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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/105—Delta proteobacteriales, e.g. Lawsonia; Epsilon proteobacteriales, e.g. campylobacter, helicobacter
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/025—Enterobacteriales, e.g. Enterobacter
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/107—Vibrio
• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
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  • C12N1/20—Bacteria; Culture media therefor
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
  • C12Q1/045—Culture media therefor
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/18—Testing for antimicrobial activity of a material
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
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Definitions

• 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.
• 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.
• important products and therapeutics e.g., new antigens for vaccines, new targets for antibiotics, and novel bacterial characteristics for diagnostic applications
• Bacterial pathogens that enter an animal host through the intestine 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 C0 2 , and low 0 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 .
• 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. Pis... 168:948-954, 1993).
• these changes were temporally associated with increased invasiveness of C. jejuni .
• PCT application publication number WO 93/22423 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.
• Campylobacter and Shigella 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 -P 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).
• 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.
• Campylobacter infections are not as well understood as that of Shigella infections.
• Cell invasion studies in vitro Konkel, et al., J. Infect. Pis.. 168:948-
• flagellar protein 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, 0 1992) .
• a flagella protein based Campylobacter vaccine may have to include the flagella protein antigen from the 8-10 most clinically relevant Lior serogroups.
• objects of the present invention include 1) in vitro culture conditions for culturing or treating enteric 5 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.
• 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.
• 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.
• 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.
• an object of the present invention is a method for producing enteric bacteria selected from the group consisting of Campylobacter sp. , Yersinia ⁇ p. , Helicobacter ⁇ p. , Gastrospirillum sp. , Bacteroides sp. , Klebsiella sp. , Enterobacter ⁇ p. , Salmonella sp. , Shigella sp. , Aeromonas sp. , Vibrio sp. , Clostridium sp. , Enterococcus sp.
• 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°C and 42°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% C0 2 with 80% to 95% air, 5% to 20% C0 2 with 80% to 95% N 2 ; or 5% to 10% 0 2 , 10% to 20% C0 2 , with 70% to 85% N 2 ; and optionally in the presence of a divalent cation chelator, such as, but not limited to 0 to 100 ⁇ M, preferably 25 ⁇ M, of BAPTA/AM, 0 to 10 mM of EGTA, and 0 to 100 ⁇ M of EGTA/AM; or b) as in a combination of conditions including: a) 0.05% to 3% bile or 0.025% to 0.6% of
• BAPTA/AM 0.5 to 10 mM of EGTA, or 1 to 100 ⁇ M of EGTA/AM, and without any bile, bile acids or bile salts.
• 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%.
• 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 ⁇ p. , Clostridium ⁇ p. , Enterococcu ⁇ ⁇ p.
• 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°C and 42°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% C0 2 with 80% to 95% air, 5% to 20% C0 2 with 80% to 95% N 2 , or 5% to 10% 0 2 with 10% to 20% C0 2 , with 70% to 85% N 2 ; and optionally a divalent cation chelator, such as, but not limited to 0 to 100 ⁇ M, preferably 25 ⁇ M, of BAPTA/AM, 0 to 10 mM of EGTA, and 0 to 100 ⁇ M of EGTA/AM, or b) as
• Another object of the invention is a vaccine comprising a whole enteric bacteria or components thereof, selected from the group consisting of: Campylobacter ⁇ p. , Yer ⁇ inia ⁇ p. , Helicobacter ⁇ p. , Ga ⁇ tro ⁇ pirillu ⁇ p. , Bacteroide ⁇ ⁇ p. , Klebsiella ⁇ p. , Enterobacter ⁇ p. , Salmonella ⁇ p. , Shigella sp. , Aeromonas ⁇ p. , Vibrio ⁇ p. , Clo ⁇ tridium sp. , Enterococcu ⁇ sp .
• 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. ,
• 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 ⁇ p.
• 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 ⁇ p. , Shigella sp. , Aeromonas sp. , Vibrio sp. , Clostridium sp. , Enterococcu ⁇ ⁇ p. and Escherichia coli, or antibodies thereto and all other essential kit components.
• enteric bacteria having enhanced antigenic properties selected from the group consisting of: Campylobacter sp. , Yersinia sp. , Helicobacter sp. , Gast
• enteric bacteria that the various aspects of the present invention relate to are Campylobacter jejuni , Campylobacter coli, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculo ⁇ i ⁇ , E ⁇ cherichia coli, Shigella flexneri, Shigella ⁇ onnei, Shigella dy ⁇ enteriae, Shigella boydii, Helicobacter pylori, Helicobacter feli ⁇ , Gastrospirillum ho inu ⁇ , Vibrio cholerae, Vibrio parahaemolyticus , Vibrio vulnificus, Bacteroide ⁇ fragili ⁇ , Clo ⁇ tridium difficile, Salmonella typhimurium, Salmonella typhi, Salmonella gallinarum, Salmonella pullorum, Salmonella choleraesuis, Salmonella enteritidis, Klebsiella pneumoniae, Enterobacter cloacae , and Enterococcu ⁇ f
• 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.
• the immune response induced by the antigenically enhanced enteric bacteria of the invention is cross-protective against a different species of enteric bacteria.
• Figures 1A, IB and 1C graphically depict the results of high-performance liquid chromatography of monosaccharides from surface extract hydrolysates of C. jejuni 81-176.
• Figure 1A Standards: Fucose "Fuc”, N-acetyl-galactosamine “GalNac”, N- acetyl-glucosamine “GlcNac”, galactose "Gal”, glucose “Glc", Mannose "Man”.
• Figure IB surface extract of conventionally grown bacteria "BHI”.
• Figure 1C surface extracts of bacteria grown according to methods of the present invention "DOC”.
• Figure 2 pictorially depicts the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showing a comparison of proteins of whole cell (columns 1, 2 and 3) or surface extracts (col 5 and 6) of C. jejuni 81-176 conventionally grown "BHI” or grown according to methods of the present invention (0.8% Oxgall bile acids "OX” or 0.1% deoxycholate "POC”) .
• SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
• Figure 3 pictorially depicts the results of western blot analysis showing a comparison of proteins bound by ferret IgA- containing mucus produced by infection with whole cell C. jejuni 81-176.
• Whole cell C. jejuni 81-176 conventionally grown, "1"; or whole cell C. jejuni 81-176 grown according to methods of the present invention: 0.8% Oxgall bile acids, "2", or 0.1% deoxycholate, "3"; or surface extracts of C. jejuni 81-176 conventionally grown, "4"; or surface extracts of C. jejuni 81-176 grown according to methods of the present invention, 0.1% deoxycholate, "5".
• Figure 4 pictorially depicts the results of western blot analysis showing a comparison of proteins bound by flagellin- specific monoclonal antibody 72c from whole cell C. jejuni 81- 176 which were grown according to methods of the present invention, "3"; conventionally grown, “2”; or grown in a fermentor according to methods of the present invention, "1".
• Figure 5 pictorially depicts the results of SPS-PAGE showing a comparison of lipopolysaccharides (LPS) of whole cell S. flexneri 2457T conventionally grown, column “1", or grown according to methods of the present invention, 0.1% deoxycholate, column "2".
• LPS lipopolysaccharides
• FIG. 6 graphically depicts the enhancement of immuno- cross reactivity of C. jejuni grown according to methods of the present invention.
• C. jejuni 81-176 cells grown conventionally or according to methods of the invention as exemplified in Example 5 (DOC) were used to induce antibodies.
• the agglutination activity of the two types of antibodies i.e., anti-C. jejuni 81-176 cultured in BHI and anti-C. jejuni 81-176 cultured in DOC
• Figures 7A, 7B and 7C graphically depict the efficacy of a vaccine comprising inactivated C.
