International Journal of
Molecular Sciences Communication
Mechanisms Underlying the Immune Response Generated by an Oral Vibrio cholerae Vaccine Danylo Sirskyj 1,2 , Ashok Kumar 1,2,3 and Ali Azizi 3, * 1 2 3
*
Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada;
[email protected] (D.S.);
[email protected] (A.K.) Children’s Hospital of Eastern Ontario (CHEO)-Research Institute, Ottawa, ON K1H 5B2, Canada Department of Pathology and Laboratory Medicine, University of Ottawa, 451 Smyth Rd, Ottawa, ON K1H 8M5, Canada Correspondence:
[email protected]; Tel.: +1-613-737-7600
Academic Editor: Ester Boix Received: 14 April 2016; Accepted: 28 June 2016; Published: 2 July 2016
Abstract: Mechanistic details underlying the resulting protective immune response generated by mucosal vaccines remain largely unknown. We investigated the involvement of Toll-like receptor signaling in the induction of humoral immune responses following oral immunization with Dukoral, comparing wild type mice with TLR-2-, TLR-4-, MyD88- and Trif-deficient mice. Although all groups generated similar levels of IgG antibodies, the proliferation of CD4+ T-cells in response to V. cholerae was shown to be mediated via MyD88/TLR signaling, and independently of Trif signaling. The results demonstrate differential requirements for generation of immune responses. These results also suggest that TLR pathways may be modulators of the quality of immune response elicited by the Dukoral vaccine. Determining the critical signaling pathways involved in the induction of immune response to this vaccine would be beneficial, and could contribute to more precisely-designed versions of other oral vaccines in the future. Keywords: oral vaccine; Toll-like receptor; humoral immunity; Vibrio cholerae
1. Toll-Like Receptors and Dukoral Vaccine The mechanisms responsible for the induction of protective immune responses from mucosal vaccines are not yet clear. Since most infections start at mucosal surfaces, an understanding of the precise mechanistic and signaling details underlying a licensed mucosal vaccine could assist in the development of new vaccines [1–3]. Dukoral is an orally administered vaccine, to protect against travellers’ diarrhea caused by ETEC, as well as for the prevention of cholera, caused by Vibrio cholera [4,5]. The Dukoral vaccine is containing V. cholerae (comprised of heat-inactivated V. cholerae 01 Inaba classic strain and Ogawa classic strain, and formalin-inactivated V. cholerae 01El Tor strain and Ogawa classic strain) along with the recombinant cholera toxin B-subunit protein (CTB) [4,5]. Included with the vaccine is a bicarbonate buffer to be ingested together with the vaccine at the time of immunization, for the purpose of neutralizing residual stomach acid in order to protect the integrity of the vaccine antigens. Mechanistic details on precise immune pathways involved in the induction of an immune response to this vaccine are largely lacking from the product monograph. Early immune responses against invading pathogens occur through the sensing of multiple microbial structures [6] by receptors, including Toll-like receptors (TLRs) [7]. TLRs are expressed predominantly on monocytes/macrophages, dendritic cells (DCs), B-cells, and T-cells [8–13]. All TLRs, excluding TLR-3, utilize the MyD88 signaling adaptor to induce the production of proinflammatory cytokines by way of the NF-κB and other transcription factors [14–16]. Alternatively, TLR-4 has been shown to utilize the MyD88 and Trif signaling adaptors, in order to induce the production of Int. J. Mol. Sci. 2016, 17, 1062; doi:10.3390/ijms17071062
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proinflammatory cytokines and type I interferons by way of NF-κB and interferon regulatory factor 3 (IRF-3) [16]. Trif has been described as such a signaling adaptor molecule in the MyD88-independent signaling pathway, stimulated by TLR-3 and TLR-4 [17,18]. To date, the precise requirement of TLR signaling for the generation of protective antibody responses to antigens from licensed commercial vaccines is not clear. Our group began uncovering such mechanistic details, by examining the requirement of TLR signaling (specifically, the requirement of MyD88, Trif, TLR-2 and TLR-4 signaling) in the induction of immune responses in mice, following immunization with the Dukoral vaccine. All subsequent animal studies utilized female mice of the C57BL/6 background, between 7 and 10 weeks of age (Jackson Laboratory, Bar Harbor, ME, USA). At the time these