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.
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
ObjectiveTo study the effects of dendritic cells (DC) transfected with recombinant vaccinia virus encoding Epstein Barr virus (EBV) latent membrane protein 2A(LMP2A) gene,and to provide evidence for further investigation on the therapeutic vaccines against EBV associated malignancies. MethodsMature DC were transfected with EBV LMP2A recombinant vaccinia virus (rVV LMP2A). Before and after the transfection,the expression of surface antigens on mature DC including CD1a,CD83,CD40,CD80,HLA DR was measured by fluorescence activated cell sorter (FACS) and the function of DC to stimulate allogeneic T cells proliferation was measured by mixed leukocyte reactions (MLR). ResultsLMP2A protein was highly expressed (66.1 %) in DC after the transfection of rVV LMP2A. No significant changes in the primary surface antigens expression and in the MLR were detected during the transfection. Transfected DC still had strong potential in stimulating the proliferation of allogeneic T cells. ConclusionRecombinant vaccinia virus was an effective and non perturbing vector to mediate the transfection of LMP2A into DC. The functions of mature DC were not affected significantly by the transfection of Vac LMP2A. This study could provide evidence for the further immunotherapy of EBV associated malignancies,e.g. nasopharyngeal carcinoma (NPC).
Type Ⅱ Epstein-Barr virus （EBV） associated malignancies such as nasopharyngeal carcinoma and non-Hodgkin＇s lymphomas consistently express latent membrane 2A （LMP2A） proteins, which have been suggested to be an ideal target for immunotherapy. In previous studies we have demonstrated that using LMP2A protein loaded dendritic cells, the most powerful antigen processing cells in the body can elicit specific and robust anti-tumor cellular immune response in vitro. In this paper, we further investigated the T cell profile of the anti-tumor immune response. We found that LMP2A specific CD4＋ and CD8＋ T cells could be stimulated by LMP2A protein loaded dendritic cells （DCs）. The Thl type immune response is dominant in the immune response mediated by LMP2A specific CD4^＋ T cells. The CD8^＋ cytotoxic T cells can lyse LMP2A bearing cells effectively and specifically. The CD8^＋ cytotoxic T cells can also secrete high level of intracellular IFN-γ, which indicates these cells are EBV-LMP2A specific cytotoxic T cells. Altogether, our studies proved that LMP2A protein loaded DCs can elicit anti-tumor cellular immune responses efficiently. This study provides a rationale for the DC-based immunotherapy against EBV-LMP2A expressing malignancies.
Yun ChenHua SunGenyan LiuBing WangFang WangBeicheng SunKun Yao
