, formula O18B, formula O18B1, formula O19, formula O20, formula O21, formula O22, Formula O23, Formula O23A, Formula O24, Formula O25, Formula O25a, Formula O25b, Formula O26, Formula O27, Formula O28, Formula O29, Formula O30, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37, Formula O38, Formula O39, Formula O40, Formula O41, Formula O42 , formula O43, formula O44, formula O45, formula O45, formula O45rel, formula O46, formula O48, formula O49, formula O50, formula O51, formula O52, formula O53, formula O54, formula O55, formula O56, formula O57, formula O58, formula O59, formula O60, formula O61, formula O62, formula 62D ₁ , formula O63, formula O64, formula O65, formula O66, formula O68, formula O69, formula O70, formula O71, formula O73, formula O73, formula O74, formula O75, formula O76, formula O77, formula O78, formula O79, formula O80, formula O81 , formula O82, formula O83, formula O84, formula O85, formula O86, formula O87, formula O88, formula O89, formula O90, formula O91, formula O92, formula O93, formula O95, formula O96, formula O97, formula O98, formula O99, formula O100 , formula O101, formula O102, formula O103, formula O104, formula O105, formula O106, formula O107, formula O108, formula O109, formula O110, formula O111, formula O112, formula O113, formula O114, formula O115 , formula O116, formula O117, formula O118, formula O119, formula O120, formula O121, formula O123, formula O124, formula O125, formula O126, formula O127, formula O128, formula O129, formula O130, formula O131, Formula O132, Formula O133, Formula O134, Formula O135, Formula O136, Formula O137, Formula O138, Formula O139, Formula O140, Formula O141, Formula O142, Formula O143, Formula O144, Formula O145, Formula O146, Formula O147, Formula O148, Formula O149, Formula O150, Formula O151, Formula O152, Formula O153, Formula O154, Formula O155, Formula O156, Formula O157, Formula O158, Formula O159, Formula O160, Formula O161, Formula A composition comprising a sugar comprising a structure selected from any one of formulas O162, O163, O164, O165, O166, O167, O168, O169, O170, O171, O172, O173, O174, O175, O176, O177, O178, O179, O180, O181, O182, O183, O184, O185, O186, and O187.

C268. The composition of claim C267, further comprising at least one saccharide derived from any one of K. pneumoniae types selected from the group consisting of O1, O2, O3, and O5.

C269. The composition according to claim C267, further comprising a sugar derived from Klebsiella pneumoniae type O1.

C270. The composition of claim C267, further comprising a sugar derived from Klebsiella pneumoniae type O2.

C271. The composition of claim C267, further comprising a sugar derived from K. pneumoniae type O3.

C272. The composition according to claim C267, further comprising a saccharide derived from Klebsiella pneumoniae type O5.

C273. The composition of claim C267, further comprising a saccharide derived from K. pneumoniae type O1 and a saccharide derived from K. pneumoniae type O2.

C274. The composition of claim 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 claim C267, further comprising a polypeptide derived from Klebsiella pneumoniae.

C276. A composition comprising a polypeptide or fragment thereof derived from FimH; and at least one saccharide derived from any one of K. pneumoniae types selected from the group consisting of O1, O2, O3, and O5.

