AU2004231485B2 - Methods for obtaining colonization factors from bacterial strains - Google Patents

Description

WO 2004/093825 PCT/US2004/007439 METHODS FOR OBTAINING COLONIZATION FACTORS FROM BACTERIAL STRAINS CROSS REFERENCE TO RELATED APPLICATIONS [01] This application claims the benefit of U.S. Provisional Patent Application No. 60/453,956, filed 13 March 2003, listing Frederick J. Cassels as the inventor, which is herein incorporated by reference in its entirety. ACKNOWLEDGMENT OF GOVERNMENT SUPPORT [021 This invention was made by employees of the United States Army. The government has rights in the invention. BACKGROUND OF THE INVENTION 1. FIELD OF THE INVENTION. [03] The present invention generally relates to the preparation of proteins. In particular, the present invention relates to the preparation and purification of large quantities of at least one colonization factor (CF) and methods of using thereof. 2. DESCRIPTION OF THE RELATED ART. [041 Diarrhea caused by enterotoxigenic Escherichia coli (ETEC), commonly referred to as travelers' diarrhea, is a common health problem among travelers visiting less developed or tropical countries. See Peltola, H., et al. (1991) Lancet 338:1285 1289 and Ericsson, C. D. et al. (1993) Clin. Infect. Dis. 16:616-626. Diarrhea caused by ETEC and other ETEC infections are important concerns for military personnel when deployed to less developed countries. See Wolf, M.K., et al. (1993) Clin. Microbiol. 31:851-856 and Bourgeois, A.L., et al. (1993) Am. J. Trop. Med. Hyg. 48:243-248. ETEC may be transmitted by food or water contaminated with animal or human feces. ETEC produces two toxins, a heat-stable toxin (ST) and a heat-labile toxin (LT). ETEC infections may cause profuse watery diarrhea, abdominal cramping, fever, nausea, vomiting, chills, loss of appetite, headache, muscle aches, and bloating. [051 . The current therapy for travelers' diarrhea is to initiate treatment with agents such as bismuth subsalicylate (Pepto-Bismol@), antidiarrheals such as diphenoxylate with atropine (Lomotil@), loperamide HCl (Immodium@), attapulgite (Kaopectate@) and the like, rehydration therapy, and combinations thereof. The majority of the treatments involve the non-specific removal of the offending agents (i.e. toxins) from the intestinal tract. Only in moderate to severe cases of diarrhea where distressing or WO 2004/093825 PCT/US2004/007439 incapacitating symptoms are reported is antimicrobial therapy recommended. ETEC is frequently resistant to common antibiotics such as trimethoprim-sulfamethoxazole and ampicillin. Fluoroquinolones such as ciprofloxacin have shown some efficacy. Antibiotics are not usually effective at reducing clinical symptoms of the disease and problems associated with antibiotic resistance can occur. Prophylactic use of antibiotics is not recommended. Thus, therapies that specifically remove ETEC from the intestine are needed to provide more effective treatments for ETEC diarrhea. [061 In order to initiate the infectious process of diarrhea, ETEC must adhere to the host intestinal epithelial cells via the binding between bacterial adhesions, colonization factors (CFs) and host receptors. This binding is commonly referred to as adhesion receptor interaction. See Beachey, E.H. (1981) J. Infect. Dis. 143:325-345; Satterwhite, T. K., et al. (1978) Lancet 2:181-184; and Warner, L. and Y. S. Kim. (1989) "Intestinal Receptors for Microbial Attachment", Eds. M. J. G. Farthing, and G. T. Kensch, ENTERIC INFECTION: MECHANISMS, MANIFESTATIONS AND MANAGEMENT, pp. 31-40. Raven Press, New York. ETEC then causes secretory diarrhea by expressing toxins, heat-labile enterotoxin (LT) and heat-stabile enterotoxin (ST). CFs interact with receptors on the host epithelial cells allowing for adherence of the ETEC to the mucosa. See Cassels, F.J. and Wolf, M.W. (1995) J. Indust. Microbiol 15:214-2263. CFs include colonization factor antigens (CFAs), coli surface (CS), and putative colonization factors (PCFs). [07] A few ETEC challenge trials in humans have been successfully undertaken. For example, ETEC strain B7A, expressing colonization factor CS6 that enables colonization of the intestine was successful in causing diarrhea in human subjects. See Levin, M.M. et al. (1982) Scand. J. Infect. Dis. Supp. 33:83-95. ETEC strain H10407, expressing CFA/I colonization factor, mediated the development of disease in 9 out of 10 humans challenged with the plate grown bacteria. See Tacket, C.O. et al. (1988) N. Engl. J. Med. 318(19):1240-3124. In a similar human challenge studies, ETEC strain E24377A, expressing colonization factor antigen II (CFA/II) containing CS1 and CS3, provided similar attack rates as in the previous studies. See Tacket, C.O. (1994) Vaccine 12(14):1270-1274. In each of these studies, the ETEC challenge strains used were cultured on either TSA or CFA solid agar plates and harvested by scraping the bacteria from the surface. [08] Unfortunately, prior art methods for large-scale production of various colonization factors (CFs) have been unsatisfactory. In particular, prior art methods WO 2004/093825 PCT/US2004/007439 resulted in low yields of bacteria and the desired CF under great risks of contamination. For example, U.S. Patent No. 5,698,416 discloses a process for fermenting and isolating CF based on an example at small scale. However, the levels of purity (70%) obtained by some prior art processes were insufficient for approval by the U.S. Food and Drug Administration (FDA) for use in some human vaccine applications. In addition, the yields obtained by prior art processes were not commercially viable. [091 Early ETEC vaccine efforts at the Walter Reed Army Institute of Research (Washington, DC) involved the growth of wild type bacteria on agar plates, and purification of the CF from bacteria recovered from the surface of these plates. See Reid et al. (1993) Vaccine 11:159. While it is possible to generate small lots of CF this way, the process does not allow scale-up manufacturing to make the process more economically feasible. The conventional wisdom at the time held that ETEC grown on plates expressed CFs readily, but when grown in broth culture, a scalable process (using fermentors), the bacteria would grow well but not express CFs. In order to generate large quantities of CF in broth culture, CS6, the CF found most commonly in the world was cloned and sequenced. See Wolf et al. (1997) FEMS Microbiol. Lett. 148:35, and Wolf (1997) Clin. Microbiol. Rev. 10:569. Recombinant CS6 was found to be suitable for vaccine manufacturing. See U.S. Patent No. 5,698,416; Guerena et al. (2002) Infect. Immun. 70:1874; and Katz et al. (2003) Vaccine 21:341. Unfortunately, however, some CFs could not be expressed in the prior art broth cultures. [101 Therefore, a need still exists for methods of expressing and producing CFs in high purities and large amounts that are commercially viable and suitable for approval from governmental regulatory agencies for administration to humans. SUMMARY OF THE INVENTION till The present invention generally relates to methods for expressing colonization factors (CFs). [12] In some embodiments, the present invention provides a method of obtaining a preparation comprising at least one colonization factor from a bacterial strain which comprises culturing or fermenting the bacterial strain in DME/F-12 broth. In some embodiments, the expression levels of the colonization factor are increased, induced, enhanced, or modulated as compared with expression levels of the bacterial strain cultured or fermented in a conventional medium, such as Luria broth, CFA agar, or CFA broth. In some embodiments, the bacterial strain is an enterotoxigenic WO 2004/093825 PCT/US2004/007439 Escherichia coli strain, preferably H10407, E9034A, or 60R75. The colonization factor is a colonization factor antigen, a coli surface protein, or a putative colonization factor, such as those belonging to the CFA/I family, the CS5 family, the Type IV family, or the distinct group of colonization factors. In some embodiments, the colonization factor is CS 1, CS3, or CFA/I. In some embodiments, the bacterial strain is cultured or fermented at about 25 'C to about 37 *C. [13] In some embodiments, the colonization factor is obtained in an amount that is greater than amounts obtained when the bacterial strain is cultured or fermented in a conventional medium, such as Luria broth, CFA agar, or CFA broth. In some embodiments, the amount obtained is about 1 to about 14 times greater than amounts obtained when the bacterial strain is cultured or fermented in the conventional medium. In some embodiments, the amount obtained is about 1 to about 7 times greater than amounts obtained when the bacterial strain is cultured or fermented in the conventional medium. In some embodiments, the amount obtained is about 1 to about 4 times greater than amounts obtained when the bacterial strain is cultured or fermented in the conventional medium. In some embodiments, the amount obtained is about 12.4 times greater than amounts obtained when the bacterial strain is cultured or fermented in the conventional medium. In some embodiments, the amount obtained is about 3.7 times greater than amounts obtained when the bacterial strain is cultured or fermented in the conventional medium. In some embodiments, the colonization factor is not obtained from the bacterial strain when cultured or fermented in Luria broth. In some embodiments, the colonization factor is not obtained from the bacterial strain when cultured or fermented in CFA broth. In some embodiments, the preparation has a protein purity of the colonization factor of about 70% or more. In some embodiments, the protein purity is about 80% or more. In some embodiments, the protein purity is about 90% or more. In some embodiments, the bacterial strain was frozen prior to cultivating or fermenting. [141 In some embodiments, the present invention provides a pharmaceutical composition comprising the preparation or the colonization factor made by the methods disclosed herein and a carrier, an adjuvant, or both. The pharmaceutical composition may be an oral formulation, an intranasal formulation, or a transcutaneous formulation. In some embodiments, the pharmaceutical composition is an injectable formulation. [151 In some embodiments, the present invention provides methods of treating, preventing or inhibiting an enterotoxigenic Escherichia coli infection in a subject

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WO 2004/093825 PCT/US2004/007439 which comprises administering at least one preparation or at least one colonization factor made by the methods disclosed herein. In some embodiments, the enterotoxigenic Escherichia coli infection is traveler's diarrhea. [16] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed. The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute part of this specification, illustrate several embodiments of the invention and together with the description serve to explain the principles of the invention. DESCRIPTION OF THE DRAWINGS [17] This invention is further understood by reference to the drawings wherein: [18] Figure 1 shows the growth curves for H10407. [191 Figure 2 shows the growth curves for 60F75. [20] Figure 3 shows the SDS-PAGE from H10407 when grown in DME/F-12, L broth, and CFA broth. [21] Figure 4 shows the Western blot for H10407. [221 Figure 5 shows the SDS-PAGE from E9034A when grown in DME/F-12, L broth, and CFA broth. [23] Figure 6 shows the Western blot for E9043A. [241 Figure 7 shows the SDS-PAGE from 60R75 when grown in DME/F-12, L broth, and CFA broth. [25] Figure 8 shows the Western blot for 60R75. [26] Figure 9A and Figure 9B show CS 1 expressing ETEC grown on CFA agar. [27] Figure 1OA and Figure lOB show CS1 expressing ETEC grown on CFA broth. [28] Figure 1 1A and Figure 11 B show CS1 expressing ETEC grown on L broth. [29] Figure 12A and Figure 12B show CS1 expressing ETEC grown on DME/F-12 broth. [30] Figure 13A and Figure 13B show CFA/I expressing ETEC grown on CFA agar. [31] Figure 14A and Figure 14B show CFA/I expressing ETEC grown on CFA broth. [321 Figure 15A and Figure 15B show CFA/I expressing ETEC grown on DME/F-12 broth.

