Construction Of Epstein-Barr Virus LMP2-LMP1Δ Fusion Gene And Study On Immunogenicity Of EBV-LMP2-LMP1Δ

In 1967, Epstein and Barr firstly isolated and identified Epstein-Barr virus from Burrkitt's lymphoma tumor cells in vitro suspension culture. Studies have showed that EBV is closely associated with several of human malignancies including Burrkitt's lymphoma (BL), Nasopharyngeal carcinoma (NPC), Hodgkin's disease (HD), T cell lymphoma and post-transplant lymphoproliferative disease (PTLD). NPC is a common malignant tumor which has a high incidence in south of China and southeast Asia. In some areas of Guangdong and Guangxi provinces, NPC mortality ranks first or second among all tumors. Thus, NPC is one of the ten main malignancies that urgently need efficient prevention and therapeutic measures in our country. Now radiotherapy is standard treatment for NPC cases. Regional recurrence and distant metastasis are mainly responsible for the failure of the treatment for advanced NPC. After disease recurs or progresses, no additional effective therapy is available, and nearly 85% of the patients die in one year and virtually all died in three years. So, it is obvious that if conventional treatment for NPC fails or the disease advanced, the outcome of NPC is extremely poor.The detection rate of the EBV is almost 100% in undifferentiated nasopharyngeal carcinoma tumor cells, while in the normal tissue around the tumor cells of the EBV detection rate was negative. The CTL response to EBV is weaker in the nasopharyngeal carcinoma patients compared with the healthy EBV carriers. So, it is critical to increase the autologous specific CTL responses, especially CD8+ CTL in clearing viral infection and tumor immune surveillance.EBV expressed different antigens in different cancers. In NPC cells, EBV mainly express Epstein-Barr virus nuclear antigen 1(EBNA1), latent membrane proteins 1 (LMP1) and latent membrane proteins 2(LMP2). Now the vaccine to EBV-associated diseases focus on the LMP1 and LMP2. But the specific CTL response induced by LMP1Δand LMP2 in some individuals is poor. Our previous data also indicated the immunogenicity of LMP2 is weak in some macaca fascicularis Based on the research of our group, we attempt to construct the fusion gene containing LMP1Δand LMP2 in order to increase the celluar immunity effect induced by single gene, and explore the possibility of cooperative effect on LMP1Δand LMP2 in different individuals. Hence we plan to construct LMP1Δand LMP2multivalent vaccine for improving specific CTL response, and then overcome the immune escape of the tumor to some extent.LMP1Δwas cloned into pCDNA3.1(+)-his. Then, restriction nuclease cleavage and sequence confirmed that the certain gene was cloned into the proper orientation and expected fragments. After transient transfecting pCDNA3.1-LMP1Δinto 293 cells, immunofluorescence assay and western blot analysis were done and the results showed that LMPΔwas effectively expressed in 293 cells.LMP2-LMP1Δwas cloned into pCDNA3.1(+)-his. Then, restriction endonuclease analysis and sequence analysis confirmed that the certain gene was cloned into the proper orientation and expected fragments. After transient transfecting pCDNA3.1-LMP2-LMP1Δinto 293 cells, immunofluorescence assay and western blot analysis were done and the results showed that LMP2-LMP1Δwas expressed effectively in 293 cells.Finally, the BalB/C mice were immunized with pCDNA3.1-LMP2-LMP1Δ, pCDNA3.1-LMP1Δ, pCDNA3.1-LMP2, rAd-LMP1Δand rAd-LMP2, alone or combined at 0,2,4 week.. IFN-y ELISPOT assays showed that alone DNA vaccine pCDNA3.1-LMP2-LMP1Δ, pCDNA3.1-LMP1Δ, pCDNA3.1-LMP2, induced poor immune effect. Alone rAd-LMP1Δvaccine also induced poor immune effect. While alone rAd-LMP2 vaccine could induce strong specific CTL responses comparing with the alone rAd-LMP2 vaccine, followed by boosting with rAd-LMP2 once with priming twice with pCDNA3.1-LMP2. The latter immune strategy could induce stronger specific CTL responses which was consistent with the relevant study. Meanwhile, the immune strategy boosting with rAd-LMP1Δand rAd-LMP2 once with priming twice with pCDNA3.1-LMP2-LMP1Δequivalent to the immune strategy boosting with rAd-LMP1Δand rAd-LMP2 once with priming twice with pCDNA3.1-LMP1Δand pCDNA3.1-LMP2, could induce equal immune effect which was stronger than the single LMP1 vaccine or the single LMP2 vaccine.In conclusion, we constructed DNA vaccine pCDNA3.1-LMP2-LMP1Δand pCDNA3.1-LMP1Δ-his, confirmed they could be expressed in 293 cells. Then we tested different immune combination, and found that the immune strategy boosting with rAd-LMP1Δand rAd-LMP2 once with priming twice with pCDNA3.1-LMP2-LMP1Δequivalent to the immune strategy boosting with rAd-LMP1Δand rAd-LMP2 once with priming twice with pCDNA3.1-LMP1Δand pCDNA3.1-LMP2, can induce equal immune effect which was stronger than the single LMP1 vaccine or the single LMP2 vaccine.