Oral immunization of mice with a glycoconjugate vaccine containing ...

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Oral immunization of mice with a glycoconjugate vaccine containing the O157 antigen of Escherichia coli O157:H7 admixed with cholera toxin fails to elicit protection against subsequent colonization by the pathogen J. Wayne Conlan, Rhonda KuoLee, Ann Webb, Andrew D. Cox, and Malcolm B. Perry

Abstract: It has been postulated that a humoral immune response directed against the O157 antigen of Escherichia coli O157:H7, and expressed in the intestine, might afford protection from colonization and consequent infection by this enteric pathogen. The present study was conducted to determine whether such an immune response can be experimentally generated in mice. To this end, mice were orally immunized with a glycoconjugate vaccine consisting of horse serum albumin and the O157 polysaccharide admixed with the mucosal adjuvant, cholera toxin. Mice consistently developed robust local and systemic immune responses to the cholera toxin adjuvant, but were far from uniformly reactive to the test vaccine. Moreover, vaccinated mice were as susceptible to transient intestinal colonization following challenge with an isolate of E. coli O157:H7 as unvaccinated control mice. These results indicate that this vaccination approach is unlikely to be straightforward in target bovine or human hosts. Key words: Escherichia coli O157:H7, glycoconjugate vaccine, mucosal immunity, mice. Résumé : On croit qu’une réponse immunitaire humorale dirigée contre l’antigène O157 d’Escherichia coli et exprimée au niveau intestinal pourrait conférer une protection contre la colonisation par ce pathogène entérique et l’infection subséquente. La présente étude a cherché à vérifier si une telle réponse immunitaire pouvait être produite expérimentalement chez la souris. À cette fin, des souris ont été immunisées par voie orale avec un vaccin glycoconjugué, soit le polysaccharide O157 et l’albumine sérique de cheval, mélangé à la toxine du choléra qui est un adjuvant au niveau des muqueuses. Les souris ont systématiquement développé une forte immunité locale et généralisée contre la toxine du choléra, mais la réponse au vaccin évalué a été très hétérogène. De plus, comparativement aux animaux témoins non vaccinés, les souris vaccinées se sont révélées aussi sensibles à la colonisation intestinale et à l’infection par un isolat d’E. coli O157:H7. Les résultats obtenus ici laissent croire que cette approche vaccinale n’est peut-être pas aussi simple chez les hôtes ciblés dont les humains et les bovins. Mots clés : Escherichia coli O157:H7, vaccin glycoconjugué, immunité des muqueuses, souris. [Traduit par la Rédaction]

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Introduction The enteric bacterium Escherichia coli O157:H7, is a harmless commensal of cattle, but a highly virulent pathogen for susceptible humans (reviewed in Su and Brandt 1995). Dissemination of this organism from the feces of the former host to the food chain of the latter host is considered to be the primary means of transmission of human infection. In humans, the pathogen colonizes the intestinal wall where it can Received August 25, 1999. Revision received November 15, 1999. Accepted November 29, 1999. J.W. Conlan,1 R. KuoLee, A. Webb, A.D. Cox, and M.B. Perry. National Research Council Canada, Institute for Biological Sciences, Ottawa, ON K1A 0R6, Canada. 1 Author to whom all correspondence should be addressed (email: [email protected]). Can. J. Microbiol. 46: 283–290 (2000)

cause a localized severe hemorrhagic colitis (Su and Brandt 1995). Additionally, a systemic toxemia, hemolytic uremic syndrome (HUS), can develop which can lead to kidney failure and death. Some epidemiological evidence suggests that treating E. coli O157:H7 infection with certain antibiotics might promote the development of HUS (reviewed in Neill 1998), though this opinion remains controversial. For this reason, other intervention strategies to combat this bacterium are being sought. In this regard, the possibility of mass vaccinating cattle, or of selectively vaccinating susceptible human populations, has been suggested (Tauxe 1998). The polysaccharide O157 somatic antigen of this pathogen has been identified as a vaccine candidate (Konadu et al. 1994). Coupled to protein carriers, the O157 antigen has been shown to elicit a systemic humoral immune response in both mice (Konadu et al. 1994; Conlan et al. 1999a) and humans (Konadu et al. 1998). Moreover, O157-specific mouse and human antisera have been © 2000 NRC Canada

