| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | April 18, 2000 |
| PATENT TITLE |
Methods for producing enhanced antigenic Helicobacter sp. |
| PATENT ABSTRACT | Methods using in vitro processes are disclosed for inducing or enhancing expression of enteric bacterial antigens or virulence factors. The methods, therefore, produce antigenically enhanced enteric bacteria. Also methods for using the antigenically enhanced bacteria are also disclosed, as well as vaccines containing the enteric bacteria. Specifically a whole enteric bacterium or components thereof are provided by Helicobacter species. Also there are other enteric bacteria which are useful for the disclosed invention; such as Campylobacter jejuni |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | May 29, 1997 |
| PATENT REFERENCES CITED |
Andrews & Maurelli, 1992, mxiA of Shigella flexneri 2a, which facilitates export of invasion plasmid antigens, encodes a homolog of the low-calcium-response protein, LcrD, of Yersinia pestis. Infect. Immun. 60:3287-3295. Baqar et al., 1995, "Immunogenicity and protective efficacy of a prototype Campylobacter killed whole-cell vaccine in mice." Infect. Immun. 63:3731-3735. Bell & Manning, 1990, "A domestic ferret model of immunity to Campylobacter jejuni-induced enteric disease." Infect. Immun. 58:1848-1852. Blaser & Gotschilch, 1990, "Surface array protein of Campylobacter fetus." J. Biol. Chem. 265:14529-14535. Caldwell et al., 1983, "Simple adult rabbit model for Campylobacter jejuni enteritis." Infect. Immun. 42:1176-1182. Chen et al., 1992, "Immunization against gastric helicobacter infection in a mouse/Heliocobacter felis model." Lancet 339:1120-1121. Durham et al., 1994, 94th Am. Soc. Microbiol. Abstract P-36, p. 386. Field et al., 1993, "Characteristics of an avirulent Campylobacter jejuni strain and its virulence-enhanced variants." J. Med. Microbiol. 38:293-300. Gilmour et al., 1991, "Vaccine containing iron-regulated proteins of Pasteurella haemolytica A2 enhances protection against experimental pasteurellosis in lambs." Vaccine 9:137-140. Grant et al., 1993, "Role of flagella in adherence, internalization, and translocation of Campylobacter jejuni in nonpolarized and polarized epithelial cell cultures." Infect. Immun. 61:1764-1771. Hale et al., 1985, "Identification and antigenic characterization of virulence-associated, plasmid-coded proteins of Shigella spp. and enteroinvasive Escherichia coli." Infect. Immun. 50:620-629. High et al., 1992, "IpaB of Shigella flexneri causes entry into epithelial cells and escape from the phagocytic vacuole." EMBO J. 11:1991-1999. Konkel et al., 1993, J. Infect. Dis. 168:948-954. Levenson et al., 1988, "Parental immunization with Shigella ribosomal vaccine elicits local Ig A response and primes for mucosal memory." Arch. Allergy Appl. Immunol. 87:25-31. J. Mekalanos, 1992 J. Bacteriol. 174:1-7. Panigrahi et al., 1992, Infect. Immun. 60:4938-4944. Pavlovskis et al., 1992, "Significance of flagella in colonization resistance of rabbits immunized with Campylobacter spp." Infect. Immun. 59:2259-2264. S.M. Payne, 1989, Mol. MicroBiol. 3:1301-1306. Payne & Finkelstein, 1977, "Imferon agar: Improved medium for isolation of pathogenic Nesseria." J. Clin. Microbiol. 6:293-297. Pope & Payne, 1993, 93rd Am. Soc. Microbiol. Abstract B-147. Robbins et al., 1991, "O-Specific side-chain toxin-protein conjugates as parentaeral vaccines for the prevention of shigellosis and related diseases." Rev. Inf. Dis. 13:S362-365. H.W. Yoder, 1989, "Congo red binding by Escherichia coli isolates from chickens." Avian Dis. 33:502-505. Czinn and Nedrud, 1991, Infection and Immunity 59:2359-2363. Sigma Chemical Co. Catalog, p. 420. Sigma Chemical Co. Catalog, p. 104. Clements et al., "Adjuvant activity of Escherichia coli heat-labile enterotoxin and effect on the induction of oral tolerance in mice to unrelated protein antigens," Vaccine, 6:269-275, Jun. 1988. Czinn et al., "Oral Immunization Protects Germ-Free mice Against Infection From Helicobacter Felis," Gut, vol. 102(4), Part 2 Suppl., Apr. 1992. Czinn et al., "Oral Immunization Against Helicobacter pylori," Infection and Immunity, 59:2359-2363, 1991. Czinn et al., "Protection of germ-free mice after infection by Helicobacter felis after active oral or passive IgA immunization," Vaccine 11:637-642, 1993. Doig et al., "Production of a Conserved Adhesin by the Human Gastroduodenal Pathogen Helicobacter pylori," Journal of Bacteriology, 174:2539-2547, Apr. 1992. Ferrero et