experiments were carried out additional mutants were only available on the Balb/C background. As such, to ensure uniformity of the experimental results and to ensure that all animals were of the same genetic background, groups of TLR-2-, TLR-4-, MyD88-, Trif-deficient mice, as well as wild type mice, were investigated. Mutant strains were described by the supplier as having large deletions of their respective TLR gene which eliminated the expression of TLR mRNA and corresponding protein. To evaluate the precise mechanistic underlying the immune responses generated by the Dukoral vaccine, the role of other TLRs (using TLR-deficient mice with the same genotype) should be also investigated. In this study, animals were housed in micro-isolator cages under specific-pathogen free conditions, with food and water provided ad libitum. All mice being orally immunized were fasted for at least 4 h prior to immunization, and first received a 100 µL dose of sodium hydrogen carbonate buffer to neutralize residual stomach acid. All neutralizing buffer and vaccine doses were administered intra-gastrically by gavage needle. To determine the optimal oral dose of the Dukoral vaccine in animals, C57BL/6 WT mice were orally immunized with various amounts of V. cholerae along with 10 µg CTB, on days 0, 10, 20 and 30. Serum and feces were collected before and after each immunization. C57BL/6 mice receiving 3 ˆ 109 V. cholerae with 10 µg CTB showed the highest V. cholerae and CTB-specific serum IgG and fecal IgA responses. Since this dose of vaccine resulted in the highest V. cholerae specific serum and fecal antibody responses, four oral immunizations with 3 ˆ 109 V. cholerae and 10 µg CTB were found to be the optimal oral dose of Dukoral vaccine for C57BL/6 mice (data not shown). Oral vaccine doses were administered in a 100 µL volume. TLR mutant mice and WT controls (n = 5 mice per group) were orally immunized on days 0, 10, 20, and 30, with 3 ˆ 109 V. cholerae and 10 µg CTB. Pre- and post-vaccination sera and feces were collected. Blood was collected via saphenous vein puncture, centrifuged to obtain serum, and stored at ´20 ˝ C until used. Fecal pellets were collected and stored at ´80 ˝ C prior to use. To extract fecal antibody, 100 mg of feces per mouse was weighted out, then dissolved in 1 mL PBS containing 2.5% non-fat milk with complete mini EDTA-free protease inhibitors (Roche Applied Science, Laval, QC, Canada). Fecal pellets were broken up using a pipette tip, vortexed, and incubated on ice for 1 h with intermittent vortexing. Next, samples were centrifuged for 15 min at 4 ˝ C to pellet debris. The supernatant was then collected and stored at ´80 ˝ C until analyzed. None of the TLR-deficient animals (MyD88´/´ , Trif´/´ , TLR-2´/´ , and TLR-4´/´ ) showed any significant impairment in the generation of V. cholerae-specific or CTB-specific serum antibodies compared to those generated in WT mice, at any time following immunization. The V. cholerae-specific antibody titers following various immunizations are shown in Figure 1. These findings suggest that TLR signaling may not be essential for the induction of IgG in sera. IgA is the predominant antibody on mucosal surfaces and is the primary line of protection against pathogens. Therefore, the requirement of TLR signaling for the generation of fecal antigen-specific IgA antibody was investigated. Fecal samples from orally immunized mice were collected and tested for the presence of vaccine antigen-specific IgA antibody responses. For measuring fecal IgA antibody responses, fecal pellets was processed and 50 µL of clarified fecal supernatant at an undiluted, 1:10, and 1:100 dilution was applied to antigen-coated plates. While none of the groups of TLR-deficient animals showed significantly impaired V. cholerae-specific fecal IgA antibody production (data not shown),
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IgA antibody production (data not shown), MyD88−/− and TLR-2−/− mice were significantly impaired ´/´ and TLR-2´/´ mice were significantly impaired in their ability to produce CTB-specific MyD88 in their ability to produce CTB-specific fecal IgA (p = 0.02 and p = 0.03 respectively) (Figure 2). These fecal IgA (p = 0.02that andTLR p = 0.03 respectively) results suggest that TLR signaling may results suggest signaling may be (Figure required2). forThese the generation of CTB-specific IgA but not be V. required for the generation of CTB-specific IgA but not V. cholerae-specific IgA antibodies. cholerae-specific IgA antibodies.