WO 2004/093825 PCT/US2004/007439 [331 Figure 16 is an SDS-PAGE of CS3 from a 300L fermentation. 16% tris-Tricine SDS-PAGE gel (Invitrogen, Carlsbad, CA) with 10 and 20 ptg loaded (lanes 2 and 3, respectively) of CS3, Lot 0963 from 300 L fermentation. The molecular weight standards (Mark 12, Invitrogen, Carlsbad, CA) are in lane 1. [34] Figure 17 is an SDS-PAGE of CFA/I from a 1OL fermentation. 16% tris Tricine SDS-PAGE gel (Invitrogen, Carlsbad, CA) with 30 pig loaded (lane 2) of CFA/I from 10 L fermentation. The molecular weight standards (Mark 12, Invitrogen, Carlsbad, CA) are in lane 1. [35] Figure 18 is an SDS-PAGE of CFA/I from a 300L fermentation. 16% tris Tricine SDS-PAGE gel (Invitrogen, Carlsbad, CA) with 20 and 10 ptg loaded (lanes 1 and 2, respectively) of CFA/I, Lot 1096 from 300 L fermentation. A CFA/I standard is included for comparison (20 tg, lane 4). The molecular weight standards (Mark 12, Invitrogen, Carlsbad, CA) are in lane 3. DETAILED DESCRIPTION OF THE INVENTION [361 The present invention generally provides methods for increasing, inducing, or enhancing the expression levels of at least one colonization factor (CF), such as CS 1 and CFA/I, as compared to prior art methods. The methods for increasing, inducing, or enhancing the expression levels of CFs as provided herein may be used in order to obtain at least one CF in high purities and large amounts that are suitable for approval from governmental regulatory agencies for administration to humans. [371 Over the years, investigators have come to realize the many problems, ambiguity, uncertainty and interference associated with data obtained when serum is used in mammalian cell tissue culture. To address these concerns, Richard Ham developed a serum-free medium called F-12 medium that was used to support the clonal growth of Chinese hamster ovary and lung cells. See Ham, R. (1965) PNAS USA 53:288-293, which is herein incorporated by reference. Later, it was demonstrated that F- 12 also supported the growth of differentiated cells from rat, rabbit and chicken embryos. More importantly, chemical analysis of the medium revealed selenium contamination that proved essential in the absence of serum. [38] Dulbecco's Modified Eagle's medium, DME and not DMEM by standard convention, is a modification of Eagle's medium (BME). See Morton, H. J: (1970) In vitro 6; and Eagle, H. (1959) Science 130, which is herein incorporated by reference. DME was developed as a basal medium for use with serum and was formulated to 'c WO 2004/093825 PCT/US2004/007439 contain 4x the amino acid and vitamin content as compared with BME. See Ham, R.G. and McKeehan, W. L. (1979) in METHODS IN ENZYMOLOGY Jacoby, W. B. and Pastan, I. H., eds. vol. 58, which is herein incorporated by reference. [391 In an effort to increase the growth of immortalized mammalian cells in serum free medium, DME and F-12 media were combined to create DME/F-12. This combination essentially increased the concentrations of components already present in F-12 by a factor of 2. See Ham R. and McKeehan, W. (1979) in METHODS IN ENZYMOLOGY Jacoby W. B. and Pastan, I. H., eds. vol. 58; and Barnes, D. and Sato, G. (1980) J. Anal. Biochem. 102:255-270, which are herein incorporated by reference. Generally, Dulbecco's Modified Eagle's Medium (DME) and Dulbecco's Modified Eagle's/Ham's Nutrient Mixture (DME/F-12) media are used in mammalian cell culture and not used to culture bacteria. [40] As provided herein, Dulbecco's Modified Eagle's/Ham's Nutrient Mixture (DME/F-12) (Sigma-Aldrich, St. Louis, MO) was used to grow enterotoxigenic Escherichia coli (ETEC) strains. When grown in DME/F-12 CFs are expressed at levels per bacterium that are higher than that of the methods known in the art such as those using CFA agar, CFA broth, and L broth. Although DME/F-12 D8900 was used for the experiments herein, other Dulbecco's Modified Eagle's/Ham's Nutrient Mixtures known in the art as well as those commercially available from Sigma-Aldrich, such as D0547, D2906, D6421, D6434, D6559, D6905, D8062, D8437, D9785, may be used according to the present invention. Therefore, the present invention provides methods for increasing, inducing, or enhancing the expression levels of at least one colonization factor (CF) which comprise culturing a bacterial strain in DME/F-12. [41] As provided herein, growing ETEC strains in DMEM/F-12 increases, induces, enhances, or modulates colonization factor expression. Based on 10-liter fermentation studies as well as a cGMP manufacturing run of 300 L, it has been shown that three ETEC strains, H10407, E9034A, and 60R75 express CFA/I, CS3, and CS1 respectively. The colonization factor expression data is based primarily on SDS-PAGE and Western blotting analysis. [42] As provided herein, the ETEC strains exhibit prodigious increases in expression levels of CFA/I, CS1, and CS3 when cultured in DME/F-12, which is contrary to the accepted dogma in the art. Recently, the method disclosed herein was used for CFA/I vaccine production in the BioProcess Production Facility (BPF) at Walter Reed Army Institute of Research and resulted in the highest level of production of any CF to date. 17 WO 2004/093825 PCT/US2004/007439 As provided herein, yields from the method of the present invention far surpasses yields of recombinant CS6 produced according to the methods disclosed in U.S. Patent No. 5,698,416 and U.S. Patent Application No. 20040005662, which are herein incorporated by reference. [43] The CFs made by the method of the present invention may be incorporated into various pharmaceutical formulations and delivered to a subject via numerous routes. See e.g. International Application Publication No. WO 02/064162, and U.S. Patent No. 6,309,669, which are herein incorporated by reference. As provided herein, the method of the present invention reduces chances for contamination during the production process and reduces the large number of assays required for quality control when small aliquots are pooled to make a final product. [441 Example 1 provides methods of making host cells comprising a DNA insert that encodes a CF of interest. Although the cultivation of certain bacterial strains are exemplified herein, other strains including wildtype strains known in the art may be used. Although the expression and purification of certain CFs are exemplified herein, various CFs, including colonization factor antigens (CFAs), coli surface (CS) proteins, and putative colonization factors (PCFs), such as those provided in Table 1 may be made according to the present invention using conventional methods known in the art. See e.g. Wolf, M.K., et al. (1997) FEMS Microbiol. Letts. 148:35-42; see also U.S. Patent No. 5,698,416, which are herein incorporated by reference. TABLE I Morphologic and Size Characteristics of Colonization Factors of Human ETEC CF CS TOXINS SUBUNIT ULTRASTRUCTURE MASS TYPE* DIAMETER (nm)** 1. CFAII Family CFNI CFNI ST+LT, ST 15,074 rod 7 CS1 CS1 ST+LT 15,233 rod 7 CS2 CS2 ST+LT 15,418 rod 7 CS4 CS4 ST+LT 14,961 rod 7 PCF0166 CS14 ST 15,029/15,541 rod 6-7 CS17 CS17 LT 15,375 rod 6-7 CS19 CS19 ST+LT 14,964 rod 7 II. CS5 Family CS5 CS5 ST 18,617 flexible 5-6 CS7 CS7 ST+LT 18,726 helical 3.5-6.5 PCF09 CS13 LT 24,753 flexible unk CS20 CS20 ST+LT 17,524 rod 7 PCF020 CS18 ST+LT 18,454 rod 7 1I. Type-IV Family CFMIll CS8 LT 21,608 rod 7-8 Longus CS21 ST+LT, ST, LT 21,446 rod 7 WO 2004/093825 PCT/US2004/007439 Arg-3 CS22 ST 15,024 flexible n.d. 8786 CS15 ST 15,346 AF IV. Distinct CS3 CS3 ST+LT 15,107/15,233 curly 2-3 CS6 CS6 ST+LT, ST, LT 15,058/15,877 AF PCF0148 CS11 ST+LT unk curly 3 PCF0159 CS12 ST+LT 17,921 rod 7 2230 CS10 ST 16,430 AF *rod: rigid rod; AF: afimbrial (no known structure) **unk: unknown; n.d.: not determined 1451 Other peptides, polypeptides, and proteins in which an antibody raised against a native CF are cross-reactive to may also be made according to the present invention using conventional methods known in the art. See e.g. U.S. Patent Application No. 20010014668, which is herein incorporated by reference. [46] It is unknown if increasing colonization factor protein expressed by ETEC will result in more efficient infectivity and more vibrant challenge strains; however, based on the positive preliminary studies in DME/F-12 bacterial strains grown under these conditions may be frozen and reconstituted directly in challenge studies using various subjects such as humans, Aotus monkeys, and cotton rats. The advantages of broth grown ETEC strains and development and implementation of frozen/reconstituted ETEC strains for use in challenge studies are numerous and include the following: [47] (1) Large-scale production fermentation technology known in the art may be used to generate large quantitites of cGMP grade virulent ETEC strains that can be frozen indefinitely and the same lots can be used in challenge studies in different clinical locations and times. In contrast, relatively small amounts of plate grown strains can be produced at one time. Furthermore, since these strains are prepared for specific challenges they may differ from study to study which introduces variability in the data. [481 (2) Frozen bacteria can be quickly and easily thawed and reconstituted anytime for use in animal or human challenge studies. However, preparing plate grown strains for challenge studies requires considerable amounts of time and energy, particularly on the day of the challenge. Personnel conducting the challenges often find bacterial preparation and manipulation difficult and distracting from the many other demands at challenge which may introduce further variability. [49] By maintaining the same lots of strains in different challenges studies meaningful comparison of rates of attack, on-set of disease, and dose dependent antibody conversion can be determined. The development and use of frozen ETEC

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WO 2004/093825 PCT/US2004/007439 strains for use in challenge studies is not unprecedented as similar methodologies have been used to produce large-scale amounts of Vibrio cholerea for use in challenge studies. See Cohen, M.B. et al. (2002) Infect. Immun. 70(4):1965-1970; Sack, D.A. et al. (1998) Infect. Immun. 66(5):1968-1972; and Pitisuttithum, et al. (2002) Vaccine 20:920-925, which are herein incorporated by reference. [50] DME/F-12 may be commercially obtained and prepared according to the manufacturer's instructions or made according to methods known in the art. As provided herein DME/F-12 powder (Sigma-Aldrich, St. Louis, MO) plus NaHCO 3 was dissolved in WFI water and sterile filtered. [51] Determination of growth curves and optimal OD600 for freezing strains cultivated in DME/F-12 may be determined according to methods known in the art. For example, a series of 1-liter pilot studies are conducted using at least one ETEC strain. Frozen seed lots previously prepared under cGMP are used to inoculate 1 liter amounts of DME/F-12 and incubated in a shake flask at 37 *C. Aliquots of each strain are collected at OD600 = 0.5 intervals during the entire growth phase and plated on Tryptic soy agar to determine the pre-freeze CFU. Optical density and CFU data are graphed verses time. The remaining fraction is frozen according to methods known in the art. Data from these initial experiments can be used to determine an optimal OD600 for freezing ETEC strains with maximum viability at thaw, balancing the desire for highest initial OD vs. maximal post-thaw viability. [52] More specifically, a fraction of each aliquot collected during the growth curve study, with different OD600 values, may be frozen in a similar fashion used to successfully freeze/thaw Vibrio cholera used in human challenge studies. See Cohen, M.B. et al. (2002) Infect. Immun. 70(4):1965-1970, which is herein incorporated by reference. Following centrifugation to remove spent medium, the bacteria may be resuspended in PBS-0.1% gelatin and then re-centrifuged. See Yoh, M. et al. (1991) J. Clin. Microbiol. 29(10):2326, which is herein incorporated by reference. The supernatant may be decanted and the pellet resuspended in tryptic soy broth (TSB)/20% glycerol. The levels of gelatin and glycerol may be adjusted depending on the viability of the thawed bacteria according to methods known in the art. A fraction of the bacteria may be enumerated by plating on TSA and the remaining portion may be aliquoted in 1 -ml amounts and then frozen to - 80 "C. [53] Bacteria previously frozen may be thawed at room temperature and diluted in sodium bicarbonate and CeraVacx@ (Cera Products, Jessup, MD). The levels of both -1 n WO 2004/093825 PCT/US2004/007439 may be adjusted to optimize ETEC viability after thawing according to methods known in the art. Thawed bacteria may be re-enumerated using OD600 values and then used in animal challenge studies according to methods known in the art. Enumeration by optical density may be confirmed by serially diluting and plating thawed bacteria on TSA plates. [54] Preliminary infectivity studies may be conducted using methods known in the art to determine whether ETEC strains cultured in DME/F- 12 are as (or more) virulent and as capable of causing disease as plate grown ETEC strains. For example, harvested, but not frozen, strains are diluted at several concentrations between 5.x 107 to 5 x 109 CFU and given intragastrically to seven-day old preweaned cotton rats according to protocols previously described. See U.S. Provisional Serial Number 60/280,736 and PCT/US02/08234, which are herein incorporated by reference. [551 More specifically, pre-maturely weaned 7 day old cotton rats (Sigmodon hispidus) may be pre-bled to confinn that naYve animals possess less than 1:10 anti CFA/I, anti-CS3 and anti-CS6 antibody. Immunologic assays may be conducted using ELISA methods known in the art. Cotton rats with low antibody titer to colonization factor proteins may be pre-treated with acidified water for several days prior to the ETEC challenge. Prior to the ETEC challenge, cotton rats may be treated with 250 pl of sodium bicarbonate intragastrically to neutralize stomach acid that could adversely impact the virulence of the ETEC challenge strains. A separate group of cotton rats may also be challenged with commensal K coli strain HS as a negative control. The on-set and development of diarrhea may be monitored and scored according to protocols known in the art. The production of antibodies against CFs may be monitored in serum and stool samples over time using ELISA and other methods known in the art. [56] Following the study, the cotton rats may be sacrificed and dissected to remove the small intestine. The small intestine may be vortex in PBS and plated on to Tryptic soy agar (TSA). A sterile filter may be applied to the plate as bacteria begin to appear and then incubated for an additional time, e.g. about 2 hours. The filter may then be screened using antibodies against CS6 produced by strain B7A, CS3 produced by E23347A and CFA/I and CFA/E produced by H10407 to confirm the presence of disease causing bacteria. [57] Additional challenge studies may be performed in Aotus monkeys according to methods known in the art. Based on previous ETEC challenges with human and 11 WO 2004/093825 PCT/US2004/007439 monkey, a little more than about 1 x 108 to about 1 x 1010 CFU is expected to be required to cause disease in Aotus monkeys. [58] Adult Aotus nancymae monkeys may be challenged intragastrically with an ETEC strain as well as strain HS, non-pathogenic control after peroral administration of a rice-based buffer (CeraVacx@) and a histamine-2 (H2)-receptor antagonist to neutralize stomach acidity. Fecal excretion of E. coli may be monitored daily after challenge by culturing fecal homogenates on MacConkey agar. The identity of presumptive ETEC or E coli HS isolates may be confirmed by colony blots, with detection by antisera specific for the challenge strains. Monkeys may be examined twice daily for a period of 10 days for evidence of diarrhea. Blood samples may be collected before and 7, 14 and 21 days after experimental infection. Using methods known in the art, immune response patterns may be studied by measuring ETEC specific IgA and IgG antibodies in serum and stool by ELISA. See Ahren, C. et al. (1998) Infect. Immun. 66(7):3311-3316; Baqar, S. et al. (1995) Vaccine 13(1):22-28; and Jertborn, M. et al. (1986) J. Clin. Microbiol. 24(2):203-209, which are herein incorporated by reference. [591 CFs produced according to the present invention may be formulated into pharmaceutical compositions and vaccines. For example, a CF of the present invention may be formulated into an oral vaccine and tested in a manner similar to that provided in Example 6, wherein an oral vaccine comprising a CF, CS6, microencapsulated in poly(DL-lactide-co-glycolide) (PLG) microspheres was tested. See Katz et al. (2003) Vaccine, 21(5-6):341-346; de Lormier, et al. (2003), Vaccine, 21:341-346, ; and see also Reid, R.H., et al. (1993) Vaccine 11:159-167, which are herein incorporated by reference. PLG is the same biodegradable material used in resorbable surgical sutures. The CS6 used in the test material was produced from genes cloned from ETEC strain E8775. See Wolf, M.K., et al. (1997) FEMS Microbiol Lett. 148(1):35-42, which is herein incorporated by reference. As provided in Example 6, the oral vaccine was safe and well tolerated in all 6 formulations. None of the vaccine-related symptoms met the definition of severe. All vaccine formulations elicited an immune response; there was at least one responder in each group. The observed immune response was similar in magnitude to experimental infection using a pathogenic strain of ETEC expressing the CF and transcutaneous administration of the CF. See Gierefua-Burguefno, F., et al. (2002) Infect. Immun. 70(4):1874-1880, which is herein incorporated by reference.

