Research Of Therapeutic Vaccine Against Chronic Hepatitis B Based On Heat Shock Protein

Hepatitis B is a public problem.There is 20 billion people in the world infected by hepatitis B virus,among which 3-4 billion people have chronic hepatitis B. 25-40% of chronic hepatitis B patients develop liver cirrhosis and cancer. There are estimated to be 1.3billion cases of chronic hepatitis B in China. Between them 30 million patients need anti-virus therapy,which result in health care and other costs in China that are estimated to be RMB 300 to 500 billion per year. There is no effective therapy against Chronic hepatitis B now.Anti-virus is the most important therapy. With the development of molecular medical science,therapeutic vaccine is the new way to therapy chronic hepatitis B.Our work aimed to develop a new therapeutic vaccine based on heat shock protein against Chronic hepatitis B.In recent years, investigators found out that HSP-peptides complex can deliver the antigen to MHC-I restricted antigen presenting pathway through receptor (CD91) mediated phagocytosis,and then, cellular immunity is activated. We select the hsp65 of M.tuberculosis to fuse with HBcAg as the therapeutic vaccine, which can present the antigen to MHC-I restricted pathway and activate cellular immunity against cells infected by hepatits B virus.Reseach found out that HBcAg is the dominant antigen in the hepatitis virus infection,and the CTL epitope of HBcAg can prime specific CTL activity against HBV.So HBcAg is used as the aim of therapeutic vaccine gradully.We fuse HBcAg to the N terminus of HSP65,which to activate CTL against HBV.Soluble fusion protein was expressed at high level in E.coli. To facilitate the purification of this protein, the protein is attached a polarized domain at C terminus. After Ammonium sulfate precipitation, cation exchanger chromatography and Gel filtration chromatography, protein sample that has the purity above 80% was achieved.Mice were immunized subcutaneously with fusion protein and HBcAg in the absence of additional adjuvant. Immune spleen cells were stimulated by ConA and HBcAg ,and the T cell multiplication analysis indicated that the fusion protein prime T cell multiplication. Effector cells were assayed against HBcAg-pulsed target cells.Lysis of target cells by effector CTL from mice given a single immunization with Hsp65-HBc was effective.In contrast, immunization with HBc alone was less effective thanHsp65-HBc in priming CTL activity. The results of these studies clearly demonstrate the potential efficacy of Hsp65-HBcAg in the immunotherapy of chronic HBV infection.