al., The Importance of Urease in Acid Protection for the Gastric-colonising Bacteria Helicobacter pylori and Helicobacter felis sp. nov., Microbial Ecology in Health and Disease, 4:121-134, 1991. Heap et al., Immunisation and Gastric Colonisation with Helicobacter Felis, VIth International Workshop on Campylobacter, Hexicobacter and Related Organisms, Sydney, New South Wales, Australia, Oct. 7-10, 1991, Microb Ecol. Health Dis. 4, 1991. Mills et al., "Lipopolysaccharde Antigens of Helicobacter Pylori," VIth International Workshop on Campylobacter Helicobacter and Related Organisms, Sydney, New South Wales, Australia, Oct. 7-10, 1991, Microb Ecol. Health Dis. 4, 1991. Pappo et al., "Effect of Oral Immunization with Recombinant Urease on Murine Helicobacter felis Gastritis," Infection and Immunity, 53:1246-1252, Apr. 1995. Pavlovskis et al., "Adjuvant Effect of Escherichia coli Heat-Labile Enterotoxin on Host Immune Response Following Vaccination with Non-Viable Campylobacter Antigens," VIth International Workshop on Campylobacter Helicobacter and Related Organisms, Sydney, New South Wales, Australia, Oct. 7-10, 1991, Microb Ecol. Health Dis. 4, 1991. Rappuoli et al., "Development of a vaccine against Helicobacter pylori: a short overview," European Journal of Gastroenterology & Hepatology, 5:576-578, 1993. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A method of producing Helicobacter bacteria having enhanced antigenic properties which comprises growing a culture of the Helicobacter bacteria in vitro in a combination of conditions comprising: a) about 0.05% to about 3% bile or about 0.025% to about 0.6% of one or more bile acids or salts thereof; b) at a temperature between about 30.degree. C. and about 42.degree. C.; c) in air or an microaerophillic condition, wherein the microaerophillic condition comprises i) about 5% to about 20% CO.sub.2 with about 80% to about 95% air; or ii) about 5% to about 10% O.sub.2 with about 10% to about 20% CO.sub.2 with about 70% to about 85% N.sub.2 ; and d) a divalent cation chelator selected from the group consisting of 0 to about 100 .mu.M of BAPTA/AM, 0 to about 10 mM of EGTA, and 0 to about 100 .mu.M of EGTA/AM, for a sufficient time so that the culture is in a growth phase at about early log phase, between early log phase and stationary phase, or at about stationary phase. 2. The method according to claim 1, wherein said bile salt or acid comprises glycocholate or glycocholic acid. 3. The method according to claim 1, wherein said Helicobacter is Helicobacter pylori or Helicobacter felis. 4. The method according to claim 3, wherein said Helicobacter is Helicobacter pylori strain 49503, NB3-2 or G1-4. 5. The method according to claim 4, wherein the combination of conditions comprises: a) about 0.05% bile salt which is glycocholate or about 0.1% to about 0.2% bile; b) the temperature is about 37.degree. C.; c) in about 10% to about 20% CO.sub.2 with about 80% to about 90% air, or about 10% CO.sub.2 with about 5% O.sub.2 with about 85% N.sub.2 ; and the culture is grown for a sufficient time so that the culture is in log phase. 6. A method of producing Helicobacter bacteria having enhanced antigenic properties which comprises growing a culture of the Helicobacter bacteria in vitro in a combination of conditions comprising: a) a divalent cation chelator selected from the group consisting of about 1.0 to about 25 .mu.M of BAPTA/AM, about 0.5 about 10 mM of EGTA, and about 1.0 to about 100 .mu.M of EGTA/AM; b) at a temperature between about 30.degree. C. and about 42.degree. C.; and c) in air or an microaerophillic condition, wherein the microaerophillic condition comprises i) about 5% to about 20% CO.sub.2 with about 80% to about 95% air; or ii) about 5% to about 10% O.sub.2 with about 10% to about 20% CO.sub.2 with about 70% to about 85% N.sub.2, for a sufficient time so that the culture is in a growth phase at about early log phase, between early log phase and stationary phase, or at about stationary phase. 7. A Helicobacter bacterium having enhanced antigenic properties, which is harvested from a culture of Helicobacter bacteria grown in vitro under a combination of conditions comprising: a) about 0.05% to about 3% bile or about 0.025% to about 0.6% of one or more bile acids or salts thereof; b) at a temperature between about 30.degree. C. and about 42.degree. C.; c) in air or an microaerophillic condition, wherein the microaerophillic condition comprises i) about 5% to about 20% CO.sub.2 with about 80% to about 95% air; or ii) about 5% to about 10% O.sub.2 with about 10% to about 20% CO.sub.2 with about 70% to about 85% N.sub.2 ; and d) a divalent cation chelator selected from the group consisting of 0 to about 25 .mu.M of BAPTA/AM, 0 to about 10 mM of EGTA, and 0 to about 100 .mu.M of EGTA/AM, wherein said Helicobacter culture is at about early log phase, between early log phase and stationary phase, or at about stationary phase. 