C277. Formula O1, Formula O1A, Formula O1B, Formula O1C, Formula O2, Formula O3, Formula O4, Formula O4:K52, Formula O4:K6, Formula O5, Formula O5ab, Formula O5ac, Formula O6, Formula O6:K2;K13;K15, Formula O6:K54, Formula O7, Formula O8, Formula O9, Formula O10, Formula O11, Formula O12, Formula O13, Formula O14, Formula O15, Formula O16, Formula O17, Formula O18, Formula O18A, Formula O18ac, Formula O18A1, Formula O18B, Formula O18B1, Formula O19, Formula O20, Formula O21, Formula O22, Formula O23, Formula O23A, Formula O 24, formula O25, formula O25a, formula O25b, formula O26, formula O27, formula O28, formula O29, formula O30, formula O32, formula O33, formula O34, formula O35, formula O36, formula O37, formula O38, formula O39, formula O40, formula O41, formula O42, formula O43, formula O44, formula O45, formula O45, formula O45rel, formula O46, formula O48, formula O49, formula O50, formula O51, formula O52, formula O53, formula O54, formula O55, formula O56, formula O57, formula O58, formula O59, formula O60, formula O61, formula O62, formula 62D ₁ , formula O63, formula O64, formula O65, formula O66, formula O68, formula O69, formula O70, formula O71, formula O73, formula O73, formula O74, formula O75, formula O76, formula O77, formula O78, formula O79, formula O80, formula O81, formula O 82, Formula O83, Formula O84, Formula O85, Formula O86, Formula O87, Formula O88, Formula O89, Formula O90, Formula O91, Formula O92, Formula O93, Formula O95, Formula O96, Formula O97, Formula O98, Formula O99, Formula O100, Formula O10 1, Formula O102, Formula O103, Formula O104, Formula O105, Formula O106, Formula O107, Formula O108, Formula O109, Formula O110, Formula O111, Formula O112, Formula O113, Formula O114, Formula O115, Formula O116, Formula O117, formula O118, formula O119, formula O120, formula O121, formula O123, formula O124, formula O125, formula O126, formula O127, formula O128, formula O129, formula O130, formula O131, formula O132, formula O13 3. Formula O134, Formula O135, Formula O136, Formula O137, Formula O138, Formula O139, Formula O140, Formula O141, Formula O142, Formula O143, Formula O144, Formula O145, Formula O146, Formula O147, Formula O148, Formula O149, formula O150, formula O151, formula O152, formula O153, formula O154, formula O155, formula O156, formula O157, formula O158, formula O159, formula O160, formula O161, formula O162, formula O163, formula O16 4. The composition according to item C276, further comprising at least one sugar comprising a structure selected from any of formulas O165, O166, O167, O168, O169, O170, O171, O172, O173, O174, O175, O176, O177, O178, O179, O180, O181, O182, O183, O184, O185, O186, and O187.

C278. The composition of claim 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 claim C277, further comprising a polypeptide derived from Klebsiella pneumoniae.

C280. At least one saccharide derived from any one of K. pneumoniae types selected from the group consisting of O1, O2, O3, and O5; and a saccharide of formula O1, O1A, O1B, O1C, O2, O3, O4, O4:K52, O4:K6, O5, O5ab, O5ac, O6, O6:K2;K13;K15, O6:K54, O7, O8, O9, O10, O11, O12, O13, O14, O15, O16, O17, O18, O18A, O18ac, O18A1, O18B, Formula O18B1, Formula O19, Formula O20, Formula O21, Formula O22, Formula O23, Formula O23A, Formula O24, Formula O25, Formula O25a, Formula O25b, Formula O2 6, Formula O27, Formula O28, Formula O29, Formula O30, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37, Formula O38, Formula O39, Formula O40, formula O41, formula O42, formula O43, formula O44, formula O45, formula O45, formula O45rel, formula O46, formula O48, formula O49, formula O50, formula O51, formula O52, formula O53, formula O54, formula O55, formula O56, formula O57, formula O58, formula O59, formula O60, formula O61, formula O62, formula 62D ₁ , formula O63, formula O64, formula O65, formula O66, formula O68, formula O69, formula O70, formula O71, formula O73, formula O73, formula O74, formula O75, formula O76, formula O77, formula O78, formula O79, formula O80, formula O81, Formula O82, Formula O83, Formula O84, Formula O85, Formula O86, Formula O87, Formula O88, Formula O89, Formula O90, Formula O91, Formula O92, Formula O93, Formula O95, Formula O96, Formula O97, Formula O98, Formula O99, Formula O100, Formula O101, formula O102, formula O103, formula O104, formula O105, formula O106, formula O107, formula O108, formula O109, formula O110, formula O111, formula O112, formula O113, formula O114, formula O115, formula O 116, Formula O117, Formula O118, Formula O119, Formula O120, Formula O121, Formula O123, Formula O124, Formula O125, Formula O126, Formula O127, Formula O128, Formula O129, Formula O130, Formula O131, Formula O13 2. Formula O133, Formula O134, Formula O135, Formula O136, Formula O137, Formula O138, Formula O139, Formula O140, Formula O141, Formula O142, Formula O143, Formula O144, Formula O145, Formula O146, Formula O147 , formula O148, formula O149, formula O150, formula O151, formula O152, formula O153, formula O154, formula O155, formula O156, formula O157, formula O158, formula O159, formula O160, formula O161, formula O162, formula A composition comprising at least one saccharide comprising a structure selected from any one of the following: O163, O164, O165, O166, O167, O168, O169, O170, O171, O172, O173, O174, O175, O176, O177, O178, O179, O180, O181, O182, O183, O184, O185, O186, and O187.

C281. The composition according to claim C280, further comprising a polypeptide derived from FimH or a fragment thereof.

C282. The composition of claim C280, wherein the E. coli saccharide comprises the formula O8.

