The Study Of Cellular Immune Responses In NPC And Recombinant Ad5F35-LMP2 Adenovirus Vaccine

Epstein-Barr virus(EBV)is a ubiquitous gamma-herpesvirus that can establish both latent and lytic infections.EBV is commonly associated with several malignant diseases,including Burkitt's lymphoma(BE),Hodgkin's disease(HD),undifferentiated nasopharyngeal carcinoma(NPC),and various T-cell lymphomas.It can also been detected in other tumors such as gastric carcinoma,lung cancer and thymic carcinoma.NPC is a common cancer in Southeastern Asia,especially in the southern part of China,with a yearly incidence rate between 10 and 50 per 100,000.It's one of the ten main malignancies that urgently need efficiently preventive and therapeutic measures in our country.Radiotherapy is so far the preferred method for NPC therapy,with which the 5-year overall survival rates are about 50～60%.Regional relapse and distant metastasis are the main reason for the failure of the treatment for advanced NPC.Cellular immune responses play an important part in the clear virus infection and tumor immunosurveillance.In the early of 1980s in China,there were researches about CTL-mediated immunity and the HLA restriction in NPC patients,and it was found that T cell in NPC patients had some specific cytotoxic effect on NPC cell.So enhancing the level of specific CTL responses to NPC cell could suppress relapse and metastasis of NPC. Viral antigen is the strongest immunogenic molecule in malignancies.The existence of EBV in the NPC tumor tissue provides a target ofgene therapy.EBV-positive NPC cells express EBNA1,LMP1 (in part NPC)and LMP2 protein.Of the three antigens,EBNA1 contains a Gly-Ala repeat sequence,which will interrupt the presentation of it through HLA I restricted pathway to T cells.LMP1 displays oncogenicity and EBV isolates recovered from different geographic regions display a high degree of genetic variation within the LMP1 gene.LMP2 is consistently detected in nasopharyngeal carcinoma tumor biopsies and can be found on the cancer cell membrane.It is not effect the efficiency of primary B-lymphocyte growth transformation and is not oncogenicity.LMP2 is the most frequently recognized protein by CTL.Many HLA class I-restricted CTL epitopes of LMP2 have been identified.HLA alleles that presented LMP2 epitopes are relatively common in the Chinese population and the epitope sequence is widely conserved among EBV strains.So it becomes the target of immunotherapy for NPC.Adenoviruses can transfer genes into a broad spectrum of cell types,including mitotic and postmitotic cells.Additionally,high titers of viruses and high level of transgene expression generally can be obtained.It has been used the research of immunotherapy for many kinds malignant.Ad type 5(Ad5)has been commonly used to prepare recombinant Ad vectors.Ad5 requires the coxsackievirus-adenovirus receptor(CAR)on the cell membrane as an initial receptor for infection, and the transduction efficiency of the Ad5-based vector closely correlates with the cell-surface density of CAR.Unfortunately,the expression levels of CAR are often low or almost lack in most of the important target cells for gene therapy,including primary cancer cells, skeletal muscle cells,lymphocytes,fibrocytes,macrophage, monocyte-derived dendritic cells,and hematopoietic stem cells,and those cells are difficult to achieve sufficient gene transfer with recombinant Ad5 vectors unless high viral titers are used.Ad5F35 adenovirus is constructed by the Ad5 modify with a type 35 fiber and can bind with CD46,which is a membrane protein receptor and expressed on a broad type cells,including human hematopoietic stem cells,DCs,malignant tumor cells and so on.It did not depend on the CAR on the cell membrane as a receptor for infection target cell.There were reports that Ad5 carrying subgroup B Ad fibers is more potent than classical Ad5 for gene transfer and expression in human DC,Ad5 modified with a type 35fiber(Ad5F35),being the most efficient vector.Our aim of the study was,on the basis of understanding the EBV-specific cell immunity condition in NPC patient,to construct recombinant adenovirus Ad5F35-LMP2 and to detect its immune effect. At first,we used a highly sensitive real-time PCR,ELISPOT assay, immunoenzymic method and flow cytometry to measure respectively the EBV-DNA load,EBV-LMP2 specific CTL responses,the antibody of EBV-VCA-IgA,T cell subgroup and the ratio of CD4⁺CD25⁺highTr cell. The results showed that the EBV-DNA load,no matter positive rate or quantitate in NPC patient serum,was statistically different from control and healthy virus carriers samples and displayed a higher the EBV-DNA load in three cases.NPC Patients with advance stage disease had significantly higher viral load compared to patients with early stage disease.The antibody titers of EBV-VCA-IgA in NPC patient also had statistically different with healthy virus carriers.EBV-LMP2 specific CTL responses of control,healthy virus carriers and NPC patient samples were