INFECTION AND IMMUNITY, JUlY 1991, p. 2359-2363 0019-9567/91/072359-05$02.00/0 Copyright C 1991, American Society for Microbiology
Vol. 59, No. 7
Oral Immunization against Helicobacter pylori STEVEN J. CZINN1* AND JOHN G. NEDRUD2 Department of Pediatrics' and Institute of Pathology,2 Case Western Reserve University, Cleveland, Ohio 44106 Received 10 December 1990/Accepted 15 April 1991
Helicobacter pylori, which has been associated with gastritis and duodenal ulcers, commonly chronically infects adults. Eradication of this microorganism, which is difficult to achieve, results in normalization of gastritis and marked reduction in the relapse rate of duodenal ulcers. Since eradication is difficult to achieve, prevention of initial colonization of the gastrointestinal tract may be a viable alternative for abrogation of H. pylori-associated gastroduodenal disease. To test the feasibility of this approach, mice and ferrets were orally immunized with killed H. pylori. Immunization induced immunoglobulin A and G anti-H. pyloni antibodies in both gastrointestinal secretions and sera of mice. These responses were enhanced when cholera toxin was included in the immunization protocol as a mucosal adjuvant. In ferrets, addition of cholera toxin resulted in significant enhancement of anti-H. pylori antibody levels in sera and intestines. Thus, oral immunization with killed H. pylori may be a feasible approach to protect hosts from this infection and the accompanying gastroduodenal disease.
Gastroduodenal disease is a major cause of morbidity and mortality in children and adults. A number of investigators have suggested a causal relationship between Helicobacter pylori infection and gastritis and peptic ulcer disease (6, 14, 19, 30). Other studies have demonstrated the difficulty in consistently eradicating H. pylori from the upper gastrointestinal tract (4, 12, 15, 27). This difficulty relates in part to our lack of understanding of the pathophysiology of H. pylori gastroduodenal disease. Trials using two antibiotics in combination with oral colloidal bismuth for 2 to 8 weeks have been successful in eradicating H. pylori in 20 to 90% of the patients studied (3, 13, 20). These reports suggest that eradication of H. pylori is associated with both resolution of the underlying gastritis and a significant decrease in the relapse rate of duodenal ulcers. However, a reliable method for long-term eradication of H. pylori does not exist, and at least two or three antimicrobial agents are necessary to achieve temporary eradication. Thus, once H. pylori infection is established, it is difficult to eradicate. Consequently, immunologically mediated prevention of H. pylori infection using oral vaccines may be the ideal way to approach this problem. Oral immunization to induce mucosal immunity against infection is a convenient and safe form of immunization. In addition, the gut contains the largest mass of mucosa-associated lymphoid tissue in the body (21). Previously, investigators have shown that repetitive oral immunizations with soluble proteins or particulate antigens, along with mucosal adjuvants, such as cholera toxin (CT), induce a gastrointestinal immune response in rodents (7, 18, 25). In addition, recent reports suggest that the ferret is an excellent animal model for Helicobacter-associated gastroduodenal disease (10). Therefore, the objective of the present study was to develop an oral immunization protocol which provides a vigorous gastrointestinal immunoglobulin A (IgA) anti-H. pylori response. Thus, oral immunization with H. pylori in the presence or absence of CT was evaluated in mice and ferrets.
*
MATERIALS AND METHODS
Animals. Although H. pylori has not been demonstrated to infect rodents, mice were used initially to examine the feasibility of oral immunization with H. pylori. Since the ferret has been shown to be a useful model for Helicobacterinduced gastroduodenal disease, in subsequent experiments ferrets were orally immunized with H. pylori. Mice. Adult male BALB/c mice (7 to 12 per group) were obtained from the Charles River Laboratory, Cambridge, Mass. Ferrets. Six-week-old male ferrets (four per group) were obtained from Marshall Farms, North Rose, N.Y. Animals were housed in the Case Western Reserve University animal facility, which is fully accredited, and they were allowed free access to standard laboratory chows and water. Bacterial strains. Bacteria recovered from gastric biopsy specimens were identified as H. pylori on the basis of colony morphology, Gram stain, and production of urease, catalase, and oxidase. The test strain of H. pylori used for these studies was a clinical isolate (P-17) from the gastric biopsy of a pediatric patient with gastritis (5). Organisms were stored in 50% phosphate-buffered saline (PBS)-25% glycerol-25% heated fetal calf serum at -70°C. The bacteria used in these studies had been passaged in vitro 5 to 10 times after isolation. Bacterial antigens. The test strain was inoculated onto Columbia agar (Difco, Detroit, Mich.) containing 5% sheep blood and incubated microaerophilically at 37°C for 96 h. The organisms were harvested in PBS, and the resulting suspensions were sonicated at 4°C and cleared of cellular debris by centrifugation. These whole-cell sonic extracts were stored as 100-.lI aliquots at -70°C until needed for oral immunization of animals (5). Outer membrane preparations. Cell envelopes were prepared from bacterial suspensions and treated with 1 mg each of RNase and DNase (Sigma Chemical Co., St. Louis, Mo.) in 0.05 M Tris-EDTA buffer (pH 7.8) before sonication and centrifugation. The envelopes were separated from the cleared lysate by ultracentrifugation at 150,000 x g for 1 h. Outer membranes were separated from the cell envelopes by differential solubilization in sodium n-lauroylsarcosine and
Corresponding author. 2359
