Development And Clinical Application Of HBV Antigen-specific CD8~+T Cell Quantitative Detection Kit

Background:Hepatitis B virus(HBV)infection is a serious public health problem in our country.Patients can develop from acute infection to chronic infection,liver cirrhosis and even liver cancer.At present,antiviral drugs based on nucleoside analogs(NAs)are mainly used for treatment but the virus cannot be completely eliminated and it is prone to relapse after therapy discontinuation.The cellular immune responses mediated by HBV antigen-specific CD8~+T cells play a critical role in the clearance of virus in vivo.The HBV-infected target cells present viral antigen peptides to CD8~+T cells through HLA class I molecules such as HLA-A,B and C.Then CD8~+T cells differentiate into CTL to initiate specific killing against target cells,achieving the purpose of eliminating virus.The alleles of HLA class I molecules in the population are highly polymorphic.Different HLA class I molecules have obvious differences in the types and ability of antigen peptide presentation.Therefore,the intensity of HBV specific CD8~+T cell immune responses is distinct in different patients.Quantitative detection of HBV antigen-specific CD8~+T cells in patients can understand the true cellular immune function status of patients with hepatitis B and provide new information for clinical monitoring of disease progression,evaluation of antiviral treatment effects,improvement of treatment options and prediction of recurrence risk after therapy discintunation,thus will facilitate immunological precision medicine.Purposes:This study aims to predict and identify the immunogenicity of HBV epitopes restricted by HLA-A0203,A0206 or A0207 molecules wich with a gene frequency of 3.9%,6.1%or 9.5%in Chinese populations;establish epitope peptide pools using the HBV epitope peptides identified in house;develop a quantitative detection kit for HBV antigen-specific CD8~+T cells using INF-γELISPOT technology to detect clinical blood samples from patients with hepatitis B followed by discussion of the clinical significance of quantitative detection of HBV-specific T cells.Methods:(1)Five epitope peptide prediction databases were used to on-silicon predict the CD8~+T cell epitopes derived from HBV proteins including HBs Ag,HBe Ag(HBc Ag),HBpol and HBx Ag,which are restricted by HLA-A0203,A0206 and A0207 molecules.Then the candidate epitopes were further selected,grouped into three epitope peptide pools,and co-clutured with the peripheral blood mononuclear cells(PBMC)isolated from peripheral blood of clinical hepatitis B patients.IFN-γELISPOT experiments were carried out to verify the patients whose PBMCs dispaled CTL positive reaction to the epitope peptide pools.Then the PBMCs were collected again from these positive patiens,and co-cultured with each single epitope peptide followed by IFN-γELISPOT experiment to identify the immunogenicity of each peptide in the peptide pools.(2)The 105 HBV CD8~+T cell epitopes with a strong immunogenicity as verified in our research group and restricted by 13 predorminant HLA-A molecules with a gene frequency more than 1%in Chinese populations were selected out and grouped into 7 peptide pools,and followed by the development of quantitative detection kit of HBV antigen-specific CD8~+T cells using IFN-γELISPOT technology.Then the kit was used to detect the number of immunoreactive and memory or active HBV specific CD8~+T cells in peripheral blood of hepatitis B patients.The repeatability and stability of the kit and the detection effect under different blood store conditions were further analyzed.(3)81 peripheral blood samples of the patients with hepatitis B were collected and used to quantify HBV antigen-specific CD8~+T cells using our kit and the reference value from patient population was initially established based on te data;13 patients undergoing antiviral treatmen due to chronic hepatitis B(CHB)were detected twice for the HBV antigen-specific CD8~+T cells at an interval of three months.The dynamic data were combined with other laboratory indicators to analyze the clinical implications of HBV antigen-specific CD8~+T cells in monitoring disease process and evaluating antiviral treatment effects.Results:(1)20 epitope peptides restricted by HLA-A0203,A0206,or A0207 molecules were on-silicon predicated and selected as candidate HBV epitopes through 5 epitope peptide prediction database.Then IFN-γELISPOT experiments were performed using candidated epitope pools and PBMCs from 150 patients with hepatitis B.The PBMCs from 19 patients displayed positive reaction against the peptide pools,thus followed by IFN-γELISPOT assay with single epitope peptide from the corresponding peptide pool.Finally,7 epitope peptides were verified with real-world immunogenicity.Of which one was restricted by HLA-A0203,two were restricted by HLA-A0206 and four were restricted by HLA-A0207,respectively,according to the HLA-A genotypes of tested blood samples and the results of virtual prediction.(2)The 105 HBV CD8~+T cell epitopes restricted by 13 predorminant HLA-A molecules and verified by our group were selected and grouped into 7 peptide pools,with which the enumeration kit of HBV antigen-specific CD8~+T cells were developed(ELISPOT,11 tests/99wells/kit).Each test included 7 experimental wells for peptide pools,1 negative control well without peptide stimulation and 1 positive control well stimulated by mitogen PHA.The kit stability could be maintained for at least 5 months under the storage condition of 4℃;The within-run coefficient variation(CV)was<15%,and the between-run CV was<20%,which is in line with the national standard of enzyme-linked immunoassay,suggesting an acceptable well-to-well reproducibility;the test results of blood specimen under different storage conditions showed that the storage prolongation of blood samples at 4℃or room temperature could reduce spot forming units,thus the storage condition of blood sample should be unified according to actual situations in different laboratories.It is recommended to start testing within6 hours at room temperature;No significant difference was found between the results of the test immediately after PBMCs isolation and the test after overnight culture of PBMCs.In the laboratory where patients’blood samples were received very late,it is recommend that patients’PBMCs could be isolated immediately but cultured overnight to avoid night shift operation.(3)81 patients with hepatitis B were tested using this kit for the enumeration of HBV specific T cells in peripheral blood.The results(SFU/2×105 PBMCs)were statistically divided into low,intermediate and high three-stage reference values:low grade 0-14,inter grade 14-140,high grade>140,accounting for 15%,70%,and 15%of patients respectively.In addition,13 CHB patients undergoing antiviral treatmen were detected twice for enumeration of HBV antigen-specific CD8~+T cells at an interval of three months.The SFU significantly increased in 8patients,no obvious changes in 2 patients,and decreased in other 3 patients.Conclusion:7 HBV candidate epitopes restricted by 3 HLA-A molecules were verified as real-world epitopes with immunogenicity;A enumerating kit for HBV antigen specific CD8~+T cell were developed successfuly;81 patients with hepatitis B were detected and preliminary clinical reference values were obtained;13 CHB patients undergoing antiviral therapy were tested dynamically at an interval of 3 months and followed by the discussion of clinical significance in monitoring disease progression and evaluating therapu effects.This is the first kit for HBV specific T cell detection in the domestic and foreign markets.