statistically different,with a gradually decrease in control, healthy virus carriers and NPC cases.And positive-rate of cellular immune responses to EBV-LMP2 was inversely associated with serum EBV-DNA load.The ratio of CD3⁺ and CD4⁺ T cell in NPC had significantly lower than control and healthy virus carriers.The ratio of CD4⁺CD25⁺highTr cell in NPC had significantly higher than control and healthy virus carriers.The antibody of EBV-LMP2 could be found in 75%NPC patients,but not be detected in control and healthy virus carriers.The second,we constructed replication defective recombinant adenovirus Ad5F35-LMP2 which containing LMP2 gene for immunotherapy vaccine for NPC.We cloned EBV-LMP2 genes into shuttle plasmids PDC316,Then the recombinant adenoviruses containing EBV-LMP2 gene was constructed using Admax system. Plamids PDC316 and adenovirus backbone pBHG-fiber5/35 were co-transfected by lipoplast into 293 cells to produce rAds.PCR test of supernant of virus showed that the LMP2 gene has been integrated into the genome of Ad5F35-LMP2.LMP2 specific mRNA was detected in Ad5F35-LMP2 infected 293 cells by RT-PCR to demonstrate the successful transduction of LMP2 gene.Indirect immunofluorescence assays further proved that LMP2 protein was expressed on Ad5F35-LMP2 infected 293 cells.To observe the LMP2 specific cellular and humoral immune responses after immunization with recombinant adenovirus Ad5F35-LMP2,we immunized rhesuses with Ad5F35-LMP2 through muscular injection in four groups of rhesuses,with four rhesus in each group, which were high dosage group(1.5×10¹⁰TCID50/each rhesus),medium dosage group(1.5×10⁹TCID50/each rhesus),low dosage group (1.5×10⁸TCID50/each rhesus),and the control group(PBS 4ml/each rhesus).They were totally immunized three times at intervals of one month.The EBV-LMP2 specific cellular immune responses were tested during the 0,4,8,12 weeks by ELISPOT after immunization respectively.And the titers of LMP2 antibody were tested by EIA at the same time.The results showed that EBV-LMP2 specific cellular and humoral immune responses,which induced by recombinant adenovirus Ad5F35-LMP2,could be found in all three dosage group.The potency of immune responses was related with the dosage of immunization. Which meant higher dosage tends to elicit more potent immune response.Among the high dosage groups rhesus,the highest immune response was 650 IFN-γpositive cells/1×10⁶ PBMCs stimulated by LMP2 polypeptide.The LMP2 antibody could be detected from 4 weeks after Ad5F35-LMP2 immunization.They would reach the peak during 8～12 weeks after immunization.And the control group could not be found the LMP2 specific cellular and humoral immune responses after immunization with PBS.To study whether the Ad5F35-LMP2 modified DCs can elicit CTL in vitro and testify whether the CTL activated in vitro can recognize and lysis tumor cells,we performed the growth surpression experiment of CNE-2 in vitro and in vivo.First,the DCs of volunteer(who has the same HLA type All with CNE-2)were isolated and cultured,then cocultured with autologous PBMC after the DCs were infected with Ad5F35-LMP2.After that,the CTL activated by Ad5F35-LMP2 modified DCs(Ad5F35-LMP2-DC)were cocultured with CNE-2 in vitro at some certain ratio,and the proliferation of CNE-2 was detected by MTT method.The results showed that the CTL activated by Ad5F35-LMP2-DC could effectively suppress the proliferation of CNE-2,compared with Ad5F35-HIV-mod-gp120.When the ratio of effecter and target was 20:1,the suppress rates of the specific CTL activated by Ad5F35-LMP2-DC reach 46.59%,and the Ad5F35-HIV-mod-gp120 control are 28.3%in vitro.On the basis of experiments in vivo,we carried on the experiments of suppress the proliferation of CNE-2 in vivo.Human PBMCs were injected to SCID mice,then, vaccinated SCID mice with Ad5F35-LMP2-DC or untransfected DC. And four days latter,CNE-2(1×10⁶ cell for each mouse)inoculated into SCID mouse subcutaneously.The tumor growth was monitored every week and the mice were executed on the 4th week to weigh up and pathologic examination.The results showed that tumor latent period is longer in Ad5F35-LMP2-DC group.Untransfected DC group and CNE-2 positive control group could be found tumor about one week,but Ad5F35-LMP2-DC group was about two weeks.And the tumor was smaller and lighter than untransfected DC group and CNE-2 positive control group.Average tumor control rate reached 89.18%. In summary,the cellular immune responses of NPC patients are lower,and EBV-DNA load displays a higher level.Replication of defective recombinant adenovirus Ad5F35-LMP2,which we constructed and contained LMP2 gene can induce EBV-LMP2 specific cellular and humoral immune responses.DC vaccine modified by recombinant adenovirus containing EBV-LMP2 gene can activate the LMP2-specific CTL in vitro and in vivo.The specific CTL can even recognize and destroy NPC tumor cell CNE-2.And it is a potential therapeutic vaccine in the immunotherapy of the EBV-associated malignancies,including NPC.