Study Of Immunologic Effect Of Recombinant MVA Virus Of Expressing EBV-LMP2

Epstein-Barr virus (EBV) can result in a number of diseases, and nasopharyngealcarcinoma (NPC) is one of the most important EBV-associated malignancicesin in theworld, which mostly occurs at high frequency in Southeast Asia, the incidence ofChina nearly accounted for80%of the world. At present, radiotherapy andchemotherapy are the main treatment method for nasopharyngeal carcinoma, but theresult of treatment is not very well. Because there was local recurrence and distantmetastasis, especially in advanced of nasopharyngeal carcinoma. After treatment, theincidence of distant metastasis is about22%～36%.EBV provides a target for immunotherapy, which exist in the tumor cells of NPC.Three viral proteins are only expressed in tumor cells of NPC, including EBV nuclearantigen1(EBNA1), latent membrane protein1(LMP1), latent membrane protein2(LMP2). There are several advantages for the gene of LMP2, which has non-cancerigenic, doesn’t cause the transformation of B lymphocytes. Not only has it theactivation epitope of the cell for HLA, but also can induced CTL response. In addition,the protein can be continually expressed in nasopharyngeal carcinoma cells. Therefore,LMP2is a good target antigen that can induce CTL for prevention and treatment ofNPC.Vaccinia virus is a virus that can be inserted the exogenous gene which is long, andthere is widely host cells. For it, immunogenicity is good, and proliferation productionis easy. There are many ways which could be used to immune vaccination. MVA isvaccinia virus that is defective for replication, which was obtained by repeatedlysubcultured from vaccinia virus Ankara strain. There were a lot of deficiencies andmutation in its genome, resulting in the loss of a host range and pathogenicity. Inaddition to its advantages of vaccinia virus, MVA also has the characteristics of highsafety, so it has been widely applied to the research of vaccine.Because the virus vector could strongly produce neutralizing antibodies against it-self, the same vector is limited which is repeatly used. Therefore, academician ZengYi proposed a strategy that multi-vector vaccines are sequentially used to immune.The results of research shows, the specific immune responses can be induced well.In this paper, using MVA as vector, MVA-LMP2was successfully constructedwhich was a recombinant vaccinia virus of replication defective. The results showedthat the correct sequence of lmp2was accurately inserted into MVA vector by themeans of PCR, enzyme digestion and sequencing. LMP2protein could also beeffectively expressed through some ways of RT-PCR, indirect immunofluorescenceassay and Western-Blot assay. The goal of the paper was to really evaluate that the specific CTL immune response can be induced in Balb/c mice by immunizedMVA-LMP2recombinant virus. With three dosages of low (2×105pfu/mouse), middle(2×106pfu/mouse), high (2×107pfu/mouse), Balb/c mice are respectively immunizedat0,2weeks by the method of intramuscular injection. At third weeks, the IFN-γELISPOT method is used for detecting EBV-LMP2specific CTL immuneresponse ofmice spleen lymphocytes. The results showed that specific cellular responses forLMP2could be generated in the three dose groups of mice, along with the increase ofimmune dosage, induced specific cellular immune response enhancement.The more immune dose is, the stronger LMP2-specific cellular immune response is.The most of releasing the cytokines IFN is up to437/106of spleen lymphocytes. Thespecific anti-LMP2antibody can be detected in mice immunized with MVA-LMP2byindirect immunofluorescence assay. The results showed that specific anti LMP2antibody was generated.In addition, some sequential vaccines was composed of pVR-LMP2of DNAvaccine, Ad5f35-LMP2, Ad5-LMP2of adenovirus vaccine and MVA-LMP2ofrecombinant vaccinia virus. Firstly, Balb/c mice were immunized with DNA vaccinetwo times, adenovirus vaccine two times again, and finally respectively wereimmunized with by Ad5-LMP2, Ad5f35-LMP2and MVA-LMP2. After a week of thelast immunization, IFN-γ ELISPOT was used for the detection of specific CTL level.The results showed that the specific CTL response induced by the immune group ofAd5f35-LMP2was higher than that of the immunized group of Ad5-LMP2.Compared with the two immune groups above, the specific CTL response induced byMVA-LMP2was best. Stimulated by the LMP2peptide, cytokines IFN released cellpopulation could up to2694in1×106spleen lymphocytes. Thus, the best effect wasthat specific CTL was induced by the vaccine composed DNA vaccine, adenovirusvaccine and recombinant vaccinia virus vaccine.