• jejuni 81-176 whole cells in protecting mice against a nasally delivered challenge of live C. jejuni 81-176 cells.
• Mice were vaccinated with phosphate buffered saline (PBS; solid line) , PBS plus LT adjuvant (Adjuvant; dash line) , formalin-inactivated C. jejuni 81-176 whole cells that were grown and harvested according to Example 5 without adjuvant (CWC; open circle/solid line) or with LT adjuvant (CWC + Adjuvant; solid circle/solid line) .
• the vaccine efficacies was examined using the intestinal colonization assay.
• Figure 7A shows the results of the protection afforded by the vaccinations using three oral doses of 10 ⁇ inactivated bacterial particles per dose.
• Figure 7B shows the results of the protection afforded by the vaccinations using three oral doses of 10 7 inactivated bacterial particles per dose.
• Figure 7C shows the results of the protection afforded by the vaccinations using three oral doses of 10 ⁇ inactivated bacterial particles per dose. See Example 34 in Section 11 for details.
• FIGS 8A, 8B and ⁇ c graphically depict the efficacy of vaccine comprising inactivated C. jejuni 81-176 whole cells in protecting mice against an orally delivered challenge of live
• Figure 8A shows the results of the protection afforded by the vaccinations using three oral doses of 10 ⁇ inactivated bacterial particles per dose.
• Figure 8B shows the results of the protection afforded by the vaccinations using three oral doses of 10 7 i•nactivated bacterial particles per dose.
• Figure 8C shows the results of the protection afforded by the vaccinations using three oral doses of l ⁇ " inactivated bacterial particles per dose. See Example 34 in Section 9 for experimental details.
• Figure 9 graphically depicts the effect of the growth phase of the Shigella flexneri culture on the invasiveness of the Shigella flexneri 2457T cells.
• Shigella flexneri 2457T cells were grown conventionally (BHI) , or according to the methods of the present invention as exemplified by Example 9 (DOC-EL) — that is harvesting the cells when the culture is in early log phase — or according to Example 9 but allowing the culture to reach late log phase before harvesting the cells (DOC-LL) .
• the invasiveness of these different preparations of cells are shown. See Example 35 in Section 12 for details.
• Figure 10 graphically depicts the enhancement of invasiveness of Shigella cells when they are cultured using the methods of the present invention. S. sonnei and S.
• dysenteriae 3818 were cultured conventionally (BHI) or according to the methods of the present invention as exemplified in Example 9. The invasiveness of these different preparations of Shigella cells against INT-407 cells are shown. See Example 35 in Section 12 for details.
• Figure 11 graphically depicts the enhancement of immuno- cross reactivity of Shigella grown according to methods of the present invention.
• S. flexneri 2457T grown according to methods of the present invention as exemplified in Example 9 was used to induce antibodies.
• the agglutination activity of the induced antibodies against S . flexneri , S. sonnei , S . dysenteriae and S . boydii grown conventionally (BHI) or according to the methods of the present invention as exemplified in Example 9 serotype ⁇ are shown. See Example 36 in Section 12 for details.
• Figure 12 graphically depicts the effect of bile concentration and the growth phase of the Helicobacter pylori culture on the adhesiveness of Helicobacter pylori NB3-2 cells.
• H. pylori NB3-2 cells were grown in culture medium containing 0%, 0.025%, 0.05% or 0.1% bile and harvested at 8, 10, 12 and 18 h after inoculation. The invasiveness of these different preparations of H. pylori NB3-2 cells against INT- 407 cells are shown. See Example 38 in Section 14 for details.
• Figure 13 graphically depicts the effect of bile concentration and the growth phase of the Helicobacter pylori culture on the adhesiveness of Helicobacter pylori Gl-4 cells. H.
• pylori Gl-4 cells were grown in culture medium containing 0%, 0.1% or 0.2% bile and harvested at 6, 8, 10, 12, 14 and 16 h after inoculation. The invasiveness of these different preparations of H. pylori Gl-4 cells against INT-407 cells are shown. See Example 38 in Section 15 for details.
• the methods of the present invention relate to growing enteric bacteria in vitro in the presence of a combination of certain conditions with certain components selected to induce or enhance the expression of antigens and/or virulence factors.
• enteric refers to bacteria normally found in or associated with any part of an animal's gastrointestinal tract and any bacteria that causes an infection in any part of an animal's gastrointestinal tract.
• enteric bacteria include both gram positive and gram negative bacteria.
• components and conditions relate to many factors associated with an enteric bacterium's natural in vivo environment and other factors. Such components and conditions include, but are not limited to, bile, bile acids or salts thereof or their biological precursors such as cholesterol, pH, microaerophillic condition, osmolarity, and harvesting or collecting the bacteria at a desired bacterial growth phase.
• antigens and its related term “antigenic” as used herein and in the claims includes antigens or antigenic characteristics including, but not limited to, macromolecules contributing to cellular morphology or cell motility; proteins; more particularly surface proteins, lipopolysaccharides and carbohydrates. Preferably said antigens are immunogenic.
• immunogenic as used herein and in the claims refers to the ability to induce antibody production in an animal after said animal is exposed to a composition comprising whole bacteria produced by the present invention or a fragment of said whole bacterium.
• antigenic state of enteric bacteria grown according to the methods of the present invention. Such bacteria have higher levels of certain immunogenic antigens and/or new immunogenic antigens as compared to the same bacteria grown using conventional methods.
• microaerophillic conditions refers to anaerobic conditions or elevated C0 2 levels, such as 5% to 20% C0 2 with 80% to 95% air; 5% to 20% C0 2 with 80% to 95% N 2 ; or 5% to 10% 0 2 with 10% to 20% C0 2 with 70% to 85% N 2 .
• viralence refers to those factors of an enteric bacteria associated with the ability to adhere to and/or to invade and/or to survive in a host and/or cause a pathological condition.
• immuno-cross protective refers to the ability of the immune response induced by one bacterial strain or serotype, whole cell or otherwise, to prevent or attenuate infection of the same host by a different bacterial strain, serotype, or species of the same genus.
• immuno-cross reactive refers to the ability of the humoral immune response (i.e., antibodies) induced by one bacterial strain or serotype, whole cell or otherwise, to cross react with (i.e., the antibody binding) a different bacterial strain, serotype, or species of the same genus.
• Immuno-cross reactivity is indicative of the bacterial immunogen's potential for immuno- cross protection and vice versa.
• the term "host” as used herein and in the claims refers to either in vivo in an animal or in vitro in animal cell cultures.
• the term "animal” as used herein and in the claims includes but is not limited to all warm-blooded creatures such as mammals and birds (e.g., chicken, turkey, duck etc.)
• the enteric bacteria in a vaccine comprising antigenically enhanced enteric bacteria or an immunogenic fragment or derivative thereof, may be either live bacteria or may be inactivated and may further comprise an adjuvant, such as, but not limited to, alum, oil- water emulsion, heat labile toxin from enterotoxigenic E .
• coli (LT) nontoxigenic forms thereof eg. mLT
• BCG Bacille Calmette-Guerin
• Fruend's adjuvant may also further comprise a suitable pharmaceutical carrier, including but not limited to saline, dextrose or other aqueous solution.
• suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences. Mack Publishing Company, a standard reference text in this field.
• the term “vaccine” also encompasses "passive vaccines,” which comprise antibodies that specifically bind pathogens against whose infections or diseases protection is sought.
• inactivated bacteria refers to enteric bacteria that are incapable of infection and/or colonization and encompasses attenuated as well as killed bacteria. Attenuated bacteria may replicate but cannot cause infection or disease. Inactivation of said bacteria may be accomplished by any methods known by those skilled in the art. For example, the bacteria may be chemically inactivated, such as by formalin fixation, or physically inactivated such as by heat, sonication or irradiation, so that they are rendered incapable of replication and/or infection and/or causing disease.
