Construction Of Recombinant Adeno-associated Virus Expressing HBV Envelope Major Protein, Large Protein And The Preliminary Study Of Their Immunogenicity

Hepatitis B, caused by hepatitis B virus (HBV) infection, is an infectious disease that has been threatening public health and has affected more than 350 million people worldwide. Among which, hepatocirrhosis, liver failure, or hepatocellular carcinoma will be developed eventually in about 15 to 40 percent. HBV is heavily endemic in China as there are approximately 10 million chronically infected patients. It is in urgent need of excogitating new HBV vaccines and searching for effective therapeutic methods since there is no panacea for chronic HBV infection to date.Due to its non-pathogenicity, the ability to infect various types of cells and the capability to mediate long-term gene expression, recombinant adeno-associated virus type 2 (rAAV-2) has attracted tremendous interest as a promising vector for gene delivery. In this study we have developed an HBV vaccine and then studied its immunogenicity by using rAAV-2 vector expressing HBV envelope major protein and large protein. Meanwhile, we have probed into the feasibility that rAAV-2 was introduced to dendritic cell (DC)-based immunotherapy of chronic hepatitis B.Hepatitis B virus (subtype ayw) envelope major protein (HBsAg) and lager protein (LHBsAg) gene were amplified from PTHBV-1 by PCR and then cloned into the adeno-associated virus vector pSNAV, the recombinant pSNAV-HBsAg and pSNAV- LHBsAg were transfected into BHK-21 cell by using Lipofectamine?2000. Both mixed cell lines, BHK-HBsAg and BHK-LHBsAg which expressing HbsAg, were isolated by using G418 selection and were then infected with recombinant herpes simplex virus (HSVl-rc/AUL2) which can package the rAAV-2. rAAV-2-HBsAg and rAAV-2-LHBsAg were obtained after purification. HPLC and SDS-PAGE were used to monitor the purity of rAAV-2-HBsAg and rAAV-2-LHBsAg,respectively. The blot hybridization was used to determine the physical titers of rAAV-2-HBsAg and rAAV-2-LHBsAg. The results of SDS-PAGE and HPLC showed that the purities of the final rAAV-2 products were higher than 99%. The physical titers of rAAV-2-HBsAg and rAAV-2-LHBsAg were 5 ×1011and 2 × 1012 virus particles/ml(vp/ml), respectively. The expression level of HBsAg in BHK-HbsAg and BHK-LHBsAg, detected by ELISA(enzyme-linked immunosorbent assay), were 28.6 ±6.7 ng/5× 106cells and 15.4±5.8 ng/5×106cells. The expression of HBsAg in BHK-21 cells and 293 cells infected with rAAV-2-HBsAg and rAAV-2-LHBsAg can be detected, and the amounts of HBsAg expression elevated following the increasing of MOI (multiplicity of infection, MOI). These results indicate that rAAV-2-HBsAg and rAAV-2-LHBsAg can transduce various types of cultured cells efficiently in vitro.The transduction efficiencies of rAAV-2-HBsAg and rAAV-2-LHBsAg in vivo and their immunogenicities were studied via intramuscular injection (ini), intravenous injection (iv), intraperitoneal injection (ip) and gastric gavage (gg). The results showed that there is no difference of HBsAg expression between rAAV-2-HBsAg and rAAV-2-LHBsAg via the same administration route. Expression levels of HBsAg are about the same after the administration of rAAV-2-HBsAg or rAAV-2-LHBsAg to Balb/c mice via different routes. One single administration (im, iv, ip and gg) of rAAV-2-HBsAg or rAAV-2-LHBsAg can induce both humoral and cellular immune response for a long time. Among the four administration routes, both rAAV-2-HBsAg and rAAV-2-LHBsAg can induce the strongest cytotoxic T lymphocyte (CTL) response via intravenous injection. To our knowledge, this is the first report that rAAV-2-mediated HBV antigen gene expression can induce CTL responses as well as anti-HBsAg antibodies. Thus, the rAAV-2 vector can be used as a promising candidate for hepatitis B vaccine, particularly as a therapeutic vaccine for chronic hepatitis B and for those patients who have non-responsive to the current HBV vaccine.Much attention has been paid to the adjuvanticity of DC in generating antigen-specific immune responses for anti-tumor and anti-infection therapeutics. First, Viral vectors have been introduced succes...