Experimental Study On Therapeutic HBV-HSP70 Fusion DNA Vaccines Which Delivered By Live Attenuated Salmonella

Chronic hepatitis B (CHB) severely threaten to the health of human beings, but the treatment options for chronic HBV carriers were still limited and less effective. The main reason of chronicity after hepatitis B virus infection was considered as the deficiency of specific cellular immunity that can entirely eliminate virus in vivo. DNA vaccine, with a predominate advantage in specific cellular immunity in vivo, has been widely predicted as a potential immunotherapy method for cure CHB. However, DNA vaccines wrer still in laboratory scale after decade's research. The capability of DNA vaccine to induce specific immunity decreased obviously with the size increased of experimental animals. So how to enhance the immune response, especially the specific cellular immune response, which induced by DNA vaccine, should be particularly focused on DNA vaccine development.Mycobacterium tuberculosis heat shock protein 70 C-terminal fragment (mtHSP70c) and mtHSP70 T cell stimulatory epitode (mtHSP70t) had the complete adjuvant function, and with the better performance by reducing self-immune responses caused by the full length mtHSP70. Their gene sequences were used to construct fusion DNA vaccine. Attenuated salmonella which given in oral route could deliver DNA plasmid to antigen presenting cells (APC), and enhanced the potency of DNA vaccine. Prime immunization by DNA vaccine-boost immunization by modified vaccinia Ankara (MVA) vector vaccine was another effective immunization strategy.Use several immunization strategies should get stronger immune response which induced by DNA vaccine. This study applied multiple molecular techniques to construct recombinant genetic vaccines encoding fusion protein of HBV preC/C-HSP70c, HBV preC/C-HSP70t, HBV preS2/S-HSP70c and HBV preS2/S-HSP70t. The constructs were verified by PCR, endonuclease digestion and sequencing. In these constructs, HBV preC/C & preS2/S was the target of the immunotherapy, and mtHSP70c & mtHSP70t help to improve the immunogenicity of the vaccines as immunoadjuvants. The recombinated DNA vaccines were transfected into HepG2 cells by liposome transfect system. The target antigen mRNA and protein levels in transfected cells were examined respectively by RT-PCR, ELISA and CLIA. The results showed that the recombinant plasmids could be expressed rightly in the cells. Then the recombinant plasmids were electrical transformed into attenuated salmonella SL7207 strains to get the recombinant salmonella which delivered the recombinant plasmids.Balb/c mice were immunized by different processes, including HBV DNA vaccines prime/rMVA boost, HBV-HSP70 DNA fusion vaccines prime/rMVA boost, attenuated salmonlla harbored HBV DNA vaccines prime/rMVA boost and attenuated salmonlla harbored HBV-HSP70 DNA fusion vaccines prime/rMVA boost, the serum and spleen cells of mice were collected. ELISA analysis was used to detect the specific antibodies in serum. Flow cytometry examined the CD3+/CD4+/CD8+T cell subgroups level to analysis nonspecfic cellular immune response, and IFN-y ELISpot to evaluate antigen-specific cellular immune response. Result:(1)For specific humoral immunity, all experiment groups besides control groups could induce specific antibodies, and there was no statistical difference of antibodies'level. (2)For cellular immunity, both of nonspecfic or specific cellular immunity, HBV-HSP70 fusion DNA vaccines created better performance. And the HBV-HSP70t fusion DNA vaccines were better than HBV-HSP70c fusion DNA vaccines. There was no statistical difference in cellular immunity between recombinant salmonlla oral groups and recombinant DNA vaccine inject groups. This experimental research provides basic evidence for the development of therapeutic HBV DNA vaccines.