• An effective amount of the vaccine should be administered, in which "effective amount" is defined as an amount of enteric bacteria or an immunogenic fragment or derivative thereof that is capable of producing an immune response in a subject.
• an effective amount of vaccine produces an elevation of anti ⁇ bacterial antibody titer to at least two times the antibody titer prior to vaccination.
• approximately 10 7 to 10 11 bacteria and preferably 10 8 to lO 1 ⁇ bacteria are administered to a host.
• vaccines comprising inactivated whole bacteria.
• an effective amount as applied to passive vaccines is an amount of antibody that is capable of preventing or attenuating a bacterial disease or infection.
• the amount needed will vary depending upon the type of antibody and the antibody titer, and the species and weight of the subject to be vaccinated, but may be ascertained using standard techniques.
• Vaccines of the present invention may be administered locally and/or systemically by any method known in the art, including, but not limited to, intravenous, subcutaneous, intramuscular, intravaginal, intraperitoneal, intranasal, oral or other mucosal routes.
• Vaccines may be administered in a suitable, nontoxic pharmaceutical carrier, may be comprised in microcapsules, and/or may be comprised in a sustained release implant.
• Vaccines may desirably be administered at several intervals in order to sustain antibody levels.
• Vaccines of the invention may be used in conjunction with other bacteriocidal or bacteriostatic methods.
• Antibodies of the invention may be obtained by any conventional methods known to those skilled in the art, such as but not limited to the methods described in Antibodies A Laboratory Manual (E. Harlow, D. Lane, Cold Spring Harbor Laboratory Press, 1989) .
• an animal a wide range of vertebrate species can be used, the most common being mice, rats, guinea pig, hamsters and rabbits
• the animal serum is assayed for the presence of desired antibody by any convenient method.
• the serum or blood of said animal can be used as the source of polyclonal antibodies.
• animals are treated as described above.
• an acceptable antibody titre is detected, the animal is euthanized and the spleen is aseptically removed for fusion.
• the spleen cells are mixed with a specifically selected immortal myeloma cell line, and the mixture is then exposed to an agent, typically polyethylene glycol or the like, which promotes the fusion of cells. Under these circumstances fusion takes place in a random selection and a fused cell mixture together with unfused cells of each type is the resulting product.
• the myeloma cell lines that are used for fusion are specifically chosen such that, by the use of selection media, such as HAT: hypoxanthine, aminopterin, and thymidine, the only cells to persist in culture from the fusion mixture are those that are hybrids between cells derived from the immunized donor and the myeloma cells.
• selection media such as HAT: hypoxanthine, aminopterin, and thymidine
• the only cells to persist in culture from the fusion mixture are those that are hybrids between cells derived from the immunized donor and the myeloma cells.
• the cells are diluted and cultured in the selective media.
• the culture media is screened for the presence of antibody having desired specificity towards the chosen antigen. Those cultures containing the antibody of choice are cloned by limiting dilution until it can be adduced that the cell culture is single cell in origin.
• the antibodies of the present invention have use as passive vaccines against enteric bacteria
• Methods for the detection of antibodies or said bacteria in a host include im unoassays.
• immunoassays are known in the art and include, but are not limited to radioimmunoassays (RIA) , enzyme-linked immunosorbent assays (ELISA) , fluorescent immunoassays, and fluorescence polarization immunoassays (FPIA) .
• RIA radioimmunoassays
• ELISA enzyme-linked immunosorbent assays
• FPIA fluorescence polarization immunoassays
• Another embodiment includes diagnostic kits comprising all of the essential reagents required to perform a desired immunoassay according to the present invention.
• the diagnostic kit may be presented in a commercially packaged form as a combination of one or more containers holding the necessary reagents.
• kit comprises an enteric bacteria of the present invention, and/or a monoclonal or polyclonal antibody of the present invention in combination with several conventional kit components.
• Conventional kit components will be readily apparent to those skilled in the art and are disclosed in numerous publications, including Antibodies A Laboratory Manual (E. Harlow, P. Lane, Cold Spring Harbor Laboratory Press, 1989) .
• kit components may include such items as, for example, roicrotiter plates, buffers to maintain the pH of the assay mixture (such as, but not limited to Tris, HEPES, etc.), conjugated second antibodies, such as peroxidase conjugated anti-mouse IgG (or any anti-IgG to the animal from which the first antibody was derived) and the like, and other standard reagents.
• roicrotiter plates such as, but not limited to Tris, HEPES, etc.
• conjugated second antibodies such as peroxidase conjugated anti-mouse IgG (or any anti-IgG to the animal from which the first antibody was derived) and the like, and other standard reagents.
• Methods of the present invention include growing enteric bacteria in a suitable basal essential culture medium, such as but not limited to commercially available brain heart infusion broth “BHI”, Luria broth “LB”, sheep blood agar “SBA”, Brucella broth, Meuller-Hinton broth, proteose peptone beef extract broth, etc.
• a suitable basal essential culture medium such as but not limited to commercially available brain heart infusion broth “BHI”, Luria broth “LB”, sheep blood agar “SBA”, Brucella broth, Meuller-Hinton broth, proteose peptone beef extract broth, etc.
• bile acids or salts thereof or biological precursors thereof such as cholesterol
• a temperature between 30 ⁇ C and 42 ⁇ 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% C0 2 with 80% to 95% air; 5% to 20% C0 2 with 80% to 95% N 2 ; or 5% to 10% 0 2 with 10% to 20% C0 2 with 70% to 85% N 2 ; and optionally in the presence of a divalent cation chelator, such as, but not limited to 0 to 100 ⁇ M, preferably 25 ⁇ M, of BAPTA/AM (2 • (ethylenedioxy)dianiline n,n,n',n'- tetraacetic acid/acetoxymethyl ester; Molecular Probes, Eugene, OR) , 0 to
• the methods of the present invention also include growing enteric bacteria as described immediately above except in the presence of a divalent cation chelator, such as, but not limited to 1.0 to 100 ⁇ M, preferrably 25 ⁇ M, of BAPTA/AM, 0.5 to 10 mM of EGTA, or 1.0 to 100 ⁇ M of EGTA/AM; but without any bile, bile acids or bile salts.
• a divalent cation chelator such as, but not limited to 1.0 to 100 ⁇ M, preferrably 25 ⁇ M, of BAPTA/AM, 0.5 to 10 mM of EGTA, or 1.0 to 100 ⁇ M of EGTA/AM; but without any bile, bile acids or bile salts.
• Bile or bile acids or salts thereof useful for the present invention include any natural bile compound secreted by the liver and normally concentrated in the gall bladder as well as synthetic bile acids known by those skilled in the art, such as but not limited to "OXGALL” (Pifco Laboratories, Detroit, Michigan) , bovine bile (Sigma Chemicals, St. Louis, Missouri) or other commercially available preparations, cholic, deoxycholic, taurocholic and glycocholic acids.
• POC deoxycholate
• GC glycocholate
• enteric bacterial cultures selected from the group of Campylobacter sp. , Yersinia sp. , Helicobacter sp. , Gastrospirillum sp. , Bacteroides sp. , Klebsiella sp. , Enterobacter sp. , Salmonella sp. , Aeromonas ⁇ p. , Vibrio ⁇ p. , Shigella ⁇ p. , Clo ⁇ tridium sp. , Enterococcus ⁇ p. , and E ⁇ cherichia coli can be prepared as frozen stocks by methods generally known to those skilled in the art and maintained at -80°C for future use.
• stocks of Campylobacter jejuni can be prepared by growing the organism on trypticase soy agar containing 5% defribinated sheep erythrocytes (SBA) , at 37°C in 5% 0 2 , 10% co 2 , 85% N 2 (microaerophilic condition, "MC") for 20 h.
