Immunodominant CD4+T Cell Responses Against Helicobacter Pylori Infection In Immunized Mice With Ureb Antigen Under Different Adjuvants

BackgroundHelicobacter pylori, a spiral-shaped, Gram-nagtive bacteria, resides in the gastricmucosa and infects more than half of the word’s population. Persistent infection of H. pyloriis linked with chronic gastritis, peptic ulcer, gastric cancer and gastric mucosa-associatedlymphoid tissue lymphoma. Current clinical treatment against H. pylori relies on twoantibiotics at least and proton-pump inhibitor. Unfortunately drawbacks to the treatment arenot satisfactory, including antibiotic resistance, re-infection, high cost and poor patientcompliance. Therefore, vaccination may be an alternative method against H. pyloriinfection.Currently, vaccines against H. pylori are being researched, including whole-cellpreparation which is a traditional vaccine type. The whole-cell vaccine which containsdiverse antigen-compnents may lead to severe side effects thought the protective effect issignificant. Vaccines composed of pure protein antigen derived from H. pylori may induceantigen-specific immune reaction more safely. The preventive effects of different vaccinesvaries if the adjuvants or delivery mechanisms are different，even the antigen is the same，which has been known, and the role of CD4+T lymphocytes in combating againstextracellular bacteria infection has been recognized. Especially, the effect of antigenspecific T lymphocytes secreting IFN-γin protection against H. pylori infection has beenwidely accepted. Epitopes, especially the immunodominant epitopes, could stimulatespecific CD4+T lymphocytes secreting IFN-γ effectively. So we suppose that adjuvantsand delivery mechanisms may affect the immune reaction of epitope-specific T cells, whichresults in different preventive effects.Urease is considered as an excellent vaccine candidate and found to be associated with the colonization, infection and survive of H. pylori in stomach. Urease is composed of Aand B subunit which is believed to be protective in mice. Thus, in this study, we took UreB,a conserved vaccine candidate, as a model antigen of H. pylori. We expandedantigen-specific CD4+T cells from spleens of mice immunized with UreB in combinationwith different adjuvants and screened the most immunodominant epitopes within9318meroverlapping synthetic peptides. Then we identified more brief epitopes within13mersynthetic peptides, and confirmed the MHC restriction profiles of the peptides by usingantigen blocking assay. The preventive effect of the immunodominant epitopes wasevaluated in BALB/c mice. Finally, we found that adjuvants and delivery mechanisms mayinfluence immunodominant T cell responses hierarchy, which results in the protectivediversity of vaccines.Aim：In this study，establishing mouse models immunized with different adjuvants, weaimed to known the reason of the different protective effects, and to achieve protectiveimmunodominant T cell epitopes instead of the whole antigen, which may avoid drawbacksof the antigen and induce more specific immune responses. These epitopes may becomecandidates for effective vaccines against H. pylori infection.Methods:1. H. pylori cultureA BALB/c mouse-adapted H. pylori strain B6was cultured on brain-heart infusionplates,then amplified in skirrow broth with5%fetal bovine serum. The concentration of H.pylori strain was adjusted to109colony forming units (CFU)/ml prior to inoculation.2. Mice, immunization and infectionBALB/c mice were immunized with rUreB protein emulsified in equivoluminalFreund’s adjuvant or AddaVax (Invivigen) subcutaneously. For the adjuvant CpG(Invivigen), mice were inoculated subcutaneously or intranasally.For peptides immunization, mice were immunized with peptides in CpG OND1826(Invivigen) by intranasal route. As controls, mice were immunized with PBS instead of theprotein or peptides following the same procedure.One week after the final boost, mice were infected with H. pylori strain. Four weeks after the last intragastric administration, mice were