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recovered by ultracentrifugation (28). The resulting pellets were suspended in 0.05 M phosphate buffer (pH 7.0), divided into aliquots, and stored at -70°C (5). Protein concentration was determined by the method of Lowry et al. (17). Experimental protocols. For intragastric immunization, whole-cell sonic extract preparations with or without CT (C-3012; Sigma) were suspended in 0.2 M NaHCO3 and 1.0-ml volumes were delivered into the stomachs of lightly etherized mice (1 mg/ml) or awake ferrets (7 mg/ml) by intubation through polyethylene tubing attached to a hypodermic syringe. This procedure will be referred to as oral immunization. At various times postimmunization, animals were killed or endoscoped and one or more of the following tissue fluids were collected: serum, gastric secretions, and intestinal secretions. These samples were then titrated for the presence of anti-H. pylori antibodies by enzyme-linked immunosorbent assay (ELISA). Collection of samples. Serum was obtained by tail vein bleeding and letting the blood clot at room temperature. Gastric and intestinal secretions were collected by a modification of the procedure of Elson et al. (9). Briefly, gastric and intestinal secretions from mice and ferrets were collected separately. For mice, stomachs and intestines were removed and injected with 2.0 ml of a polyethylene glycolbased lavage solution. For ferrets, 5 ml of this solution was injected into the stomachs and intestines (25). ELISA. Murine or ferret samples were assayed for Helicobacter-specific antibodies by a modification of the method described by Booth et al. (2). Ninety-six-well polystyrene microtiter plates were coated with appropriate outer membrane proteins at 100 ,u per well (20 p.g/ml) and incubated overnight at 4°C. Nonspecific binding sites were blocked with 1% bovine serum albumin in PBS for 90 min at room temperature, and then the plates were washed with 0.1% bovine serum albumin in PBS. Samples were tested in duplicate at dilutions ranging from neat to 1:512,000, and 100 pL of each dilution per well was added to the antigen-coated plates. For competitive ELISAs, a single dilution of serum from a ferret immunized with H. pylori was mixed with serial dilutions of either H. pylori or H. mustelae outer membrane protein. Following incubation at room temperature for 90 min, the plates were washed three times with 0.1% bovine serum albumin in PBS and 100 RI of a 1:1,000 dilution of either goat anti-ferret IgG-alkaline phosphatase conjugate (see below) or goat anti-mouse IgA- or IgG-alkaline phosphatase conjugate (Zymed, San Francisco, Calif.) was added to each well for 90 min. To detect anti-ferret antibodies, goat anti-ferret IgG (heavy and light chain specific; KPL, Gaithersburg, Md.) was used. This is the only anti-ferret immunoglobulin reagent available. This reagent is raised against a purified immunoglobulin fraction of ferret serum which contains IgG, as well as IgM and IgA, and reacts with ferret IgG and a number of other ferret serum proteins thought to be IgA and IgM (4a). After being washed, the plates were developed with 100 p1 of a 1-mg/ml solution of p-nitrophenyl phosphate in glycine buffer (pH 9.6) per well for 1 h. The A410 in each well was measured by using a Dynatech MR 700 Microtiter Plate Reader. The antibody titer was defined as the reciprocal of the highest dilution yielding an A410 of 0.05 above wells which contained antigen and which were incubated with the antibody conjugate but without the primary antibody sample (25). Statistical analysis. Comparisons among experimental groups were evaluated by one-way analysis of variance (ANOVA) with Fisher's protected-least-difference test, as well as by Kruskall-Wallis using Statview 512 software
INFECT. IMMUN. 6
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FIG. 1. Murine antibody titers after oral immunization with H. pylori (HP). Seronegative BALB/c mice (7 to 12 per group) were intragastrically immunized with four doses of 1 mg of H. pylori lysate either in the presence or in the absence of 10 ,ug of CT. Five days after the final immunization, the animals were sacrificed and anti-H. pylori ELISA titers were determined for gastrointestinal secretions and sera. The error bars show standard deviations.
(Brainpower, Calabasas, Calif.) for the Apple Macintosh computer. ANOVA was used for multiple inferences. RESULTS In the first experiment, we investigated the murine immune response to oral immunization with H. pylori lysate alone or in the presence of CT (a mucosal adjuvant). We have previously shown that optimal immunization of murine mucosa-associated lymphoid tissue can be achieved by intragastrically immunizing mice four times over a 4-week period (25). Additionally, mucosal adjuvants, such as CT, when mixed with protein antigens, can enhance gastrointestinal immune responses (8, 16, 18, 25). In this experiment, seronegative mice were actively immunized with 1 mg of H. pylori lysate with or without 10 pug of CT. As shown in Fig. 1, oral immunization without CT induced detectable serum IgA and IgG H. pylori-specific antibodies. In this experiment, nonimmunized controls had antibody levels below the limit of detection (data not shown). When CT was included in the immunization protocol, these responses were significantly enhanced. Specifically, there was a 5-fold increase of IgA in serum (F = 73.9, H = 20.9, P < 0.001) and a 16-fold increase of IgA in intestinal secretions (F = 52.9, H = 20.0, P < 0.001) compared with animals which did not receive CT. Elevations of IgG in serum (F = 14.2, H = 13.8, P = 0.001) and intestinal secretions (F = 7.3, H = 9.4, P < 0.009) were also noted. It has not been possible to infect rodents with H. pylori, but recent results suggest that the ferret may be a useful model for the study of Helicobacter-induced chronic gastritis. Therefore, ferrets were orally immunized against H. pylori lysate with or without CT. Preliminary dose-response studies were used to determine a safe dose of CT for ferrets. No untoward side effects, such as weight loss or diarrhea, were noted in any ferrets that received CT doses of up to 60 pug. Therefore, in subsequent experiments, 60 p.g of CT was used per dose of antigen. Ferrets are naturally colonized shortly after birth with another member of the Helicobacter genus, H. mustelae. Since these two organisms are quite similar histologically (and perhaps antigenetically as well), it was necessary to demonstrate that they could be distinguished serologically by using purified outer membranes as an antigen in an
ORAL IMMUNIZATION AGAINST H. PYLORI
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0.6 H. mustelae outer membrane protein c-
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.0 a
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COMPETING ANTIGEN (jg/ml) 0
FIG. 2. ELISA using outer membrane proteins can distinguish between H. pylori and H. mustelae. ELISA plates were coated with H. pylori outer membrane protein, a single dilution of serum from a ferret immunized with H. pylori was mixed with serial dilutions of either H. pylori (0) or H. mustelae (0) outer membrane proteins, and a competitive ELISA was run as described in Materials and Methods. The data are from one experiment and are representative of four independently tested sera.