• SBA defribinated sheep erythrocytes
• Escherichia coli, Salmonella typhimurium, Helicobacter pylori and Shigella flexneri can be prepared by growing the organism in brain heart infusion broth ("BHI") .
• Bacteria can be harvested for freezing by any known method, for instance by swabbing the culture and resuspending in BHI containing 30% glycerol.
• Cultures for analytical experiments or for production fermentations can be prepared by any generally known methods, such as by growing the organism on BHI with 1.5% agar at 37 ⁇ C under MC or atmospheric conditions and then transferring a single colony to broth and culturing according to methods of the present invention described herein.
• Bacteria can be harvested for use by any method generally known to those skilled in the art, such as by centrifugation.
• antigenically enhanced cells of Campylobacter sp. preferably of the species jejuni or coli and most preferably of the jejuni strain 81-176, are grown in a basal essential culture medium, preferably BHI broth, additionally comprising about 0.1% POC or about 0.8% bile at 37°C in a mixture of about 10 to 20% C0 2 with about 80 to 90% air and harvested after the growth of the culture has reached about late log phase to about stationary phase, typically about 20 h after inoculation.
• antigenically enhanced cells of Shigella sp. preferably of the species flexneri or dysentariae and most preferably of the flexneri strain 2457T, are grown in a basal essential culture medium, preferably BHI broth, additionally comprising about 0.1% POC or about 0.8% bile at 37 ⁇ C in air and harvested after the growth of the culture has reached about early log phase, typically about 30 min after inoculation with a culture that is at early to mid log phase.
• antigenically enhanced cells of Helicobacter pylori are grown in a basal essential culture medium, preferably BHI broth, additionally comprising about 0.05% to about 0.2% bile or about 0.05% glycocholate (GC) at 37°C in a mixture of about 5% to 20% C0 2 with about 80% to 95% air, or about 10% C0 2 with about 5% 0 2 with about 85% N 2 and harvested after the growth of the culture has reached about log or about stationary phase.
• the cells are harvested after the culture has reached about log phase.
• Enteric bacteria cultured according to the methods of the present invention have altered morphologies, and/or cell motilities and/or produce certain new proteins, lipopolysaccharides and/or carbohydrates and/or such macromolecules at altered levels compared to cells cultured in basal medium alone.
• Optimum cultural conditions that enhance cell yield and the indices of pathogenicity can be identified. Utilizing these cultural conditions, virulence-associated antigens that are enhanced or induced can be identified.
• Motility and gross morphological changes can be seen by microscopic examination of either untreated or stained bacteria. It is possible that other morphological changes might result from methods of the present invention as could be seen through electron microscopy or fluorescence microscopy.
• the morphology and mucus-like characteristics of the enteric bacteria cultured according to methods of the present invention suggest that capsule and/or surface layer expression might be induced.
• phenol extracts of surface components such as proteins, carbohydrates and lipopolysaccharides, can be prepared.
• the enhanced carbohydrates can be seen by high pressure liquid chromatography (HPLC) .
• Protein profiles of outer membranes prepared from enteric bacteria grown under virulence enhancing growth condictions of the present invention can be characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and compared to those from organisms grown in conventional media. SDS-PAGE is conducted to evaluate changes induced in bacterial cellular and extracted proteins in response to antigen enhancing or altering conditions. These data offer qualitative and quantitative information concerning surface changes associated with increased invasiveness or altered antigenicity.
• the immunogenic potential of induced or altered protein antigens can be identified by Western Blotting. Im unogenicity of induced or altered bacterial proteins identified by SDS-PAGE can be evaluated by the generally accepted techniques of Western Blotting as described below.
• Any source of antibody can be used, such as convalescent immune rabbit or ferret sera (source of antibody from animals infected orally with live organisms grown conventionally or according to methods of the present invention) , intestinal mucus (source of IgA antibody) , polyclonal antisera, or a monoclonal antibody, for instance one that is cross-reactive with C. jejuni flagellin, for assaying bacterial antigens.
• convalescent immune rabbit or ferret sera source of antibody from animals infected orally with live organisms grown conventionally or according to methods of the present invention
• intestinal mucus source of IgA antibody
• polyclonal antisera or a monoclonal antibody, for instance one that is cross-reactive with C. jejuni flagellin, for
• Congo red dye binding Assays for virulence and is described below and in Andrews et al., (Infect. Immun.. 60:3287-3295, 1992); and Yoder (Avian Pis.. 33:502-505, 1989).
• Bacterial binding of Congo red indicates ability to bind hemin, this ability to bind hemin is correlated with virulence.
• Congo red binding also correlates with enhanced bacterial invasion of epithelial cells.
• Methods of the present invention for production of antigenically enhanced enteric bacteria correlate to enhanced virulence in small animal models.
• Several domestic animals can be used as models of Campylobacter disease in humans.
• the most studied from the standpoint of immunization efficacy is the reversible intestinal tie, adult rabbit diarrhea (RITARD Model) of Caldwell, et al., (Infect. Immun.. 42:1176-1182, 1983) .
• This model has also demonstrated the association of Lior serotypes with cross-strain protection. However, this model measures resistance to colonization rather than resistance to disease.
• the ferret model of Bell et al. Infect. Immun..
• the antigenically enhanced enteric bacteria produced by the methods of the present invention are used to prepare prototype killed whole-cell or subunit vaccines. These vaccines when administered co animals can be shown to induce antibodies and thus establish the vaccines' protective potential. Elevation or induction of these antigens in a bacterial cell produces cells that make more efficacious vaccines.
• Vaccine candidate preparations produced by the methods of the present invention can be used with various mucosal immunization strategies to induce an intestinal immune response. Successful immunization protocols can then be used to protect animals challenged with the pathogens with or without enhanced antigenicities. Also vaccines can be formulated and tested as combined vaccines, for example Shigella and Campylobacter cells or components thereof can be combined as a single vaccine.
• Campylobacter jejuni strain 81-176 was streaked on blood agar plates (containing trypticase soy agar, plus 5% defibrinated sheep erythrocytes) and incubated in a microaerophilic GasPak jar (BBL, Cockeysville, MP) for 20 h at 37°C. Lawns of bacteria were removed by swabbing and inoculated into flasks containing 1 liter of BHI medium pre- equilibrated to 10% C0 2 with 0.01% to 0.1% sodium deoxycholate (DOC) .
• BBL microaerophilic GasPak jar
• Campylobacter jejuni strain 81-176 was streaked on blood agar plates (containing trypticase soy agar, plus 5% defibrinated sheep erythrocytes) and incubated in a microaerophilic GasPak jar (BBL, Cockeysville, MO) for 20 h at 37°C. Lawns of bacteria were removed by swabbing and inoculated into flasks containing 1 liter of Brucella broth pre-equilibrated to 10% C0 2 with 0.01% to 0.1% sodium deoxycholate. Cultures were incubated for 20 h with shaking at 37 ⁇ C in 5% 0 2 , 10% C0 2 , 85% N 2 and then harvested as described above.
• Campylobacter jejuni strain 81-176 was streaked on blood agar plates (containing trypticase soy agar, plus 5% defibrinated sheep erythrocytes) and incubated in a microaerophilic GasPak jar (BBL, Cockeysville, MP) for 20 h at 37°C. Lawns of bacteria were removed by swabbing and inoculated into flasks containing 1 liter of Mueller-Hinton broth pre-equilibrated to 10% C0 2 , with 0.01% to 0.1% sodium deoxycholate. Cultures were incubated for 20 h with shaking at 37 ⁇ C in 5% 0 2 , 10% C0 2 , 85% N 2 and then harvested as described above.
