Analysis of Hepatitis B in novel primary hepatocyte cultures

Abstract

Current model systems for the study of HBV infection and replication in vitro and in vivo are mainly based on transgenic ectopic expression of the HBV genome in either human hepatoma cell lines or transgenic mice. Failure to establish a durable cell culture using primary or tumor derived cells lines is thought to be due to the poor levels of hepatocyte differentiation. A novel primary hepatocyte culture system (LiverChip), which preserves primary hepatocyte differentiation and mimics the in-vivo microenvironment of the human liver, was used in this study to address this limitation. LiverChip provides cylindrical wells and a three- dimensional microfluidics system to culture primary hepatocytes and has been shown to reproduce microstructural features such as bile canaliculi between hepatocytes. This study aimed to create and evaluate a cell culture system for HBV using the LiverChip system. The objectives were: • To determine whether infection could be established reproducibly by exposure of cells to infected human serum • To demonstrate the full replication cycle of HBV in the cell culture system • To prove that viral progeny were infectious in naïve hepatocytes • To establish whether susceptibility to infection is broadly similar across a range of hepatocyte donors • To investigate whether infection could be established using serum from patients carrying either the HBeAg positive or the HBeAg negative variants of HBV • To investigate the interferon response to HBV infection in cell culture Described in this study, is a novel culture system for HBV based on the culture of primary human hepatocyte in a three-dimensional microfluidics system. Each experiment was conducted over 10 to 21 days. Multiple hepatocyte donors were i used with infection from HBeAg+/- patient isolates (serum). Infection with HBeAg+ patient serum leads to high levels of HBV DNA being secreted longitudinally. This model system proves the infection is sustained for at least 40 days in culture. HBV infection leads to the accumulation of HBV replication intermediates inside the cell and pregenomic(pg) RNA is detectable, increasing over the course of infection. LiverChip is also able to show the secretion of high level of HBV surface antigen (HBsAg) indicating virus replication. PHH in these cultures are permissive to both HBeAg +/- patient isolates. Accumulation of covalently closed circular DNA (cccDNA), replication intermediates, pregenomic RNA as well as de novo production of significant titers of infectious virus progeny, as determined by HBsAg secretion and reinfection of naïve cells, confirms that the complete HBV life cycle is supported in vitro. In addition to HBeAg-positive isolates, infection is successfully launched in liver microtissues using HBeAg- negative patient isolates, and viral replication is inhibited upon treatment with direct acting antiviral drugs. This project also studied in parallel to compare the robustness and susceptibility of LiverChip along with other established tissue culture system. The molecular virological characteristics of both model systems with a focus on viral kinetics and infection frequencies are explained. DNA and RNA from cultured PHH were extracted for detailed analysis, to investigate persistence of cccDNA and to evaluate the replication process of HBV. Host response to innate immune activity using NK cell/ Kupffer cell co-cultures and human cytokines production analysis was also explored using samples from LiverChip. These results were analyzed to provide a better understanding of HBV infection and the role of cccDNA in the viral cycle in disease progression of chronically infected patients. With these proven results, further research is now possible towards the cure of HBV.