Study On Therapeutic HBV Vaccines Which Compose With Recombinant Fusion Protein CS1 And HBsAg

Chronic hepatitis B is the global disease which serious threaten human's health. Currently the effective medicine hasn't been discovered, and the vaccine still has the key function for the prophylaxis of the disease. Currently hepatitis surface antigen-based vaccines have been widely used by inducing effective humoral immunity. But the major shortcoming of these vaccines is the disability to induce cellular immunity, which is essential for eliminating the endocellular virus and breaking immunologic tolerance which exist widely in chronic hepatitis B patients.We transformed the recombinant fusion HBV core antigen and PreS1 gene (CS1) prokaryotic expression vector (this HBV genome belongs to adr subtype of Chinese epidemic strain) which constructed by institute of virology in Chinese academy of preventive medicine into E. coli, and expressed by IPTG induction. The expressed protein was proved the same as target protein we wanted by SDS-PAGE electrophoresis, western blot and antigenicity investigation. The protein purified by DEAE-Sepharose 4FF ion exchange chromatography and Sephadex-G75 gel filtration, and which became more than 98% purity, higher immunogenicity and can brought strong level antibody against HBcAg.BALB/C mice were immunized with the combined vaccine recombinant -- CS1 which were purified in this study and CHO-HBsAg which were produced by the third vaccine department of LanZhou Institute of Biological Products, immunized separately with HBcAg and HBsAg as the contrast, and we investigated the immunization effect of several regimens involving different components, different potency and different times of vaccination in BALB/C mice. After immunization, we prepared mice serum to detect the concentration of antibody by ELISA and prepared splenic mononuclear cells (MNC) of mice to detect specific lymph proliferation by MTT. The result as detection criterion, we screen out the best immunization protocol.Afterwards, we immunized the BALB/C mice again with appropriate potency combinatorial vaccine with CS1 and HBsAg (CS1+S), and Al(OH)₃ which finally concentration is 1mg/ml as adjuvant, separate immunization with HBcAg and HBsAg asthe contrast. After immunization, we prepared mice serum to detect the concentration of antibody by ELISA and prepared splenic mononuclear cells (MNC) of mice to detect the concentration of IFN-y by ELISA and analysis the T lymphocyte subpopulations through FACS. Finally, DTH response was detected by subcutaneous injecting antigen into BALB/C mice. It is reported that the concentration of IFN-y and the count of CD3+ CD4+ splenocyte of mice brought by the mouse immunized by CSl+S was distinct higher than others (P<0.05), and the counts of CD8+ splenocyte of mice brought by CSl+S plus Al(0H)3 adjuvant group was distinct higher than controls (P<0.05);and CSl+S group can induce obvious DTH (PO.05). Concentration of the anti-HBsAg brought by CSl+S group as much as twice dose HBsAg group. The effect brought by antigen of plus or not plus A1(OH)3 adjuvant have no distinct difference (P>0.05).In general, it was shown that the combinatorial vaccine with HBsAg and HBcAg can induce strong and homogenous humoral and cellular responses, and conduce to eliminate the endocellular HBV virus and break immunologic tolerance which existed durative in chronic hepatitis B patients. So it can offer the new thought and theory foundation for the further study to exploit the Hepatitis therapeutic vaccine.