• Campylobacter jejuni strain 81-176 was streaked on blood agar plates (containing trypticase soy agar, plus 5% defibrinated sheep erythrocytes) and incubated in a microaerophilic GasPak jar (BBL, Cockeysville, MO) for 20 h at 37°C. Lawns of bacteria were removed by swabbing and inoculated into flasks containing 1 liter of BHI medium pre- equilibrated to 10% C0 2 , with 0.1% sodium deoxycholate. Cultures were incubated for 20 h with slow stirring at 37 ⁇ C in 10% C0 2 , 90% air and then harvested as described above.
• Vibrio cholerae is streaked on BHI agar plates (containing trypticase soy agar, plus 5% defibrinated sheep erythrocytes) and incubated in air for 20 h at 37°c. Lawns of bacteria are removed by swabbing and inoculated into flasks containing 1 liter of BHI medium with 0.1% sodium deoxycholate. Cultures are incubated for 20 h with shaking at 37°C in air, then harvested as described above.
• BHI agar plates containing trypticase soy agar, plus 5% defibrinated sheep erythrocytes
• Lawns of bacteria are removed by swabbing and inoculated into flasks containing 1 liter of BHI medium with 0.1% sodium deoxycholate. Cultures are incubated for 20 h with shaking at 37°C in air, then harvested as described above.
• Example 7 Salmonella cholerasius is streaked on BHI agar plates and incubated in air for 20 h at 37 ⁇ C. Lawns of bacteria are removed by swabbing and inoculated into flasks containing 1 liter of BHI medium with 0.1% sodium deoxycholate. Cultures are incubated for 20 h with shaking at 37 ⁇ C in air, then harvested as described above.
• Example 8. Salmonella typhimurium is streaked on Luria broth agar plates and incubated for 20 h at 37 ⁇ c in air. Lawns of bacteria are inoculated into flasks containing 1 liter of BHI medium with 0.1% sodium deoxycholate.
• Cultures are incubated for 20 h with shaking at 37°C in 10% C0 2 , 90% air.
• One colony is transferred into 1 liter of LB containing 0.1% POC and incubated in a closed top flask at 37°C with slow 5 shaking.
• 60 ml of the culture is diluted into 1 liter of the same fresh prewarmed medium and incubated a further 30 min, and then is harvested as described above.
• Campylobacter jejuni 81-176, 81-116, or HC in BHI with 30% glycerol was rapidly thawed and plated on
• the bacterial suspension was inoculated into 1 liter of BHI broth alone or BHI broth containing 0.1% POC, pre- equilibrated to 10% C0 2 , 90% air, in a 2 liter flask.
• the inoculum was added to preequilibrated medium until the 00 62J is equal to 0.05.
• the inoculated flask was returned to the 10%
• Example 11 Helicobacter pylori was added to BHI broth plus 4% bovine calf serum. After inoculation the flasks were flushed with 5% 0 2 , 10% C0 2 , 85% N 2 and incubated for 22 h at
• Example 12 Salmonella typhimurium (in LB with 30% glycerol) was streaked on a LB agar plate and cultured for 18- 20 h at 37°C in air. One colony was picked and transferred into 1 liter of LB or LB containing 0.1% POC in flasks that are flushed with 10% C0 2 , 5% C0 2 , 85% N 2 , sealed and incubated for 12 h at 37 ⁇ C with shaking. The bacteria were then diluted in the same media to OD ⁇ of 0.17 and incubated under identical conditions until the culture reaches early log phase, typically 30 min after the dilution. Cells were harvested as described above.
• Example 13 Salmonella typhimurium is streaked on a LB agar plate and cultured for 18-20 h at 37°C in air. One colony is picked and transferred into 1 liter of LB or LB containing 0.1% DOC and incubated for 12 h at 37°C in air. The culture is then diluted (1/5) in the same fresh media and incubated a further 4 hours under identical conditions. The cultures are then diluted in the same fresh media to OD ⁇ of 0.17 and incubated under identical conditions until the culture reaches log phase, typically 30 minutes after the dilution. Cells are harvested as described above.
• Klebsiella pneumoniae is streaked on a BHI agar plate and incubated 18-20 h at 37°C in air. One colony is picked and inoculated into 1 liter of BHI or BHI containing 0.1% DOC and shaken for 12 h at 37°C in air. The bacteria are then diluted in the same media to OO ⁇ o of 0.17 and grown for 30 min further and then harvested as described above.
• Example 15 Enterobacter cloacae is streaked on a BHI agar plate and incubated at 37°C in air for 18-20 h. One colony is inoculated into 1 liter of BHI or BHI containing o.l% DOC and shaken for 12 h at 37°C. The bacteria are then diluted in the same media to OP ⁇ o of 0.17 and grown for 30 min further and then harvested as described above.
• Example 16 Escherichia coli strain 0157:H7 was streaked on sheep blood agar plate and incubated at 37°C in air for 18- 20 h. One colony was inoculated into 1 liter BHI or BHI containing 0.1% to 0.2% POC flask and shaken for 12 h at 37°C. The bacteria were then diluted to OO ⁇ o of 0.17 and grown for 30 min further and then harvested as described above.
• Example 17 Enterococcu ⁇ faecali ⁇ is streaked on sheep blood agar plate and incubated at 37°C in air for 18-20 h. One colony is inoculated into 1 liter BHI or BHI containing 0.1% POC flask and shaken for 12 h at 37°C. The bacteria are then diluted to O ⁇ of 0.17 and grown for 30 min further and then harvested as described above.
• Example 18. Clo ⁇ tridium difficile (modified chopped meat medium with 30% glycerol) is streaked on a plate of Beef liver medium for anaerobes containing 1.5% agar and cultured at 37°C under microaerophillic conditions (5% C0 2 and 95% N 2 ) . One colony is transferred to 1 liter of modified chopped meat medium or same medium containing 0.1% POC. The bacteria are cultured under microaerophillic conditions at 37 ⁇ C for 12 h, and harvested as described above.
• Example 19 Bacteroide ⁇ fragili ⁇ (modified chopped meat medium with 30% glycerol) is streaked on a modified chopped meat medium agar plate and cultured at 37°C under microaerophillic conditions (5% C0 2 and 95% N 2 ) . One colony is transferred to 1 liter of modified chopped meat medium or same medium containing 0.1% POC. The bacteria are cultured under microaerophillic conditions at 37°C for 12 h, and harvested as described above.
• Example 20 Yer ⁇ inia pseudotuberculosi ⁇ (Luria broth containing 30% glycerol) is streaked on a Luria broth agar plate and incubated at 30 ⁇ C. One colony is transferred to 1 liter of LB and incubated for 12 h at 30°C. This culture is diluted (1/5) in LB or LB containing 0.1% POC and incubated 4 h at 37 ⁇ C. Subsequently, the cultures are diluted in the same media to 00 ⁇ of 0.17 and incubated a further 30 min and then harvested as described above.
• Example 21 Helicobacter pylori was added to BHI broth plus 4% bovine calf serum. After inoculation the flasks were flushed with 5% 0 2 , 10% C0 2 , 85% N 2 and incubated for 22 h at 37 ⁇ C with shaking. After this incubation, 2.5 ml of the culture was transferred to a flask containing BHI broth with 4% bovine calf serum or the same medium additionally containing about 0.1% to about 0.2% bovine bile. These cultures were again flushed with the microaerophilic gas mixture (5% 0 2 , 10% C0 2 85% N 2 ) , and incubated 20-24 h at 37 ⁇ c The cells were harvested as described above.
• ENHANCED ANTIGENIC BACTERIA Example 22. Microscopic examination of wet mounted bacteria was utilized to observe motility and gross morphology. Surface layers were observed by capsule staining in india ink (nigrosine) . After air drying, the cells were counter-stained with crystal violet. All observations were at 1000 x magnification.