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ELISA. As shown in Fig. 2, soluble H. pylori outer membrane proteins added to aliquots of serum from a ferret immunized with H. pylori effectively blocked the binding of ferret antibodies to H. pylori outer membrane proteins coated on an ELISA plate. In contrast, equal concentrations of soluble purified H. mustelae outer membrane proteins added to the serum exhibited only a small amount of antibody-blocking activity, even at high outer membrane protein concentrations. Figure 3A shows antibody titers to both H. pylori and H. mustelae in serum in an experiment wherein groups of four 6-week-old ferrets were orally immunized weekly for 5 weeks with 7 mg of H. pylori lysate with or without 60 ,ug of CT per dose. By nonparametric Kruskall-Wallis analysis, the anti-H. pylori antibody titers were significantly different at all time points (P c 0.02). As in the murine model, ferrets immunized with H. pylori with CT demonstrated elevated anti-H. pylori antibody titers in serum compared with ferrets immunized with H. pylori alone (P s 0.05 by ANOVA) or control animals (P s 0.0001 by ANOVA) at all time points. Since these animals were all naturally infected with H. mustelae, all were also found to have serum anti-H. mustelae antibodies which were not enhanced relative to those of nonimmunized control ferrets (Fig. 3B). Our long-term goals are to immunize the gut-associated lymphoid tissue and provide protection against Helicobacter-associated gastritis. Thus, the ferrets from this experiment were sacrificed and the gastric and intestinal antibody responses were determined by ELISA. The results (Fig. 4) show that oral immunization with H. pylori lysate in the presence of CT induced significant intestinal anti-H. pylori antibodies (P < 0.05 compared with the control by ANOVA). Although not statistically significant, gastric anti-H. pylori levels were also increased when CT was used. DISCUSSION
Gastritis and peptic ulcer disease are significant causes of morbidity and mortality. Recent studies from a number of laboratories have improved our understanding of gastroduo-
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FIG. 3. Ferret anti-Helicobacter antibody titers in serum after immunization with H. pylori plus CT (O). 0, H. pylori; 0, control. Groups of four ferrets were immunized five times (arrows) with 7 mg of H. pylori lysate in the presence or absence of 60 ,ug of CT. Anti-H. pylori and H. mustelae ELISA antibody titers were determined for serially obtained serum samples. (A) Ferret anti-H. pylori antibody titers in serum. (B) Ferret anti-H. mustelae antibody titers in
serum.
denal disease. Specifically, investigators have demonstrated an association between gastric infection with H. pylori, formerly called Campylobacter pylori, and gastritis and peptic ulcer disease, offering new insights into the diagnosis and treatment of these common disorders. Immunologically mediated prevention of H. pylori infection with oral vaccines may be the ideal way to approach this problem. Numerous studies have demonstrated that pre-existing secretory IgA can interfere with the ability of enteric pathogens, such as enterotoxigenic Escherichia coli, to attach to epithelial cells and that oral immunization can reduce Streptococcus mutans colonization and prevent dental caries (1, 11, 22-24, 29). 14
HP.C
12
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-8
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Gatic Wash Intestinal Wash Titers vs H. mustels
FIG. 4. Ferret anti-Helicobacter mucosal antibody titers after immunization with H. pylori (HP) plus CT. The three groups of ferrets whose serum responses are presented in Fig. 3 were sacrificed after 15 weeks, and anti-H. pylori titers were determined for gastric and intestinal washes.
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Furthermore, because H. pylori infection is very rarely seen in children, vaccination could be undertaken prior to the onset of infection. The ideal H. pylori vaccine would act locally in the upper gastrointestinal tract and prevent the initial adherence to the gastric mucosa which is necessary for colonization. By using CT as an adjuvant, we successfully induced both IgA and IgG responses in the sera and gastrointestinal secretions of mice. Similar mucosal immunization protocols using attenuated viruses alone or killed virus plus CT have induced good gastrointestinal immune responses and been able to induce protective immunity (25, 26). Since the gastrointestinal tracts of ferrets are anatomically and physiologically very similar to those of humans, ferrets have been used to study other enteric infections and may be useful to further our understanding of H. pylori infection; recently, Fox et al. showed that the ferret could serve as a useful animal model for Helicobacter-associated gastritis (10). We conducted oral immunization studies with H. pylori antigens in ferrets colonized with H. mustelae, a Helicobacter organism indigenous to ferrets. H. mustelae and H. pylori are immunologically distinct, such that immunization with and measurement of antibodies to H. pylori were not affected by the presence of indigenous H. mustelae in the ferrets. In these experiments, we were able to induce anti-H. pylori antibodies in serum. The peak serum response was noted 3 weeks after the last immunization, with a 6- to 10-fold increase of anti-H. pylori antibodies. When 60 ,ug of CT was added to each oral immunization, significant enhancement of anti-H. pylori antibody titers in serum and gastrointestinal secretions occurred. These results suggest that it is possible to develop an oral immunization protocol for prevention of H. pylori infection and associated gastritis. ACKNOWLEDGMENTS This research was supported by National Institute of Health grants AI-25818 and HL-37117 and grant Z063 9-1 from the Cystic Fibrosis Foundation. We thank Howard Carr for technical assistance and Katie Chipps for manuscript assistance. REFERENCES 1. Abraham, S. N., and E. H. Beachey. 1985. Host defense against adhesion of bacteria to mucosal surfaces. Adv. Host Def. Mech. 4:63-88. 2. Booth, L., G. Holdstock, H. McBride, P. Hawtin, J. R. Gibson, A. Ireland, J. Banforth, E. DuBoulay, R. S. Lloyd, and A. D. Pearson. 1986. Clinical importance of Campylobacter pyloridis and associated serum IgG and IgA antibody responses in patients undergoing upper gastrointestinal endoscopy. J. Clin. Pathol. 39:216-219. 3. Borody, T., P. Cole, S. Noonan, A. Morgan, G. Ossip, J. Maysey, and S. Brandl. 1988. Long-term Campylobacter pylori recurrence post-eradication. Gastroenterology 94:A43. 4. Borsch, G. 1989. Searching for the best way to eradicate Campylobacter pylori. Long-term studies are still needed. Fortschr. Med. 107:471-472. 4a.Czinn, S. J. Unpublished data. 5. Czinn, S. J., H. Carr, L. Sheffler, and S. Aronoff. 1989. Serum IgG antibody to the outer membrane proteins of Campylobacter pylori in children with gastroduodenal disease. J. Infect. Dis.