C283. The composition of claim C280, wherein the E. coli saccharide comprises the formula O9.

C284. The composition according to claim C280, further comprising a polypeptide derived from Klebsiella pneumoniae.

C285. The composition of any one of items C267 to C284, wherein the sugar is covalently attached to a carrier protein.

C286. The composition of claim C285, wherein the sugar further comprises a 3-deoxy-d-manno-octa-2-ulosonic acid (KDO) moiety.

C287. The composition according to claim C285, wherein the sugar comprises lipid A.

C288. The composition of any one of claims C285 to C287, wherein the sugar is synthetically synthesized.

C289. 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 (EPA) from P. aeruginosa, maltose binding protein (MBP), detoxified hemolysin A from S. aureus, clumping factor A, clumping factor B, cholera toxin B subunit (CTB), Streptococcus pneumoniae, The composition of item C285, wherein the antibacterial agent is selected from any one of C. pneumoniae pneumolysin and detoxified variants thereof, C. jejuni AcrA, C. jejuni native glycoprotein and Streptococcal C5a peptidase (SCP).

C290. A method for eliciting an immune response against Escherichia coli in a mammal, comprising administering to the mammal an effective amount of a composition described in any one of items C267 to C289.

C291. The method of claim C290, wherein the immune response comprises opsonophagocytic antibodies against E. coli.

C292. The method of claim C290, wherein the immune response protects the mammal from E. coli infection.

C293. A method for eliciting an immune response against Klebsiella pneumoniae in a mammal, comprising administering to the mammal an effective amount of a composition according to any one of items C267 to C289.

C294. The method of claim C293, wherein the immune response comprises opsonophagocytic antibodies against Klebsiella pneumoniae.

C295. The method of claim C293, wherein the immune response protects the mammal from Klebsiella pneumoniae infection.

C296. The composition and method of any one of paragraphs C1 to C266, further comprising at least one saccharide derived from any one of K. pneumoniae types selected from the group consisting of O1, O2, O3, and O5.

C297. The composition and method of claim C296, wherein the K. pneumoniae serovar O1 comprises variant O1V1 or O1V2.

C298. The composition and method of claim C296, wherein the K. pneumoniae serovar O2 comprises variant O2V1 or O2V2.

C299. Use of a composition described in any one of items C1 to C298 herein.

C300. A conjugate comprising: (i) a polypeptide derived from fimbrial H (FimH) or a fragment thereof; and (ii) a carrier protein covalently linked to a saccharide, wherein the saccharide is selected from the group consisting of Formula O1, Formula O1A, Formula O1B, Formula O1C, Formula O2, Formula O3, Formula O4, Formula O4:K52, Formula O4:K6, Formula O5, Formula O5ab, Formula O5ac, Formula O6, Formula O6:K2;K13;K15, Formula O6:K54, Formula O7, Formula O8, Formula O9, Formula O10, Formula O11, Formula O12, Formula O13, Formula O14, Formula O15, Formula O16, Formula O17, Formula O18, Formula O18A, Formula O18ac, Formula O18A1, Formula O18B, Formula O1 8B1, formula O19, formula O20, formula O21, formula O22, formula O23, formula O23A, formula O24, formula O25, formula O25a, formula O25b, formula O26, Formula O27, Formula O28, Formula O29, Formula O30, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37, Formula O38, Formula O39, Formula O 40, Formula O41, Formula O42, Formula O43, Formula O44, Formula O45, Formula O45, Formula O45rel, Formula O46, Formula O48, Formula O49, Formula O50, Formula O51, formula O52, formula O53, formula O54, formula O55, formula O56, formula O57, formula O58, formula O59, formula O60, formula O61, formula O62, formula 62D ₁ , formula O63, formula O64, formula O65, formula O66, formula O68, formula O69, formula O70, formula O71, formula O73, formula O73, formula O74, formula O75, formula O76, formula O77, formula O78, formula O79, formula O80, formula O81, formula O82, formula O83, formula O84, formula O85, formula O86, formula O87, formula O88, formula O89, formula O90, formula O91, formula O92, formula O93, formula O95, formula O96, formula O97, formula O98, formula O99 , formula O100, formula O101, formula O102, formula O103, formula O104, formula O105, formula O106, formula O107, formula O108, formula O109, formula O110, formula O111, formula O112, formula O113, formula O 114, formula O115, formula O116, formula O117, formula O118, formula O119, formula O120, formula O121, formula O123, formula O124, formula O125, formula O126, formula O127, formula O128, formula O129, Formula O130, Formula O131, Formula O132, Formula O133, Formula O134, Formula O135, Formula O136, Formula O137, Formula O138, Formula O139, Formula O140, Formula O141, Formula O142, Formula O143, Formula O1 44, Formula O145, Formula O146, Formula O147, Formula O148, Formula O149, Formula O150, Formula O151, Formula O152, Formula O153, Formula O154, Formula O155, Formula O156, Formula O157, Formula O158, Formula O159, formula O160, formula O161, formula O162, formula O163, formula O164, formula O165, formula O166, formula O167, formula O168, formula O169, formula O170, formula O171, formula O172, formula O173, formula O174, formula O175, formula O176, formula O177, formula O178, formula O179, formula O180, formula O181, formula O182, formula O183, formula O184, formula O185, formula O186, and formula O187
(wherein n is an integer from 1 to 100).
A composition comprising one or more conjugates comprising a structure selected from the group consisting of:

C301. The composition of claim C300, wherein the sugar is selected from the group consisting of formula O25b, formula O1A, formula O2, formula O6, formula O8 and formula O9.

C302. The composition of claim C301, wherein the sugar is selected from the group consisting of formula O25b, formula O1A, formula O2, and formula O6.

C303. A conjugate comprising: a polypeptide derived from pilus antigen H (FimH) or a fragment thereof; and (a) a carrier protein covalently bound to a saccharide comprising the formula O25b, wherein n is an integer between 31 and 90.
(b) a conjugate comprising a carrier protein covalently attached to a saccharide comprising the formula O1A, where n is an integer from 31 to 90;
(c) a conjugate comprising a carrier protein covalently attached to a saccharide comprising formula O2, where n is an integer from 31 to 90;
(d) a conjugate comprising a carrier protein covalently attached to a saccharide comprising formula O6, where n is an integer from 31 to 90;
(e) a conjugate comprising a carrier protein covalently attached to a saccharide comprising formula O8, wherein n is an integer between 31 and 90; and (f) a composition comprising a conjugate comprising a carrier protein covalently attached to a saccharide comprising formula O9, wherein n is an integer between 31 and 90.

C304. A conjugate comprising: a polypeptide derived from pilus antigen H (FimH) or a fragment thereof; and (a) a carrier protein covalently bound to a saccharide comprising the formula O25b, wherein n is an integer between 31 and 90.
(b) a conjugate comprising a carrier protein covalently attached to a saccharide comprising the formula O1A, where n is an integer from 31 to 90;
The composition according to item C303, comprising: (c) a conjugate comprising a carrier protein covalently bound to a saccharide comprising formula O2, wherein n is an integer from 31 to 90; and (d) a conjugate comprising a carrier protein covalently bound to a saccharide comprising formula O6, wherein n is an integer from 31 to 90.

C305. The composition of claim C303 or C304, further comprising one or more conjugates having a sugar selected from the group consisting of formula O15, formula O16, formula O17, formula O18, and formula O75, where n is an integer from 31 to 90. 7. The composition of any one of claims 1 to 6, wherein the sugar further comprises any one of an E. coli R1 moiety, an E. coli R2 moiety, an E. coli R3 moiety, an E. coli R4 moiety, and an E. coli K-12 moiety.

C306. The composition of any one of items C300 to C304, wherein the sugar does not further comprise any one of an E. coli R1 portion, an E. coli R2 portion, an E. coli R3 portion, an E. coli R4 portion, and an E. coli K-12 portion.

C307. The composition of claim C306, wherein the sugar does not further comprise an E. coli R2 moiety.

C308. The composition of any one of items C300 to C307, wherein the sugar further comprises a 3-deoxy-d-manno-octa-2-ulosonic acid (KDO) moiety.

C309. 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, or exotoxin A from Pseudomonas aeruginosa; detoxified exotoxin A (EPA) from P. aeruginosa, maltose binding protein (MBP), detoxified hemolysin A from S. aureus, clumping factor A, clumping factor B, cholera toxin B subunit (CTB), Streptococcus pneumoniae, The composition of any one of items C300 to C308, wherein the C5a peptidase is selected from the group consisting of C. pneumoniae pneumolysin and detoxified variants thereof, C. jejuni AcrA, C. jejuni native glycoprotein, and Streptococcal C5a peptidase (SCP) or a variant thereof.

C310. The composition according to claim C309, wherein the carrier protein is CRM ₁₉₇ .

C311. The composition according to claim C309, wherein the carrier protein is a tetanus toxoid.

C312. The composition of any one of claims C300 to C311, wherein the sugar to protein ratio is at least 0.5 and at most 2.