• EHANCED bacteria Morphology of bacteria cultured according to methods of the present invention (hereinafter referred to as "ENHANCED” bacteria) was altered compared to those cultured in basal media alone (conventionally grown) .
• “ENHANCED” c. jejuni aggregated, and formed large clumps of cells, while conventionally grown cells were predominantly solitary. It was apparent from capsule staining (data not shown) that a change in the bacterial surface was effected by culturing using methods of the present invention. This surface alteration actually resulted in the increased binding by "ENHANCED" cells of the nigrosine particles from the stain.
• the "ENHANCED” bacteria remained highly motile.
• Example 23 C. jejuni surface components were analyzed by phenol extraction. Extracts were made from C. jejuni 81- 176 grown conventionally or cultured according to Example 2 above. C. jejuni cells were harvested from culture medium by centrifugation as described above. The cell pellet was extracted for 2 h at room temperature with 1% phenol. Intact cells were separated from extracted materials by centrifugation for 45 min. The supernatant containing extracted bacterial surface components was dialyzed against distilled water overnight. The retentate was centrifuged 105,000 x g for 3 h at 4°C. The extract pellet was redissolved in 10% NaCl and precipitated with two volumes of cold 95% ethanol. The precipitation was repeated, and the sample was lyophilized. Subsequently, the sample was dissolved in water at 1 mg/ml for further analysis.
• Carbohydrate content of the extract was assayed with the generally accepted phenol-sulfuric acid method utilizing glucose as a standard. Uronic acid content of the extract was measured with the method of Dische using the carbazole reagent. Total protein content of the phenol extract was evaluated with the biccichinoic acid assay kit (Pierce Chem. Co. , Rockford, IL) .
• the carbohydrate to protein ratio of the extracts is shown in Table 1 below.
• the extract Upon rehydration, the extract had a high geling capacity in water rendering the solution highly viscous and mucus-like, which was similar in character to the aggregated bacteria.
• the functionality of the extract resembled mucin-like glycoproteins.
• Example 24 Bacterial proteins were analyzed by SDS-PAGE and Western Blotting.
• the gel system of Lugtenberg, et al. (EEfiS Letters 58:254-258, 1975) was used.
• the gel system is a discontinuous gel consisting of a low acrylamide (typically 4%) stacking gel pH 6.8, and a higher percentage acrylamide separation gel pH 8.8. SDS (0.1%) was included in both gels and all buffers used.
• Protein separation was according to molecular size, and 8 or 12% acrylamide separation gels were used. Visualization of separated proteins was by silver staining of fixed gels, and molecular size determinations were made based on the M r values of known proteins used as standards.
• C. jejuni cell proteins were separated by SDS-PAGE and visualized by silver staining.
• Four proteins including a 62 kDa protein were induced or enhanced in cells cultured with DOC ( Figure 2) .
• Example 25 S . flexneri LPS was analyzed by phenol extraction.
• S . flexneri cells grown conventionally or according to the present invention as exemplified in Example 9 above were harvested from culture medium by centrifugation as described above.
• Lipopolysaccharides (LPS) were extracted by the method of Westphal and Jann (In: R. Whistler, ed.. Methods in Carbohydrate Chemistry, vol 5; p. 83, 1965). Briefly, cells cultured in BHI or as exemplified in Example 9 above were harvested by centrifugation and washed once in PBS. The cells were then extracted for 15 min at 68°C with 45% phenol in water. The extract was cooled to 10°C and centrifuged.
• the LPS-containing upper aqueous phase was aspirated off and dialyzed against distilled water.
• the retentate was centrifuged 7 h at 80,000 x g, 4°C once, and three times for 3 h each at 105,000 x g.
• the final pellet was lyophilized. Prior to analysis the LPS was resuspended in water (1 mg/ml) .
• the purified LPS was characterized as to carbohydrate content as described above, and by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SPS-PAGE) as described below and shown in Figure 5.
• Example 26 Immunogenicity of proteins was determined by Western Blots. Proteins from bacteria grown conventionally or according to the methods of the present invention as exemplified in Example 1 or 5 above were separated by SPS- PAGE, then were electrophoretically transferred to nitrocellulose or PVPF membranes and were blocked with a standard blocking agent [3% BSA, 50 mM Tris (pH 8.5), 50 mM NaCl, 0.2% TWEEN 20]. Primary antibody was applied in blocking buffer, the blot was then washed and a secondary antibody reporter cognate was applied. Following washing, the blot was visualized with light or chromophore producing substrates. The reporter moiety used was horse radish peroxidase or alkaline phosphatase.
• Figure 3 depicts proteins from Western Blotting with immune rabbit mucus containing IgA.
• the 62 kPa protein was the immunodominant antigen.
• the antigenicity of the 62 kPa protein was greatly enhanced in cells cultured with POC or bile. This protein was also the predominant antigen in the surface extract of cells cultured with POC.
• Example 27 Congo red dye binding was used to measure virulence. Enteric bacteria grown conventionally (BHI) or according to the methods of the present invention ("ENHANCEP”) on BHI agar plates and containing 0.025% Congo red were resuspended in distilled water and extracted with acetone for 10 min. Cellular debris was pelleted by centrifugation, and the 0P 488 of the dye was measured with a blank solution of 40% acetone, 60% water. The dye absorbance was compared to the cell absorbance at 660 nm and expressed as the ratio of OD 488 /OP 660 . The data are shown in Table 2 below.
• Example 2 shows that for several species of enteric bacteria cultured according to methods of the present invention, Congo red dye binding was enhanced. These results show that in vitro methods of the present invention are useful to induce virulence and other characteristics known to correlate with in vivo pathogenesis for other bacterial species.
• Example 28 Bacterial adhesion to cultured epithelial cells was analyzed. Bacterial adhesion was assayed as described by Galan and Curtiss (Proc. Natl. Acad. Sci. USA. 86:6383-6387, 1989).
• Tissue culture cells (INT-407 or Henle cells (ATCC # CCL6) , and CaCo-2 (ATCC # HTB37) human intestinal cell lines) were cultured in 24-well tissue culture plates (37°C, 5% C0 2 ) to a confluency of 60-80%.
• the medium is dependent on the cell line used, but Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 50 mg/ml each of penicillin G and streptomycin was used for Henle cells, and RPMI 1640 medium with 10% fetal bovine serum and 50 mg/ ⁇ l each of penicillin G and streptomycin was used for the culture of CaCo-2 cells.
• HBSS Hank's balanced salt solution
• the bacteria were prepared as follows. For slow growing enteric bacteria such as Campylobacter and Helicobacter the bacterial culture density was diluted to an OD 62j of 0.1 with fresh, preequilibrated medium and then used in the assay. For Shigella and other fast growing enteric bacteria, the bacterial culture was diluted to 0.17 at OD 62J with fresh, preequilibrated medium and then used in the assay. The bacteria were added to the epithelial cells at a multiplicity of infection of 10 bacteria per cell to avoid saturation. The number of bacteria inoculated into the tissue culture well was calculated by plate counting.
• Percent adhesion is expressed as the number of colony forming units (CFU) recovered from the monolayer divided by the number of CFU inoculated onto the monolayer multiplied by 100.
• epithelial cells were grown and prepared according to the methods described above for the adhesion assay. Bacteria grown conventionally or according to methods of the present invention were added to the epithelial cells at a multiplicity of infection of 10 bacteria per cell to avoid saturation. The number of bacteria inoculated into the tissue culture well was calculated by plate counting. Following 2 h infection under 5% C0 2 for Campylobacter, and 30 min for Shigella , the infecting bacteria were aspirated off, and the monolayer was overlaid with growth medium containing genta icin to kill any extracellular bacteria. Any culturable bacteria remaining at this point have invaded the epithelial cell monolayer. The incubation continued under C0 2 for 3 h in the case of C.