WO 2004/093825 PCT/US2004/007439 Most peak responses were seen after the third dose of the vaccine, and support the use of three doses in subsequent studies. [60] Subjects of Group II, who received 1 mg of microencapsulated CF in buffer, did demonstrate the best immune response to the vaccine. Comparing the ASC response of Group II to its non-encapsulated counterpart (Group II) rendered a p = 0.17. The microencapsulated formulation displayed the greatest reactogenicity of all of the groups. The microencapsulated CF in buffer formulation elicited the best mucosal and systemic immune response. Further experiments in mice show that the addition of an adjuvant to microencapsulated CF improves the immune response to CF (results unpublished). Thus, the present invention also provides compositions comprising microencapsulated CF and an adjuvant, such as LT, LT R192G, CT, and the like, and methods of treating diseases and disorders associated with ETEC comprising administering a therapeutically effective amount of CF and an adjuvant. [61] A CF made by the method of the present invention may be formulated into a variety of formulations suitable for administration. For example, a CF of the present invention may be formulated into a patch or biodegradable/biosorbable microspheres according to methods known in the art. In preferred embodiments, at least one CF may be incorporated into a trancutaneous patch or incorporated into a lactide-coglycolide biodegradable microsphere (PLGA) for oral administration. A CF made by the method of the present invention may be used as one component of a multivalent vaccine against ETEC infections. In preferred embodiments, the CF is CS1, CS6, or CFA/I. Other suitable components of the multivalent vaccine include other colonization factors which include colonization factor antigens (CFAs), coli surface (CS) proteins, and putative colonization factors (PCFs), such as CS1-CS5, CFA/I-CFA/IV, and the like. [62] As used herein, "antibody" refers to innunoglobulin molecules and immunologically active portions that comprise an antigen binding site which specifically binds an antigen, such as a CF. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab') 2 fragments which may be generated by treating the antibody with enzymes such as pepsin and papain. Polyclonal and monoclonal antibodies against the polypeptides of the present invention may be made by conventional methods known in the art. Antibodies of the present invention may be produced by conventional methods known in the art. See e.g., Coligan (1991) CURRENT PROTOCOLS IN IMMUNOLOGY. Wiley/Greene, NY; and Harlow and Lane (1989) ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor

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WO 2004/093825 PCT/US2004/007439 Press, NY; Stites, et al. (1986) BASIC AND CLINICAL IMMUNOLOGY. 4th ed. Lange Medical Publications, Los Altos, CA; Goding (1986) MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE. 2d ed. Academic Press, New York, NY; and Kohler and Milstein (1975) Nature 256:495-497, which are herein incorporated by reference. Therapeutic antibodies may be produced specifically for clinical use in humans by conventional methods known in the art. See Chadd, H.E. and S.M. Chamow (200 1) Curr. Opin. Biotechnol. 12:188-194 and references therein, all of which are herein incorporated by reference. [63] The antigens or antibodies the present invention may be administered, preferably in the form of a pharmaceutical composition, to a subject. Preferably the subject is mammalian, more preferably, the subject is human. Preferred pharmaceutical compositions are those comprising at least one CF made by the method of the present invention or at least one antibody against a given CF, and a pharmaceutically acceptable vehicle. The immunogenic composition may be an active immunizing agent, such as a CF of the present invention, or a passive immunizing agent, such as an antibody raised against a given CF of the present invention. The immunogenic composition may elicit an immune response that need not be protective or the immunogenic composition may provide passive immunity. A vaccine elicits a local or systemic immune response that is protective against subsequent challenge by the immunizing agent such as a given CF. Accordingly, as used herein, an "immunogenic composition" can refer to vaccines as well as antibodies. A "protective immune response" may be complete or partial, i.e. a reduction in symptoms as compared with an unvaccinated mammal. As used herein, an "immunogenic amount" is an amount that is sufficient to elicit an immune response in a subject and depends on a variety of factors such as the immunogenicity of the antigen, the manner of administration, the general state of health of the subject, and the like. The typical immunogenic amounts of a given CF for initial and boosting immunization for therapeutic or prophylactic administration ranges from about 0.001 mg to about 50 mg per about 65-70 kg body weight of a subject with a preferred range of 0.01 to 10 mg. For example, the typical immunogenic amount for initial and boosting immunization for therapeutic or prophylactic administration for a human subject ranges from about 0.05 mg to about 5 mg. Examples of suitable immunization protocols include initial immunization injections at time 0 and 4 or initial immunization injections at 0, 2, and 4 weeks, which 1 A WO 2004/093825 PCT/US2004/007439 initial immunization injections may be followed by further booster injections at 1/2 or 1 years. [641 As used herein, a "therapeutically effective amount" refers to an amount of an antigen that may be used to treat, prevent, or inhibit an infection caused by an organism expressing the antigen in a subject as compared to a control. Thus, a "therapeutically effective amount" may be an "immunogenic amount". For example, a "therapeutically effective amount" of a CF refers to an amount of CF that may be used to treat, prevent, or inhibit an ETEC infection in a subject as compared to a control. Again, the skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including the severity of exposure to the organism, previous treatments, the general health and age of the subject; and the like. A therapeutically effective amount may be readily determined by conventional methods known in the art. It should be noted that treatment of a subject with a therapeutically effective amount of a CF of the present invention can include a single treatment or, preferably, can include a series of treatments. [65] The pharmaceutical compositions of the present invention may include an adjuvant. As used herein, an "adjuvant" refers to any substance which, when administered with or before the CF the present invention, aids the CF in its mechanism of action. Thus, an adjuvant in a vaccine is a substance that aids the immunogenic composition in eliciting an immune response. Suitable adjuvants include cholera toxin (CT), heat-labile toxin (LT), incomplete Freund's adjuvant, alum, aluminum phosphate, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, nor-MDP), N acetylmuramyl-Lalanyl-D-isoglutaminyl-L-alanine-2-(l'-2'-dipa-lmitoyl-sn-glycero-3 hydroxyphosphoryloxy)-ethylamine (CGP 19835A, MTP-PE), and RIBI, which comprise three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (NPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. The effectiveness of an adjuvant may be determined by conventional methods in the art. [661 The compositions of the present invention may be administered to a subject by any suitable route including oral, transdermal, intranasal, inhalation, intramuscular, and intravascular administration. It will be appreciated that the route of administration and pharmaceutical formulation will vary with the condition and age of the subject, the nature of the condition to be treated, the therapeutic effect desired, and the particular WO 2004/093825 PCT/US2004/007439 CF used. In preferred embodiments, the route of administration is oral, intranasal, parenteral, or transcutaneous. [67] As used herein, a "pharmaceutically acceptable vehicle" or "pharmaceutically acceptable carrier" refers to and includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. Pharmaceutically acceptable vehicles include those known in the art. See e.g. REMiNGTON: THE SCIENCE AND PRACTICE OF PHARMACY 20 th ed. (2000) Lippincott Williams & Wilkins. Baltimore, MD, which is herein incorporated by reference. [681 The pharmaceutical compositions of the present invention may be provided in dosage unit forms. "Dosage unit form" as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of the active agent calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active agent and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active agent for the treatment of individuals. [691 The pharmaceutical formulations of the invention comprise at least one CF of the present invention and may be prepared in a unit-dosage form appropriate for the desired mode of administration. The pharmaceutical formulations of the present invention may be administered for therapy by any suitable route including oral, rectal, nasal, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous and intradermal). It will be appreciated that the preferred route will vary with the condition and age of the recipient, the nature of the condition to be treated, and the chosen CF of the present invention. [701 It will be appreciated that the actual dosages of the CF used in the pharmaceutical formulations of this invention will vary according to the particular CF being used, the pharmaceutical formulation, the mode of administration, and the like. Optimal dosages for a given set of conditions may be ascertained by those skilled in the art using conventional dosage determination tests in view of the experimental data for a given CF. [71] Pharmaceutical formulations of this invention comprise a therapeutically effective amount of at least one CF of the present invention, and an inert, 1 / WO 2004/093825 PCT/US2004/007439 pharmaceutical or diluent. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical or cosmetic administration. The pharmaceutical or cosmetic carrier employed may be either a solid or liquid. Exemplary of solid carriers are lactose, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Exemplary of liquid carriers are syrup, peanut oil, olive oil, water and the like. Similarly, the carrier or diluent may include time-delay or time release material known in the art, such as glyceryl monostearate or glyceryl distearate alone or with a wax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate and the like. The use of such media and agents for pharmaceutically or cosmetically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with a given CF, use thereof in the formulation is contemplated. [72] Supplementary active compounds can also be incorporated into the formulations. Supplementary active compounds include antimalarials, antibacterials, antiprotozoal agents, antifungal agents, and antidiarrheals and other compounds commonly used to treat bacterial, protozoal, and fungal infections, preferably ETEC infections. Supplementary active compounds include: [731 Antibiotics such as penicillin, cloxacillin, dicloxacillin, methicillin, nafcillin, oxacillin, ampicillin, amoxicillin, bacampicillin, azlocillin, carbenicillin, mezlocillin, piperacillin, ticarcillin, azithromycin, clarithromycin, clindamycin, erythromycin, lincomycin, demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline, quinolone, cinoxacin, nalidixic acid, fluoroquinolone, ciprofloxacin, enoxacin, grepafloxacin, levofloxacin, lomefloxacin, norfloxacin, ofloxacin, sparfloxacin, trovafloxacin, bacitracin, colistin, polymyxin B, sulfonamide, trimethoprim sulfamethoxazole, co-amoxyclav, cephalothin, cefuroxime, ceftriaxone, vancomycin, gentamicin, amikacin, metronidazole, chloramphenicol, nitrofurantoin, co-trimoxazole, rifampicin, isoniazid, pyrazinamide, and the like; [741 Antiprotozoal agents such as chloroquine, doxycycline, mefloquine, metronidazole, eplornithine, furazolidone, hydroxychloroquine, iodoquinol, pentamidine, mebendazole, piperazine, halofantrine, primaquine, pyrimethamine sulfadoxine, doxycycline, clindamycin, quinine sulfate, quinidine gluconate, quinine dihydrochloride, hydroxychloroquine sulfate, proguanil, quinine, clindamycin, atovaquone, azithromycin, suramin, melarsoprol, eflornithine, nifurtimox, amphotericin 1'7 WO 2004/093825 PCT/US2004/007439 B, sodium stibogluconate, pentamidine isethionate, trimethoprim-sulfamethoxazole, pyrimethamine, sulfadiazine, and the like; [751 Antifungal agents such as amphotericin B, fluconazole, itraconazole, ketoconazole, potassium iodide, flucytosine, and the like; and [761 Antidiarrheal such as diphenoxylate, codeine phosphate, paregoric (camphorated opium tincture), loperamide hydrochloride, anticholinergics such as belladonna tincture, atropine, propantheline, kaolin, pectin, activated attapulgite, and the like. [771 A pharmaceutical formulation of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., ingestion), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. [78] A variety of pharmaceutical forms can be employed. Thus, if a solid carrier is used, the preparation can be tableted, placed in a hard gelatin capsule in powder or pellet form or in the form of a troche or lozenge. The amount of solid carrier may vary, but generally will be from about 25 mg to about 1 g. If a liquid carrier is used, the preparation will be in the form of syrup, emulsion, soft gelatin capsule, sterile injectable solution or suspension in an ampoule or vial or non-aqueous liquid suspension. [79] To obtain a stable water-soluble dose form, a pharmaceutically acceptable salt of at least one CF is dissolved in an aqueous solution of an organic or inorganic acid, such as 0.3M solution of succinic acid or citric acid. If a soluble salt form is not available, the agent may be dissolved in a suitable co-solvent or combinations of co solvents. Examples of suitable co-solvents include, but are not limited to, alcohol, 10 WO 2004/093825 PCT/US2004/007439 propylene glycol, polyethylene glycol 300, polysorbate 80, glycerin and the like in concentrations ranging from 0-60% of the total volume. The pharmaceutical formulation may also be in the form of a solution of a salt form of the active ingredient in an appropriate aqueous vehicle such as water or isotonic saline or dextrose solution. [801 The pharmaceutical formulations of the invention may be manufactured in manners generally known for preparing pharmaceutical compositions, e.g., using conventional techniques such as mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing. Pharmaceutical formulations may be formulated in a conventional manner using one or more physiologically acceptable carriers, which may be selected from excipients and auxiliaries that facilitate processing of a given CF into preparations which can be used pharmaceutically. [811 Proper formulation is dependent upon the route of administration chosen. For injection, the CFs of the invention may be formulated into aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. [82] For oral administration, the CFs of the present invention can be formulated readily by combining with pharmaceutically acceptable carriers known in the art. Such carriers enable the CFs of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained using a solid excipient in admixture with at least one CF, optionally grinding the resulting mixture, and processing the mixture of granules after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients include: fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; and cellulose preparations, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as crosslinked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. [83] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally comprise gum horoi, -1 f% WO 2004/093825 PCT/US2004/007439 polyvinyl pyrrolidone, Carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compounds and agents. [841 Pharmaceutical formulations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can comprise the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active agents may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration. For buccal administration, the formulations may take the form of tablets or lozenges formulated in conventional manner. [851 Oral formulations generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, a given CF can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral formulations can also be prepared using a fluid carrier for use as a mouthwash, wherein the CF in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can comprise any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. Preferred formulations for oral formulations include microcrystalline tablets, gelatin capsules, or the like. [861 For administration intranasally or by inhalation, a CF of the present invention may be conveniently delivered in the form of an aerosol spray presentation from syringe-based fine mist particle generators (e.g. Accuspray, Bectin-Dickinson), pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon 1A WO 2004/093825 PCT/US2004/007439 dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of gelatin for use in an inhaler or insufflator and the like may be formulated comprising a powder mix of at least one CF and a suitable powder base such as lactose or starch. [87] The CF of the present invention may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit-dosage form, e.g, in ampoules or in multi-dose containers, with an added preservative. The formulations may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may comprise formulatory agents such as suspending, stabilizing and/or dispersing agents. [88] Pharmaceutical formulations suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Aqueous injection suspensions may comprise substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also comprise suitable stabilizers or agents which increase the solubility of the CF to allow for the preparation of highly concentrated solutions. Additionally, suspensions of the active agents may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. [891 For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium comprising, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid horoidsene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, 21 WO 2004/093825 PCT/US2004/007439 chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the formulation. Prolonged absorption of the injectable compositions can be brought about by including in the formulation an agent which delays absorption, for example, aluminum monostearate and gelatin. [901 Sterile injectable solutions can be prepared by incorporating a therapeutically effective amount of at least one CF of the present invention in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the CF of the present invention into a sterile vehicle which comprises a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the CF plus any additional desired ingredient from a previously sterile-filtered solution thereof. [911 Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the CF may be formulated into ointments, salves, gels, foams, powders, sprays, aerosols or creams as generally known in the art. [921 For example, for topical formulations, pharmaceutically acceptable excipients or cosmetically acceptable carriers and additives include solvents, emollients, humectants, preservatives, emulsifiers, and pH agents. Suitable solvents include ethanol, acetone, glycols, polyurethanes, and others known in the art. Suitable emollients include petrolatum, mineral oil, propylene glycol dicaprylate, lower fatty acid esters, lower alkyl ethers of propylene glycol, cetyl alcohol, cetostearyl alcohol, stearyl alcohol, stearic acid, wax, and others known in the art. Suitable humectants include glycerin, sorbitol, and others known in the art. Suitable emulsifiers include glyceryl monostearate, glyceryl monoleate, stearic acid, polyoxyethylene cetyl ether, polyoxyethylene cetostearyl ether, polyoxyethylene stearyl ether, polyethylene glycol stearate, propylene glycol stearate, and others known in the art. Suitable pH agents include hydrochloric acid, phosphoric acid, diethanolamine, triethanolamine, sodium I99 WO 2004/093825 PCT/US2004/007439 hydroxide, monobasic sodium phosphate, dibasic sodium phosphate, and others known in the art. Suitable preservatives include benzyl alcohol, sodium benzoate, parabens, and others known in the art. Alternatively, CF of the present invention may be in powder form for constitution with a suitable vehicle, e.g, sterile pyrogen-free water, before use. The CF of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., comprising conventional suppository bases such as cocoa butter or other glycerides. [93] In addition to the formulations described above, CF of the present invention may also be formulated as a depot preparation. Such long-acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the CF of the present invention may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion-exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. [94] A pharmaceutical carrier for hydrophobic CF is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The cosolvent system may be a VPD co-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD co-solvent system (VPD:5W) comprises VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic CFs well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co solvent system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied, for example: other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may be substituted for dextrose. [95] Alternatively, other delivery systems for hydrophobic pharmaceutical formulations may be employed. Liposomes and emulsions are known examples of delivery vehicles or carriers for hydrophobic drugs and cosmetics. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity. Additionally, the CF may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers comprising the WO 2004/093825 PCT/US2004/007439 therapeutic agent. Various sustained-release materials have been established and are known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the CFs for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed. [96] The pharmaceutical formulations also may comprise suitable solid- or gel-phase carriers or excipients. Examples of such carriers or excipients include calcium carbonate, calcium phosphate, sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. [971 Some of the CF of the invention may be provided as salts with pharmaceutically compatible counter ions. Pharmaceutically compatible salts may be formed with many acids, including hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, and the like. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free-base forms. [981 In one embodiment, a CF of the present invention is prepared with a carrier that will protect the CF against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation, Boeringer-Ingelheim Corp., and Nova Pharmaceuticals, Inc. Liposomal suspensions can also be used as pharmaceutically or cosmetically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent Nos. 4,522,811 and 6,309,669, and International Application No. PCT/US91/03328, which are herein incorporated by reference. [99] Toxicity and therapeutic efficacy of the compositions of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. For example, one may determine the LD 50 (the dose lethal to 50% of the population), and the ED 5 o (the dose therapeutically effective in 50% of the population) by conventional methods in the art. The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio

LD

5 0/ED 5 o. CFs which exhibit large therapeutic indices are preferred. While compounds or agents that exhibit toxic side effects may be used, care should be taken n~ A WO 2004/093825 PCT/US2004/007439 to design a delivery system that targets such compounds or agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. [1001 The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of the compositions of the present invention lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any antigen used in the method of the invention, the therapeutically effective dose can be estimated initially from assays known in the art. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the

IC

50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined by conventional assays. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. 11011 Immune responses to the CFs of the present invention may be determined using conventional methods known in the art. See e.g. Gierefia-Burguef5o, F., et al. (2002) Infect. Immun. 70(4):1874-1880; Reid, R.H., et al. (1993) Vaccine 11:159-167; Yu et al. (2002) Infect. Immun. 70(3):1056-1068; and Katz et al. (2003) Vaccine, 21(5 6):341-346, which are herein incorporated by reference. [1021 The following examples are intended to illustrate but not to limit the invention. Example 1 Construction of Host Cells Expressing CS6 Antigen [103] Host cells producing CS6 were made according to the procedures described in United States Patent Serial No. 09/479,877, filed 10 January 2000, which is herein incorporated by reference. See also Wolf, et al. (1997) FEMS Microbiology Letts. 148:35-42, which is herein incorporated by reference. [1041 Generally, the genes for CS6 expression were from enterotoxigenic Escherichia coli (ETEC) strain E8775 tox~ of serotype 025:H42 which was a gift from Alejandro Cravioto. E8775 tox~ is a derivative of E. coli strain E8775 which was originally isolated from Bangladesh. DH5a which was purchased from Bethesda Research Laboratories, Inc. (Gaithersburg, MD). pUC19 was originally purchased from P-L 25 WO 2004/093825 PCT/US2004/007439 Biochem. The antibiotic resistance gene encodes resistance to kanamycin and was purchased from Pharmacia, Uppsala, Sweden (Kan@ Genblock@). However, other kanamycin resistance genes known in the art as well as genes for resistance to other antibiotics known in the art may be used. E. coli HB101 (ATCC 33694 and batch 91-1) was purchased from the American Type Culture Collection (Rockville, MD). [1051 The plasmid containing the CS6 genes, the pUC19 origin of replication, and the gene for kanamycin resistance was transformed into E. coli HB 101 by transformation using conventional methods known in the art. The transformants were selected by growth on L agar supplemented with 0.004% X-gal and 50 pg/ml kanamycin sulfate and optionally 50 pg/ml ampicillin. The plasmid comprising the CS6 gene was isolated from the strain and examined by agarose gel electrophoresis. [1061 While the above protocol was used for obtaining a host cell that expresses CS6, other host cells, genes encoding other CFs, other antibiotic resistance genes, and conventional methods known in the art may be employed in accordance with the present invention. Example 2 Growth Comparisons of 3 Bacterial Strains in 3 Different Liquid Broths [1071 The growth curves of the three bacterial strains, H 10407 (expresses CFA/I), E9034A (expresses CS3), and 60R75 (expresses CS 1) in three different liquid broths was examined in order to show that the amounts of the CFs obtained and studied herein is due to expression level of the bacterial strains rather than the quantity or amount of bacterial cells grown. SDS-PAGE, Western blot, and mass spectroscopy analysis were conducted according to methods known in the art. See Kahlil, et al., (1999) Infect. Immun. 67:4019-4026; Hess, S. et al. (2002) Anal. Biochem. 302(1):123-130; and Kahlil, S., et al. (1999) Infect. Immun., 67 (8): 4019-4026, which are herein incorporated by reference. [1081 Specifically, for SDS-PAGE 20 ptg of each sample was loaded in sample buffer onto a 16% tris-Tricine SDS-PAGE gel (Invitrogen, Carlsbad, CA) and run for 90 minutes at 125 volts. Gels were stained in 0.125% Coomassie blue. For Western blot, after separation by SDS-PAGE, the samples were electrotransferred onto a nitrocellulose membrane at 4 *C at 125 volts at constant voltage for 18 hours in transfer buffer and developed using 0.5 ptg/ml of rabbit anti -CS6 or -CS3 antibody, and 2 pig/ml of rabbit antibody to CFA/I.