159:586-589. 6. Czinn, S. J., and W. T. Speck. 1989. Campylobacter pylori: a new pathogen. Adv. Pediatr. Infect. Dis. 5:221-237. 7. Elson, C. O., and W. Ealding. 1984. Generalized systemic and mucosal immunity in mice after mucosal stimulation with chol-
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era toxin. J. Immunol. 132:2736-2741. 8. Elson, C. O., and W. Ealding. 1984. Cholera toxin feeding did not induce oral tolerance in mice and abrogated oral tolerance to an unrelated protein antigen. J. Immunol. 133:2892-2897. 9. Elson, C. O., W. Ealding, and J. Lefkowitz. 1984. A lavage technique allowing repeated measurement of IgA antibody in mouse intestinal secretions. J. Immunol. Methods 67:101-108. 10. Fox, J. G., C. Pelayo, N. S. Taylor, A. Lee, G. Otto, C. Murphy, and R. Rose. 1990. Helicobacter mustelae-associated gastritis in ferrets: an animal model of Helicobacter pylori gastritis in humans. Gastroenterology 99:352-361. 11. Fubara, E., and R. Freter. 1973. Protection against enteric bacterial infection by secretory IgA antibodies. J. Immunol. 111:395-403. 12. Gilman, R., R. Leon-Barva, A. Ramirez-Ramos, D. Morgan, S. Recavarron, and W. Spira. 1987. Efficacy of nitrofurans in the treatment of antral gastritis with Campylobacter pyloridis. Gastroenterology 92:1405. 13. Goodwin, C. S., B. J. Marshall, J. R. Warren, S. Blackbourn, and E. D. Blincow. 1989. Clearance of Campylobacter pyloridis and reduced duodenal ulcer relapse with bismuth and tinidazole compared to cimetidine, p. 368-369. In B. Kaijser and E. Falsen (ed.), Campylobacter IV. University of Goteborg, Goteborg, Sweden. 14. Graham, D. Y. 1989. Campylobacter pylori and peptic ulcer disease. Gastroenterology 96:615-625. 15. Hirschl, A. M., E. Hentschel, K. Schutze, H. Nemec, R. Potzi, A. Gangl, W. Weiss, M. Pletschette, G. Stanek, and M. Rotter. 1988. The efficacy of antimicrobial treatment in Campylobacter pylori associated gastritis and duodenal ulcer. Scand. J. Gastroenterol. 23:76-81. 16. Liang, X., M. E. Lamm, and J. G. Nedrud. 1989. Cholera toxin as a mucosal adjuvant for respiratory antibody responses in mice. Reg. Immunol. 2:244-248. 17. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 18. Lycke, N., and J. Holmgren. 1986. Strong adjuvant properties of cholera toxin on gut mucosal immune responses to orally presented antigen. Immunology 23:611-616. 19. Marshall, B., and J. R. Warren. 1984. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet i:1311-1314. 20. Marshall, B. J., K. R. Dye, M. Plankey, H. Frierson, S. Hoffman, R. Guerrant, and R. McCallum. 1988. Eradication of Campylobacter pylori infection with bismuth subsalicylate and antibiotic combinations. Am. J. Gastroenterol. 83:1035. 21. Mestecky, J., and J. R. McGhee. 1987. Immunoglobulin A (IgA): molecular and cellular interactions involved in IgA biosynthesis and immune response. Adv. Immunol. 40:153-245. 22. Mestecky, J., J. R. McGhee, R. R. Arnold, S. M. Michalek, S. J. Prince, and J. L. Babb. 1978. Selective induction of an immune response in human external secretions by ingestion of bacterial antigen. J. Clin. Invest. 61:731-737. 23. Michalek, S. M., J. R. McGhee, and J. L. Babb. 1978. Effective immunity to dental caries: dose-dependent studies of secretory immunity by oral administration of Streptococcus mutans to rats. Infect. Immun. 19:217-224. 24. Morisaki, I., S. M. Michalek, C. C. Harmon, M. Torii, S. Hamada, and J. R. McGhee. 1983. Effective immunity to dental caries: enhancement of salivary anti-Streptococcus mutans antibody responses with oral adjuvants. Infect. Immun. 40:577591. 25. Nedrud, J. G., X. P. Liang, N. Hague, and M. E. Lamm. 1987. Combined oral/nasal immunization protects mice from Sendai virus infection. J. Immunol. 139:3484-3492. 26. Ogra, P. L., D. T. Karzon, F. Righthand, and M. MacGillivray. 1968. Immunoglobulin response in serum and secretions after immunization with live and inactivated polio vaccine and natural infection. N. Engl. J. Med. 279:895-900. 27. Rauws, E. A. J., W. Langenberg, H. J. Houthoff, H. C. Zanen, and G. N. S. Tytgat. 1988. Campylobacter pyloridis-associated chronic antral gastritis. A prospective study of its prevalence
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and the effects of antibacterial and antiulcer treatment. Gastroenterology 94:33-40. 28. Sawai, T., R. Hiruma, N. Kawana, M. Kaneko, F. Tarriyasu, and A. Inami. 1982. Outer membrane permeation of beta-lactam antibiotics in Escherichia coli, Proteus mirabilis, and Enterobacter cloacae. Antimicrob. Agents Chemother. 22:585-592.
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29. Tagliabue, A., L. Villa, M. T. de Magistris, M. Romano, S. Silvestri, D. Boraschi, and L. Nencioni. 1986. IgA-driven T cell-mediated anti-bacterial immunity in man after live oral Ty 21a vaccine. J. lmmunol. 137:1504-1510. 30. Warren, J. R. 1983. Unidentified curved bacilli on gastric epithelium in chronic gastritis. Lancet i:1273-1275.
