Identification Of HLA Restricted CD4+T-cell Epitopes From HpaA Of Helicobacter Pylori And Study Of Their Immunodominant Responses

BackgroundHelicobacter pylori (H. pylori) infection is one of the most common chronic infectionsin human which involve more than50%global population and more than half billion peoplein China. H. pylori infection is the major cause of the chronic gastritis, peptic ulcers,mucosa-associated lymphoid tissue lymphoma (MALT), and closely related to gastriccancer. The current anti-infection therapy relies on antibiotic combine with proton-pumpinhibitor. This strategy became much inefficient due to the increasing antibiotic resistancestrains and the patients’ poor compliance. The peptide-based vaccine provides us a newstrategy for preventing and treating H. pylori infection. It has been well studied in multipleanti-virus and anti-tumor vaccines with pretty gratifying results. However, the developmentof epitope-based vaccine against H. pylori is just beginning.H. pylori infection induced a CD4+T cell predominant T cell response which was alsoproved to be critical in the protective immunity against H. pylori. However, current studieson H. pylori specific CD4+T cell responses were limited in the whole bacteria or singleprotein antigen and ignored the differences between epitopes. Based on the ability ofepitopes in stimulating T cell responses, the epitopes were classified asimmunodominant epitopes and subdominant epitopes. The antigen-specific CD4+T cellresponses were dominated by those immunodominant epitope-specific CD4+T cellresponses. However, due to the HLA polymorphism, the immunodominant or subdominantCD4+T cell epitopes of one antigen were normally different between individuals. To makethe vaccine protect more people, we need to find out the HLA alleles with the highestfrequencies firstly and then identify the epitopes restricted by these high-frequency HLAalleles before we use them as candidates for further vaccine design. H. pylori adhesion A subunit (HpaA) is indispensable for H. pylori colonization.Because it is highly conserved amongst many strains, HpaA has been considered as apotential protective antigen and a vaccine candidate. Studies in mice indicated that oralimmunization with recombinant HpaA stimulated therapeutic immunity against H. pyloriand the protection was strongly related to HpaA-specific mucosal CD4+T cell responses.However, further studies on the immunodominant epitopes recognized by HpaA specificCD4+T cell responses and their immunodominance hierarchies were required.Aim:1. To define the high-frequency HLA alleles in Chinese Han population. To predictpotential HpaA CD4+T cell epitopes restricted to the high-frequency HLA alleles viacomputer software. And to certificate their ability in stimulating CD4+T cell responsesthrough T cells responding assay.2. To systematically analyze the characteristics of immunodominant HpaA-specificCD4+T cell responses in H. pylori infected subjects with diverse HLA alleles expressing.Methods:1.13C Urea Breath Test and serum anti-H. pylori antibody enzyme-linkedimmunosorbent assay (ELISA) were used to screen the H. pylori infected subjects.HLA-DRB1genotyping was performed through PCR-SBT. The HLA alleles with higherfrequencies in Chinese Han population were analyzed via the data in the Allele FrequencyNet data base. NetMHCIIpan was used to predict the potential CD4+T cell epitopes.HpaA-specific CD4+T cell lines were established by stimulating PBMCs withrecombination HpaA-antigen; Synthetic peptides were used to evaluate the immunogenicityof these potential epitopes; Dentritic cells were cultured in vitro and pulsed withrecombinant HpaA protein to identify whether these epitopes can be natural processed andpresented.2. HpaA-specific CD4+T cell lines were established by stimulating PBMCs withrecombination HpaA-antigen; immunodominant epitopes were identified by a serial ofoverlapping synthetic peptides; PCR-SBT genotyping, antibody blocking test and epitopepresenting assay by BCLs were used to determine the HLA restriction of theimmunodominant epitope; Sequence conservancy of the immunodominant epitope wasanalyzed by multi-sequence alignment. Results:1. The top three higher-frequency HLA-DRB1alleles in Chinese Han population are:DRB1*0901(14.4%), DRB1*1202(13.3%) and DRB1*1501(10.8%).2. NetMHCIIpan prediction showed us the8potential epitopes with the strongestbinding affinity to these three high-frequency HLA alleles were: HLA-DRB1*0901restricted HpaA39-53; HpaA42-56; HpaA78-92; HpaA85-99; HpaA92-106; HpaA117-131; HpaA175-189and HpaA194-208、HLA-DRB1*1202restricted HpaA56-70; HpaA60-74; HpaA219-233; HpaA38-52;HpaA89-103; HpaA192-206; HpaA222-236and HpaA189-203and HLA-DRB1*1501restrictedHpaA56-70; HpaA87-101; HpaA38-52; HpaA223-237; HpaA219-233; HpaA78-92; HpaA36-50andHpaA191-205.3. HpaA-specific CD4+T cell lines were successful established through the PBMCsfrom H. pylori infected subjects, while failed through the PBMCs from H. pylori uninfectedsubjects.4. Three immunogenic HpaA-specific CD4+T cell epitopes were identified by T cellstimulating assay, they were: HpaA39-53(HLA-DRB1*0901)、 HpaA89-103(HLA-DRB1*1202) and HpaA87-101(HLA-DRB1*1501).5. All of these three epitopes can be naturally processed and presented by antologousDCs.6. HpaA-specific CD4+T cell lines were successfully established and significantdifferences of the immunodomiant epitopes were observed between different subjects withdifferent HLA allels.22immunodominant epitopes were identified through overlappingpeptides, amongst them, HpaA142-159specific CD4+T cell response got the highestfrequency (23/66) in HLA-DRB1*1501-negative individuals.7. HpaA144-156is the core sequences of epitope HpaA142-159. It was restricted toHLA-DRB1*0901.8. HpaA144-156specific CD4+T cell response was dominant in6subjects of8H. pyloriinfected and HLA-DRB1*0901positive subjects.9. Epitope HpaA144-156can be naturally processed and presented by BLCLs.10. HpaA144-156was highly conseved among various H. pylori strains.Conclusion:1. Epitopes HpaA39-53(HLA-DRB1*0901), HpaA89-103(HLA-DRB1*1202) and HpaA87-101(HLA-DRB1*1501) can be responded by HpaA specific CD4+T cells with thecorresponding HLA alleles. They also can be naturally processed and presented.2. HpaA142-159specific CD4+T cell response got the highest frequency inHLA-DRB1*1501-negative individuals. HpaA144-156is the core sequence of epitopeHpaA142-159. It was restricted to HLA-DRB1*0901and can be naturally processed andpresented by antigen presenting cells. HpaA144-156specific CD4+T cell response wasdominant in HLA-DRB1*0901positive subjects and the sequence of HpaA144-156washighly conseved among various H. pylori strains and it can be used as a candidate epitopein vaccine design.3. Computer prediction can be used to discovery some immunogenic CD4+T cellepitopes, but it was not accurate enough and unable to identify the immunodominantepitopes. Systematic epitope mapping through overlapping systematic peptides was theoptimum way to identify the immunodominant epitopes recognized by HpaA-specificCD4+T cells and analyze their immunodominant hierarchies.Significance：We evaluated the application of computer prediction in epitopes identification, and wefurther systematic analyzed the HpaA-specific CD4+T response in H. pylori subjectsthrough overlapping synthetic peptides in this study. Some naturally presented epitopeswere identified here which may provide help for the development of new H. pylori vaccine.Our study will also help us choosing the best method to identify the immunodominantepitopes and provide insight into the immundominance hierarchies of antigen specificCD4+T cells in H. pylori infection.