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shown to be capable of killing E. coli O157:H7 in vitro in the presence of xenogeneic rabbit complement (Konadu et al. 1994; Konadu et al. 1998). Additionally, there are several precedents showing that antibodies directed against lipopolysaccharides of other enteric bacterial pathogens afford protection against the respective organisms in vivo (Michetti et al. 1992; Winner et al. 1991). Finally, because the O157 antigen is essentially specific for the pathogen, the possibility of immune elimination of potentially beneficial commensal strains of E. coli is curtailed. However, because E. coli O157:H7 is not an enteroinvasive pathogen, in nature, an effective O157antigen-based vaccine against this organism presumably will have to provide protective antibodies locally at sites of infection in the intestinal mucosa. Some believe that systemic antibodies will naturally transude to such sites from the blood (Konadu et al. 1998). However, a recent study from this laboratory implies that this is unlikely to be the case (Conlan et al. 1999a). Instead, it is widely believed that local enteric antibodies, essentially secretory IgA, can only be produced by stimulating the common mucosal immune system by immunization of either gut-associated or other more distal mucosa-associated lymphoid tissues (McGhee and Kiyono 1994). Traditionally, it has been assumed that direct immunization of the gut associated lymphoid tissue by oral vaccination affords the best means of eliciting this type of protective immunity against enteric and enteroinvasive pathogens (reviewed in O’Hagan 1994; Shalaby 1995). This strategy is very effective when live attenuated vaccines are employed (reviewed in Curtiss 1990; Shalaby 1995). By contrast, nonviable oral vaccines need to be administered with potent mucosal adjuvants such as cholera toxin (CT) in order to elicit useful enteric immune responses (reviewed in Hornquist et al. 1994; Jackson et al. 1993). In light of this, the present study was undertaken to assess the immunogenicity and protective potential of an O157antigen-containing glycoconjugate oral vaccine adjuvanted with cholera toxin using a mouse model of benign transient colonization by the pathogen. All of the methods used in the present study have been described in detail elsewhere (Conlan et al. 1998, 1999a). Briefly, 8-week-old specific pathogen free BALB/c and C57BL/6 mice, which have been shown to be susceptible to transient intestinal colonization by E. coli O157:H7 (Conlan et al. 1998), were obtained from Charles Rivers Laboratories (St. Constant, Que). Mice were maintained and used in accordance with the recommendations of the Canadian Council Guide to the Care and Use of Experimental Animals (1993). Mice entered experiments when they were 10 weeks old. The O157-antigen-horse-serum-albumin glycoconjugate was prepared as previously described (Conlan et al. 1999a), and has been shown to elicit serum antibodies in mice against O157 antigen when administered parenterally in incomplete Freund’s adjuvant. Briefly, O-antigen (5 mg/mL in saline) on ice was adjusted to pH 10.5–11.0 with 0.1 M NaOH. An equal weight of cyanogen bromide (1 g/mL in acetonitrile) was added, and the mixture maintained on ice for 6 min at pH 10.5–11.0. Next, an equal volume of 0.8 M adipic acid dihydrazide (ADH; Sigma Chemical Co.) in 0.5 M NaHCO3 was added, and the pH adjusted to 8.5. This reaction mixture was stirred overnight at 3–8°C, then