• Invasion is expressed as the percent of cell entering the monolayer, as determined by the number of colony forming units (CFU) recovered from the monolayer after gentamycin treatment divided by the number of CFU inoculated onto the monolayer multiplied by 100.
• CFU colony forming units
• Shigella grown according to the methods of the present invention had greatly enhanced abilities of both adhesion and invasion.
• Example 29 A rapid slide agglutination assay was used to show immuno-cross reactivity.
• C. jejuni strains grown conventionally or according to the methods of the present invention as exemplified in Example 5 were exposed to serum IgG from animals immunized with C. jejuni 81-176 (Lior 5) grown either conventionally, or according to the present invention (e.g., Example 5).
• the IgG antibodies were immobilized on Protein A coated latex beads. If there are cross reactive epitopes between the test serotype and the antibodies generated against the Lior 5 serotype, then nearly immediate clumping (i.e. agglutination) of the cells is visible.
• This clumping is rated based on a scale of 0 to 3 after allowing the reaction to proceed for a short period of time, where 0 means no observable clumping and 3 means a high degree of agglutination.
• 0 means no observable clumping
• 3 means a high degree of agglutination.
• jejuni grown according to methods of the present invention cross reacted with antibodies to Lior 5 serotype strain (see Table 16 below) .
• the results show that methods of the present invention significantly extend the number of Lior serotypes which cross react with anti-serum from animals immunized with Lior 5 serotype strain of C. jejuni .
• VACCINE EFFICACY Example 30 The ferret model for studying Campylobacter pathogenesis can be used as a model to evaluate vaccine efficacy in protecting against colonization and/or disease because infection of ferrets reproducibly generates two of the three disease manifestations seen in humans.
• ferrets were anesthetized with ACE promazine- Ketamine and challenged orally with a 10 ml PBS solution containing live C . jejuni 81-176 (lxlO 10 CFU). Thereafter the animals were monitored daily for mucoid diarrhea, bacteremia, fecal shedding of Campylobacter, weight changes, occult blood, and fecal leukocytes. Bactere ia was detected by drawing l to 2 ml of blood from the jugular vein of anesthetized ferrets and incubating the specimen in a vented trypticase soy broth culture.
• Subcultures to blood agar plates were taken at 2, 5 and 7 days post challenge. Serum samples were collected prior to immunization (baseline) , one week after the second immunization, and at the time of challenge, and one week post challenge to determine IgG titres.
• Occult blood was detected by testing fecal material on a Hemacult card. Fecal material was smeared on a slide and stained with methylene blue to detect fecal leukocytes. Fecal shedding of Campylobacter was established by culturing smears from rectal swabs on CampyloJbacter-selective medium plates (trypticase soy agar, 5% sheep blood, trimethoprim, vancomycin, poly yxin B, cephalothin, and amphotericin B, Remel, Lenexa, KS) . Results from these experiments are presented in Tables 10 and 11 below.
• Table 10 shows that upon live challenge, animals immunized with a killed-whole cell vaccine of the present invention were protected against colonization and disease.
• the data in Table 11 show that a much greater IgG antibody titre results from vaccines of the present invention
• Groups of 10 female Balb/c mice about sixteen weeks old were immunized orally with phosphate-buffered saline (PBS) C. jejuni conventionally grown (BHI) or C. jejuni grown according to Example 5 (ENHANCED) in doses of about 10' CFU or 10 9 CFU, then challenged.
• IgA titres from intestinal mucus in each group were determined by ELISA methods and are presented in Table 12 below.
• a Lavage titre indicates the mean anti-C. jejuni IgA titre obtained for each individual group of mice.
• Responders are defined as those animals whose endpoint titres exceeded 2 standard deviations above the mean of the animals receiving PBS alone
• Table 12 shows that animals immunized with bacteria grown according to the methods of the present invention have a higher intestinal IgA antibody titre as presented by a greater percentage of responders than animals immunized with bacteria grown conventionally.
• Example 32 Although not intending for the present invention to be limited to any particular mechanism of action, the present inventors have obtained evidence that suggests that deoxycholate (DOC) appears to have a two fold action in altering or enhancing the antigenicity of enteric bacteria.
• DOC deoxycholate
• Several bacterial genera that are susceptible to antigenic enhancement or alteration by bile or bile salts such as DOC have genes homologous to low calcium response (lcr) genes from Yersinia .
• the lcr locus is known to regulate virulence of Yersinia in response to low calcium levels.
• Two Campylobacter genes involved in flagellin expression and assembly which are required for invasion are regulated in part by the lcr product.
• the C. jejuni fla and flbA mutants do exhibit significantly enhanced Congo red binding and immuno-cross reactivity when cultured with DOC (see Table 15 and Figure 6, respectively) .
• the results shown in Table 15 and Figure 6 were obtained using C. jejuni cultured conventionally or according to the methods of the present invention as exemplified in Example 5 above.
• the Congo red dye binding assays, whose results are shown in Table 15, were carried out as described in Example 26 above.
• the immuno-cross reactivity, whose results are shown in Figure 6, were carried out as described in Example 29 above.
• Table 15 Congo Red Dye Binding by C jejuni Mutants Grown Conventionally (BHI) or According to Methods of the Invention (ENHANCED)
• the flak mutant is unable to express flagellin.
• the flaA mutant and a _flaA-_flaB double mutant (received from C. Grant, NIH) are both noninvasive even after treatment with DOC, indicating that flagellin is required for invasion.
• the normally exhibited (i.e., non-DOC induced) immuno-cross reactivity observed between the isogenic parent strain of these fla mutants and certain other Lior serotypes of C. jejuni is absent in the mutants.
• DOC treatment can induce these flagellin-less mutants to exhibit enhanced immuno-cross reactivity and Congo red binding.
• Campylobacter jejuni grown according the methods of the present invention was examined using the rapid slide agglutination assay.
• the assay used intestinal mucus from immunized and non-immunized rabbits to determine the effects of altering culture conditions on the cross-reactivity of heterologous strains of Campylobacter.
• the rabbits were immunized with live C . jejuni 81-176 grown conventionally.
• the agglutination activity of the mucus antibodies were tested against twenty-four Campylobacter strains, comprising eighteen serotypes, grown conventionally in BHI-YE medium or according to the methods of the invention as exemplified in Example 5.
• the results of the agglutination assays show that the cross-agglutination of heterologous Campylobacter strains was broader and, in many cases, stronger when the strains were grown according to the methods of the invention than when they were grown conventionally. Specifically, there was an over two fold increase in heterologous agglutination reactivity: 6 of 24 conventionally-grown heterologous strains agglutinated at level + or greater in the anti-81-176 immune mucus, whereas 14 of the same 24 strains grown under DOC conditions agglutinated at level + or greater.
• heterologous strains demonstrated weak (+) or no agglutination when conventionally grown
• eleven of these same strains showed an enhanced agglutination response when grown under ENHANCED culture conditions (e.g., DOC containing medium) .
• Table 16 illustrates cross-reactivity of anti-81-176 immune rabbit mucus against 19 heterologous Lior serotypes consisting of 22 strains grown conventionally (BHI-YE) or using the methods of Example 5 (ENHANCED) . Even though this experiment assayed only a fraction of the known Lior serotypes, the results demonstrate that the methods of the invention induce substantial immuno-cross reactivity between Lior serotypes. The results further show that the DOC enhanced or induced antigens in Campylobacter appear to be important in the secretory IgA response associated with resistance to and recovery from intestinal infection by Campy 1 obacter . It should be further noted that strains of Lior serotype 8 are of a different species, Campylobacter coli .