Figure 1. TLR signaling is dispensable for V. cholerae-specific antibody production following oral Figure 1. TLR signaling is dispensable for V. cholerae-specific antibody production following oral immunization with Dukoral vaccine. TLR mutant mice and WT controls (n = 5 mice per group) were immunization with Dukoral vaccine. TLR mutant mice and WT controls (n = 5 mice per group) were orally immunized on days 0, 10, 20, and 30, with 39 × 109 V. cholerae and 10 μg CTB. Serum was orally immunized on days 0, 10, 20, and 30, with 3 ˆ 10 V. cholerae and 10 µg CTB. Serum was collected collected 9 days after each vaccination and V. cholerae-specific antibody titer against A, IgG1, 9 days after each vaccination and V. cholerae-specific antibody titer against A, IgG1, B, IgG2a, and6 C, B, IgG2a, and C, IgG-Fc was measured by ELISA. The plates were coated with 50 μL of 12 × 10 IgG-Fc was measured by ELISA. The plates were coated with 50 µL of 12 ˆ 106 V. cholerae/mL. The V. cholerae/mL. The next day, coating buffer was decanted and plates were washed once in PBS. next day, coating buffer was decanted and plates were washed once in PBS. Plates were then blocked Plates were then blocked for 2 h at 37 °C with 2% FCS-PBS. After blocking, plates were washed once for 2 h at 37 ˝ C with 2% FCS-PBS. After blocking, plates were washed once in PBS and diluted samples in PBS and diluted samples were added to duplicate wells. After incubating for 1 h at 37 °C, plates were added to duplicate After incubating at °C 37 ˝with C, plates washed with PBS and were washed with PBSwells. and then incubated for 1for h 1ath37 50 μLwere of secondary horseradish ˝ C with 50 µL of secondary horseradish peroxidase (HRP)-conjugated then incubated for 1 h at 37 peroxidase (HRP)-conjugated secondary antibody in 2% FCS-PBS (goat anti-mouse A. IgG1, B. secondary in 2% were FCS-PBS anti-mouse A. IgG1, IgG2a, C. IgG-Fc). Plates were then IgG2a, C.antibody IgG-Fc). Plates then (goat washed and developed withB.3,3′,5,5′-tetramethylbenzidine (TMB) washed and developed with 3,31substrate. ,5,51 -tetramethylbenzidine (TMB)by one component HRP microwell one component HRP microwell The reaction was stopped stop solution and plates were substrate. reaction stopped stop and platesline. were read on 450 nm. The naïve read on The at 450 nm. Thewas naïve controlby data aresolution shown as dashed Results are at shown as the mean control data are shown as dashed line. Results are shown as the mean log of O.D 450 nm ˘ SEM. P0V log of O.D 450 nm ± SEM. P0V denotes pre-vaccination serum. P1V−P4V denotes post-1st vaccination denotes pre-vaccination serum. P1V´P4V denotes post-1st vaccination through post-4th vaccination through post-4th vaccination time points. The production of IgG, IgG1, and IgG2c was not time points. The production of IgG, IgG1, and IgG2c was not statistically significant (p ě 0.05). statistically significant (p ≥ 0.05).
study involvementofofTLR TLRsignaling signaling in in the the generation serum ToTo study thethe involvement generationof ofprotection protectionby bythe thevaccine, vaccine, serum antibodies were evaluated using an in vitro agglutination assay using live V. cholerae. Samples were antibodies were evaluated using an in vitro agglutination assay using live V. cholerae. Samples were tested against three 01 Inaba strains, two 01 Ogawa strains, and one 01 Inaba El Tor strain, for a total tested against three 01 Inaba strains, two 01 Ogawa strains, and one 01 Inaba El Tor strain, for a total of six strains. The agglutination assay was performed in the National Microbiology Laboratory at of six strains. The agglutination assay was performed in the National Microbiology Laboratory at Public Health Agency of Canada (Winnipeg, MB, Canada). The results demonstrated that serum Public Health −/− Agency of Canada (Winnipeg, MB, Canada). The results demonstrated that serum from −/− animals was unable to agglutinate live V. cholerae, despite those animals from TLR-2 and TLR-4 ´ / ´ ´ / ´ TLR-2 and TLR-4 animals was unable to agglutinate live V. cholerae, despite those animals being being unimpaired in their ability to generate V. cholerae or CTB specific serum IgG antibodies. unimpaired in their ability generate V. MyD88 cholerae−/−orand CTBTrif specific serum −/− mice Surprisingly, serum fromtoimmunized was IgG ableantibodies. to induce Surprisingly, V. cholerae ´ / ´ ´ / ´ serum from immunized MyD88 and Trif mice was able to induce V. cholerae agglutination.
