Screening Of Epstein-Barr Virus Latent Membrane Protein 2A-specific CTL Epitope And Study Of Immune Function

Epstein–Barr virus (EBV) belongs to the gama-herpesvirinae subfamily and is closely related to human tumors. EBV associated NPC (nasopharyngeall carcinoma) is a malignant tumor derived from the epithelial cells and endemic in the Southeast Asia and South of China. Recently, the incidence of NPC has been increasing in Jiangsu province. NPC usually are not suitable for surgery and tumor cells are not eliminated effectively and completely by radiotherapy and chemotherapy. Relapse and metastasis may occur in a portion of NPC patients and side effect arising from conventional therapeutic methods will affect self immune system greatly. Therefore, it is necessary to develop immunotherapeuic approaches for NPC except for conventional therapy. In the EBV-associated NPC patients, the proteins of EBV expressed on tumor cells are very limited, and only type?latency EBV antigens such as the latent EBV nuclear antigens (EBNA1) and latent membrane proteins (LMP1 and LMP2A, 2B) can be detected on NPC cells. Among EBV encoded proteins, LMP2A is not transformed protein and it's transcripts are frequently expressed not only in primary NPC but also in metastatic tumors. LMP2A protein is conserved and contains HLA-restrict cytotoxic T lymphocyte (CTL) target epitopes which can elicit strong specific CTL response. So LMP2A is known as an important target antigen for T cell therapy of EBV.In our previous work, LMP2A-specific CTL could be induced by DC loaded with LMP2A protein or transfected with rAd-LMP2A in healthy EBV carriers in vitro. In the previous study, six HLA-A2 restricted CTL candidate epitopes of LMP2A were predicted by SYFPEITHI, NetMHC and MHCPred methods combined with the polynomial method. Subsequently, biological functions of these peptides were tested by experiments (enzyme-linked immunospot assay (ELISPOT), intracellular cytokine staining assay(ICS) and cytotoxicity assays) in healthy EBV carriers and NPC patients in vitro. Finally, only three peptides, which could induce CTL response to LMP2A in vitro and effectively kill specific HLA-A2-expressing target cells, were identified as LMP2A-specific CTL epitopes. They were LMP2A264 (QLSPLLGAV), LMP2A426 (CLGGLLTMV) and LMP2A356 (FLYALALLL). To our knowledge, peptide LMP2A264 was a novel LMP2A-specific CD8+T cell epitope identified in Chinese population. In addition, of three epitope specific T cell clones were established by the liquid microwell limiting dilution technique (LDT), two was specific to peptide CLGGLLTMV and one was specific to peptide QLSPLLGAV. Then the phenotype and function of T cell clones were analyzed initially.In summary, a combination of bioinformatics tools and in vitro assays was used to screen and select antigen sequences as potential LMP2A-specific CD8+T-cell epitopes, and three epitopes were identified; These epitopes could induce immune response in both healthy EBV carriers and NPC patients; Three T cell clones specific for two of three epitopes were established by LDT. It would be useful to clarify immune response toward EBV and to develop a vaccine against EBV-correlative NPC. Moreover, this research provides experimental and theoretical basis for other virus related tumor immunotherapy.