Mechanisms of hepadnavirus persistence and role of interferon gamma in modulation of anti-viral immune response

Hepatitis B virus (HBV) is a major pathogen that induces chronic hepatitis in approximately 400 million people worldwide frequently leading to liver cirrhosis and hepatocellular carcinoma. Woodchucks infected with woodchuck hepatitis virus (WHV) demonstrate virological and immunological profiles of infection and liver disease patterns highly comparable to those seen in human hepatitis B. Many attributes of HBV and the immune system interactions remain unknown, particularly how HBV persists and causes chronic liver disease. In the current studies, we investigated several aspects of the hepadnavirus-host immune system interaction and examined a role in these interactions of interferon gamma (IFNγ), which is known to be a potent antiviral and immune system regulatory cytokine. The specific objectives of our studies were to: (1) clone and express woodchuck IFNγ and tumor necrosis factor alpha (TNFα) in the baculovirus and Escherichia coli (E.coli) expression systems; (2) establish an in vitro model in which the effect of WHV genome expression on the hepatocyte surface presentation of the class 1 major histocompatibility complex (MHC) molecules can be investigated and, by using this system, to determine which WHV translation product interferes with the class 1 antigen display and whether this interference can be reversed by hepatocyte treatment with IFNγ, and (3) investigate the modulator effect of IFNγ on a DNA vaccine in preventing WHV infection. In the course of these studies, we have successfully produced biologically active recombinant woodchuck IFNγ and TNFα. The data showed that the baculovirus-derived IFNγ and TNFα had considerably greater biological potency than the same cytokines produced in E. coli. In a subsequent study, we established an in vitro model to examine hepadnavirus interference with hepatocyte class 1 MHC expression. The results revealed that the class 1 antigen presentation was significantly inhibited on hepatocytes transcribing the complete WHV genome, similarly as it was previously reported by this laboratory for hepatocytes in chronic WHV hepatitis. WHV envelope preS2 protein was found to be responsible for the class 1 MHC antigen suppression, while transcription of WHV X gene significantly augmented the class 1 antigen display on the hepatocytes surface. This defect in class 1 MHC presentation may diminish the susceptibility of hepatocytes to virus-specific T cell-mediated elimination, hamper hepadnavirus clearance, and contribute to the establishment of chronic hepatitis. Importantly, we also demonstrated that IFNγ, a stimulator of class 1 MHC expression, can reverse this WHV-induced inhibitory effect and restore class 1 MHC presentation on hepatocytes, suggesting that the same could be true for hepatocytes in chronic hepadnaviral hepatitis. Further, our results demonstrated that DNA vaccination with combined WHV core gene and IFNγ DNA provided significantly better protection against serologically evident WHV infection and hepatitis than WHV core DNA vaccine alone. However, our study revealed that protection against the virus is never complete. The data also showed that IFNγ can serve as an adjuvant and can augment immune response against hepadnavirus, as well as possibly clear infection in a non-cytopathic manner. In summary, the results and the investigative tools generated should advance our understanding of the complexity of molecular interactions occurring between hepadnavirus, hepatocytes and the host immune system which govern development of HBV persistence and chronic hepatitis. They may also aid in the development of novel more effective therapies against HBV.