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Int. J. Mol. Sci. 2016, 17, 1062 4 of 6 agglutination. While it is not yet clear how MyD88−/− mice showed agglutination to live V. cholerae ´/´ mice showed agglutination to live V. cholerae while TLR-2´/´ −/− and While it is not yet clear how−/−MyD88 while TLR-2 TLR-4 animals did not, we assume that the Dukoral vaccine might activate −/− mice showed agglutination to live V. cholerae agglutination. Whiledid it isother notwe yet clear how MyD88 and TLR-4´/´receptors animals not, assume that the Dukoral vaccineTLR10), might activate additional receptors additional or TLRs (e.g., TLR1, TLR5, TLR6, subsequently activating −/− −/− while TLR-2 and TLR-4 animals did not, we assume that the Dukoral vaccine might activate ornon-MyD88 other TLRs pathways (e.g., TLR1, TLR5, TLR10), non-MyD88 (data (data not TLR6, shown). Due to subsequently the limitationsactivating in the volume of serum pathways collected, the additional receptors or other (e.g., TLR1, TLR5, TLR10), subsequently activating agglutination assay could only beTLRs performed once. experiments should be executed to not shown). Due to the limitations in the volume ofAdditional serum TLR6, collected, the agglutination assay could non-MyD88 pathways (data not shown). Due to the limitations in the volume of serum collected, the confirm the obtained data. only be performed once. Additional experiments should be executed to confirm the obtained data. agglutination assay could only be performed once. Additional experiments should be executed to confirm the obtained data.
Figure TLRsignaling signalingmight might mediate mediate the fecal IgA antibody production. Figure 2.2.TLR the generation generationofofCTB-specific CTB-specific fecal IgA antibody production. −/−, TLR-4−/−, MyD88−/− and Trif−/−) and WT controls (n = 5 per group) were TLR mutant (TLR-2 ´/´ ´/´ ´/´ ´/´ TLR mutant (TLR-2 , TLR-4 , MyD88 and Trif ) and WT controls (n = 5 per group) were Figure 2. TLR signaling might of CTB-specific fecal IgA antibody production. immunized orally on days 0, 10,mediate 20, andthe 30 generation with Dukoral (3 × 109 9V. cholerae with 10 μg CTB). Fecal immunized orally on days 0, 10,−/−20, and 30−/−with Dukoral (3 ˆ 10 V. cholerae with 10 µg CTB). Fecal −/−, TLR-4 −/−) and TLR mutant (TLR-2 , MyD88 and Trif WT controls (n = 5 per group) were pellets were collected pre-vaccination and 9 days after the last vaccination. Fecal supernatants were pellets were collected pre-vaccination and 9 days after the last vaccination. supernatants were 9 V. choleraeFecal immunized orally on days 0, 10, 20, and 30 with Dukoral (3 × 10 with 10 μg CTB). Fecal extracted and fecal CTB-specific IgA antibodies were measured by ELISA at the indicated dilutions. extracted and fecal CTB-specific IgA antibodies were measured by ELISA at the indicated dilutions. pelletsshown were collected pre-vaccination and 9mean daysO.D after450 thenm last±vaccination. FecalThe supernatants were Results are the post-4th vaccination SEM. * p ≤ 0.05. naïve control Results shown are theCTB-specific post-4th vaccination meanwere O.D measured 450 nm ˘by SEM. * p at ď the 0.05. The naïve control extracted and fecal IgA antibodies ELISA indicated dilutions. data are shown as dashed line. dataResults are shown as are dashed line. shown the post-4th vaccination mean O.D 450 nm ± SEM. * p ≤ 0.05. The naïve control data are shown as dashed line. To evaluate CD4+ T-cell immune responses, spleens from immunized animals were collected 2 To after evaluate CD4+ T-cell immune responses, spleens from with immunized were weeks the last vaccination, labelled with 5 μM CFSE, stimulated V. choleraeanimals for 5 days, To CD4+ T-cell spleens immunized animals were collected 2 evaluate weeks after the last immune vaccination, withfrom 5 µM CFSE, stimulated withcollected V. cholerae and proliferation was evaluated by responses, flowlabelled cytometry by the degree of CFSE-dilution on 2 weeks last vaccination, labelled with 5show μMcytometry CFSE, stimulated with cholerae for 5 days,on CD4+ was T-cells. The results that CD4+ fromV.of orally immunized forfluorescently-labelled 5 days,after andthe proliferation evaluated by flow byT-cells the degree CFSE-dilution and proliferation was evaluated by flow cytometry by the degree of CFSE-dilution on −/− −/− TLR-2 (p = 0.0002) and MyD88 < 0.0001) mice were significantly inhibited their immunized ability to fluorescently-labelled CD4+ T-cells.