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dialysed against saline for 24 h, and then water for 2 days. The retentate was lyophilised. On ice, the ADH-activated O-antigen was dissolved in saline (5 mg/mL), and an equal weight of horse serum albumin (HSA; Sigma Chemical Co.) was added, and the pH adjusted to 5.1–5.5. Next, 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDAC; Sigma Chemical Co.) was added to the reaction mixture to a final concentration of 0.05 M, and the pH maintained at 5.1 to 5.5 for 4 h. Then, the reaction mixture was dialysed against saline for 2 days. After this time, the retentate was lyophilised, re-dissolved in saline and eluted through a Sephadex G-100 column (100 × 2.5 cm) and the eluate was monitored for protein at 280 nm absorbance. Column fractions were individually screened for carbohydrate by the method of Dubois et al. (1956), and fractions positive for both carbohydrate and protein were pooled, lyophilised, re-dissolved in water, and dialysed against water for 24 h to remove NaCl. The retentate was lyophilised yielding the conjugate O157 antigen-HSA vaccine. The glycoconjugate contains 40% w/w carbohydrate all of which is provided by the O157 antigen. In the present study, some mice (group A) were vaccinated by gavage at intervals with 125 µg of glycoconjugate dissolved in PBS, and admixed with 5 µg CT (Cedarlane Laboratories, Hornby, Ont.). Other mice (group B) were similarly vaccinated with 75 µg HSA (equivalent to the amount of HSA present in the conjugate) admixed with 5 µg CT, whilst a third group (group C) received no treatment. The streptomycin-resistant isolate of E. coli O157:H7 employed, its method of propagation for inoculation into mice, and the method for its isolation from mouse feces have been fully described in earlier publications (Conlan et al. 1998, 1999a). Briefly, for each experiment, fresh inocula were prepared in brain heart infusion broth containing streptomycin (SBHI). Flask inocula were incubated at 37°C with shaking to an absorbance at 600 nm of approximately 1.1. Inocula were harvested by centrifugation at 7200 g at ambient temperature and resuspended in phosphate buffered saline (PBS) to a concentration of approximately 1011 CFU/mL. In each case, the viability of the inoculum was determined by plating serial dilutions of it on SBHI agar. Mice were inoculated with the E. coli O157:H7 isolate (0.2 mL inoculum/mouse) by gavage using a 1-mL tuberculin syringe fitted with a 20 g gavage needle. At various intervals post-challenge, fresh fecal pellets expressed during a 15-min collection time were obtained from individual animals and transferred into preweighed tubes of sterile PBS. Feces were thoroughly resuspended by vortex mixing and trituration using a 1-mL serological pipette, then serially diluted and plated on SBHI agar. Plates were incubated overnight at 37°C and E. colilike colonies were counted. The identity of random colonies was confirmed by a slide agglutination test using a monoclonal antibody specific for the O157 antigen (Perry et al. 1988). Bacterial burdens per unit weight of feces were compared among various groups of mice using the Mann Whitney rank sum test to assess statistically significant (P < 0.05) differences. Fecal shedding periods between groups were also compared for statistically significant differences. For individual mice, the fecal shedding period was calculated as the last day of examination on which the pathogen © 2000 NRC Canada

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Fig. 1. Specific serum (A), and fecal (B) antibodies in vaccinated mice. BALB/c and C57BL/6 (BL6) mice were vaccinated with either the conjugate or HSA alone, or were left untreated. Five days after the third vaccination, sera and fecal extracts were assayed for the presence of O157-specific IgG (䊉) and IgA (䊊), HSA-specific IgG (䊏) and IgA (ⵧ), and CT-specific IgG (䉱) and IgA (䉭). The means of duplicate samples from individual mice are shown (in all cases duplicate samples gave results that varied from each other by less than 50%).

