The Preliminary Research Of The Cellular Immunology Of The New Replicon Vaccine PSVK-HBVA For The Treatment Of Chronic Hepatitis B

Background: Chronic hepatitis B virus (HBV) infection is one of the worldwideproblem of public health, and about1million people die from hepatic failure，livercirrhosis and hepatocellular carcinoma caused by HBV infection each year. Currenttherapies for chronic hepatitis caused by HBV are limited. Therapeutic Vaccination is anefficient new technique to prevent and treat for chronic HBV through stimulating thebody’s own defense systems. Our lab had developed a new type of replicon based DNAvaccine vector pSVK, which can stimulate an efficient cellular immune response through1/100does of regular DNA vaccine. In view of the difficult problem of ‘immunetolerance’, many kinds of replicon based DNA vaccine vector pSVK-HBVA wereconstructed, which have the advantage of the intake, expression, divilery of target antigensand the activation of immune response. This study was to investigate the cellullarimmunologic response after the treatment with immune vaccine pSVK-HBVA in mice and to construct recombinant plasmid pIRES-neo-HBAg which included multiple HBVantigen genes.Objectives: We investigated the cellullar immunologic response and the effect ofimmunotherapy after the treatment with immune vaccine pSVK-HBVA in mice.Furthermore, we constructed eukaryotic expression plasmid pIRES-neo-HBAg whichincluded many HBV antigen genes, which could lay the foundation for vitro model ofHBV and effective CTL cytotoxicity by DNA vaccine pSVK-HBVA.Methods: The BALB/C mice were immunized by DNA vaccine pSVK-HBVA whichwas constructed in our lab. A week later from the last immune, spleen lymphocytes wereseparated, and then the activity of CD8+T cells were detected by ELISPOT, and theproliferation of leukomonocyte after stimulation by specific hepatitis B antigen wasdetected through the method CCK-8, cytokines including IL-2、IFN-γ、IL-4、IL-10weredetected by ELISA. Furthermore, pVAX1-HBVA was used as a template and the targetgenes of S1, S2, HBsAg And HBcAg were amplified by PCR, which was cloned intoeukaryotic expression vector pIRES-neo and developed a new recombinant expressionvector pIRES-neo-HBAg. Then the plasmid was confirmed by restriction enzymedigestion and DNA sequencing. The recombinant plasmid pIRES-neo-HBAg wastransfected into293T cells, and the expression were detected by methods of Westernblotting, flow cytometry, and immunofluorescence. Growing transfected cells wereselected by G418, expression of HBV fusion antigen was detected by flow cytometry.Results： New type of DNA vaccine pSVK-HBVA showed a strong cellularimmunological activity only at the does of1μg. The release of IFN-γand the activity ofCD8+T cells in leukomonocyte was increased significally, and the proliferation of spleenlymphocytes were observably increased through CCK8detection, and Th1cytokines weremarkedly increased by ELISA. Furthermore, pIRES-neo-HBAg was successfullyconstructed through confirmation by restriction enzyme digestion and DNA sequencing.The gene HBV was highly expressed after transient transfections of293T cells, and therate of positive cells was20.43%; The immunofluorescence results showed thatpIRES-neo-HBAg could highly expressed, then growing transfected cells were selected by 800μg/ml G418, and positive cells was86.20%。Conclusion: New type of DNA vaccine pSVK-HBVA could show a strong cellularimmunological activity at a low immunizing dose and this immune response was skewingto Th1response, which could improve the lack of immunogenicity and overcome theimmune tolerance of chronic hepatitis B. In addition, pIRES-neo-HBAg could expressmulti HBV antigens, which could lay the foundation to establish the vitro model of HBVand the tumor-bearing mice model.