(p The results show that CD4+ T-cells frominorally fluorescently-labelled CD4+ T-cells. The results show that CD4+ T-cells from orally immunized ´/´ (p in proliferate responseand to stimulation V. cholera, compared toinhibited wild type mice to TLR-2 = 0.0002) MyD88´−/−/´with (p < whole-cell 0.0001) mice were significantly incontrol their ability TLR-2−/− (p = 0.0002) and MyD88 (p from < 0.0001) mice werewas significantly inhibited in their ability to −/− mice (Figure 3). Proliferation of CD4+ T-cells TLR-4 also decreased, but not significantly. proliferate in response to stimulation with whole-cell V. cholera, compared to wild type control mice proliferate inofresponse to stimulation with whole-cell V. cholera, to wild type control mice −/−compared ´/´from No inhibition proliferation was seen from in CD4+ T-cells mice,decreased, suggesting that CD4+ T cell (Figure 3). Proliferation of CD4+ T-cells TLR-4 miceTrif was also but not significantly. −/− mice was also decreased, but not significantly. (Figure 3). Proliferation of CD4+ T-cells from TLR-4 response to V. cholerae stimulation occurs independently of suggesting Trif signaling. Noproliferation inhibition ofinproliferation was seen in CD4+ T-cells from Trif´/´ mice, that CD4+ T cell No inhibition of proliferation was seen in CD4+ T-cells from Trif−/− mice, suggesting that CD4+ T cell proliferation in response to V. cholerae stimulation occurs independently of Trif signaling. proliferation in response to V. cholerae stimulation occurs independently of Trif signaling.
Figure 3. CD4+ T-cell proliferation in response to stimulation by V. cholerae. Splenocytes from immunized mice were collected 2 weeks after the last vaccination, labelled with 5 μM CFSE and Figure CD4+ T-cellproliferation proliferation responsewas stimulation by V.cytometry cholerae. Splenocytes stimulated with V. cholerae for 5 days. Proliferation evaluated byby flow by the degreefrom of Figure 3. 3. CD4+ T-cell ininresponse totostimulation V. cholerae. Splenocytes from immunized mice were collected 2 weeks after the last vaccination, labelled with 5 μM CFSE CFSE-dilution fluorescently-labelled T-cells. Cell proliferation is shown % and ofand immunized miceonwere collected 2 weeks CD4+ after the last vaccination, labelled with 5 as µMthe CFSE stimulated with V. cholerae for 5 days. Proliferation was by flowstimulated cytometrywith by the degree of CFSE-diluted from for 10,000 gatedProliferation events ± SEM. −, evaluated unstimulated; V.the cholerae stimulated withevents V. cholerae 5 days. was evaluated by +, flow cytometry by degree CFSE-dilution on fluorescently-labelled CD4+ T-cells. Cell proliferation is shown as the % of a ratio of 1:2000. p < 0.001, ns, not significant. ofatCFSE-dilution on*** fluorescently-labelled CD4+ T-cells. Cell proliferation is shown as the % of CFSE-diluted events from 10,000 gated events ± SEM. −, unstimulated; +, stimulated with V. cholerae CFSE-diluted events from 10,000 gated events ˘ SEM. ´, unstimulated; +, stimulated with V. cholerae at a ratio of 1:2000. *** p < 0.001, ns, not significant. at a ratio of 1:2000. *** p < 0.001, ns, not significant.
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2. Conclusions In summary, our results indicate that TLR pathways may have important roles in regulating the quality of immune response induced following oral immunization with the Dukoral vaccine. The results also suggest that proliferation of both CD4+ T-cells in response to V. cholerae is mediated via MyD88/TLR signaling, and independently of Trif signaling. Our results represent novel findings regarding the role of TLR signaling in the induction of humoral immune responses to the Dukoral vaccine. Acknowledgments: We would like to thank Helen Tabor and Morganne Jerome for the neutralization assays in the National Microbiology Laboratory at Public Health Agency of Canada. We thank Rebecca Malott for critical reading of our manuscript. Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations CTB TLR ETEC V. cholera
Cholera toxin B subunit Toll-Like Receptors Enterotoxigenic Escherichia coli Vibrio cholera
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