Prediction And Identification Of HLA-A Restricted HBV Epitope Peptide And Establishment Of Personalized ELISPOT Detection Method

Background:Hepatitis B virus(HBV)infection is a serious public health problem in China.It can progress from acute to chronic hepatitis,cirrhosis,and even hepatocellular carcinoma.Antiviral therapy is the most fundamental treatment for chronic hepatitis B.Nucleoside(acid)analogues(NAs)are currently the most important drugs,but can only inhibit HBV replication rather than the complete elimination of HBV,thus the recurrence has become a serious problem.Liver inflammation induced by HBV mainly depends on the immune responses that are closely associated with the clearance of HBV,the occurrence of hepatitis and the prognosis of the disease.The immune responses mediated by HBV antigen-specific CD8~+T cells are particularly critical for host anti-virus infection.HLA class I molecules(such as HLA-A,B,and C)expressed by virus-infected liver cells present the HBV antigenic peptides to specific CD8~+T cells,thus iniate the activation,proliferation and differentiation of cytotoxic T cells.The high polymorphism of HLA class I alleles in population leads to the variability of T cell epitomes restricted by HLA class I molecules and the later immune responses.Therefore,it is a key step for monitoring the personalized anti-virus immune responses to verify the HBV antigenic epitopes restricted by various HLA class I molecules.It will be useful for detecting the number and reactivity of HBV antigen-specific CD8~+T cells in peripheral blood of patients before and after relapse of drug withdrawal,and beneficial for designing the combined therapy regimen of NAs treatment with immunotherapy and evaluating the therapeutic efficacy.Purposes:To identify the HBV antigenic epitopes restricted by HLA-A0201,A1101,and A2402 molecules(with a total gene frequency greater than 50%in population),respectively;and to establish the ELISPOT method and clinical ready-to-use HBV antigen-specific CTL ELISPOT kit for the personalized quantification of HBV antigen-specific CD8~+T cells in peripheral blood of HBV-infected patients.These detections will be able to provide new laboratory indicators for the diagnosis,treatment planning,clinical efficacy evaluation and chronic outcome prediction of hepatitis B,thus promoting the implementation of precision medicine.Methods:(1)The epitopes derived from four HBV proteins(HBs Ag,HBc Ag,HBpol,HBx)and restricted,respectively,by 13 HLA-A alleles(HLA-A0101,A0201,A0203,A0206,A0207,A0301,A1101,A1102,A2402,A2601,A3001,A3101,A3303,a gene frequency greater than 1%for each allele)were virtually predicted and screened,by using six epitope prediction data banks(SYFPEITHI,BIMAS,SVMHV,IEDB,EPIJEN,NETMHC).(2)Peripheral blood samples were collected from the in-patients with chronic hepatitis B in the Department of Clinical Laboratory of Nanjing Second Hospital affiliated to Southeast University,and processed into peripheral blood mononuclear cells(PBMCs).The predicted epitopes restricted by HLA-A0201,A1101,or A2402 were mixed into one group as the peptide cocktail,and co-cultured with PBMCs for 40 hrs followed by IFN-γELISPOT assay.(3)The fresh peripheral blood samples were collected again from the in-patients who’s PBMCs presented positive CTL responses to the peptide cocktail.Then the the immunogenicity of each type of peptide in the peptide cocktail was identified using the the same method.Meanwhile,HLA-A genotyping was carried out for the patients with positive CTL responses using polymerase chain reaction-sequencing-based typing(PCR-SBT)method.(4)The in-house ELISPOT kit based on HLA-A0201,A1101 and A2402 molecules was generated for HBV antigen-sepcific CTL detection.The sensitivity of kit was compared with two commercial counterparts using mitogen and healthy peripheral blood.Then,mixing three HLA-A molecularly restricted HBV antigen T cell epitope peptides into three groups,using the peripheral blood from in-patients with chronic hepatitis B were detected using the ELISPOT kit and followed by HLA-A genetyping.Results:(1)After the virtual prediction,8-9 epitopes derived from four HBV proteins with the highest virtual affinity were selected as candidates of antigenic peptides restricted by each HLA-A molecule,according to the scoring criteria of each prediction method.(2)Fourteen in-patients with positive CTL responses to HBV peptide cocktail were screened from 126in-patients with chronic hepatitis B by IFN-γELISPOT assay.The immunogenicity of 21epitopes were identified,which derived from four kinds of HBV proteins and restricted by HLA-A0201,A1101 or A2402 molecules.Of them,eleven epitopes have not been reported before.Thirteen epitopes were restricted and presented by HLA-A0201,sixteen epitopes were restricted and presented by A1101,and eleven epitopes were restricted and presented by A2402.(3)The in-house ELISPOT kit for HBV antigen-specific CTL detection was successfully generated and 50 in-patients were detected using this in-house kit.Of them,14patitents presented positive CTL responses to the peptide cocktail,and 36 paitents did not presented.Conclusion:Twenty–one epitopes derived from HBV antigens and presented by three HLA-A molecules were identified.The in-house ELISPOT kit was generated for the personalized detection of HBV antigen-specific CD8~+T cells in peripheral blood of HBV virus-infected patients.