Development of a non-replicative, single shot DNA-based vaccine expressing all hepatitis B viral antigens

Abstract

Introduction: The activity of a broad based cellular immune response to different HBV antigens is an important factor contributing to virus elimination. Our aim was to develop a single shot DNA vaccine expressing all HBV antigens but lacking replication. Therefore, a CMV-promoter based plasmid producing a HBV pregenome lacking the first 43 nt resulting in a mutated epsilon-signal and subsequently a defective pregenome encapsidation (pCH3143) was employed. Materials and methods: pCH3143 was transfected in G8 murine myoblast and Huh–7 human hepatoma cells. Expression of HBcAg, HBsAgs, HBeAg, polymerase, and HBxAg was determined by Western-blot and ELISA techniques. HBV replication and RNA analysis was assessed by Southern blot as well as RNAse protection assays and Northern blot. Balb/c and C57BL/6 mice were immunized once with 100mg pCH3143 i.m. and, subsequently, antibody as well as cytotoxic T lymphocyte (CTL) responses against all viral gene products were determined. Protective immunity was assessed by re-challenge of pCH3143-immunized Balb/c mice with syngeneic tumor cells expressing HBcAg, HBeAg, HBsAgs, and polymerase. Results: High level expression of HBeAg, polymerase, and all HBsAgs (small, pre-S2, pre-S1) could be detected in transfected cells. Expression of x-antigen was weak. No HBV replication or encapsidation of pregenomic RNA could be observed. Immunization of Balb/c and C57BL/6 mice resulted in strong anti-HBs and anti-HBc antibody responses. Only low-titer antibodies against polymerase and no anti-HBx could be detected. Strong CTL responses were observed against all viral antigens in a hierarchical manner (HBcAg>HBsAg>polymerase>HBxAg). Corresponding to the in vitro CTL activity, pCH3143 vaccinated animals were protected against a s.c. challenge with syngeneic tumors expressing HBcAg, HBsAg, and polymerase. Contrary, there was a rapid growth of x-antigen expressing tumors in pCH3143 immunized animals. Discussion: This is the first demonstration of a safe single shot vaccine expressing all HBV antigens, lacking HBV replication and inducing strong humoral and cellular immune responses against all viral proteins except HBxAg. It may be of value for the treatment of chronic viral hepatitis, for vaccine non-responders, and the prophylaxis of HCC.