8. The Helicobacter bacterium according to claim 7, wherein said bile salt or acid comprises glycocholate or glycocholic acid. 9. The Helicobacter bacterium according to claim 7, wherein said Helicobacter is Helicobacter pylori or Helicobacter felis. 10. The Helicobacter bacterium according to claim 9, wherein said Helicobacter is Helicobacter pylori strain 49503, NB3-2 or G1-4. 11. The Helicobacter bacterium according to claim 7, wherein the combination of conditions comprises: a) about 0.05% bile salt which is glycocholate or about 0.1 to about 0.2% bile; b) the temperature is about 37.degree. C.; c) in about 10% to about 20% CO.sub.2 with about 80% to about 90% air, or about 10% CO.sub.2 with about 5% O.sub.2 with about 85% N.sub.2 ; and the culture is at about log phase. 12. The Helicobacter bacterium according to claim 11, wherein said Helicobacter is Helicobacter pylori or Helicobacter felis. 13. A Helicobacter bacterium having enhanced antigenic properties, which is harvested from a culture of Helicobacter bacteria grown in vitro under a combination of conditions comprising: a) a divalent cation chelator, selected from the group consisting of about 1.0 to about 25 .mu.M of BAPTA/AM, about 0.5 to about 10 mM of EGTA, and about 1.0 to about 100 .mu.M of EGTA/AM; b) at a temperature between about 30.degree. and about 42.degree. C.; and c) in air or an microaerophillic condition, wherein the microaerophillic condition comprises i) about 5% to about 20% CO.sub.2 with about 80% to about 95% air; or ii) about 5% to about 10% O.sub.2 with about 10% to about 20% CO.sub.2 with about 70% to about 85% N.sub.2, wherein said Helicobacter culture is at about early log phase, between early log phase and stationary phase, or at about stationary phase.18. A method for producing anti-Helicobacter antibodies in an animal which comprises immunizing the animal with an effective amount of an immunogen comprising the Helicobacter bacterium of claim 7, 9, 11, 12 or 13 wherein the anti-Helicobacter antibodies bind said Helicobacter bacterium or a component thereof. 19. A method of stimulating an immune response in an animal which comprises administering to the animal an effective amount of an immunogen comprising the Helicobacter bacterium of claim 7, 9, 11, 12 or 13 wherein said immune response prevents, attenuates or cures Helicobacter infections or diseases in the animal |
| PATENT DESCRIPTION |
1 FIELD OF THE INVENTION This invention relates generally to in vitro methods for inducing or enhancing expression of enteric bacterial antigens and/or virulence factors thereby producing antigenically enhanced enteric bacteria, to methods for using antigenically enhanced enteric bacteria and to vaccines comprising antigenically enhanced enteric bacteria. 2 BACKGROUND OF THE INVENTION It is widely recognized that bacteria cultured in vitro using conventional media and conditions express characteristics that are different from the characteristics expressed during growth in their natural habitats, which includes in vivo growth of normal microflora or pathogens in an animal host. Therefore, such in vitro grown pathogenic bacteria might not be good for use as vaccine components. However, if it were possible to define conditions that trigger or enhance expression of virulence factors, relevant physiology, or antigens including outer-surface antigens then important products and therapeutics (e.g., new antigens for vaccines, new targets for antibiotics, and novel bacterial characteristics for diagnostic applications) could be rapidly identified. Several environmental factors have been identified which influence expression of virulence determinants in bacteria (Mekalanos, J. J., J. Bacteriol. 