C313. The composition of any one of claims C300 to C312, wherein the conjugate is prepared by reductive amination.

C314. The composition of any one of claims C300 to C312, wherein the sugar is conjugated to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer.

C315. The composition of any one of claims C300 to C314, wherein the sugar is a single-end-linked conjugated sugar.

C316. The composition according to claim C308, wherein the sugar is conjugated to the carrier protein via a 3-deoxy-d-manno-octa-2-ulosonic acid (KDO) residue.

C317. The composition of any one of claims C300 to C313, wherein the conjugate is prepared by CDAP chemistry.

C318. The composition of any one of claims C300 to C317, wherein the polypeptide is derived from E. coli fimbrial H (FimH).

C319. The composition according to claim C318, wherein the polypeptide comprises a phenylalanine residue at the N-terminus of the polypeptide.

C320. The composition of claim C318, wherein the polypeptide comprises a phenylalanine residue within the first 20 residues of the N-terminus.

C321. The composition according to claim C314, wherein the polypeptide comprises a phenylalanine residue at position 1 of the polypeptide.

C322. The composition according to item C317, wherein the polypeptide does not contain a glycine residue immediately preceding the phenylalanine residue at position 1 of the polypeptide.

C323. The composition according to item C314, wherein the polypeptide does not contain an N-glycosylation site at position 7 of the polypeptide.

C324. The composition according to C318, wherein the polypeptide does not contain an Asn residue at position 7 of the polypeptide.

C325. The composition of claim C320, wherein the polypeptide comprises a residue at position 7 selected from the group consisting of Ser, Asp, Thr, and Gln.

C326. The composition according to item C317, wherein the polypeptide does not contain an N-glycosylation site at position 70 of the polypeptide.

C327. The composition according to claim C322, wherein the polypeptide does not contain an Asn residue at position 70 of the polypeptide.

C328. The composition according to claim C322, wherein the polypeptide does not contain a Ser residue at position 70 of the polypeptide.

C329. The composition of claim 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 claim C325, wherein the N-glycosylation site comprises position N235 of the polypeptide.

C331. The composition according to claim C325, wherein the N-glycosylation site comprises position N228 of the polypeptide.

C332. The composition according to item C325, wherein the N-glycosylation sites include positions N235 and N228 of the polypeptide.

C333. The composition of claim C314, wherein the polypeptide comprises SEQ ID NO:2.

C334. The composition of claim C314, wherein the polypeptide comprises SEQ ID NO:3.

C335. The composition according to item 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 claim 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 claim C314, wherein the polypeptide comprises an aliphatic hydrophobic amino acid residue at position 1 of the polypeptide.

C338. The composition according to item C333, wherein the aliphatic hydrophobic amino acid residue is selected from the group consisting of Ile, Leu, and Val.

C339. The composition of claim C314, wherein the polypeptide comprises a fragment of FimH.

C340. The composition according to claim C335, wherein the polypeptide comprises a lectin domain of FimH.

C341. The composition of claim C336, wherein the lectin domain has a mass of about 17022 daltons.

C342. The composition according to claim C314, wherein the polypeptide is complexed with a FimC polypeptide or a fragment thereof.

C343. The composition according to claim C338, wherein the FimC polypeptide or fragment thereof comprises a glycine residue at position 37 of the FimC polypeptide or fragment thereof.

C344. The composition according to claim C314, wherein the polypeptide is in a low affinity conformation.

C345. The composition according to claim C314, wherein the polypeptide is stabilized by FimG.

C346. The composition according to claim C314, wherein the polypeptide is stabilized by a donor strand peptide of FimG (DsG).

C347. The composition according to claim C346, wherein the polypeptide further comprises a linker.

C348. The composition according to item C343, wherein the linker comprises at least 4 amino acid residues and at most 15 amino acid residues.

C349. The composition according to claim C343, wherein the linker comprises at least 5 amino acid residues and at most 10 amino acid residues.

C350. The composition according to claim C343, wherein the linker comprises 7 amino acid residues.

C351. The composition of claim C314, wherein the polypeptide does not contain a signal peptide selected from the group consisting of a native FimH leader peptide, an influenza hemagglutinin signal peptide, and a human respiratory syncytial virus A (A2 strain) fusion glycoprotein F0 signal peptide.

C352. The composition according to claim C314, wherein the polypeptide comprises a mouse IgK signal peptide sequence.

C353. The composition according to item C314, wherein the polypeptide comprises any one of a signal peptide sequence selected from the human IgG receptor FcRn large subunit p51 signal peptide and the human IL10 protein signal peptide.