WO 2004/093825 PCT/US2004/007439 [109] Figure 1 shows the growth curves for H10407 and Figure 2 shows the growth curves for 60F75. [110] For the lab scale culture of the ETEC strains examined herein, the following procedures were followed: A. Culture: [111] Inoculation of ETEC strains were conducted by thawing a frozen vial of a given ETEC strain and pipeting 800 d of thawed contents into 200 ml of the desired culture media, i.e. DME/F-12, L Broth, or CFA Broth. ETEC strains were cultured at 37 C at 200 rpm in a shaking incubator (Innova 4000, New Brunswick Scientific, New Brunswick, NJ) for about 5 to about 16 hours (about 5 to about 9 hours for late log phase growth, about 16 hours for complete growth curve). The bacteria were harvested by centrifugation at 8000 X g for 20 minutes at 4*C (RC 5C Plus, Sorvall/Dupont, Wilmington, DE). A. Strain H10407 [1121 The following was conducted to determine the yield of strain H 10407 when grown in three different broths and harvested at 7 hours. Table 2 provides the results as follows: Table 2 pH OD 600 Weight of Bacteria DME/F-12 4.66 1.344 1.97 g CFA Broth 7.71 1.572 1.25 g L Broth 7.19 1.712 1.11 g [1131 The final product purity and yield refer to amount of CFA/I after harvest, heat extraction, ammonium sulfate precipitation at 25% saturation, centrifugation, resuspension of the precipitate, and dialysis. For heat extraction and purification of the given CF, the bacterial pellet was suspended by adding 20 ml of PBS. The bacterial suspension was placed in a preheated water bath at 60 *C for 30 minutes, then centrifuged at 8000 X g for 20 minutes. The given CF was purified by adjusting the supernatant to 25% saturation of ammonium sulfate, incubated at room temperature for about 2 to about 6 hours or overnight at 4 *C. The ammonium sulfate precipitate was harvested at 25,000 X g and then subjected to dialysis against PBS three times. Table 3 shows the final CF products from 200 ml as follows: WO 2004/093825 PCT/US2004/007439 Table 3 Concentration Volume Purity Total Yield DMEIF-12 696.1 pg/ml 2 ml 70 % 974.4 pg CFA Broth 118.1 pg/mI 2 ml 70% 165.3 pg L Broth 111.0 pg/ml 2 ml 2% 44.4 pg [114] Material from the resultant 2 ml sample was applied to SDS-PAGE gels for purity determination, and also run in gels and transferred to nitrocellulose and developed with CFA/I specific antibody in the Western blot. The protein concentration of the 2 ml sample was determined by Lowry assay. Final purity was determined from the SDS-PAGE gels and total yield calculated by multiplying the concentration by the volume (2 ml) times the purity for the total yield of CFA/I in the final column. [1151 As provide in Figure 3, 16% tricine SDS-PAGE shows that CFA/I results from H10407 when grown in DME/F-12 (lane 6), L-broth (lane 7), and CFA broth (lane 8), although the yield of CFA/I from L-broth appears very low. As shown in Figure 3, lane 1 is a marker, Mark 12, (Novex/Invitrogen, Carlsbad, CA), lane 2 is 25% PE (centrifugation pellet, protein precipitated at 25% saturation of ammonium sulfate) of 1933D in DME/F-12 with NaHCO 3 , lane 3 is 25% PE of 1933D in L Broth, lane 4 is 25% PE of 1933D in CFA Broth, lane 5 is Mark 12, lane 6 is 25% PE of H10407 in DME/F-12 with NaHCO 3 , lane 7 is 25% PE of H10407 in L Broth, and lane 8 is 25% PE of H10407 in CFA Broth. [116] As provided in Figure 4, the Western blot verifies that (lanes 6, 7, and 8 of the SDS-PAGE show the CFA/I band) CFA/I is present in L-broth grown 110407. As shown in Figure 4, lane 1 is CFA/1, lane 2 is 25% PE of 1933D in DME/F-12 and NaHCO 3 , lane 3 is 25% PE of 1933D in L Broth, lane 4 is 25% PE of 1933D in CFA Broth, lane 5 is 25% PE of H10407 in DME/F-12 and NaHCO 3 , lane 6 is 25% PE of H10407 in CFA Broth, lane 7 is 25% PE of H10407 in L Broth, and 8 is a marker, See Blue (Novex/Invitrogen, Carlsbad, CA). [117] Yield data CFA/I- total yield column- demonstrates that the most CFA/I is produced when grown in DME/F-12 , less so in CFA/I broth, and very little from L broth. Growth of H10407 in DME/F-12 yielded about 494.6 ptg of purified CF per gram of bacteria. Growth of H10407 in CFA broth yielded about 132.2 pg of purified CF per gram of bacteria. Growth of H110407 in L broth yielded about 40.0 p.g of purified CF per gram of bacteria. Therefore, fermenting the bacteria in DME/F-12 WO 2004/093825 PCT/US2004/007439 yielded an amount of purified CF about 3.7 times the amount yielded with CFA broth and about 12.4 times the amount yielded with L broth. B. Strain E9034A [118] The following was conducted to determine the yield of strain E9034A when grown in three different broths and harvested at 7 hours. Table 4 provides the results as follows: Table 4 pH OD 600 Weight of Bacteria DMEI/F-12 5.07 1.086 1.26 g CFA Broth 6.48 2.200 1.81 g L Broth 5.28 2.460 1.86 g [1191 The final product purity and yield refer to amount of CS3 after harvest, heat extraction, ammonium sulfate precipitation at 25% saturation, centrifugation, resuspension of the precipitate, and dialysis. For heat extraction and purification of the given CF, the bacterial pellet was suspended by adding 20 ml of PBS. The bacterial suspension was placed in a preheated water bath at 60 0 C for 30 minutes, then centrifuged at 8000 X g for 20 minutes. The given CF was purified by adjusting the supernatant to 25% saturation of ammonium sulfate, incubated at room temperature for about 2 to about 6 hours or overnight at 4 'C. The ammonium sulfate precipitate was harvested at 25,000 X g and then subjected to dialysis against PBS three times. Table 5 shows the final CF products from 200 ml as follows: Table 5 Concentration Volume Purity Total Yield DMEIF-12 486 pg/mI 1 ml 80 % 388.8 pg CFA Broth 1327 pg/ml 1 ml 0% 0 pg L Broth 1009 pg/ml I ml 0 % 0 pg [120] Material from the resultant 1 ml sample was applied to SDS-PAGE gels for purity determination, and also run in gels and transferred to nitrocellulose and developed with CFA/I specific antibody in the Western blot. The protein concentration of the 2 ml sample was determined by Lowry assay. Final purity was determined from the SDS-PAGE gels and total yield calculated by multiplying the concentration by the volume (2 ml) times the purity for the total yield of CS3 in the final column. [121] Growth of E9034A in DME/F-12 yielded about 308.6 ptg of purified CF per gram of bacteria. Growth of E9034A in CFA broth yielded about 0.0 p.g of purified CF WO 2004/093825 PCT/US2004/007439 per gram of bacteria. Growth of E9034A in L broth yielded about 0.0 ptg of purified CF per gram of bacteria. Therefore, fermenting the bacteria in DME/F-12 yields quantifiable amounts of purified CF, whereas growth of E9034A in CFA broth did not result in any detectable amount of purified CF and growth E9034A in L broth did not result in any detectable amount of purified CF. [122] As provided in Figure 5, 16% tricine SDS-PAGE shows that CS3 results only from E9034A when grown in DME/F-12. As shown in Figure 5, lane 1 is Mark 12, lane 2 is CS3 marker, lane 3 is 25% PE of E9034A in DME/F-12 and NaHCO 3 , lane 4 is 25 % PE of E9034A in L Broth, and lane 5 is 25% PE of E9034A in CFA Broth. As provided in Figure 6, the Western blot verifies that lanes 4 and 5 of the SDS-PAGE do not show the CS3 band and lanes 4 and 5 of the Western blot are negative when detecting with antibody. As shown in Figure 6, lane 1 is See Blue, lane 2 is CS3, lane 3 is 25% PE of E9034A in DME/F-12 with NaHCO 3 , lane 4 is 25% PE of E9034 in L Broth, and lane 5 is 25% PE of E9034A in CFA Broth. C. Strain 60R75 [123] The following was conducted to determine the yield of strain 60R75 when grown in three different broths and harvested at 7 hours. Table 6 provides the results as follows: Table 6 PH OD 600 Weight of Bacteria DME/F-12 5.42 0.969 1.68 g CFA Broth 7.27 1.345 1.21 g L Broth 7.04 1.568 1.43 g [1241 The final product purity and yield refer to amount of CS 1 after harvest, heat extraction, ammonium sulfate precipitation at 25% saturation, centrifugation, resuspension of the precipitate, and dialysis. For heat extraction and purification of the given CF, the bacterial pellet was suspended by adding 20 ml of PBS. The bacterial suspension was placed in a preheated water bath at 60 *C for 30 minutes, then centrifuged at 8000 X g for 20 minutes. The given CF was purified by adjusting the supernatant to 25% saturation of ammonium sulfate, incubated at room temperature for about 2 to about 6 hours or overnight at 4 *C. The ammonium sulfate precipitate was harvested at 25,000 X g and then subjected to dialysis against PBS three times. Table 7 shows the final CF products from 200 ml as follows: WO 2004/093825 PCT/US2004/007439 Table 7 Concentration Volume Purity Total Yield DMEM 1477.6 pg/ml 2 ml 85 % 2460 pg CFA Broth 367 pg/ml 2 ml 0 % 0 pg L Broth 1246 pg/ml 2 ml 0% Opg [125] Material from the resultant 2 ml sample was applied to SDS-PAGE gels for purity determination, and also run in gels and transferred to nitrocellulose and developed with CS 1 specific antibody in the Western blot. The protein concentration of the 2 ml sample was determined by Lowry assay. Final purity was determined from the SDS-PAGE gels and total yield calculated by multiplying the concentration by the volume (2 ml) times the purity for the total yield of CS 1 in the final column. [126] Growth of 60R75 in DME/F-12 yielded about 1464.3 pg of purified CF per gram of bacteria. Growth of 60R75 in CFA broth yielded about 0.0 pig of purified CF per gram of bacteria. Growth of 60R75 in L broth yielded about 0.0 pig of purified CF per gram of bacteria. Therefore, fermenting the bacteria in DME/F-12 yields quantifiable amounts of purified CF, whereas growth of 60R75 in CFA broth did not result in any detectable amount of purified CF and growth 60R75 in L broth did not result in any detectable amount of purified CF. [127] As provided in Figure 7, 16% tricine SDS-PAGE shows that CS1 results only from 60R75 when grown in DME/F-12. As shown in Figure 7, lane 1 is Mark 12, lane 2 is 25% PE of 60R75 in DME/F-12 and NaHC0 3 , lane 3 is 25 % PE of 60R75 in L Broth, and lane 4 is 25% PE of 60R75 in CFA Broth. As provided in Figure 8, the Western blot verifies that lanes 3 and 4 of the SDS-PAGE do not show the CSI band and lanes 4 and 5 of the Western blot are negative when detecting with antibody. As shown in Figure 8, lane 1 is See Blue, lane 2 is CS1, lane 3 is 25% PE of 60R75 in DME/F-12 with NaHCO 3 , lane 4 is 25% PE of 60R75 in L Broth, and lane 5 is 25% PE of 60R75 in CFA Broth. Example 3 Agglutination Studies [128] The following agglutination studies were conducted as follows. Bacteria were recovered from broth at late log phase (about 5 hours) by centrifugation (30 min. 7,000 x g), and the pellet suspended in phosphate buffered saline (PBS) to an optical density of 5.0 at 600 nm. Bacteria from CFA plates were scraped into PBS, and suspended as WO 2004/093825 PCT/US2004/007439 above. 8 pl of the bacterial suspension was mixed with the same volume of purified antibody from rabbits before (preimmune) and after (postimmune) immunization, as well as with 8 pl of PBS. The degree of agglutination was observed visually and graded based on the time of agglutination as well as the clump size. Score of agglutination: 4, agglutination less than about 10 seconds; 3, agglutination about 10 to about 30 seconds; 2, agglutination about 0.5 to about 1 minute; 1, agglutination about 1 to about 2 minutes; 0, no agglutination within about 2 minutes or more. [129] (ii) For slide agglutination suspend bacteria in minimal volume of PBS, read absorbance at OD 600 nm, adjust to OD 600 nm of 5.0. [130] The media used was as follows: DME/F-12 (D8900, Sigma-Aldrich, St. Louis, MO) supplemented with 0.12% sodium bicarbonate, L broth containing 1% trypton, 0.5% yeast extract, and 1% sodium chloride, CFA broth containing 1% casamino acids, 0.15% yeast extract, 0.0005% manganese chloride tetrahydrate, and 0.005% magnesium sulfate heptahydrate, and CFA agar containing 1% casamino acids, 0.15% yeast extract, 0.0005% manganese chloride tetrahydrate, 0.005% magnesium sulfate heptahydrate, and 2% granulated agar. [131] For CFA/I and CS1 detection, pre- and post-immune antibody was affmity purified on Hi-Trap Protein-G Sepharose (Amersham, Piscataway, NJ) from pooled sera from rabbits 92319 (CFA/I) and 94859 (CS 1). [132] The following Table 8 illustrates that when grown in different broths and when compared to plate grown bacteria, the DME/F-12 grown bacteria are equal or better than other broths by this criteria (by antibody detection). Table 8 Antibody Agglutination of CFA/I- and CS1 Expressing ETEC Grown in DME-F12-, L-, and CFA-broths, as well as CFA plates Agglutination Score ETEC Strain Post Immune Ab Preimmune Ab PBS Growth Media H10407 DME/F-12 3 0 0 L broth 0 0 0 CFA broth 1 0 0 CFA agar 2 0 0 1933D DMEF-12 4 0 0 L broth 4 0 0 CFA broth 4 0 0 CFA agar 4 0 0 WO 2004/093825 PCT/US2004/007439 60R75 DME/F-12 4 0 0 L broth 0 0 0 CFA broth 0 0 0 CFA agar 2 0 0 [133] As provided above, it should be noted that strain 1933D expressed CFA/I very well in all broths. Although the expression of CFA/I should be turned off in L broth, it is not, thereby indicating a factor independent of growth media conditions that causes an up-regulation of the expression of CFA/I in 1933D. Example 4 Electronmicroscopy of CF Expression [1341 Electron microscopy was conducted using methods known in the art. See Harris, R., et al. (1991) ELECTRON MICROSCOPY IN BIOLOGY "Negative Staining" CRC Press, Boca Raton, FL, pp 328-342; and Cassels, F.J., et al. (1992) Infect. Immun. 60:2174-2181, which are herein incorporated by reference. Cell analyzed were according to Example 2. About 1 to about 2 ml samples from growing cultures were obtained and submitted to an electron microscopy facility without centrifugation (neat no concentration or dilution) for electronmicroscopy according to methods known in the art. [1351 Figure 9A and Figure 9B show CS 1 expressing ETEC grown on CFA agar. [136] Figure 1OA and Figure 1OB show CS1 expressing ETEC grown on CFA broth. [137] Figure 11 A and Figure 1 1B show CS1 expressing ETEC grown on L broth. [138] Figure 12A and Figure 12B show CS1 expressing ETEC grown on DME/F-12 broth. [139] Figure 13A and Figure 13B show CFA/I expressing ETEC grown on CFA agar. [140] Figure 14A and Figure 14B show CFA/I expressing ETEC grown on CFA broth. [141] Figure 15A and Figure 15B show CFA/I expressing ETEC grown on DME/F-12 broth. [1421 Table 9 as follows summarizes the electron microscopic analysis of CS 1 and CFA/I expression.