Vol. 59, No. 7
Oral Immunization against Helicobacter pylori STEVEN J. CZINN1* AND JOHN G. NEDRUD2 Department of Pediatrics' and Institute of Pathology,2 Case Western Reserve University, Cleveland, Ohio 44106 Received 10 December 1990/Accepted 15 April 1991
Helicobacter pylori, which has been associated with gastritis and duodenal ulcers, commonly chronically infects adults. Eradication of this microorganism, which is difficult to achieve, results in normalization of gastritis and marked reduction in the relapse rate of duodenal ulcers. Since eradication is difficult to achieve, prevention of initial colonization of the gastrointestinal tract may be a viable alternative for abrogation of H. pylori-associated gastroduodenal disease. To test the feasibility of this approach, mice and ferrets were orally immunized with killed H. pylori. Immunization induced immunoglobulin A and G anti-H. pyloni antibodies in both gastrointestinal secretions and sera of mice. These responses were enhanced when cholera toxin was included in the immunization protocol as a mucosal adjuvant. In ferrets, addition of cholera toxin resulted in significant enhancement of anti-H. pylori antibody levels in sera and intestines. Thus, oral immunization with killed H. pylori may be a feasible approach to protect hosts from this infection and the accompanying gastroduodenal disease.
Gastroduodenal disease is a major cause of morbidity and mortality in children and adults. A number of investigators have suggested a causal relationship between Helicobacter pylori infection and gastritis and peptic ulcer disease (6, 14, 19, 30). Other studies have demonstrated the difficulty in consistently eradicating H. pylori from the upper gastrointestinal tract (4, 12, 15, 27). This difficulty relates in part to our lack of understanding of the pathophysiology of H. pylori gastroduodenal disease. Trials using two antibiotics in combination with oral colloidal bismuth for 2 to 8 weeks have been successful in eradicating H. pylori in 20 to 90% of the patients studied (3, 13, 20). These reports suggest that eradication of H. pylori is associated with both resolution of the underlying gastritis and a significant decrease in the relapse rate of duodenal ulcers. However, a reliable method for long-term eradication of H. pylori does not exist, and at least two or three antimicrobial agents are necessary to achieve temporary eradication. Thus, once H. pylori infection is established, it is difficult to eradicate. Consequently, immunologically mediated prevention of H. pylori infection using oral vaccines may be the ideal way to approach this problem. Oral immunization to induce mucosal immunity against infection is a convenient and safe form of immunization. In addition, the gut contains the largest mass of mucosa-associated lymphoid tissue in the body (21). Previously, investigators have shown that repetitive oral immunizations with soluble proteins or particulate antigens, along with mucosal adjuvants, such as cholera toxin (CT), induce a gastrointestinal immune response in rodents (7, 18, 25). In addition, recent reports suggest that the ferret is an excellent animal model for Helicobacter-associated gastroduodenal disease (10). Therefore, the objective of the present study was to develop an oral immunization protocol which provides a vigorous gastrointestinal immunoglobulin A (IgA) anti-H. pylori response. Thus, oral immunization with H. pylori in the presence or absence of CT was evaluated in mice and ferrets.
*
MATERIALS AND METHODS
Animals. Although H. pylori has not been demonstrated to infect rodents, mice were used initially to examine the feasibility of oral immunization with H. pylori. Since the ferret has been shown to be a useful model for Helicobacterinduced gastroduodenal disease, in subsequent experiments ferrets were orally immunized with H. pylori. Mice. Adult male BALB/c mice (7 to 12 per group) were obtained from the Charles River Laboratory, Cambridge, Mass. Ferrets. Six-week-old male ferrets (four per group) were obtained from Marshall Farms, North Rose, N.Y. Animals were housed in the Case Western Reserve University animal facility, which is fully accredited, and they were allowed free access to standard laboratory chows and water. Bacterial strains. Bacteria recovered from gastric biopsy specimens were identified as H. pylori on the basis of colony morphology, Gram stain, and production of urease, catalase, and oxidase. The test strain of H. pylori used for these studies was a clinical isolate (P-17) from the gastric biopsy of a pediatric patient with gastritis (5). Organisms were stored in 50% phosphate-buffered saline (PBS)-25% glycerol-25% heated fetal calf serum at -70°C. The bacteria used in these studies had been passaged in vitro 5 to 10 times after isolation. Bacterial antigens. The test strain was inoculated onto Columbia agar (Difco, Detroit, Mich.) containing 5% sheep blood and incubated microaerophilically at 37°C for 96 h. The organisms were harvested in PBS, and the resulting suspensions were sonicated at 4°C and cleared of cellular debris by centrifugation. These whole-cell sonic extracts were stored as 100-.lI aliquots at -70°C until needed for oral immunization of animals (5). Outer membrane preparations. Cell envelopes were prepared from bacterial suspensions and treated with 1 mg each of RNase and DNase (Sigma Chemical Co., St. Louis, Mo.) in 0.05 M Tris-EDTA buffer (pH 7.8) before sonication and centrifugation. The envelopes were separated from the cleared lysate by ultracentrifugation at 150,000 x g for 1 h. Outer membranes were separated from the cell envelopes by differential solubilization in sodium n-lauroylsarcosine and
Corresponding author. 2359