C354. The composition of claim C314, wherein the polypeptide comprises an arginine to proline mutation at amino acid position 60 (R60P) according to the numbering of SEQ ID NO:3.

C355. The composition of any one of claims C300 to C354, comprising at most about 25% free sugars compared to the total amount of sugars in the composition.

C356. The composition of any one of items C300 to C354, further comprising one or more conjugates, the conjugates comprising a carrier protein covalently bound to a K. pneumoniae O antigen selected from O1 and O2.

C357. The composition of claim C356, wherein the K. pneumoniae O antigen is selected from the group consisting of a) serotype O1 subtype v1 (O1v1), b) serotype O1 subtype v2 (O1v2), c) serotype O2 subtype v1 (O2v1), and d) serotype O2 subtype v2 (O2v2).

C358. (i) one or more conjugates comprising a carrier protein covalently attached to a K. pneumoniae O antigen selected from the group consisting of serotype O1 subtype v1 (O1v1), serotype O1 subtype v2 (O1v2), serotype O2 subtype v1 (O2v1), and serotype O2 subtype v2 (O2v2); and (ii) a conjugate comprising a carrier protein covalently attached to a saccharide, wherein the saccharide is selected from the group consisting of Formula O1, Formula O1A, Formula O1B, Formula O1C, Formula O2, Formula O3, Formula O4, Formula O4:K52, Formula O4:K6, Formula O5, Formula O5ab, Formula O5ac, Formula O6, Formula O6:K2;K13;K15, Formula O6:K54, Formula O7, Formula O8, Formula O9, Formula O10, Formula O11, Formula O12, formula O13, formula O14, formula O15, formula O16, formula O17, formula O18, formula O18A, formula O18ac, formula O18A1, formula O18B, formula O18B1, formula O19, formula O20, formula O21, formula O22, formula O23, formula O23A, formula O24, formula O25, formula O25a, formula O25b, formula O26, formula O27, formula O28, formula O29, formula O30, formula O32, formula O33, formula O34, formula O35, formula O36, formula O37, formula O38, formula O39, formula O40, formula O41, formula O42, formula O43, formula O44, formula O45, formula O45, formula O45rel, formula O46, formula O48, formula O49, formula O50, formula O51, formula O52, formula O53, formula O54, formula O55, formula O56, formula O57, formula O58, formula O59, formula O60, formula O61, formula O62, formula 62D ₁ , formula O63, formula O64, formula O65, formula O66, formula O68, formula O69, formula O70, formula O71, formula O73, formula O73, formula O74, formula O75, formula O76, formula O77, formula O78, formula O79, formula O80, formula O81, formula O82, formula O83, formula O84, formula O85, formula O86, formula O87, formula O88, formula O89, formula O90, formula O91, formula O92, formula O93, formula O95, formula O96, formula O97, formula O98, formula O99 , formula O100, formula O101, formula O102, formula O103, formula O104, formula O105, formula O106, formula O107, formula O108, formula O109, formula O110, formula O111, formula O112, formula O113, formula O 114, formula O115, formula O116, formula O117, formula O118, formula O119, formula O120, formula O121, formula O123, formula O124, formula O125, formula O126, formula O127, formula O128, formula O129, Formula O130, Formula O131, Formula O132, Formula O133, Formula O134, Formula O135, Formula O136, Formula O137, Formula O138, Formula O139, Formula O140, Formula O141, Formula O142, Formula O143, Formula O1 44, Formula O145, Formula O146, Formula O147, Formula O148, Formula O149, Formula O150, Formula O151, Formula O152, Formula O153, Formula O154, Formula O155, Formula O156, Formula O157, Formula O158, Formula O159, formula O160, formula O161, formula O162, formula O163, formula O164, formula O165, formula O166, formula O167, formula O168, formula O169, formula O170, formula O171, formula O172, formula O173, formula O174, formula O175, formula O176, formula O177, formula O178, formula O179, formula O180, formula O181, formula O182, formula O183, formula O184, formula O185, formula O186, and formula O187
(wherein n is an integer from 1 to 100).
A composition comprising one or more conjugates comprising a structure selected from the group consisting of:

C359. The composition of claim C358, wherein the sugar is selected from the group consisting of formula O25b, formula O1A, formula O2, formula O6, formula O8, and formula O9.

C360. The composition of claim C359, wherein the sugar is selected from the group consisting of formula O25b, formula O1A, formula O2, and formula O6.