WO 2004/093825 PCT/US2004/007439 Table.9 ELECTRON MICROSCOPIC ANALYSIS OF CSI AND CFAIl EXPRESSION ON AGAR AND BROTH Bacterial Fimbrial Growth Diameter Length Diameter Length Fimbriated Distance 1 (pm) (pm) (pm) (pm) Bacteria 2 CS1 CFA Agar 0.4-1.5 0.8-5.0 7 0.18-1.2 50% DME/F-12 0.45-1.8 0.7-5.5 7 0.35-1.7 75-80 % some > 2.5 CFA Broth 0.5-1.5 0.75-5.0 - - 0 % L Broth 05-1.5 0.5-5.0 - - 0% CFAll 3 CFA Agar 0.5-1.0 0.6-4.0 7 0.12-1.2 85% DME/F-12 0.5-1.5 0.6-5.5 7 0.1-1.3 95% some > 3.0 CFA Broth 0.5-1.5 0.6-6.0 7 0.2-0.5 5-10% 1 CFA Broth: 1% casamino acids, 0.15% yeast extract, 0.0005% manganese chloride tetrahydrate, 0.0005% magnesium sulfate heptahydrate; CFA Agar: same as CFA broth and 2% agar; L Broth: 1% tryptone, 0.5% yeast extract, 1% sodium chloride. 2 Calculated from the visualization of about 300 to about 400 individual bacteria. 3 L Broth not done. Example 5 Analysis of Strain E9034A (Expresses CS3) [1431 Strain 1933D which expresses CFA/I and strain E9034A which expresses CS3 were cultured in 10 L and 300 L volumes of DME/F-12 broth as follows: 10 L fermentation and purification of CFA/I from strain 1933D A. Culture and fermentation [1441 A frozen vial of ETEC strain 1933D was thawed and 800 pl were pipetted into 1000 ml of DME/F-12 and then cultured at 37 *C, 200 rpm in a shaking incubator (Innova 4000, New Brunswick Scientific, New Brunswick, NJ) for about 5 to about 7 hours to an absorbance of about 0.9 to about 1.1 optical density unit at 600 nm. Then 100 ml of culture were aseptically removed to inoculate a fermentor containing 10 L DME/F-12 and 1 ml antifoam (New Brunswick Scientific, New Brunswick, NJ) and fermented for about 6 to about 8 hours or until dissolved oxygen rapidly increases from a low level (about 5% to about 40%) to about 15% above the lowest level. The cells were harvested by centrifugation (8,000 x g for 30 minutes). The bacterial pellet was suspended in PBS at about 3 to about 4 ml/g wet weight of bacteria. I A WO 2004/093825 PCT/US2004/007439 B. Heat extraction for recovery of CFA/I; purification by tangential flow filtration and ammonium sulfate precipitation. [1451 The bacteria suspension was placed in a pre-heated water bath at about 60 0 C to about 70 'C for 30 minutes. Then the suspension was centrifuged at 8000 X g for 20 minutes. The supernatant was recovered for tangential flow filtration per the manufacturer's instructions using an Amersham (Piscattaway, NJ) AG/T CFP-2-E-6A 0.2 micron filter. CFA/I passes through the filter to be collected in a reservoir for ultrafiltration. The 0.2 um filter retains remaining cells and large cell debris. The reservoir solution containing CFA/I was passed over an Amersham (Piscattaway, NJ) AG/T UFP-300-C-6A filter per the manufacturer's instructions. CFA/I was retained on the filter as spent media passes through the filter to waste. Retentate of UFP-300-C-6A was adjusted to 25% saturation of ammonium sulfate and incubated at room temperature overnight at 4 "C. The ammonium sulfate precipitate was harvested at 25,000 X g. Then the 25% precipitate was dialfiltered against PBS using an Amersham AG/T UFP-300-C-4A filter per manufacturer's instructions, with CFA/I recovered from the retentate. 10 L fermentation and purification of CS3 from strain E9034A A. Culture and fermentation [146] A frozen vial of ETEC strain E9034A was thawed and 800 pL were pipetted into 1000 ml of DME/F-12 and then cultured at 37 "C, 200 rpm in a shaking incubator (Innova 4000, New Brunswick Scientific, New Brunswick, NJ) for about 5 to about 7 hours to an absorbance of about 0.9 to about 1.1 optical density unit at 600 nm. Then 100 ml of culture were aseptically removed to inoculate a fermentor containing 10 L DME/F-12 and 1 ml antifoam (New Brunswick Scientific, New Brunswick, NJ) and fermented for about 6 to about 8 hours or until dissolved oxygen rapidly increases from a low level (about 5% to about 40%) to about 15% above the lowest level. The cells were harvested by centrifugation (8,000 x g for 30 minutes). The bacterial pellet was suspended in PBS at about 3 to about 4 ml/g wet weight of bacteria. B. Heat extraction for recovery of CS3; purification by tangential flow filtration and ammonium sulfate precipitation. [1471 The bacteria suspension was placed in a pre-heated water bath at about 60 'C to about 70 *C for 30 minutes. Then the suspension was centrifuged at 8000 X g for 20 WO 2004/093825 PCT/US2004/007439 minutes. The supernatant was recovered for tangential flow filtration per the manufacturer's instructions using an Amersham (Piscattaway, NJ) AG/T CFP-2-E-6A 0.2 micron filter. CS3 passes through the filter to be collected in a reservoir for ultrafiltration. The 0.2 um filter retains remaining cells and large cell debris. The reservoir solution containing CS3 was passed over an Amershan (Piscattaway, NJ) AG/T UFP-500-C-6A filter per the manufacturer's instructions. CS3 was retained on the filter as spent media passes through the filter to waste. Retentate of UFP-500-C-6A was adjusted to 25% saturation of ammonium sulfate and incubated at room temperature overnight at 4 "C. The ammonium sulfate precipitate was harvested at 25,000 X g. Then the 25% precipitate was dialfiltered against PBS using an Amersham AG/T UFP-500-C-4A filter per manufacturer's instructions, with CS3 recovered from the retentate. 300 L fermentation and purification of CFA/I from strain 1933D A. Culture and fermentation [148] Three frozen vials of ETEC strains 1933D were thawed and 800 pd were pipetted into each of three 1000 ml of DME/F-12 and cultured at 37 'C, 200 rpm in a shaking incubator (Innova 4000, New Brunswick Scientific, New Brunswick, NJ) for about 5 to about 7 hours to an absorbance of about 0.9 to about 1.1 optical density unit at 600 nm. The contents of the three flasks of culture were aseptically transferred to inoculate a fermentor containing 300 L DME/F-12 and 30 ml antifoam (New Brunswick Scientific, New Brunswick, NJ) and fermented for about 6 to about 8 hours or until dissolved oxygen rapidly increased from a low level (about 5% to about 40%) to about 15% above the lowest level. The cells were harvested by continuous flow centrifugation (Sharples AS-26SP) (12,000 rpm at a flow rate of about 1.5 to about 2.0 L per minute). Then the bacterial pellet was suspended in PBS at about 3 to about 4 ml/g wet weight of bacteria. B. Heat extraction for recovery of CFA/I; purification by tangential flow filtration and ammonium sulfate precipitation. [149] The bacterial suspension was aseptically pumped into a 30 L New Brunswick fermentor and heated at about 60 *C to about 70 "C for 30 minutes. The cells were harvested by continuous flow centrifugation (Sharples AS-26SP) (12,000 rpm at a flow rate of about 0.75 to about 1.5 L per minute). The supernatant was recovered for 11IC WO 2004/093825 PCT/US2004/007439 tangential flow filtration and conducted per the manufacturer's instructions using an Amersham (Piscattaway, NJ) AG/T CFP-2-E-65 0.2 micron filter. CFA/I passes through the filter to be collected in a reservoir for ultrafiltration. The 0.2 pm filter retains remaining cells and large cell debris. The reservoir solution containing CFA/I was passed over an Amersham (Piscattaway, NJ) AG/T UFP-300-C-55 filter per the manufacturer's instructions. CFA/I was retained on the filter as spent media passes through the filter to waste. Retentate of UFP-300-C-55 was adjusted to 25% saturation of ammonium sulfate and incubated at room temperature for 2 hours followed by about 18 to about 72 hours at 4 "C. The ammonium sulfate precipitate was harvested at 25,000 X g (50 minutes). The 25% precipitate was dialfiltered against PBS using an Amersham AG/T UFP-300-C-4A or UFP-300-C-5A filter per the manufacturer's instructions, with CFA/I recovered from the retentate. 300 L fermentation and purification of CS3 from strain E9034A A. Culture and fermentation [150] Three frozen vials of ETEC strains E9034A were thawed and 800 pL were pipetted into each of three 1000 ml of DME/F-12 and cultured at 37 'C, 200 rpm in a shaking incubator (Innova 4000, New Brunswick Scientific, New Brunswick, NJ) for about 5 to about 7 hours to an absorbance of about 0.9 to about 1.1 optical density unit at 600 nm. The contents of the three flasks of culture were aseptically transferred to inoculate a fermentor containing 300 L DME/F-12 and 30 ml antifoam (New Brunswick Scientific, New Brunswick, NJ) and fermented for about 6 to about 8 hours or until dissolved oxygen rapidly increased from a low level (about 5% to about 40%) to about 15% above the lowest level. The cells were harvested by continuous flow centrifugation (Sharples AS-26SP) (12,000 rpm at a flow rate of about 1.5 to about 2.0 L per minute). Then the bacterial pellet was suspended in PBS at about 3 to about 4 ml/g wet weight of bacteria. B. Heat extraction for recovery of CS3; purification by tangential flow filtration and ammonium sulfate precipitation. [151] The bacterial suspension was aseptically pumped into a 30 L New Brunswick fermentor and heated at about 60 *C to about 70 *C for 30 minutes. The cells were harvested by continuous flow centrifugation (Sharples AS-26SP) (12,000 rpm at a flow rate of about 0.75 to about 1.5 L per minute). The supernatant was recovered for WO 2004/093825 PCT/US2004/007439 tangential flow filtration and conducted per the manufacturer's instructions using an Amersham (Piscattaway, NJ) AG/T CFP-1-E-65 0.1 micron filter. CS3 passes through the filter to be collected in a reservoir for ultrafiltration. The 0.1 pm filter retains remaining cells and large cell debris. The reservoir solution containing CS3 was passed over an Amersham (Piscattaway, NJ) AG/T UFP-500-C-55 filter per the manufacturer's instructions. CS3 was retained on the filter as spent media passes through the filter to waste. Retentate of UFP-500-C-55 was adjusted to 25% saturation of ammonium sulfate and incubated at room temperature for 2 hours followed by about 18 to about 72 hours at 4 *C. The ammonium sulfate precipitate was harvested at 25,000 X g (50 minutes). The 25% precipitate was dialfiltered against PBS using an Amersham AG/T UFP-500-C-5A filter per the manufacturer's instructions, with CS3 recovered from the retentate. [1521 Table 10 summarizes the harvest parameters, yield and purity of CFA/I and CS3 in DME/F-12 broth as compared with those of a CS6 expressing strain according to Example 1. Table 10 HARVEST PARAMETERS, YIELD AND PURITY OF CS3 AND CFAI FROM 10L AND 300L FERMENTATIONS CF-Vol cGMP pellet wet yield CF mg CF/g mg CF/L Purity Lot # wt (g) (mg) wet wt broth CS3-10L -- 101 349 3.4 35 95% CS3-300L 0963 1460 5289 3.6 17.6 95.3% CFAII-10L -- 98 450 4.6 45 >93% CFAll- 1096 1271 12,809 10.1 42.7 98.4% 300L I _II CS6-300L 1051 5980 4201 0.7 14.0 99.5% [153] Figure 16 is an SDS-PAGE of CS3 from a 300L fermentation. 16% tris-Tricine SDS-PAGE gel (Invitrogen, Carlsbad, CA) with 10 and 20 pg loaded (lanes 2 and 3, respectively) of CS3, Lot 0963 from 300 L fermentation, strain E9034A grown in DME-F-12. The molecular weight standards (Mark 12, Invitrogen, Carlsbad, CA) are in lane 1. [154] Figure 17 is an SDS-PAGE of CFA/I from a 10L fermentation. 16% tris Tricine SDS-PAGE gel (Invitrogen, Carlsbad, CA) with 30 pg (lane 2) of CFA/I from 10 L fermentation, strain 1933D grown in DME/F-12. The molecular weight standards (Mark 12, Invitrogen, Carlsbad, CA) are in lane 1.