• the anti-81-176 mucus were obtained from rabbits infected with live C. jejuni 81-176 grown conventionally
• the non-immune mucus were obtained from uninfected rabbits c
• the agglutination responses range from negative (-) , to very weak ( ⁇ ) , to very strong (++++)
• Example 34 The protective efficacy of formalin-fixed whole cell Campylobacter jejuni grown according to the methods of the present invention was determined using the mouse colonization model reported by Baqar (Infect. & Immun.. 63:3731-3735, 1995).
• C. jejuni 81-176 was grown and harvested according to Example 5 and inactivated with 0.075% formalin as described above.
• Groups of five 6 to 8 week old female Balb/c mice were administered three oral doses (0.25 ml/dose in endotoxin-free PBS) containing either 10 ⁇ , 10 7 , or 10 9 inactivated bacterial particles alone or in combination with 25 ⁇ q of the heat labile enterotoxin from E . coli (LT) .
• Doses were given at 48 hour intervals and immediately after two 0.5 ml doses of 5% sodium bicarbonate solution (pH 8.5) were given at 15 minute intervals, to neutralize gastric acidity.
• Campylobacter GasPak BBL
• Colonization results are expressed as the percentage of animals shedding Campylobacter organisms on a given sample day.
• the protective efficacy of formalin-fixed whole cell Campylobacter jejuni grown according to the methods of the present invention was also evaluated.
• groups of 20 female Balb/c mice were administered a single dose of 1.3X10 10 , 2.5X10 9 , 5.0X10 8 , 1.0X10 8 or 2.0X10 7 inactivated C. jejuni particles in 0.5 ml endotoxin-free PBS without adjuvant.
• the animals were challenged 14 days later with a single lethal dose of live C. jejuni 81-176 (approximately 1.0X10 10 CFU in endotoxin-free PBS) delivered intraperitoneally. Animals were monitored daily for 4 days for mortality.
• Example 35 The invasiveness of Shigella sp . grown in vitro is affected by the culture's growth phase.
• the invasiveness of Shigella flexneri 2457T cells grown conventionally (BHI) , or according to the methods of the invention as exemplified by Example 9 (DOC-EL) (wherein the cells are from an early log phase culture) , or according to Example 9 but allowing the culture to reach late log phase before harvesting the cells (DOC-LL) was tested according to the procedures described in Example 28. The results show that culturing with DOC enhances invasiveness and that the maximum enhancement is achieved during early log phase of growth (see Figure 9) .
• Culturing with DOC according to the methods of the invention also enhances the invasiveness of other Shigella species, S. sonnei , and S . dysentariae (see Figure 10).
• S. sonnei the enhanced invasiveness of Shigella was observed only when the epithelial cells were infected basolaterally by the bacteria. This finding is consistant with the invasion process observed in vivo .
• Comparitive studies show that Shigella grown according to the methods of the present invention are nearly 10-fold more invasive than Shigella prepared according to the procedure described by Pope et al. (Infect. & Immun.. 63:3642- 3648 1995) .
• Shigella cultured according to the methods of the present invention also exhibit enhanced Congo red binding.
• Shigella is divided into four species and various serotypes.
• the immuno-cross reactivity of Shigella flexneri grown according the methods of the present invention was examined using the agglutination assay as described in Example 28.
• the assay used antiserum from immunized rabbits to determine the effects of culture conditions on the immuno- cross reactivity of different Shigella species.
• the rabbits were immunized with formalin-fixed shigella flexneri 2457T grown according to the methods of Example 9.
• the agglutination activity of the IgG antibodies obtained from the immunized animals were tested against all four Shigella species grown conventionally in BHI medium or according to the methods of the invention (e.g.. Example 9).
• results of the agglutination assays show that growth with DOC significantly enhanced the agglutination activity of the homologous Shigella flexneri as well as those of the three heterologous Shigella species to anti-S. flexneri antibody (see Figure 11) .
• Example 37 The protective efficacy of formalin-fixed whole cell Shigella flexneri grown according to the methods of the present invention was determined using the mouse nasal challenge model developed by C.P. Mallett et al. (Vaccine. 11:190-196, 1993). Briefly, Shigella flexneri was grown and harvested according to the methods exemplified in Example 9 and inactivated with 0.075% formalin as described in Example 30. Approximately 10 7 inactivated bacterial particles were used to vaccinate 14-16 week-old female Balb/c mice. The inactivated 5.
• flexneri was suspended in sterile, endotoxin- free PBS at a concentration of 10 8 particles/ml and 35 ⁇ l of this material was administered nasally to groups of 10 lightly anesthetized animals. A total of three immunizations were given at 14 day intervals.
• mice were immunized using a suspension that contained the inactivated vaccine and 5 ⁇ g of the heat-labile enterotoxin from E. coli (LT) .
• LT heat-labile enterotoxin from E. coli
• mice in each group were immunized 3X nasally with vaccine comprsing 10 inactivated S . flexneri grown according to Example 9
• mice immunized with vaccine comprising inactivated S flexneri grown according to the methods of the invention were protected against challenge with live S . flexneri organisms. Those mice suffered less weight loss and underwent more rapid weight recovery as compared to unvaccinated mice, i.e. the PB sham control group.
• the S . flexneri vaccine also protected the mice against challenge with live s. sonnei .
• animals receiving the vaccine alone without th LT adjuvant were as well protected against the homologous S. flexneri challenge as animals that received the adjuvantized vaccine.
• the S . flexneri vaccine alone also conferred protection to heterologous challenges by S. sonnei .
• the inclusion of the LT adjuvant however, noticibly enhanced the protection against the challenge by S . sonnei .
• Example 38 The adherence of H . pylori is enhanced by growth in glycocholate or bile. Cells of H. pylori strain
• Example 39 The protective efficacy of formalin-fixed whole cell Helicobacter pylori grown according to the methods of the present invention was determined using the mouse Helicobacter felis gastric colonization model described by Chen et al. (Lancet. 339:1120-1121, 1992) .
• Helicobacter pylori strain Gl-4 was grown as a seed culture for about 22 h at 37°C under 10% C0 2 , 90% air in BHI media containing 4% bovine calf serum. An aliqout of this culture was used to inoculate a 10-fold volume of the same media containing 0.1% (v/v) bovine bile.
• the cells are harvested by centrifugation and resuspended in 1/10 of the original volume of Hank's Balanced Salts Solution (HBSS) at room temperature. Cells were recentrifuged and again suspended in 1/100 of the original volume of HBSS. To the buffered cell suspension, formalin was added to a concentration of 0.075% and the cells inactivated by stirring the suspension at room temperature for 6 h then cooling the solution at 4°C for 18 hours.
• HBSS Hank's Balanced Salts Solution
• Protection potential was routinely measured by administering 3 doses of this inactivated whole cell vaccine orally to 6-8 week old female Balb/c Helico acter-free mice at days 0, 7 and 14 or at days 0, 7 and 21. Doses of 10 9 bacterial particles per dose were evaluated in combination with the heat labile enterotoxin of E. coli . Fourteen days after the third immunizing dose, animals were challenged orally with a single dose (10 7 CFU/dose) of live H. feli ⁇ .
• Urease activity was determined by incubating antral tissue samples in 0.5 ml Stuart's Urease Broth (Remel) at room temperature for 4-24 hours. A color change from clear to red occuring within this period was taken as a positive urease result.
• the methods of the present invention produce bacteria capable of inducing an immunogenic response which is protective and therefore are useful as vaccines.
• the indicationa liatM below will be autxnmed to trw international Bureau later ' .Specify the oenerel nature of the indicationa e. ⁇ .. "Accaasion Number of Oepoait'l

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