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recovered by ultracentrifugation (28). The resulting pellets were suspended in 0.05 M phosphate buffer (pH 7.0), divided into aliquots, and stored at -70°C (5). Protein concentration was determined by the method of Lowry et al. (17). Experimental protocols. For intragastric immunization, whole-cell sonic extract preparations with or without CT (C-3012; Sigma) were suspended in 0.2 M NaHCO3 and 1.0-ml volumes were delivered into the stomachs of lightly etherized mice (1 mg/ml) or awake ferrets (7 mg/ml) by intubation through polyethylene tubing attached to a hypodermic syringe. This procedure will be referred to as oral immunization. At various times postimmunization, animals were killed or endoscoped and one or more of the following tissue fluids were collected: serum, gastric secretions, and intestinal secretions. These samples were then titrated for the presence of anti-H. pylori antibodies by enzyme-linked immunosorbent assay (ELISA). Collection of samples. Serum was obtained by tail vein bleeding and letting the blood clot at room temperature. Gastric and intestinal secretions were collected by a modification of the procedure of Elson et al. (9). Briefly, gastric and intestinal secretions from mice and ferrets were collected separately. For mice, stomachs and intestines were removed and injected with 2.0 ml of a polyethylene glycolbased lavage solution. For ferrets, 5 ml of this solution was injected into the stomachs and intestines (25). ELISA. Murine or ferret samples were assayed for Helicobacter-specific antibodies by a modification of the method described by Booth et al. (2). Ninety-six-well polystyrene microtiter plates were coated with appropriate outer membrane proteins at 100 ,u per well (20 p.g/ml) and incubated overnight at 4°C. Nonspecific binding sites were blocked with 1% bovine serum albumin in PBS for 90 min at room temperature, and then the plates were washed with 0.1% bovine serum albumin in PBS. Samples were tested in duplicate at dilutions ranging from neat to 1:512,000, and 100 pL of each dilution per well was added to the antigen-coated plates. For competitive ELISAs, a single dilution of serum from a ferret immunized with H. pylori was mixed with serial dilutions of either H. pylori or H. mustelae outer membrane protein. Following incubation at room temperature for 90 min, the plates were washed three times with 0.1% bovine serum albumin in PBS and 100 RI of a 1:1,000 dilution of either goat anti-ferret IgG-alkaline phosphatase conjugate (see below) or goat anti-mouse IgA- or IgG-alkaline phosphatase conjugate (Zymed, San Francisco, Calif.) was added to each well for 90 min. To detect anti-ferret antibodies, goat anti-ferret IgG (heavy and light chain specific; KPL, Gaithersburg, Md.) was used. This is the only anti-ferret immunoglobulin reagent available. This reagent is raised against a purified immunoglobulin fraction of ferret serum which contains IgG, as well as IgM and IgA, and reacts with ferret IgG and a number of other ferret serum proteins thought to be IgA and IgM (4a). After being washed, the plates were developed with 100 p1 of a 1-mg/ml solution of p-nitrophenyl phosphate in glycine buffer (pH 9.6) per well for 1 h. The A410 in each well was measured by using a Dynatech MR 700 Microtiter Plate Reader. The antibody titer was defined as the reciprocal of the highest dilution yielding an A410 of 0.05 above wells which contained antigen and which were incubated with the antibody conjugate but without the primary antibody sample (25). Statistical analysis. Comparisons among experimental groups were evaluated by one-way analysis of variance (ANOVA) with Fisher's protected-least-difference test, as well as by Kruskall-Wallis using Statview 512 software
INFECT. IMMUN. 6
0
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0) 0 -j
0 -J
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0
I3 1-
>1-
v I2 2o
0
0
.0 C
4
4 0
Intestinal IgA Intestinal IgG Serum IgA
1
Serum IgG
FIG. 1. Murine antibody titers after oral immunization with H. pylori (HP). Seronegative BALB/c mice (7 to 12 per group) were intragastrically immunized with four doses of 1 mg of H. pylori lysate either in the presence or in the absence of 10 ,ug of CT. Five days after the final immunization, the animals were sacrificed and anti-H. pylori ELISA titers were determined for gastrointestinal secretions and sera. The error bars show standard deviations.
(Brainpower, Calabasas, Calif.) for the Apple Macintosh computer. ANOVA was used for multiple inferences. RESULTS In the first experiment, we investigated the murine immune response to oral immunization with H. pylori lysate alone or in the presence of CT (a mucosal adjuvant). We have previously shown that optimal immunization of murine mucosa-associated lymphoid tissue can be achieved by intragastrically immunizing mice four times over a 4-week period (25). Additionally, mucosal adjuvants, such as CT, when mixed with protein antigens, can enhance gastrointestinal immune responses (8, 16, 18, 25). In this experiment, seronegative mice were actively immunized with 1 mg of H. pylori lysate with or without 10 pug of CT. As shown in Fig. 1, oral immunization without CT induced detectable serum IgA and IgG H. pylori-specific antibodies. In this experiment, nonimmunized controls had antibody levels below the limit of detection (data not shown). When CT was included in the immunization protocol, these responses were significantly enhanced. Specifically, there was a 5-fold increase of IgA in serum (F = 73.9, H = 20.9, P < 0.001) and a 16-fold increase of IgA in intestinal secretions (F = 52.9, H = 20.0, P < 0.001) compared with animals which did not receive CT. Elevations of IgG in serum (F = 14.2, H = 13.8, P = 0.001) and intestinal secretions (F = 7.3, H = 9.4, P < 0.009) were also noted. It has not been possible to infect rodents with H. pylori, but recent results suggest that the ferret may be a useful model for the study of Helicobacter-induced chronic gastritis. Therefore, ferrets were orally immunized against H. pylori lysate with or without CT. Preliminary dose-response studies were used to determine a safe dose of CT for ferrets. No untoward side effects, such as weight loss or diarrhea, were noted in any ferrets that received CT doses of up to 60 pug. Therefore, in subsequent experiments, 60 p.g of CT was used per dose of antigen. Ferrets are naturally colonized shortly after birth with another member of the Helicobacter genus, H. mustelae. Since these two organisms are quite similar histologically (and perhaps antigenetically as well), it was necessary to demonstrate that they could be distinguished serologically by using purified outer membranes as an antigen in an
ORAL IMMUNIZATION AGAINST H. PYLORI
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0.6 H. mustelae outer membrane protein c-
0.5
z
0.4
-
-a
0.3
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CL
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0
0.2
H. pylori outer membrane protein
.0 a
0.1 0
0.0 0
10
20
30
40
50 C
COMPETING ANTIGEN (jg/ml) 0
FIG. 2. ELISA using outer membrane proteins can distinguish between H. pylori and H. mustelae. ELISA plates were coated with H. pylori outer membrane protein, a single dilution of serum from a ferret immunized with H. pylori was mixed with serial dilutions of either H. pylori (0) or H. mustelae (0) outer membrane proteins, and a competitive ELISA was run as described in Materials and Methods. The data are from one experiment and are representative of four independently tested sera.
1=.