WO 2004/093825 PCT/US2004/007439 [155] Figure 18 is an SDS-PAGE of CFA/I from a 300L fermentation. 16% tris Tricine SDS-PAGE gel (Invitrogen, Carlsbad, CA) with 20 and 10 tg (lanes 1 and 2, respectively) of CFA/I, Lot 1096 from 300 L fermentation, strain 1933D grown in DME/F-12. A CFA/I standard is included for comparison, Lot 0633 (20 pg, lane 4). The molecular weight standards (Mark 12, Invitrogen, Carlsbad, CA) are in lane 3. Example 6 Oral Formulation Comprising Microencapsulated CS6 [1561 Vaccine preparations comprising a CF, such as CS6, have been tested in subjects. See e.g. Yu et al. (2002) Infect. Immun. 70(3):1056-1068 (transcutaneous mice); Gierefna-Burguefuo, F., et al. (2002) Infect. Immunity 70(4):1874-1880 (transcutaneous human); de Lorimier, et al. (2003) Vaccine 21(19-20):2548-2555, (intranasally mice); and Katz et al. (2003) Vaccine, 21(5-6):341-346 (oral microencapsulated human); and Bryd and Cassels (2003) Vaccine 21:1884-1893, (intranasally and intragastrically mice), which are herein incorporated by reference. A. CLINICAL STUDY 1 [157] The safety and immunogenicity of CS6, a multi-subunit protein commonly found on the surface of ETEC, was evaluated according to the following experiments. Generally, the formulations tested included 1 and 5 mg doses of CS6, either encapsulated in biodegradable polymer poly (D,L)-lactide-co-glycolide, or as free protein, administered orally in a solution of either normal saline or a rice-based buffer. Three doses of CS6 were given at 2 week intervals. Blood was collected immediately before and 7 days after each dose. All formulations were well tolerated. Four of five subjects who received 1mg CS6 in PLG microspheres with buffer had significant IgA ASC responses (median= 30 ASC per 106 PBMC) and significant serum IgG responses (median = 3.5 fold increase). Oral administration of this prototype ETEC vaccine is safe and can elicit an immune response. The ASC, serum IgA, and serum IgG responses to CS6 are similar in magnitude to the responses after challenge with wild type ETEC (Coster et al., unpublished). [158] Healthy male and female subjects, age 18-45, were recruited from the Washington, DC, metropolitan area. All subjects were assigned a code to maintain confidentiality. Subjects were excluded from participation in the study if they met any of the following criteria: travel to a developing country within 1 year of study WO 2004/093825 PCT/US2004/007439 participation and experienced diarrhea, received a cholera vaccination within 5 years of the study, participated in previous enteric vaccine protocols, had a clinically significant illness, history of chronic gastrointestinal illness, positive pregnancy test, was unable or unwilling to submit blood samples, use of stomach acid neutralizer within 2 days of the study, had occupational exposure to ETEC or Vibrio cholerae, or participated in another vaccine challenge study within 30 days of this protocol. [1591 Six (6) formulations of the vaccine as shown in Table 11 were tested. Table 11 Group Form Buffer Dose Number I Non-encap Yes 1 mg CS6 4 Il PLG Yes 1 mg CS6 5 Ill PLG No 1 mg CS6 3 IV Non-encap Yo 5 mg CS6 4 V PLG Yes 5 mg CS6 5 VI PLG No 5 mg CS6 5 [1601 CS6 and CS6 microencapsulated in PLG microspheres (CS6-PLG) were produced under current good manufacturing practices (cGMP) at the WRAIR Pilot Bioproduction Facility, Silver Spring, MD. The bacterial strain used for the production of CS6 was constructed from E. coli (HB 101) containing a recombinant plasmid carrying the four genes necessary for CS6 expression. See Wolf, M.K., et al. (1997) FEMS Microbiol Lett. 148(1):35-42, which is herein incorporated by reference. The major components in the production included: bacterial fermentation, recovery of the CS6 from the fermentation broth by tangential flow filtration, ammonium sulfate precipitation, buffer exchange from PBS into 4% sucrose, microencapsulation by a solvent evaporation procedure using an emulsion of CS6/sucrose and PLG in methylene chloride, homogenization in polyvinal alcohol, removal of methylene chloride by evaporation, lyophilization, and storage at -80 *C. Unencapsulated CS6 was produced in an identical manner to the microencapsulated CS6, but the material was stored in PBS at -80 *C. [1611 The vaccine was administered orally in three doses. There were 6 vaccine groups (Table 3). Groups I-III (low dose; 1 mg each dose) were vaccinated on days 0, 14, and 28. Groups IV-VI received their three doses (high dose; 5 mg, 4 mg, and 4 mg, respectively) on days 7, 21, and 35. On the day of vaccination, all subjects were observed 90 minutes prior to and after vaccine administration. The subjects drank from a cup which contained either CS6 or CS6-PLG in 100 ml of either normal saline or WO 2004/093825 PCT/US2004/007439 buffer (Cera Products LLC, Jessup, MD). Subjects then drank from a second cup containing 50 ml of either normal saline or buffer (to match the solution in the first cup). [162] Subjects kept a diary to record any symptoms that occurred for the seven days following each dose. The diary collected solicited and unsolicited symptoms and intensities. Symptom presence and intensity was graded: not present; mild (noticeable, but did not interfere with routine activities); moderate (interfered with routine activities); or severe (unable to perform routine activities). Fever was defined as a temperature greater than 100.5 'F and documented by Temp-dot (3M, Rochester, MN) disposable thermometers. The number of vomiting episodes were recorded as were the number of episodes diarrhea (defined as about 3 or more loose stools over a 24 hour period), and loose stools. The subjects returned on the day after vaccination and seven days post-vaccination for clinical assessment and to monitor for any possible side effects. The diaries were reviewed by the study physicians with the subjects. [163] CS6-specific antibody-secreting cell (ASC) immune responses to the vaccine antigen were chosen as a surrogate of intestinal mucosal immune response. See Wenneras, C., et al. (1992) Infect Immun. 60:2605-11, which is herein incorporated by reference. Whole blood was collected for ASC's weekly from the day of the first vaccination (prior to receiving the vaccine) until 2 weeks after the third vaccination (a total of 7 samples). Blood specimens were collected using EDTA treated tubes (Becton Dickinson Vacutainer Systems, Rutherford, NJ). Peripheral blood mononuclear cells (PBMC) were isolated from the blood sample by gradient centrifugation on Ficoll Hypaque (Sigma Co., St. Louis, MO) and were assayed for total and CS6-specific numbers of IgA ASC by the ELISpot technique. See Wenneras, C., et al. (1992) Infect Immun. 60:2605-11 and Czerkinsky, C., et al. (1998) J. Immunol. Methods 115:31-37, which are herein incorporated by reference. Individual wells of nitrocellulose-bottom 96-well plates (Millititer HA; Millopore Corp., Bedford, MA) were coated with 0.1 ml of purified CS6 (20 tg/ml) as the solid phase antigen, and incubated overnight at 4 *C. After being washed with PBS, the plates were blocked with complete Iscove's medium (GibcoBRL, Grand Island, NY) supplemented with 5% fetal calf serum (GibcoBRL) and 50 tg/ml gentamycin (GibcoBRL). The PBMC were adjusted to 2 x 107 viable cells/ml in complete Iscove's medium. A final 0.1 ml suspension containing 106 PBMC was added to each well, and plates were incubated for 4 hours at 37 *C in 7.5 % WO 2004/093825 PCT/US2004/007439

CO

2 . Plates were washed, incubated overnight at 4 *C with goat anti-human IgA horseradish peroxidase (IgA) (Southern Biotech Associates, Birmingham, AL), and exposed to chromogen-enzyme substrate (Sigma, St. Louis, MO). Spots, corresponding to a zone of antibodies secreted by individual cells, were enumerated in triplicate wells under 40X magnification, with data expressed as the number of spot-forming cells per 106 PBMC. [1641 A positive ASC response was defined as about a 2-fold or more increase over baseline value of the ASC's per 106 PBMC, when the number of ACS's was about 0.5 per 106 PBMC or more in the baseline sample. If the number of pre-iminune ASC's was less than 0.5 per 106 PBMC, a value of greater than about 1.0 per 106 PBMC after dosing was considered a positive response. Serum was collected weekly simultaneously with whole blood (for the ASC assay). IgA and IgG antibody titers against the CS6 antigen were determined by ELISA. See Hall, E.R., et al. (2001) Infect. Immun. 69(5):2853-7, and Jertborn, M., et al. (1998) Vaccine 16:255-260, which are herein incorporated by reference. Individual microtiter wells (Nunc immunoplates, Roskilde, Denmark) were coated with 0.1 ml of a 1.0 pg/ml preparation of CS6 at 37 'C overnight. The plates were washed with PBS and then blocked with 0.1% bovine serum albumin (Sigma, St. Louis, MO). Serum samples were diluted 1:5 then 3 fold serially and incubated at room temperature for 90 minutes. Bound antibodies were visualized by addition of rabbit anti-human IgA or IgG conjugated with horseradish peroxidase (Jackson ImmunoResearch Laboratories, Westgrove, PA) and incubated at room temperature for 90 minutes followed by addition of o phenylenediamine (OPD)-H 2 0 2 (Sigma, St. Louis, MO). The endpoint titers were assigned as the interpolated dilutions of the samples giving an absorbance value at 450 nm of 0.4 above background. Titers were adjusted in relation to a reference specimen included in each test to compensate for day-to-day variation. Pre- and post-dosing serum samples from the same individual were tested side by side. The antibody titer ascribed to each sample represented the geometric mean of duplicate determinations performed on different days. Reciprocal endpoint titers less than 5 were assigned a value of 2.5 for computations. Based on calculations of the methodological error of each ELISA, a response was defined as about a two-fold increase or more in endpoint titer between pre- and post-immunization, with the added criterion that the post immunization reciprocal titer be about 10 or more. Seroconversion after any dose was defined as a positive response.