was cultured from the feces. For each group, fecal shedding periods were plotted as survival curves which were then compared for statistical differences by log-rank test. The ELISA assays were performed as previously described (Conlan et al. 1998, 1999a) using E. coli O157 LPS or HSA as coating antigen at a concentration of 1 µg/well, or cholera toxin at 0.25 µg/well. Sera were prepared from tail

blood, and were assayed at 1/100 dilution in duplicate for the presence of IgG or IgA against each test antigen. Fecal supernatants for copro-antibody determinations were prepared as described in previous publications (Conlan et al. 1998, 1999a) and were assayed at a dilution of 1/10 as for sera above. Following the addition of ELISA substrate, the colour that developed was read at 410 nm, and served as a mea© 2000 NRC Canada

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Fig. 2. Fecal burdens of E. coli O157:H7 following oral challenge with the pathogen. BALB/c mice (A) and C57BL/6 mice (B) were immunized with test (ⵧ) or control (䊊) vaccine admixed with cholera toxin, or were left unvaccinated (䉭). Following vaccination, mice were challenged by gavage with 2.84 × 1010 cfu/mouse of the test isolate of E. coli O157:H7. On days 1, 3, 7, and 10 postchallenge, the bacterial burdens in the feces were determined. Bacterial levels for individual mice are shown. Horizontal broken lines depict the lower detection limits.

sure of specific antibody levels. Positive and negative control sera or fecal extracts were included on each plate as appropriate. For each sample, the duplicate readings were averaged, and the groups were then compared for statistical differences using the Mann-Whitney test. At the start of the study, 15 mice per group (90 mice in total) of each strain were vaccinated with either the glycoconjugate (group A) plus CT, or the carrier protein plus CT (group B), and a further 15 mice of each strain were left un-

treated (group C). All group A and group B mice received booster vaccinations at 14 days, and again 28 days later. Blood and feces were collected from a representative five mice per group 5 days after the third vaccination, and antibody levels were determined. The results are presented in Fig. 1. Local IgA and systemic IgG and IgA antibodies against CT were found in all group A and B mice. In the case of vaccination with the glycoconjugate admixed with cholera toxin, C57BL/6 mice generally displayed higher se© 2000 NRC Canada

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Fig. 3. Specific serum antibody levels in mice following immunization with test and control vaccines and oral challenge with E. coli O157:H7. On day 9 of bacterial challenge, sera were prepared from the BALB/c (A) mice and C57BL/6 (B) mice depicted in Fig. 2. These sera were diluted 100-fold, then analysed for the presence of O157-specific IgG (䊉) and IgA (䊊), HSA-specific IgG (䊏) and IgA (ⵧ), and CT-specific IgG (䉱) and IgA (䉭). Sera from immunized, but unchallenged mice were also analysed. Following the addition of ELISA substrate, the coloured product was read at 410 nm after 40 min incubation at ambient temperature. The means of duplicate samples from individual mice are shown (in all cases duplicate samples gave results that varied from each other by less than 50%).

rum IgG and IgA and fecal IgA anti-CT responses, than did BALB/c mice, though these differences were not always statistically significant. This was generally the case too in mice vaccinated with HSA plus CT. Some BALB/c, but no C57BL/6 mice displayed measurable systemic or local anti-

bodies to either HSA or O157 antigen. The relative contribution of locally-produced secretory IgA versus hepatobiliarytransported (Mestecky et al. 1991) systemic IgA to the total levels of detectable copro-antibodies is unknown. IgG isotype antibodies were never detected in fecal extracts. Se© 2000 NRC Canada

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Fig. 4. Specific copro-antibody levels in mice following immunization and bacterial challenge. On day 9 of bacterial challenge, fecal extracts were prepared from the BALB/c (A) mice and C57BL/6 (B) mice depicted in Fig. 2. These extracts were diluted 10-fold and were analysed for the presence of O157-specific IgG (䊉) and IgA (䊊), HSA-specific IgG (䊏) and IgA (ⵧ), and CT-specific IgG (䉱) and IgA (䉭). Feces from immunized, but unchallenged mice were also analysed. Following the addition of ELISA substrate, the coloured product was read at 410 nm after 30 min incubation at ambient temperature. The means of duplicate samples from individual mice are shown (in all cases duplicate samples gave results that varied from each other by less than 50%).

