An hepatitis D virus infection model using human pluripotent stem cell-derived hepatocytes for virus-host interactions and antiviral evaluation

An estimated ~12 million people worldwide suffer from chronic hepatitis D virus (HDV) infection, which can cause the most aggressive form of viral hepatitis when co-infected with hepatitis B virus (HBV), leading to an accelerated process of liver dysfunction towards hepatic fibrosis, cirrhosis and hepatocellular carcinoma. HDV is a satellite virus and relies on its helper HBV to provide HBV surface antigen (HBsAg) for viral assembly and subsequent secretion. Currently, the sole antiviral drug approved by the European Medicines Agency specifically for HDV treatment is the entry inhibitor bulevirtide, however, it is only available in certain European countries. Despite this significant burden, our knowledge of HDV biology remains limited, thus restricting the development of targeted antiviral therapies. This is partly due to the lack of reliable HDV cell culture systems that mimic the physiological status of hepatocytes in vivo. In this thesis, I have demonstrated the use of stem cell-derived hepatocyte-like cells (HLCs) as a novel cell culture model for the study of HDV. HLCs are fully susceptible to HDV infection across various tested genotypes. They endogenously express the cell entry receptor the sodium taurocholate co-transporting polypeptide at levels sufficient to mediate HBV/HDV entry. When co-infected with HBV or ectopically expressing HBsAg through adeno-associated virus transduction (HLCsHBsAg), HLCs are able to effectively produce and release infectious progeny virions, thus recapitulating the entire HDV life cycle. Using the HLCsHBsAg system, I also demonstrated that the system supports the extracellular spread of HDV, which occurs in vivo but has been so far challenging to replicate in vitro. This allowed me to test available anti-HDV regimens that target HDV spread, underscoring the utility of HLCs as a platform for drug candidate evaluation. By challenging the cells along the differentiation with HDV infection, I observed an increased susceptibility to HDV infection in fully matured HLCs. Using transcriptomic analysis and further confirmation studies, I identified CD63 as a novel HDV co-entry factor, which was found to be rate-limiting for HDV infection in immature hepatocytes. While this finding augments our understanding of HBV/HDV infection imminently, it also provides a guideline on how to use stem cell differentiation and stem cell-derived culture models to identify host factors of other viruses. To conclude, my study provides a comprehensive and quantitative assessment of HDV infection, showing that HLCs can recapitulate the entire HDV life cycle and support extracellular spread, as well as revealing the identification of novel host co-factor(s), such as the entry co-factor CD63. As an alternative to hepatoma cells and primary human hepatocytes, HLCs represent a renewable, physiologically relevant and genetically tractable system for HDV-host interaction studies and anti-viral drug evaluation.