C361. One or more conjugates comprising a carrier protein covalently attached to a K. pneumoniae O antigen selected from the group consisting of serotype O1 subtype v1 (O1v1), serotype O1 subtype v2 (O1v2), serotype O2 subtype v1 (O2v1), and serotype O2 subtype v2 (O2v2); and (a) a conjugate comprising a carrier protein covalently attached to a saccharide comprising the formula O25b, wherein n is an integer between 31 and 90;
(b) a conjugate comprising a carrier protein covalently attached to a saccharide comprising the formula O1A, where n is an integer from 31 to 90;
(c) a conjugate comprising a carrier protein covalently attached to a saccharide comprising formula O2, where n is an integer from 31 to 90;
(d) a conjugate comprising a carrier protein covalently attached to a saccharide comprising formula O6, where n is an integer from 31 to 90;
(e) a conjugate comprising a carrier protein covalently attached to a saccharide comprising formula O8, wherein n is an integer between 31 and 90; and (f) a composition comprising a conjugate comprising a carrier protein covalently attached to a saccharide comprising formula O9, wherein n is an integer between 31 and 90.

C362. (i) one or more conjugates comprising a carrier protein covalently attached to a K. pneumoniae O antigen selected from the group consisting of serotype O1 subtype v1 (O1v1), serotype O1 subtype v2 (O1v2), serotype O2 subtype v1 (O2v1), and serotype O2 subtype v2 (O2v2); and (a) a conjugate comprising a carrier protein covalently attached to a saccharide comprising the formula O25b, wherein n is an integer between 31 and 90;
(b) a conjugate comprising a carrier protein covalently attached to a saccharide comprising the formula O1A, where n is an integer from 31 to 90;
The composition according to item C361, comprising: (c) a conjugate comprising a carrier protein covalently bound to a saccharide comprising formula O2, wherein n is an integer from 31 to 90; and (d) a conjugate comprising a carrier protein covalently bound to a saccharide comprising formula O6, wherein n is an integer from 31 to 90.

C363. The composition according to item C361 or C362, further comprising one or more conjugates having a sugar selected from the group consisting of formula O15, formula O16, formula O17, formula O18 and formula O75, where n is an integer from 31 to 90.

C364. The composition of any one of claims C358 to C363, wherein the sugar further comprises any one of an E. coli R1 moiety, an E. coli R2 moiety, an E. coli R3 moiety, an E. coli R4 moiety, and an E. coli K-12 moiety.

C365. The composition of any one of items C358 to C363, wherein the sugar does not further comprise any one of an E. coli R1 portion, an E. coli R2 portion, an E. coli R3 portion, an E. coli R4 portion, and an E. coli K-12 portion.

C366. The composition of claim 67, wherein the sugar does not further comprise an E. coli R2 moiety.

C367. The composition of any one of items C358 to C366, wherein the sugar further comprises a 3-deoxy-d-manno-octa-2-ulosonic acid (KDO) moiety.

C368. 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, or exotoxin A from Pseudomonas aeruginosa; detoxified exotoxin A (EPA) from P. aeruginosa, maltose binding protein (MBP), detoxified hemolysin A from S. aureus, clumping factor A, clumping factor B, cholera toxin B subunit (CTB), Streptococcus pneumoniae, The composition of any one of items C358 to C367, wherein the antibody is selected from the group consisting of C. pneumoniae pneumolysin and detoxified variants thereof, C. jejuni AcrA, C. jejuni native glycoprotein, and Streptococcal C5a peptidase (SCP) or a variant thereof.

C369. The composition according to item C368, wherein the carrier protein is CRM ₁₉₇ .

C370. The composition according to claim C368, wherein the carrier protein is a tetanus toxoid.

C371. The composition of any one of claims C358 to C370, wherein the sugar to protein ratio is at least 0.5 and at most 2.

C372. The composition of any one of items C358 to C371, wherein the conjugate is prepared by reductive amination.

C373. The composition of any one of claims C358 to C371, wherein the sugar is conjugated to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer.

C374. The composition of any one of claims C358 to C373, wherein the sugar is a single-end-linked conjugated sugar.

C375. The composition according to claim C367, wherein the sugar is conjugated to the carrier protein via a 3-deoxy-d-manno-octa-2-ulosonic acid (KDO) residue.

C376. The composition of any one of claims C358 to C372, wherein the conjugate is prepared by CDAP chemistry.

C377. The composition of any one of claims C358 to C376, comprising at most about 25% free sugars compared to the total amount of sugars in the composition.

C378. The composition of any one of claims C300 to C377, further comprising an adjuvant.

C379. The composition of claim C378, wherein the adjuvant comprises aluminum.

