Screening And Immunological Identification Of HLA-A0201 Restricted Epitope From Hepatitis B Virus X Protein

Viral hepatitis is a common and frequent disease in China and the chronic infection of hepatitis B virus (HBV) is one of major risk factors to the development of hepatocellular carcinoma (HCC). The HBV genome is highly variable containing four open reading frames (ORFs) (denoted S, C, P, and X). Thereinto, X protein (HBx) encoded by X ORF is a multifunctional protein. Many researches indicate that HBx is indispensable to the transcription of virus genome and it could affect many relevant physiological events such as virus replication, mismatch repair, cell kinetics and apoptosis. Therefore, further researches on HBx possess remarkable academic and clinical significance.Cytotoxic T lymphocyte (CTL), acting as a crucial element in cell-mediated immune response, can kill target cells via the interaction between T-cell receptor (TCR) and major histocompatibiry complex (MHC) class I carrying heterologous protein fragments. The heterologous protein fragments are so-called CTL epitope, which is the linear fragment or the space conformation structure recognized specifically by immune cell and plays a key role in CTLs activation. It is the elementary event of inducing immune responses and it decides CTL specific cytotoxic effects, therefore, selection of antigen epitope makes the pivotal step to identification of specific antibodies. The epitope would be presented by different MHC class I such as HLA-A2, HLA-A3, HLA-A24 and so on. More than 50% of the Chinese population is found to be the HLA-A2 positive. In this case, it is more significant to screen and identify HLA-A0201 restricted CTL epitopes from HBx special for Chinese.There are altogether 8 genotypes (A-H) and various serotypes so far. In this study, HBx gene sequence of Hepatitis B virus genotypes B/C and serotypes adw/adr, whose frequency are highest in Chinese, were computed and analyzed by websites and general principles (peptide supermotif, extended motif and quantitative motif prediction). Four ideal nine-peptides (HBx1: VLCLRPVGA, HBx2: CLFKDWEEL, HBx3: VLHKRTLGL, HBx4: HLSLRGLPV) were selected as candidate peptides. It has already been proved that the predictions are not always be consistent with the reality and the actual immune response could not be judged merely by the binding between peptides and MHC-I molecule ex vivo (2). As a result, it is quite V necessary to identify these predicted peptides with the affinity and immunity by experiments. Therefore, HBc: FLPSDFFPSI (HLA-A0201) and HBc: EYLVSFGVW (HLA-A2402) were selected as positive and negative control separately.The expression of empty HLA-A0201 molecule on the surface of T2-cells membrane is instable; however, it will not be stable until it is bound to exogenous peptides, which would make T2 cells to be one of most powerful tools in affinity identification between peptides and MHC-I molecule. The four candidate peptides were evaluated the affinity by T2 binding assay and the binding stability by DC50 assay using flow cytometry. Our results indicated that HBx2 might be a potential HLA-A0201 restricted CTL epitope from HBx.For further immunological identification of peptides, the HLA-A2.1/kb transgenic mice were used to simulate the natural environment in vivo. Then abilities of four candidate peptides to stimulate mouse spleen lymphocytes to secret IFN-γand derive them into peptide-specific CTLs were all detected by Elispot and LDH detection analysis. HBx2 was proved to be a potential CTL epitope once again. Where after, we also validated abilities of HBx2 to induce immune responses with PBMCs were isolated from patients and healthy donor by detecting secretion of IFN-γand cytotoxicity of peptide-specific CTLs. Our results also indicated that HBx2 must be a HLA-A0201 restricted CTL epitope from HBx with high immunogenicity. According to the immunological identification in or ex vivo, we constructed soluble MHC-I-peptide tetramer with HBx2 and make preparations for the clinical diagnosis and therapy.In conclusion, HBx2: CLFKDWEEL is a HLA-A0201 restricted CTL epitope from HBx with potential clinical usability. We make the groundwork for further researches on mechisum of HBx involving in hepatitis B or hepatocellular carcinoma and clinical diagnosis or therapy.