WO 2004/093825 PCT/US2004/007439 [1651 The Fishers exact test was used to compare proportions. Graphs were constructed to contrast each group (formulation). There were no significant differences in the frequency or the magnitude of the serum antibody or ASC response to CS6 between the 6 groups. Therefore, data were pooled for further presentation. [166] The ages of the 29 subjects who participated in this study ranged from 20 to 44 years of age. Nineteen of the subjects were African American, six were Caucasian, one was Hispanic, and three were of other nationalities/ethnicities. Among the 29 subjects who received a studyagent, three subjects received one dose, three subjects received two doses, and 23 received all three doses. Only one subject withdrew because the subject was unable to drink the vaccine (lmg CS6 in buffer) due to the taste of the buffer. Five subjects did not receive the full three doses. Specifically, two did not receive the full three doses due to scheduling conflicts, two did not receive the full three doses due to lack of follow-up, and one did not receive the full three doses due to illness. Only the 26 subjects that received 2 or more doses of the vaccine were included in the safety and immune data analysis. [1671 In general, the vaccine was well tolerated. Half of the subjects (13 of 26) reported minor symptoms that were possibly vaccine-related, such as abdominal gurgling (3 1%), headache (27%), abdominal cramps (19%), nausea (19%), diarrhea (12%), and malaise (12%). Twelve of 26 subjects reported mild symptoms, five reported moderate symptoms of abdominal cramps, abdominal gurgling, headache or malaise. None reported severe symptoms. Group II (1 mg CS6-PLG in buffer) had the greatest proportion of subjects reporting symptoms. See Table 12. Table 12 The Number of Subjects Reporting Symptoms Possibly Related to the Vaccine Group Number Mild Moderate Severe 4 2 0 0 || 5 4 1 0 Ill 3 2 1 0 IV 4 1 0 0 V 5 2 2 0 VI 5 2 1 0 [1681 Over half of the subjects reported symptoms judged not to be vaccine-related based on clinical context. One subject met the definition of diarrhea about 2 to about 3 days after the third dose. This was linked to ingestion of copious amounts of Mylanta. Two subjects experienced two loose stools, one 2 days after the second dose and one within 24 hours of the third dose. Three subjects experienced one loose stool, one 5 WO 2004/093825 PCT/US2004/007439 days after the third dose, one 2 weeks after the third dose, and one 8.5 hours and 6 days after the third dose. [1691 Ten of 26 (40%) subjects showed an ASC response to CS6 (ranging from 1.33 120). There was no apparent difference for ASC response to microencapsulated (7 of 18) and unencapsulated (3 of 8) CS6. There was a better response at the low dose when looking at encapsulated forms regardless of the buffer being used. There were more ASC responses in the groups with buffer (8 of 18) than with normal saline (2 of 8). The best responses were seen in Group II. Applying Fishers exact test to Group II and its non-encapsulated counter part (Group I) rendered a p = 0.17. Of the responders in Group 11 (4 of 5), one subject had a peak ASC response after the first dose, one after the second dose, and two after the third dose. [1701 Six of 26 (24%) subjects showed a serum IgA response to CS6 (ranging from 9.8-174 fold increase). Serum IgA responses to microencapsulated CS6 (5 of 18) was greater than the response to unencapsulated CS6 (1 of 8), and responses to vaccines given with buffer (5 of 18) were greater than responses given in normal saline (1 of 8). 11711 Eleven of 26 (44%) subjects showed a serum IgG response to CS6 under the criteria of a 2-fold increase (ranging from 2.3-288). There was no apparent difference for serum IgG response to microencapsulated (8 of 18) and unencapsulated (3 of 8) CS6. There were more IgG responses in the groups with buffer (9 of 18) than with normal saline (2 of 8). B. CLINICAL STUDY 2 [1721 As provided herein an oral vaccine against ETEC was studied. The formulations tested included CS6 encapsulation in microspheres in 1 mg and 5 mg doses, and a rice-based buffer. The microspheres are biodegradable and permit slow and continued release of antigen for increased exposure to the immune system. The microspheres were made of biodegradable polymer poly(D,L-lactide-co-glycolide) (PLG). The test vaccine was produced using Good Manufacturing Practices (GMP) at the Walter Reed Army Institute of Research Bioproduction Facility in Silver Spring, . Maryland. Three doses of CS6 in PLG microspheres were given in normal saline (NS) or CeraVacx (CV) a rice-based bicarbonate solution to neutralize stomach acid. Human subjects were divided into groups and administered the formulations according to Table 13.

WO 2004/093825 PCT/US2004/007439 Table 13 Group Form Buffer Dose 1 Doses 2 and, 3 N I - CV 1maCS6 1ma CS6 4 Il PLG CV 1 ma CS6 1 ma CS6 5 Ill PLG NS 1 ma CS6 1 ma CS6 3 IV .- CV 5 ma CS6 4 ma CS6 4 V PLG CV 5mgCS6 4mqCS6 5 VI PLG NS 5 mg CS6 4 mq CS6 5 [1731 Subjects fasted for 90 minutes, swallowed 100 ml of CV or NS containing CS6, swallowed another 50 ml of CV or NS containing CS6, and fasted an additional 90 minutes. The subjects received additional doses two and four weeks later. Subjects were interviewed and provided with diary sheets to record symptoms for the five days following each vaccination. Whole blood was collected from the subjects weekly for 7 weeks to measure immune responses to CS6. CS6-specific IgA production by antibody-secreting cells (ASC) was measured as a surrogate ofinucosal immune response, and CS6-specific immunoglobulins IgA and IgG levels in sera were determined. [174] All formulations were well tolerated; none of the subjects reported symptoms definitely related to the vaccines, while half of the subjects reported possible vaccine related symptoms. The most common symptoms were abdominal gurgling (10), headache (7) abdominal cramps (5), nausea (5), loose stools (5), and malaise (2). Five subjets reported moderate symptoms that interfered with some aspect of their daily routines: two reported abdominal cramps, two reported abdominal gurgling, one reported a headache, and one reported malaise. The subjects were not clustered around any dose or associated with any group. [1751 No subject reported diarrhea that was judged to be vaccine-related. Five reported one or two loose stools that were possibly vaccine related. Three of the subjects reported loose stools after the third dose, one after the first dose, and one after the second. The subjects were not clustered in any group. [1761 Table 14 summarizes the number of subjects reporting symptoms possibly related to the vaccination. Table 14 Group Diarrhea Loose Stools Mild Moderate Severe 1 0 1 2 0 0 11 0 1 4 1 0 Ill 0 1 2 1 0 IV 0 0 1 0 0 WO 2004/093825 PCT/US2004/007439 V 0 1l2 2 0 VI 0 1 2 1 0 [177] Immunogenicity and immune responses tested by conventional methods known in the art. Table 15 summarizes the frequency of the immune responses to CS6. Table 15 Group N ASC Serum I.qA Serum IgG I Any _ 4 1 0 0 1 II 5 4 3 4 4 1i1 3 1 0 0 1 IV 4 2 1 3 3 V 5 1 1 2 2 VI 5 1 1 2 2 Any 26 10 6 11 13 [1781 Only Group II (1 mg CS6 in PLG microcapsules delivered in CV) had median peak responses to CS6 above the baseline for all three immune parameters. Group IV (5 mg CS6 delivered in CV) had a median peak serum IgG response above the baseline. The median responses of all other groups were not above the baseline. [1791 Most of the subjects that had ASC or serum IgG responses to CS6 responded before the third dose while their first serum IgA response was after the third dose. Most subjects had peak responses to CS6 after the third dose of vaccine. [180] The frequency and magnitude of immune responses to CS6 was similar to those measured after challenge of subjects with ETEC strain B7A, that expresses CS6 (Coster et al., unpublished). [1811 To the extent necessary to understand or complete the disclosure of the present invention, all publications, patents, and patent applications mentioned herein are expressly incorporated by reference therein to the same extent as though each were individually so incorporated. [182] Having thus described exemplary embodiments of the present invention, it should be noted by those skilled in the art that the within disclosures are exemplary only and that various other alternatives, adaptations, and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments as illustrated herein, but is only limited by the following claims. 46/1 - 46/2 Throughout the specification and claims, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. 5 Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness. 10 Reference to cited material or information contained in the text should not be understood as a concession that the material or information was part of the common general knowledge or was known in Australia or any other country.

Claims (27)

1. A method of obtaining a preparation comprising at least one colonization factor from a bacterial strain which comprises culturing or fermenting the bacterial strain in DME/F-12 broth. 5

2. The method of claim 1, wherein expression levels of the colonization factor are increased, induced, enhanced, or modulated as compared with expression levels of the bacterial strain cultured or fermented in a conventional medium.

3. The method of claim 1, wherein the bacterial strain is an enterotoxigenic Escherichia coli strain. 10

4. The method of claim 3, wherein the enterotoxigenic Escherichia coli strain is H 10407, E9034A, or 60R75.

5. The method of claim 1, wherein the colonization factor is a colonization factor antigen, a coli surface protein, or a putative colonization factor.

6. The method of claim 1, wherein the colonization factor belongs to the CFA/l 15 family, the CS5 family, the Type IV family, or the distinct group of colonization factors.

7. The method of claim 1, wherein the colonization factor is CSI, CS3, or CFA/I.

8. The method of claim 1, wherein the bacterial strain is cultured or fermented at about 25 'C to about 37 'C.

9. The method of claim 1, wherein the colonization factor is obtained in an amount 20 that is greater than amounts obtained when the bacterial strain is cultured or fermented in a conventional medium.

10. The method of claim 2 or 9, wherein the conventional medium is Luria broth, CFA agar, or CFA broth.

11. The method of claim 9, wherein the amount obtained is about 1 to about 14 times 25 greater than amounts obtained when the bacterial strain is cultured or fermented in the conventional medium. -48

12. The method of claim 9, wherein the amount obtained is about I to about 7 times greater than amounts obtained when the bacterial strain is cultured or fermented in the conventional medium.

13. The method of claim 9, wherein the amount obtained is about I to about 4 times 5 greater than amounts obtained when the bacterial strain is cultured or fermented in the conventional medium.

14. The method of claim 9, wherein the amount obtained is about 12.4 times greater than amounts obtained when the bacterial strain is cultured or fermented in the conventional medium. 10

15. The method of claim 9, wherein the amount obtained is about 3.7 times greater than amounts obtained when the bacterial strain is cultured or fermented in the conventional medium.

16. The method of claim 1, wherein the colonization factor is not obtained from the bacterial strain when cultured or fermented in Luria broth or when cultured or fermented 15 in CFA broth.

17. The method of claim 1, wherein the preparation has a protein purity of the colonization factor of about 70% or more.

18. The method of claim 17, wherein the protein purity is about 80% or more.

19. The method of claim 18, wherein the protein purity is about 90% or more.

20 20. The method of claim 1, wherein the bacterial strain was frozen prior to cultivating or fermenting.

21. A pharmaceutical composition comprising the preparation or the colonization factor made by the method of claim 1 and a carrier, an adjuvant, or both.

22. The pharmaceutical composition of claim 21, wherein the pharmaceutical 25 composition is an oral formulation, an intranasal formulation, or a transcutaneous formulation.

23. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition is an injectable formulation. -49

24. A method of treating, preventing or inhibiting an enterotoxigenic Escherichia coli infection in a subject which comprises administering at least one preparation or at least one colonization factor made by the method of claim 1.

25. The method of claim 15, wherein the enterotoxigenic Escherichia coli infection is 5 traveler's diarrhea.

26. A method according to claim 1 as herein before described with reference to the Examples.

27. Use of a pharmaceutical composition comprising the preparation or the colonization factor made by the method of claim I in the preparation of a medicament for 10 the treatment of enterotoxigenic Escherichia coli infection.