174:1-7, 1992). For instance, there is a long history of research on the relationship between iron and virulence of bacteria, in particular Shigella (Payne, Mol. MicroBiol., 3:1301-1306, 1989), Neisseria (Payne and Finkelstein, J. Clin. Microbiol., 6:293-297, 1977) and Pasteurella (Gilmour, et al., Vaccine, 9:137-140, 1991). Other environmental signals that have been shown to control the expression of coordinately regulated virulence determinants of a wide variety of bacteria in plants and animals include phenolic compounds, monosaccharide, amino acids, temperature, osmolarity, and other ions (Mekalanos, J. Bacteriol., 174:1-7, 1992). Bacterial pathogens that enter an animal host through the intestine (i.e., oral route) encounter numerous host environment components and conditions that may affect bacterial physiology and expression of virulence factors. These components and conditions include bile, bile acids or salts, stomach pH, microaerophillic conditions (the intestine has high CO.sub.2, and low O.sub.2), osmolarity and many others yet undefined. Invasive enteric pathogens require de novo protein synthesis to accomplish internalization (Headley and Payne, Proc. Natl. Acad. Sci., USA, 87:4179-4183, 1990). Therefore, bacteria may optimally produce these invasive factors only in response to certain environmental signals not ordinarily present in vitro. This hypothesis is supported by the recent report that antisera raised against conventionally grown C. jejuni had only a marginal effect on blocking in vitro internalization (Konkel, et al., J. Infect. Dis., 168:948-954, 1993). However, immunization of rabbits with extracts of Campylobacter grown in the presence of epithelial cell monolayers, a condition enhancing invasiveness, resulted in production of an antiserum that markedly inhibited the internalization of the bacteria. Researchers have been studying growth of bacteria in the intestinal environment to identify relevant virulence factors. For example, Campylobacter strain 81-176 grown in rabbit ileal loops expresses proteins not expressed under conventional laboratory in vitro culture conditions (Panigrahi, et al., Infect. Immun., 60:4938-4944, 1992). New or enhanced synthesis of proteins has been seen in Campylobacter cultivated with INT 407 cell monolayers as compared to bacteria cultured in the absence of the epithelial cells (Konkel, et al., J. Infect. Dis., 168:948-954, 1993). Furthermore, these changes were temporally associated with increased invasiveness of C. jejuni. Other changes such as cellular morphology, loss of flagella, expression of a new outer membrane protein and alteration in cell-surface carbohydrates were induced or enhanced in an avirulent strain of C. jejuni when passed intravenously and chorio-allantoically through chick embryos (Field, et al., J. Med. Microbiol., 38:293-300, 1993). Other intestinal components, such as bile acids or salts, are known to be inhibitory for some bacteria, but the bile acids may play another role by affecting virulence expression by the bacterium. Pope and Payne (93rd Am. Soc. Microbiol., B-147, 1993) reported that Shigella flexneri cultured in broth containing sodium chenodeoxycholate demonstrated 3 to 5-fold enhanced infectivity of HeLa cell monolayers. They reported, however, that other bile salts and detergents including cholate, glycocholate, taurodeoxycholate, the CHAPS series, digitonin and Triton X100 and sodium salts thereof, had no effect on the invasiveness of S. flexneri. Moreover, their broth containing chenodeoxycholate also had no effect on the invasiveness of E. coli or other avirulent strains of Shigella. Synthesis of new proteins by S. flexneri is also induced by altering pH, temperature and ionic composition of the growth medium (Mekalanos, J. Bacteriol., 174:1-7, 1992). PCT application publication number WO 93/22423, published Nov. 11, 1993, discloses methods for growing bacteria on lipids, such as phosphatidylserine, or mucus and for the isolation of proteins whose expression is enhanced by growth in the presence of phosphatidylserine. This reference neither discloses nor suggests methods of the present invention for producing enteric bacteria having enhanced virulence or antigenic properties. Vaccines against many enteric pathogens, such as Campylobacter and Shigella, are not yet available but the epidemiology of these disease agents makes such vaccines an important goal. Shigellosis is endemic throughout the world and in developing countries it accounts for about 10 percent of the 5 million childhood deaths annually due to diarrhea. Campylobacter, although only recently identified as an enteric pathogen is now recognized as one of the major causes of diarrheal disease in both the developed and underdeveloped countries. An estimated 400 to 500 million Campylobacter diarrheas occur yearly, and over 2 million cases occur in the United