| Epstein-Barr virus(EBV)is the first virus was found associated with human tumors.It has been clearly associated with malignant tumors such as nasopharyngeal carcinoma(NPC),Hodgkin's(HD),non-Hodgkin's lymphoma Burkitt lymphoma(BL),Burkitt's lymphoma(BL).Most patients infected with EBV were latent infection pattern.Different latent infection states produce the different virus products.Nasopharyngeal carcinoma(NPC)is an EBV-infected type ? recessive infection.The infected cells mainly expressed viral protein such as transcripts of LMP1.LMP2,EBNA1.EBER and BamHIA fragments.LMP2 were divided into LMP2A and LMP2B subtypes.LMP2A gene was not carcinogenic and introduced the transformation of B lymphocytes.Most CTL epitopes are restricted by HLA I type have been identified and induced significantly specific cytotoxic T lymphocyte response(CTL),thus LMP2 was considered as an important target antigen for EBV-associated malignancies.MVA(Modified Vaccinia virus Ankara)has been widely used in vector vaccine research since safety and immunogenicity.Therefore,we used MVA as a vector to construct the MVA-LMP2 recombinant vaccine carrying the EBV LMP2 target antigen.Evaluation of its induced cellular and humoral immune response,and specific anti-tumor effects.There is no relevant report on the construction of LMP2 target tumor models in mice,which limits the evaluation of EBV LMP2-associated vaccines in inhibiting tumor effects.Therefore,LMP2-associated tumor models were constructed in our study to fill gaps in the LMP2-related tumor cell model for related vaccines.The tumor cell model(TC-1-GLUC-LMP2)was constructed in this experiment.PCR and RT-PCR identification results showed that the tumor model cells effectively carried and transcribed EBV LMP2 and GLUC genes.Western blot,and flow cytometry identification results showed that 30 passaged TC-1-GLUC-LMP2 cells stably express LMP2 and GLUC protein,and the positive expression level of LMP2 protein can still reach 98.7%.TC-1-GLUC-LMP2 cells were inoculated with 4×106 mice subcutaneously,and the in vivo imaging system(IVIS)showed the results.With the prolongation of tumor cell inoculation,the photon number of tumor cells gradually increased,suggesting that the tumor tissue increased continuously.After 14 days of inoculation,the average weight and volume of the detected tumor tissue were 0.41 g and 220 mm3,respectively.Moreover,the tumor tissue could effectively expression of LMP2 protein.The above results indicated that the TC-1-GLUC-LMP2 tumor cells model in this experiment stably expressed the LMP2 and GLUC proteins,and the tumor tissue formed after inoculation in mice.The stable expression of GLUC protein in mice in vivo.Real-time observation of tumor tissue provides convenience.In conclusion,the construction of TC-1-GLUC-LMP2 provides a candidate tumor cell model for the evaluation of the anti-tumor effect of LMP2-related vaccines.At the same time,the MVA-LMP2 recombinant vaccine expressing the full-length gene of EBV LMP2 was constructed to investigate the specific cellular and humoral immune response and specific anti-tumor effects induced by the EBV LMP2 gene.The results of PCR and Western blot showed that MVA-LMP2 carried LMP2A gene and expressed LMP2 protein.According to different doses,different times of inoculation and different detection times,MVA-LMP2 recombinant virus was evaluated,and the results showed MVA-LMP2 could induce LMP2-specific immune response,the immunization dose was 2×107 PFU,the specific immune response induced was the highest(p<0.01,n=6);specific immune response could be achieved the peak of the response within 3 weeks after the last immunization,and the specific immune response was still be detected 10 months after immunization.Continuous inoculation 4 times,increased immunity after interval 2w,the effect of specific immune response was no longer increased.In addition,the flow cytometry results showed that the percentages of IFN-? and TNF-? cytokines in CD3+CD8+ CTL cells were 1.51%and 1.42%,respectively,which were significantly higher than those in CD3+CD4+ CTL cells.ELISA detection showed that MVA-LMP2 could induced LMP2-specific antibody,suggesting that the vaccine could induce LMP2-specific humoral immune response.MVA-LMP2 vaccine immunized C57BL/6 mice and inoculated TC-1-GLUC-LMP2 tumor cells.all tumor cells in mice inoculated after 14 days were disappeared.These results suggest that MVA-LMP2 vaccine infected cells could express LMP2 protein and inducesd effective CD8+ T cell-dependent LMP2-specific cellular and humoral immune responses.Moreover,specific CTLs induced by MVA-LMP2 could effectively kill target cells.In addition,the specific immune responses induced by the vector itself limited the repeated use of the same vector vaccine.The results showed that the specific immune response induced by the one-boost immunization strategy of three-vector vaccine(MVA-LMP2.Ad5-LMP2 and pVR-LMP2)was significantly higher than that of two or a single vector vaccine,indicating that the one-boost immunization of vector vaccines(including MVA-LMP2)clould effectively increase the specific immune response of LMP2.In summary,this experiment constructed a TC-1-GLUC-LMP2 mouse tumor model carrying LMP2 and GLUC genes,which could effectively express LMP2 and GLUC proteins,and provided important references for evaluating the anti-tumor effect of LMP2-associated vaccine and real-time monitoring the tumor cells in mice.Successfully established MVA-LMP2 recombinant virus carrying LMP2A full-length gene,effective expression of LMP2 protein,providing a vaccine candidate for the treatment of LMP2-associated malignancy.MVA-LMP2 vaccine significantly induced LMP2-specific immune responses.The reaction effectively inhibited and killed tumor cells in vivo,and the application of the MVA-LMP2 vaccine significantly improved the effect of LMP2-specific one-boost immune responses.In conclusion,the construction and evaluation of MVA-LMP2 provided the foundation of the treatment of tumors associated with LMP2-related malignancy. |
PDF Full Text Request