0
0
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0
ELISA. As shown in Fig. 2, soluble H. pylori outer membrane proteins added to aliquots of serum from a ferret immunized with H. pylori effectively blocked the binding of ferret antibodies to H. pylori outer membrane proteins coated on an ELISA plate. In contrast, equal concentrations of soluble purified H. mustelae outer membrane proteins added to the serum exhibited only a small amount of antibody-blocking activity, even at high outer membrane protein concentrations. Figure 3A shows antibody titers to both H. pylori and H. mustelae in serum in an experiment wherein groups of four 6-week-old ferrets were orally immunized weekly for 5 weeks with 7 mg of H. pylori lysate with or without 60 ,ug of CT per dose. By nonparametric Kruskall-Wallis analysis, the anti-H. pylori antibody titers were significantly different at all time points (P c 0.02). As in the murine model, ferrets immunized with H. pylori with CT demonstrated elevated anti-H. pylori antibody titers in serum compared with ferrets immunized with H. pylori alone (P s 0.05 by ANOVA) or control animals (P s 0.0001 by ANOVA) at all time points. Since these animals were all naturally infected with H. mustelae, all were also found to have serum anti-H. mustelae antibodies which were not enhanced relative to those of nonimmunized control ferrets (Fig. 3B). Our long-term goals are to immunize the gut-associated lymphoid tissue and provide protection against Helicobacter-associated gastritis. Thus, the ferrets from this experiment were sacrificed and the gastric and intestinal antibody responses were determined by ELISA. The results (Fig. 4) show that oral immunization with H. pylori lysate in the presence of CT induced significant intestinal anti-H. pylori antibodies (P < 0.05 compared with the control by ANOVA). Although not statistically significant, gastric anti-H. pylori levels were also increased when CT was used. DISCUSSION
Gastritis and peptic ulcer disease are significant causes of morbidity and mortality. Recent studies from a number of laboratories have improved our understanding of gastroduo-
20
80
60
40
DAY
FIG. 3. Ferret anti-Helicobacter antibody titers in serum after immunization with H. pylori plus CT (O). 0, H. pylori; 0, control. Groups of four ferrets were immunized five times (arrows) with 7 mg of H. pylori lysate in the presence or absence of 60 ,ug of CT. Anti-H. pylori and H. mustelae ELISA antibody titers were determined for serially obtained serum samples. (A) Ferret anti-H. pylori antibody titers in serum. (B) Ferret anti-H. mustelae antibody titers in
serum.
denal disease. Specifically, investigators have demonstrated an association between gastric infection with H. pylori, formerly called Campylobacter pylori, and gastritis and peptic ulcer disease, offering new insights into the diagnosis and treatment of these common disorders. Immunologically mediated prevention of H. pylori infection with oral vaccines may be the ideal way to approach this problem. Numerous studies have demonstrated that pre-existing secretory IgA can interfere with the ability of enteric pathogens, such as enterotoxigenic Escherichia coli, to attach to epithelial cells and that oral immunization can reduce Streptococcus mutans colonization and prevent dental caries (1, 11, 22-24, 29). 14
HP.C
12
F=
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0 HP
1 0 ccNrTRoL. ~~~~~~~~~~~~~~~~~~~~~~~1
-
1
10 -
-10
8-
-8
6-
-6
4-
-4
2-2 Gastic Wash Intes nal Wash Titer vs H. Pylor
Gatic Wash Intestinal Wash Titers vs H. mustels
FIG. 4. Ferret anti-Helicobacter mucosal antibody titers after immunization with H. pylori (HP) plus CT. The three groups of ferrets whose serum responses are presented in Fig. 3 were sacrificed after 15 weeks, and anti-H. pylori titers were determined for gastric and intestinal washes.
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CZINN AND NEDRUD
Furthermore, because H. pylori infection is very rarely seen in children, vaccination could be undertaken prior to the onset of infection. The ideal H. pylori vaccine would act locally in the upper gastrointestinal tract and prevent the initial adherence to the gastric mucosa which is necessary for colonization. By using CT as an adjuvant, we successfully induced both IgA and IgG responses in the sera and gastrointestinal secretions of mice. Similar mucosal immunization protocols using attenuated viruses alone or killed virus plus CT have induced good gastrointestinal immune responses and been able to induce protective immunity (25, 26). Since the gastrointestinal tracts of ferrets are anatomically and physiologically very similar to those of humans, ferrets have been used to study other enteric infections and may be useful to further our understanding of H. pylori infection; recently, Fox et al. showed that the ferret could serve as a useful animal model for Helicobacter-associated gastritis (10). We conducted oral immunization studies with H. pylori antigens in ferrets colonized with H. mustelae, a Helicobacter organism indigenous to ferrets. H. mustelae and H. pylori are immunologically distinct, such that immunization with and measurement of antibodies to H. pylori were not affected by the presence of indigenous H. mustelae in the ferrets. In these experiments, we were able to induce anti-H. pylori antibodies in serum. The peak serum response was noted 3 weeks after the last immunization, with a 6- to 10-fold increase of anti-H. pylori antibodies. When 60 ,ug of CT was added to each oral immunization, significant enhancement of anti-H. pylori antibody titers in serum and gastrointestinal secretions occurred. These results suggest that it is possible to develop an oral immunization protocol for prevention of H. pylori infection and associated gastritis. ACKNOWLEDGMENTS This research was supported by National Institute of Health grants AI-25818 and HL-37117 and grant Z063 9-1 from the Cystic Fibrosis Foundation. We thank Howard Carr for technical assistance and Katie Chipps for manuscript assistance. REFERENCES 1. Abraham, S. N., and E. H. Beachey. 1985. Host defense against adhesion of bacteria to mucosal surfaces. Adv. Host Def. Mech. 4:63-88. 2. Booth, L., G. Holdstock, H. McBride, P. Hawtin, J. R. Gibson, A. Ireland, J. Banforth, E. DuBoulay, R. S. Lloyd, and A. D. Pearson. 1986. Clinical importance of Campylobacter pyloridis and associated serum IgG and IgA antibody responses in patients undergoing upper gastrointestinal endoscopy. J. Clin. Pathol. 39:216-219. 3. Borody, T., P. Cole, S. Noonan, A. Morgan, G. Ossip, J. Maysey, and S. Brandl. 1988. Long-term Campylobacter pylori recurrence post-eradication. Gastroenterology 94:A43. 4. Borsch, G. 1989. Searching for the best way to eradicate Campylobacter pylori. Long-term studies are still needed. Fortschr. Med. 107:471-472. 4a.Czinn, S. J. Unpublished data. 5. Czinn, S. J., H. Carr, L. Sheffler, and S. Aronoff. 1989. Serum IgG antibody to the outer membrane proteins of Campylobacter pylori in children with gastroduodenal disease. J. Infect. Dis.
