Development of experimental cell culture and animal models to recapitulate persistent hepatitis B virus infection

Abstract

Persistent infection with hepatitis B virus (HBV) affects approximately 240 million individuals worldwide and can lead to fibrosis, cirrhosis, and hepatocellular carcinoma. Current antiviral treatments for HBV are effective in reducing viral load, but they are incapable of curing the disease. Development of more effective therapies has been impeded by the scarcity of suitable in vitro and in vivo platforms for HBV infection. Commonly used hepatoma cell lines do not adequately model the complex physiology of adult hepatocytes, complicating in vitro HBV studies. Though cultures of primary human hepatocytes (PHHs) provide a physiologically and clinically relevant system to study HBV, it is challenging to maintain hepatocyte function in vitro. Additionally, in vivo studies of HBV have been notoriously difficult due to the limited species and cellular tropism of HBV, which only productively replicates in human and chimpanzee hepatocytes. The lack of in vivo systems susceptible to HBV has led to the search for small animal models that can be used for developing curative therapies. Human liver chimeric mice have been shown to be persistently infected with HBV, but engraftment of PHHs in current models is difficult to control. To this end, the Ploss lab has established two novel platforms, which can serve as precise drug-testing platforms for HBV: an in vitro self-assembling primary human hepatocyte co-culture (SACC-PHH) system and an immunodeficient human liver chimeric mouse with a disruption in the fumarylacetoacetate hydrolase (FAH) gene. Using SACC-PHHs, we have established long-term HBV infection using both cell culture-derived HBV and patient plasma-derived HBV without the use of innate immune inhibitors. Furthermore, we demonstrate the scalability of SACC-PHHs to a high-throughput format and use direct-acting antivirals and putative host-targeting antivirals in a proof-of-concept experiment to exhibit the utility of SACC-PHHs as a drug-testing platform. In the novel human liver chimeric mouse model, we show stable engraftment of PHHs in a FAHΔexon9 NOD Rag1-/- IL2RγNULL (FAHΔexon9 NRG) mouse and demonstrate persistent HBV infection. Additionally, drug treatment kinetics observed in clinical settings were exhibited upon treatment of FAHΔexon9 NRG mice with entecavir, a reverse transcriptase inhibitor. Together, these systems provide a basis upon which drug discovery and testing may be performed, progressing towards the ultimate aim of curing HBV.