Studying Of Specific HLA-DR Alleles Resticted CD4~+T Cell Epitope From Chronic Hepatitis B Virus Infection

Background:Hepatitis B virus(HBV) is the most prevalent virus that leads to liver injury and inflammation. approximately one third of populations have been infected, among which 3–5% of adults and more than 90 % of children developed to chronic HBV infection. The T cell response of the host play essential roles in the outcome of HBV infection. CD4+T cells is an important part of the host T cell immune response. with the progress on the research of the CD4+T cells, the CD4+T cells are divided into many subgroups. Different subgroup has different function, even for the same subgroup,different studies have different conclusions. This may be related to the different epitopes of HBV and diversity of HLA class Ⅱ molecule. Therefore the research of HBV specific CD4+T cells must go deep into the level of HBV epitopes and HLA-Ⅱ alleles. Combining our previous study that he HLA-DR * 0803 towards the immune protection, and the HLA-DR*1202 to immune injury. We assume that it can be achieved by that identification of different epitopes and inducing different CD4+T polarizations.Objective:Compare the difference between the the whole HBV epitopes resticted to two alleles and the difference of CD4+T polarization caused by the two alleles.Methods:Our project combined methods of mass spectrum,microarray assay,and the functional assay. We first use the NCBI to determine the HBV consensus sequence of the genotype B, C, including P, S, X, C protein. Then we Synthetic peptide pool(41 peptides, 15 aa length,overlapping 10 aa) of HBc Ag and Customized microarray chip(631 peptides, 15 aa length,overlapping 11 aa) of P, S, X, C protein based on the sequence. After the BLCL were pulsed with the peptide pool, we elute the peptides binding to the cell surface by isotonic citric acid buffer, then the peptides were dealed with filtration, desalination, vacuum concentration and analyzed by liquid chromatography-mass spectrometry. Ones the resultscome out, Combining the tool Net MHCIIpan 3.0 Server,we choose the peptides meet the condition of “Ion Score≥20, IC50≤500”to determine the HLA-DR resticted epitope from HBc Ag. Meanwhile, we purified the HLA-DR molecule by using a Dynabeads® Protein A Immunoprecipitation Kit. The peptide isolated by the purified HLA-DR were analyzed by mass spectrometry, and for the HLA-DR,we use it do the microarray assay with the Customized chip. Finally, we choose 4 peptides(prec21-35 、 HBc12-26 screened by microarray assay, HBc82-96 screened by mass spectrum and control peptide HBc42-56) to do the T cell reponse experiment with the CHB patients of the same type of HLA-DR.Results:1.Using by mass spectrum combined with the predicted tool Net MHCIIpan 3.0 Server, we find 8 HLA-DR*0803 restricted epitopes: HBc17-31,HBc22-36,HBc27-41, HBc82-96, HBc87-101,HBc92-106, HBc117-131,HBc122-136; 9 HLA-DR*1202 restricted epitopes: HBc17-31,HBc22-36,HBc27-41, HBc82-96,HBc87-101,HBc92-106, HBc122-136, HBc137-151,HBc142-156. And 7 of them is common.2.We only find 2 HBc Ag souce peptides from the peptides isolated from purified HLA-DR by mass spectrum. But we found hundreds of human source peptides derived from heat shock protein, transferrin receptor protein 1, cathepsin D, matrix metalloproteinases inhibitor and so on.3. From the scan of the microarray chip, we find 205 peptides restricted to HLA-DR*0803, and 169 peptides restricted to HLA-DR*1202, 114 of them are common restricted to the two alleles. Among them, the number of HBc Ag source peptides ar e 30 and 28 respective, 22 of them are common. Compared with the result analyzed by mass spectrum before, we find the spots in the chip corresponding to HBc82-96, HBc87-101, HBc92-106, HBc137-151, HBc142-156 have no corresponding fluorescence intensity. And the 8 known HBc Ag epitope, also have one epitope HBc 147-156 in the chip don’t have the corresponding fluorescence intensity.4.We collected the PBMC from 9 cases of CHB patients, and respectively get the DRB1* 0803 and DRB1 * 1202 positive patients in 2 cases(2/9). The PBMC from the 4 cases of patients were stimulated with the selected peptide(prec21-35、HBc12-26、HBc42-56、HBc82-96) 3 times repectively. We find the pepitide HBc12-26 apear strong IFN-γ release action in 2 cases of patients, the other is one patient does’ nt have a significantresponse, and one didn’t stimulated with HBc12-26. The rest three peptide didn’t have a obvious T cell response. But, in adition, we find the level of IL-17 release action stimulated by HBc Ag whole peptide pool in two patients is much higher than by any one peptide stimulated alone.Conclusions:The result of the microarray may be relatively better.than the mass spectrometry. Combined with the Bioinformatic methods, the analysis of mass spectrometry is reliable and purposeful. The HBc12-26 for HLA- DR * 0803/1202 common Th1 epitope, and the HBc42-56 for HLA- DR * 1202 allelic specific Th2 epitope. Besides, there is HLA- DR * 0803/1202 specific Th17 epitope in the HBc Ag peptide pool.