C380. The composition according to claim C378, wherein the adjuvant comprises QS21.

C381. The composition of claim C378, wherein the adjuvant comprises MPLA.

C382. The composition according to claim C380 or C381, wherein the adjuvant is a liposomal adjuvant.

C383. A method for eliciting an immune response against Escherichia coli in a mammal, comprising administering to the mammal an effective amount of a composition described in any one of items C300 to C382.

C384. The method of claim C383, wherein the immune response comprises opsonophagocytic antibodies against E. coli.

C385. The method of claim C383, wherein the immune response protects the mammal from E. coli infection.

C386. Use of a composition according to any one of claims C300 to C382 for inducing an immune response against E. coli.

C387. Use of a composition according to any one of items C300 to C382 in the manufacture of a medicament for inducing an immune response against E. coli.

C388. The use of claim C386 or C387, wherein the immune response comprises opsonophagocytic antibodies against E. coli.

C389. The use of items C386 or C387, wherein the immune response protects a mammal against E. coli infection.

C390. A method for eliciting an immune response against Klebsiella pneumoniae in a mammal, comprising administering to the mammal an effective amount of a composition according to any one of items C300 to C382.

C391. The method of claim C390, wherein the immune response comprises opsonophagocytic antibodies against K. pneumoniae.

C392. The method of claim C390, wherein the immune response protects the mammal from K. pneumoniae infection.

C393. Use of a composition according to any one of claims C300 to C382 for inducing an immune response against K. pneumoniae.

C394. Use of a composition according to any one of claims C300 to C382 in the manufacture of a medicament for inducing an immune response against K. pneumoniae.

C395. The use of claim C386 or C387, wherein the immune response comprises opsonophagocytic antibodies against K. pneumoniae.

C396. The use according to claim C386 or C387, wherein the immune response protects a mammal against K. pneumoniae infection.

C397. A nucleic acid comprising nucleotides encoding a polypeptide according to any one of items C1 to C382.

C398. The nucleic acid according to claim C397, wherein the nucleic acid is RNA.

C399. Nanoparticles containing a nucleic acid according to item C397 or C398.

C400. The composition according to any one of items 303, 304, 305, 359, 360, 361, 362 or 363, further comprising one or more conjugates having a sugar selected from the group consisting of formula O4, formula O11, formula O13, formula O21 and formula O86, wherein n is an integer from 1 to 100, preferably from 31 to 90.

Claims (7)

(i) a polypeptide derived from E. coli fimbrial antigen H (FimH), wherein said polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112 and SEQ ID NO:113, or an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112 and SEQ ID NO:113; and (ii) a conjugate comprising a carrier protein covalently linked to a sugar, wherein said conjugate comprises
(a) Formula O25b as described in Table 1, where n is an integer from 31 to 70.

a conjugate comprising a carrier protein covalently attached to a sugar comprising
(b) a conjugate comprising a carrier protein covalently attached to a saccharide comprising formula O1A as set forth in Table 1, where n is an integer between 31 and 50;
(c) a conjugate comprising a carrier protein covalently linked to a saccharide comprising formula O2 as set forth in Table 1, wherein n is an integer between 31 and 70; and (d) a conjugate comprising a carrier protein covalently linked to a saccharide comprising formula O6:K2, formula O6:K13, formula O6 : K15 or formula O6:K54 as set forth in Table 1, wherein n is an integer between 31 and 60, wherein said carrier protein is a carrier protein other than E. coli fimbrial H (FimH).
A composition comprising: The carrier protein may be 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 (EPA) from P. aeruginosa, maltose binding protein (MBP), detoxified hemolysin A from S. aureus, clumping factor A, clumping factor B, cholera toxin B subunit (CTB), Streptococcus pneumoniae, or the like. 2. The composition of claim 1, wherein the antibacterial agent is selected from the group consisting of C. pneumoniae pneumolysin and detoxified variants thereof, C. jejuni AcrA, C. jejuni native glycoprotein, and Streptococcal C5a peptidase (SCP) or a variant thereof. The composition of claim 1, wherein the polypeptide is stabilized by a donor strand peptide of FimG (DsG). The composition of claim 3, wherein the polypeptide further comprises a linker. The composition according to any one of claims 1 to 4, further comprising an adjuvant. A drug for eliciting an immune response against Escherichia coli in a mammal, comprising an effective amount of a composition according to any one of claims 1 to 5. A composition comprising a nucleic acid comprising a nucleotide encoding the polypeptide of claim 1 and one or more conjugates of claim 1.

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