States. Shigellosis is a consequence of bacterial invasion of the colonic mucosa. The invasion is associated with the presence of a plasmid found in all invasive isolates (Sansonetti et al., Infect. Immun., 35:852-860, 1982). A fragment of this plasmid contains the invasion plasmid antigen (Ipa) genes, Ipa A, -B, -C, and -D. Ipa B, -C, and -D proteins are essential for the entry process (Baudry et al., J. Gen. Microbiol., 133:3403-3413, 1987). Ipa proteins are logical vaccine candidates although their protective efficacy has not been clearly established. Ipa B and Ipa C are immunodominant proteins (Hale, et al., Infect. Immun., 50:620-629, 1985). Furthermore, the 62 kDa Ipa B protein (the invasin that initiates cell entry and functions in the lysis of the membrane-bound phagocytic vacuole) (High, et al., EMBO J., 11:1991-1999, 1992) is highly conserved among Shigella species. The prolonged illness observed in malnourished children who have no significant mucosal antibody to Shigella Ipa suggests that the presence of mucosal antibody to Ipa may limit the spread and severity of infection. Though a number of vaccine candidates for Shigella have been tested in animals and humans, a successful one has not been found. In spite of the potential significance of Ipa proteins in virulence, most vaccine candidates developed against shigellosis are based on the lipopolysaccharide antigen, which carries the serotype-specific determinants. A parenterally administered polysaccharide-protein conjugate vaccine has also been developed, but is yet to show significant protection in animals (Robbins et al., Rev. Inf. Dis., 13:S362-365, 1991). A similarly administered ribosomal vaccine does induce mucosal immunity, but its protective efficacy remains to be demonstrated (Levenson et al., Arch. Allergy Appl. Immunol., 87:25-31, 1988). The pathogenesis of Campylobacter infections is not as well understood as that of Shigella infections. Cell invasion studies in vitro (Konkel, et al., J. Infect. Dis., 168:948-954, 1993) and histopathologic examinations (Russell, et al., J. Infect. Dis., 168:210-215, 1993) suggest that colonic invasion is also important. This conclusion is consistent with the observation that diarrhea caused by Campylobacter may be severe and associated with blood in the stool. These activities may be associated with the immunodominant 62 kDa flagellin protein. A recent report indicates that the presence of flagella is essential for Campylobacter to cross polarized epithelial cell monolayers (Grant et al., Infect. Immun., 61:1764-1771, 1993). No specific Campylobacter antigens have been established as protective. However, the low molecular weight (28-31 kDa) proteins, or PEB proteins, and the immunodominant flagellar protein are thought to hold promise in this regard (Pavlovskis et al., Infect. Immun., 59:2259-2264, 1992; Blaser and Gotschilch, J. Bio. Chem., 265:14529-14535, 1990). The importance of the flagellar protein is indicated by its association with colonization of the intestine and with the cross-strain protection against infection within Lior subgroups (Pavlovskis et al., Infect. Immun., 59:2259-2264, 1992). However, a flagella protein based Campylobacter vaccine may have to include the flagella protein antigen from the 8-10 most clinically relevant Lior serogroups. Therefore, objects of the present invention include 1) in vitro culture conditions for culturing or treating enteric bacteria which optimally induce or enhance invasive activities and/or certain cellular characteristics including cell surface characteristics; 2) correlated altered invasiveness or cellular characteristics including surface characteristics with changes in antigenic profiles; 3) increased virulence of these organisms in small animal models; and 4) antisera against organisms with enhanced invasiveness or altered characteristics including surface characteristics that are more effective in neutralizing live organisms used for in vitro or in vivo challenges than antisera prepared against conventionally grown bacteria. This invention addresses these needs and others. None of the references discussed above teach or suggest the in vitro methods of the present invention nor the vaccines of the present invention comprising antigenically enhanced enteric bacteria. Citation or identification of any reference in this section or any other section of this application shall not be construed as indicative that such reference is available as prior art to the invention. 