Epstein-Barr virus （EBV） associated nasopharyngeal carcinoma （NPC） is a high incidence tumor in Southeast Asia. Among EBV encoded proteins, latent membrane protein 2A （LMP2A） is an important antigen for T cell therapy of EBV. In this study, we predicted six HLA-A2 restricted CTL candidate epitopes of LMP2A by SYFPEITHI, NetMHC and MHCPred methods combined with the polynomial method. Subsequently, biological functions of these peptides were tested by experiments in vitro. In ELISPOT assay, the positive response of the LMP2A specific CTL stimulated by three （LMP2A264.272, LMP2A426-434 and LMP2A3s6.364） of six peptides respectively showed that the numbers of spots forming cells （SFC） ranged from 55.7 to 80.6 SFC/5 x 104 CO8^＋ T cells and the responding index （RI） ranged from 5.4 to 7. These three epitope-specific CTLs could effectively kill specific HLA-A2- expressing target cells. As a result, LMP2A264.272 （QLSPLLGAV）, LMP2A426.434 （CLGGLLTMV） and LMP2A356.364 （FLYALALLL） were identified as LMP2A-specific CD8^＋ T-cell epitopes. It would be useful to clarify immune response toward EBV and to develop a vaccine against EBV-correlative NPC.
Epstein-Barr virus infection is strongly associated with a number of malignancies.The EBV latent membrane protein 2A has been implicated as one of the most attractive candidates for immunotherapy of related malignancies.In previous studies,the T cell epitopes of LMP2A have been identified systematically.However,the epitope-based vaccine generally meets inefficient immunogenicity when used in vivo directly,which could be overcome by combination with appropriate adjuvants.Heat shock protein is a natural chaperon,which is able to activate the classical major histocompatibility complex class I antigen-processing pathway(cross-presentation).In this study,a minigene encoding LMP2A356-364(FLYALALLL)was genetically fused to the carboxy-terminal of mycobacterial heat shock protein 70.The epitope fusion protein was expressed and purified,and the cross-presentation of LMP2A_(356-364) by monocyte-derived dendritic cells pulsed with the epitope fusion protein was evaluated.Results showed that the epitope fusion protein-pulsed mDCs were much more efficient than the single peptide-pulsed mDCs on CTL activation.Immunization of HLA-A2.1 transgenic mice with MtHsp70-LMP2A_(356-364) generated peptide specific CTL more effectively than a single peptide plus incomplete Freund's adjuvant(IFA).Growth of LMP2A expressing B16 melanoma tumor cells was suppressed in the vaccinated groups.Our results suggested that MtHsp70-LMP2A_(356-364) fusion protein was more effective than the CD8^(+)T cell epitope alone on anti-tumor immunity.As a result,the MtHsp70-LMP2A_(356-364) fusion protein is considered to be a promising candidate vaccine for EBV related malignancies.
Genyan LiuKun YaoBing WangYun ChenFeng ZhouYidi GuoJian XuHongzhen Shi
Epstein-Barr virus （EBV）, a potential oncogenic herpesvirus, has been found to be associated with several malignancies. It＇s critical to elicit cellular immunity of the body to fight against EBV-associated tumor development. Using dendritic cells （DCs） loaded with latent membrane protein 2A （LMP2A） to elicit T cell response against tumor may be one of the most direct and safest immunotherapy approaches. The present study aimed to develop DCs-based cancer vaccine （DC loaded with LMP2A protein） and study its biological characteristics and immune functions. Purified LMP2A protein was extracted from a cell line L929/LMP2A stably expressing LMP2A. LMP2A could be loaded on DCs with no significant changes of the DC surface markers and cytomorphology. The percentage of DCs loaded with LMP2A was above 80%. LMP2A-loaded DCs markedly enhanced the proliferation of antigen-specific CD8^＋ T and CD4^＋ T cells by 3H-TdR incorporation assay. Besides, the specific cytotoxicity of the CTLs against LMP2A target cells was also significantly increased. These results indicated that DC-based vaccine loaded with virus antigen could elicit potent CTL response and provide a foundation for further study on the DC-based immunotherapy for nasopharygeal carcinoma and other EBV associated tumors. Cellular ＆ Molecular Immunology.