sacrificed. Immune responses and the H.pylori colonization were assessed.3. Assay for specific antibodiesThe specific antibodies levels of mice post H. pylori infection were analyzed byELISA.4. Determination of H. pylori colonizationH. pylori colonization was assessed by real-time quantitative PCR, analyzing16SrDNA of H. pylori.5. Specific T cell bulk cultureSpleens were harvested from immunized mice, and mononuclear cells were isolatedfrom spleens by Ficoll-Hypaque (TBDscience, Tianjin, China) gradient. The mononuclearcells were pulsed with rUreB protein (0.5uM)/peptides (5uM) and stimulated with rmIL-2in vitro. The cells were harvest at the right moment.6. Generation of bone marrow-derived dendritic cells and co-culture withimmunodominant epitope-specific T cellsBone marrow was harvested from the femurs of wild BALB/c mice. After treatmentwith erythrocyte lysing buffer, cells were cultured in RP-10supplemented with recombinantmurine GM-CSF and recombinant murine IL-4.8days later, Dendritic cells were harvestedand pulsed with rUreB antigen, then co-cultured with epitope-specific T cells at a ratio of1:5for5hours in the presence of brefeldin A.7. Intracellular cytokine staining (ICS)Cultured lymphocytes were harvested and incubated with appropriate stimulate for5hin the presence of brefeldin A, then cells were collected and stained with fluorescentantibody. One hundred thousand cells were acquired on a FACS CantoⅡ flow cytometer(Becton Dickinson) and FCS files were analyzed with Flowjo software.8. Statistical analysisThe results of H. pylori colonization in stomachs of mice immunized with rUreB wereanalyzed by unpaired student’s t-test. Other data were analyzed by one-way ANOVAfollowed by Newmann-Keuls testing for dependent variables. All data were expressed asmeans±standard deviation (S.D.). Values of p≤0.05were considered to be statistically significant.Results1. Different protections against H. pylori colonization were detected by immunizationwith rUreB in combination with different adjuvants. Both immunization with rUreBcombinated with Freund’s adjuvant subcutaneously and CpG intranasally dramaticallyreduced the H. pylori load in the stomachs compared with PBS controls（P<0.01）.2. Expansion of rUreB antigen-specific CD4+T lymphocytes in vitro from spleens ofimmunized mice. After bulk culture following the method described above, the frequency ofUreB-specific CD4+T lymphocytes could reach up to10%. However, specific CD4+Tcells could not be detected in PBS control mice.3. Screening immunodominant Th1epitopes of UreB from immunized mice. Miceinoculated with different adjuvants presented various capacities against H. pylori infection.Antigen-specific T cells were expanded in vitro as mentioned above. Then the Th1epitopesof rUreB were identified. At last, four novel epitopes UreB317-329（MLMVCHHLDKSIK）、UreB373-385（ITRTWQTADKNKK）、UreB409-421（YTINPAIAHGISE）and UreB485-497（AKYDANITFVSQA） were achieved from different immunized groups respectively.4. All the four epitopes could be naturally processed and presented byantigen-presenting cells and the responds to the epitopes of specific T cells could beinhabited by MHC-Ⅱ（I-A）antibody..5. The protective effects of the four immunodominant epitopes were different fromeach other. Only UreB317-329（MLMVCHHLDKSIK） and UreB373-385（ITRTWQTADKNKK） could dramatically reduced the H. pylori load in the stomachscompared with PBS controls（P<0.001）.Conclusion1. Adjuvants may influence immunodominant T cell responses hierarchy, which resultsin the protective diversity of vaccines.2. Inoculation routes of vaccines may influence immunodominant T cell responseshierarchy.3. By using a systematic approach, four novel immunodominant Th1epitopes,UreB317-329（MLMVCHHLDKSIK）、UreB373-385（ITRTWQTADKNKK）、UreB409-421 （YTINPAIAHGISE）and UreB485-497（AKYDANITFVSQA）, of UreB antigen wereidentified and the MHC restriction profiles were characterized.4. The two novel immunodominant epitopes UreB317-329（MLMVCHHLDKSIK）andUreB373-385（ITRTWQTADKNKK） from the four could protect mice from H. pyloriinfection obviously.