3 SUMMARY OF THE INVENTION This invention provides defined culture conditions and components incorporated into growth media of enteric bacteria to induce or enhance the presence of virulence factors and other antigens. Preferably, such antigens are immunogenic. More preferably, such immunogenic antigens correlate with indices of virulence. Enteric bacteria are grown in the presence of conditions and components simulating certain in vivo conditions to which the organisms are exposed in nature. Methods of the present invention produce antigenically enhanced enteric bacteria with phenotypic changes such as increased total protein per cell, new or increased individual proteins, altered or increased surface carbohydrates, altered surface lipopolysaccharides, increased adhesive ability, increased invasive ability and/or increased intracellular swarming. Moreover, methods of the present invention are adaptable to practical scale-up fermentations for commercial uses. Said antigenically enhanced enteric bacteria can be used to produce protective vaccines, such as inactivated whole cell or subunit vaccines, or for diagnostic purposes such as for the production of antibodies and detection of pathogenic enteric bacteria or to produce antibiotics. Further, the antibodies induced by the enhanced enteric bacteria of the present invention may be used as passive vaccines. Therefore, an object of the present invention is a method for producing enteric bacteria selected from the group consisting of Campylobacter sp., Yersinia sp., Helicobacter sp., Gastrospirillum sp., Bacteroides sp., Klebsiella sp., Enterobacter sp., Salmonella sp., Shigella sp., Aeromonas sp., Vibrio sp., Clostridium sp., Enterococcus sp. and Escherichia coli, having enhanced antigenic properties comprising: growing enteric bacteria in vitro with a combination of conditions including: a) 0.05% to 3% bile or 0.025% to 0.6% of one or more bile acids or salts thereof, at a temperature between 30.degree. C. and 42.degree. C., until a growth phase at about early log phase, between early log and stationary phases, or at about stationary phase, in air or microaerophillic conditions, such as 5% to 20% CO.sub.2 with 80% to 95% air, 5% to 20% CO.sub.2 with 80% to 95% N.sub.2 ; or 5% to 10% O.sub.2, 10% to 20% CO.sub.2, with 70% to 85% N.sub.2 ; and optionally in the presence of a divalent cation chelator, such as, but not limited to 0 to 100 .mu.M, preferably 25 .mu.M, of BAPTA/AM, 0 to 10 mM of EGTA, and 0 to 100 .mu.M of EGTA/AM; or b) as in a) except in the presence of a divalent cation chelator, such as 1.0 to 100 .mu.M, preferably 25 .mu.M, of BAPTA/AM, 0.5 to 10 mM of EGTA, or 1 to 100 .mu.M of EGTA/AM, and without any bile, bile acids or bile salts. According to the present invention, concentrations of any individual bile acid or salt thereof include 0.025% to 0.6%, preferably 0.05% to 0.5%, more preferably 0.05% to 0.2%, most preferred is 0.05% or 0.1%. Preferred are methods wherein said bile acid or salt thereof comprises deoxycholate or glycocholate. A further object of the invention is enteric bacteria selected from the group consisting of: Campylobacter sp., Yersinia sp., Helicobacter sp., Gastrospirillum sp., Bacteroides sp., Klebsiella sp., Enterobacter sp., Salmonella sp., Shigella sp., Aeromonas sp., Vibrio sp., Clostridium sp., Enterococcus sp. and Escherichia coli, wherein said enteric bacteria are grown in vitro under a combination of conditions to promote enhanced antigenic properties, said conditions comprising: a) 0.05% to 3% bile or 0.025% to 0.6% of one or more bile acids or salts thereof, at a temperature between 30.degree. C. and 42.degree. C., until a growth phase at about early log phase, between early log and stationary phases, or at about stationary phase; in air or under microaerophillic conditions, such as 5% to 20% CO.sub.2 with 80% to 95% air, 5% to 20% CO.sub.2 with 80% to 95% N.sub.2, or 5% to 10% O.sub.2 with 10% to 20% CO.sub.2, with 70% to 85% N.sub.2 ; and optionally a divalent cation chelator, such as, but not limited to 0 to 100 .mu.M, preferably 25 .mu.M, of BAPTA/AM, 0 to 10 mM of EGTA, and 0 to 100 .mu.M of EGTA/AM, or b) as in a) except in the presence of a divalent cation chelator, such as 1.0 to 100 .mu.M, preferably 25 .mu.M, of BAPTA/AM, 0.5 to 10 mM of EGTA, 1.0 to 100 .mu.M of EGTA/AM, and without any bile, bile acids or bile salts. Another object of the invention is a vaccine comprising a whole enteric bacteria or components thereof, selected from the group consisting of: