Screening and Identification of HBV Epitopes Restricted by Multiple Prevalent HLA-A Allotypes

Abstract

Although host T cell immune responses to hepatitis B virus (HBV) have been demonstrated to have important influences on the outcome of HBV infection, the development of T cell epitope-based vaccine and T cell therapy and the clinical evaluation of specific T cell function are currently hampered markedly by the lack of validated HBV T cell epitopes covering broad patients. This study aimed to screen T cell epitopes spanning overall HBsAg, HBeAg, HBx and HBpol proteins and presenting by thirteen prevalent human leukocyte antigen (HLA)-A allotypes which gather a total gene frequency of around 95% in China and Northeast Asia populations. 187 epitopes were in silico predicted. Of which, 62 epitopes were then functionally validated as real-world HBV T cell epitopes by ex vivo IFN-γ ELISPOT assay and in vitro co-cultures using peripheral blood mononuclear cells (PBMCs) from HBV infected patients. Furthermore, the HLA-A cross-restrictions of each epitope were identified by peptide competitive binding assay using transfected HMy2.CIR cell lines, and by HLA-A/peptide docking as well as molecular dynamic simulation. Finally, a peptide library containing 105 validated epitopes which cross-binding by 13 prevalent HLA-A allotypes were used in ELISPOT assay to enumerate HBV-specific T cells for 116 patients with HBV infection. The spot forming units (SFUs) was significantly correlated with serum HBsAg level as confirmed by multivariate linear regression analysis. This study functionally validated 62 T cell epitopes from HBV main proteins and elucidated their HLA-A restrictions and provided an alternative ELISPOT assay using validated epitope peptides rather than conventional overlapping peptides for the clinical evaluation of HBV-specific T cell responses.

Keywords: ELISPOT; HLA-A; T cell epitope; antigen-specific T cell detection; bioinformatics analysis; hepatitis B virus.