159:586-589. 6. Czinn, S. J., and W. T. Speck. 1989. Campylobacter pylori: a new pathogen. Adv. Pediatr. Infect. Dis. 5:221-237. 7. Elson, C. O., and W. Ealding. 1984. Generalized systemic and mucosal immunity in mice after mucosal stimulation with chol-
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era toxin. J. Immunol. 132:2736-2741. 8. Elson, C. O., and W. Ealding. 1984. Cholera toxin feeding did not induce oral tolerance in mice and abrogated oral tolerance to an unrelated protein antigen. J. Immunol. 133:2892-2897. 9. Elson, C. O., W. Ealding, and J. Lefkowitz. 1984. A lavage technique allowing repeated measurement of IgA antibody in mouse intestinal secretions. J. Immunol. Methods 67:101-108. 10. Fox, J. G., C. Pelayo, N. S. Taylor, A. Lee, G. Otto, C. Murphy, and R. Rose. 1990. Helicobacter mustelae-associated gastritis in ferrets: an animal model of Helicobacter pylori gastritis in humans. Gastroenterology 99:352-361. 11. Fubara, E., and R. Freter. 1973. Protection against enteric bacterial infection by secretory IgA antibodies. J. Immunol. 111:395-403. 12. Gilman, R., R. Leon-Barva, A. Ramirez-Ramos, D. Morgan, S. Recavarron, and W. Spira. 1987. Efficacy of nitrofurans in the treatment of antral gastritis with Campylobacter pyloridis. Gastroenterology 92:1405. 13. Goodwin, C. S., B. J. Marshall, J. R. Warren, S. Blackbourn, and E. D. Blincow. 1989. Clearance of Campylobacter pyloridis and reduced duodenal ulcer relapse with bismuth and tinidazole compared to cimetidine, p. 368-369. In B. Kaijser and E. Falsen (ed.), Campylobacter IV. University of Goteborg, Goteborg, Sweden. 14. Graham, D. Y. 1989. Campylobacter pylori and peptic ulcer disease. Gastroenterology 96:615-625. 15. Hirschl, A. M., E. Hentschel, K. Schutze, H. Nemec, R. Potzi, A. Gangl, W. Weiss, M. Pletschette, G. Stanek, and M. Rotter. 1988. The efficacy of antimicrobial treatment in Campylobacter pylori associated gastritis and duodenal ulcer. Scand. J. Gastroenterol. 23:76-81. 16. Liang, X., M. E. Lamm, and J. G. Nedrud. 1989. Cholera toxin as a mucosal adjuvant for respiratory antibody responses in mice. Reg. Immunol. 2:244-248. 17. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 18. Lycke, N., and J. Holmgren. 1986. Strong adjuvant properties of cholera toxin on gut mucosal immune responses to orally presented antigen. Immunology 23:611-616. 19. Marshall, B., and J. R. Warren. 1984. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet i:1311-1314. 20. Marshall, B. J., K. R. Dye, M. Plankey, H. Frierson, S. Hoffman, R. Guerrant, and R. McCallum. 1988. Eradication of Campylobacter pylori infection with bismuth subsalicylate and antibiotic combinations. Am. J. Gastroenterol. 83:1035. 21. Mestecky, J., and J. R. McGhee. 1987. Immunoglobulin A (IgA): molecular and cellular interactions involved in IgA biosynthesis and immune response. Adv. Immunol. 40:153-245. 22. Mestecky, J., J. R. McGhee, R. R. Arnold, S. M. Michalek, S. J. Prince, and J. L. Babb. 1978. Selective induction of an immune response in human external secretions by ingestion of bacterial antigen. J. Clin. Invest. 61:731-737. 23. Michalek, S. M., J. R. McGhee, and J. L. Babb. 1978. Effective immunity to dental caries: dose-dependent studies of secretory immunity by oral administration of Streptococcus mutans to rats. Infect. Immun. 19:217-224. 24. Morisaki, I., S. M. Michalek, C. C. Harmon, M. Torii, S. Hamada, and J. R. McGhee. 1983. Effective immunity to dental caries: enhancement of salivary anti-Streptococcus mutans antibody responses with oral adjuvants. Infect. Immun. 40:577591. 25. Nedrud, J. G., X. P. Liang, N. Hague, and M. E. Lamm. 1987. Combined oral/nasal immunization protects mice from Sendai virus infection. J. Immunol. 139:3484-3492. 26. Ogra, P. L., D. T. Karzon, F. Righthand, and M. MacGillivray. 1968. Immunoglobulin response in serum and secretions after immunization with live and inactivated polio vaccine and natural infection. N. Engl. J. Med. 279:895-900. 27. Rauws, E. A. J., W. Langenberg, H. J. Houthoff, H. C. Zanen, and G. N. S. Tytgat. 1988. Campylobacter pyloridis-associated chronic antral gastritis. A prospective study of its prevalence
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and the effects of antibacterial and antiulcer treatment. Gastroenterology 94:33-40. 28. Sawai, T., R. Hiruma, N. Kawana, M. Kaneko, F. Tarriyasu, and A. Inami. 1982. Outer membrane permeation of beta-lactam antibiotics in Escherichia coli, Proteus mirabilis, and Enterobacter cloacae. Antimicrob. Agents Chemother. 22:585-592.
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29. Tagliabue, A., L. Villa, M. T. de Magistris, M. Romano, S. Silvestri, D. Boraschi, and L. Nencioni. 1986. IgA-driven T cell-mediated anti-bacterial immunity in man after live oral Ty 21a vaccine. J. lmmunol. 137:1504-1510. 30. Warren, J. R. 1983. Unidentified curved bacilli on gastric epithelium in chronic gastritis. Lancet i:1273-1275.