Campylobacter sp., Yersinia sp., Helicobacter sp., Gastrospirillum sp., Bacteroides sp., Klebsiella sp., Enterobacter sp., Salmonella sp., Shigella sp., Aeromonas sp., Vibrio sp., Clostridium sp., Enterococcus sp. and Escherichia coli, or an immunogenic fragment or derivative thereof, having enhanced antigenic properties; and optionally a pharmaceutically acceptable carrier or diluent. Preferred is the vaccine comprising whole, inactivated antigenically enhanced enteric bacteria. A further object of the invention is a vaccine further comprising an adjuvant. A further object of the present invention is directed to antibodies (including but not limited to antisera, purified IgG or IgA antibodies, Fab fragment, etc.) which are capable of specifically binding to at least one antigenic determinant of an enteric bacteria of the present invention. Such polyclonal and monoclonal antibodies are useful as immunoassay reagents for detecting enteric bacteria in an animal or biological sample therefrom. The polyclonal and monoclonal antibodies of the present invention are also useful as passive vaccines for use in protecting against enteric bacteria infections and diseases. A further object of the invention is an in vitro method for assaying potential antimicrobial agents comprising the steps of contacting enteric bacteria having enhanced antigenic properties selected from the group consisting of: Campylobacter sp., Yersinia sp., Helicobacter sp., Gastrospirillum sp., Bacteroides sp., Klebsiella sp., Enterobacter sp., Salmonella sp., Shigella sp., Aeromonas sp., Vibrio sp., Clostridium sp., Enterococcus sp. and Escherichia coli, with said potential agents and assaying the bacteriocidal or bacteriostatic effects. Still a further object of the invention is an in vitro method for detecting a host's production of antibodies or for the detection of enteric bacteria in an animal or biological sample therefrom, comprising the steps of contacting a biological sample from a host with enteric bacteria of the present invention having enhanced antigenic properties selected from the group consisting of: Campylobacter sp., Yersinia sp., Helicobacter sp., Gastrospirillum sp., Bacteroides sp., Klebsiella sp., Enterobacter sp., Salmonella sp., Shigella sp., Aeromonas sp., Vibrio sp., Clostridium sp., Enterococcus sp. and Escherichia coli, antigens thereof or antibodies thereto and screening for antibody:antigen interactions. Another object of the present invention relates to a diagnostic kit for detecting a host's production of antibodies to enteric bacteria or for detecting enteric bacteria, comprising enteric bacteria having enhanced antigenic properties selected from the group consisting of: Campylobacter sp., Yersinia sp., Helicobacter sp., Gastrospirillum sp., Bacteroides sp., Klebsiella sp., Enterobacter sp., Salmonella sp., Shigella sp., Aeromonas sp., Vibrio sp., Clostridium sp., Enterococcus sp. and Escherichia coli, or antibodies thereto and all other essential kit components. Preferred enteric bacteria that the various aspects of the present invention relate to are Campylobacter jejuni, Campylobacter coli, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Escherichia coli, Shigella flexneri, Shigella sonnei, Shigella dysenteriae, Shigella boydii, Helicobacter pylori, Helicobacter felis, Gastrospirillum hominus, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Bacteroides fragilis, Clostridium difficile, Salmonella typhimurium, Salmonella typhi, Salmonella gallinarum, Salmonella pullorum, Salmonella choleraesuis, Salmonella enteritidis, Klebsiella pneumoniae, Enterobacter cloacae, and Enterococcus faecalis. Preferred Escherichia coli include but are not limited to entero-toxic, entero-hemorrhagic, entero-invasive, entero-pathogenic or other strains. The present invention is based, in part, on the surprising discovery that antigenically enhanced enteric bacteria of the invention induce immune responses that are cross-protective against a broader range of strains or serotypes of the same bacterial species than that induced by the same enteric bacteria but grown using conventional culturing conditions. In at least one instance, the immune response induced by the antigenically enhanced enteric bacteria of the invention is cross-protective against a different species of enteric bacteria. |
| PATENT EXAMPLES | Available on request |
| PATENT PHOTOCOPY | Available on request |
|
Want more information ? Interested in the hidden information ? Click here and do your request. |