rum or copro-antibodies to the three test antigens were never detected in group C control mice. Next, to determine whether vaccination with the glycoconjugate had elicited any protective immunity uncorrelated to coproantibody levels, five representative BALB/c mice

and C57BL/6 mice from groups A–C were each challenged by gavage 10 days following the third vaccination with 2.84 × 1010 CFU of the test isolate of E. coli O157:H7. Subsequently, fecal burdens (Fig. 2) and fecal shedding periods of the pathogen were monitored over a 10 day period and © 2000 NRC Canada

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were analysed statistically as previously described (Conlan et al. 1998, 1999a). No significant differences were found in either the loads or the periods of fecal shedding of E. coli O157:H7 between group A and group C mice of either strain. Blood and feces were collected from challenged and unchallenged mice nine days following exposure of the former to E. coli O157:H7, and were analysed for specific antibody levels (Figs. 3 and 4). Compared to their unvaccinated counterparts, or mice vaccinated with carrier protein, BALB/c mice vaccinated with the glycoconjugate consistently developed measurable systemic O157-specific IgG antibodies following challenge with E. coli O157:H7 (Fig. 3). These mice also consistently produced systemic O157-antigen-specific IgA antibodies, but so too did challenged mice vaccinated with the carrier protein, suggesting that this was more a reflection of the primary response to infection than a result of prior specific vaccination. The systemic humoral immune response of C57BL/6 mice to infection was far less consistent. In the feces, very little O157-specific or HSA-specific IgA was detected in either vaccinated or unvaccinated challenged or unchallenged mice of either strain (Fig. 4). Interestingly, challenge appeared to suppress the local (P < 0.05), but not the systemic anti-CT response in both strains of mice. Cohen et al. (1990) have provided a possible explanation for this phenomenon. Namely, that IgA-secreting B cells are susceptible to cytolysis by shiga-like toxins. To verify the aforementioned findings, mice not challenged with E. coli O157:H7 received booster inoculations of the appropriate vaccine on day 79, and a group of each was challenged five days later with 5.6 × 1010 CFU of the test isolate of E. coli O157:H7. Fecal burdens of the pathogen were determined on days 3, 7, 10, and 14 postchallenge. Blood and feces for antibody determinations were collected on day 8 from both challenged mice and their unchallenged counterparts (data not shown). Again, only antiCT antibodies were consistently detected in the sera and feces of unchallenged BALB/c mice and C57BL/6, and there was no obvious evidence that vaccination with the glycoconjugate conferred any protection, measured as a decrease in fecal load or period of fecal shedding, against colonization of E. coli O157:H7. Overall the findings of this study indicate that oral vaccination with a glycoconjugate containing the O157 antigen, even when administered with a known potent mucosal adjuvant, is not a straightforward means of eliciting the desired local humoral immune response, namely O157-antigen-specific IgA. This finding is in keeping with that of others, and might be a reflection of the poor immunogenicity of the carbohydrate moiety of the test antigen (Childers et al. 1990). Alternatively, the glycoconjugate might not have survived the hydrolytic gastrointestinal environment. Such barriers could be overcome by packaging the vaccine in liposomes (Childers et al. 1990), or proteosomes (Orr et al. 1993), or surpassed by vaccinating via a different mucosal route such as the intranasal route (Orr et al. 1993). Regardless, even if more elaborate formulations of such a vaccine displayed the desired immunological profile, for agricultural use, any such sophisticated vaccine would be less economically viable than a simple vaccine. By contrast, a recently developed live

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Salmonella-based vaccine (Conlan et al. 1999b) does elicit the desired local anti-O157 immune response, can be produced at low cost, and is currently undergoing field trials to assess its efficacy.

Acknowledgements We thank the animal resources staff of the Institute for Biological Sciences for the quality husbandry services provided.

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