Human Liver Organoids as a Patient-derived Model for HBV Infection and Cellular Response

Background & Aims: Current HBV in vitro model systems suffer from many physiological limitations that restrict understanding of complex viral-host interactions and thus prohibit prediction of disease in vivo. We developed and assessed adult stem cell (AdSC) derived liver organoids as a novel model system for characterisation of the HBV lifecycle, the cellular response to infection and demonstrate their utility in assessing antiviral and immunomodulator response. This model system has the potential to be used in predicting individual HBV responses to antivirals and viral reactivation in the setting of immunosuppressive agents. Methods: Ductal stem cells were isolated from healthy tissue acquired from liver resections or biopsy (n=12). Wnt3a & RSPO-1 containing medium was used to stimulate ductal stem cell expansion into organoids which were subsequently differentiated into hepatocyte-like cells. Mature hepatocyte metabolic markers (albumin, CYP3A4) and HBV entry receptor (Na-taurocholate co-transporting polypeptide, NTCP) expression were evaluated throughout differentiation using qRT-PCR and confocal microscopy. We assessed the organoids culture conditions required for HBV infection and HBV life cycle using HepAD38 (genotype D) and plasma derived HBV (genotype B & C). HBV infection was confirmed using immunofluorescence staining (HBcAg), qRT-PCR (RNA, cccDNA, extracellular DNA) and ELISA (HBsAg and HBeAg). We also assessed drug responsiveness using antivirals and an immunosuppressive agent, and cellular responses (interferon-stimulated genes) using interferon-aplha and viral mimic (PolyI:C). Results: Following differentiation, organoids underwent structural remodelling and changes in cellular polarity, accompanied with an increase in albumin, CYP3A4 and NTCP mRNA expression. Optimal HBV infection was achieved in well-differentiated organoids using spinoculation of at least 200 copies/cell of AD38 derived HBV. Infected organoids demonstrate time and donor dependent increase in HBV RNA, cccDNA, extracellular DNA, HBe and HBsAg consistent with viral replication and antigen secretion. Using these markers we assessed drug-responsiveness to the HBV entry inhibitor, Myrcludex B and the JAK inhibitor, Baricitinib. Despite having a very robust interferon stimulated gene response to interferon-alpha and PolyI:C stimulation, HBV infection in liver organoids did not reveal innate immune activation. Conclusions: AdSC derived liver organoids support the full life cycle of HBV with significant donor dependent variation in viral replication and cellular responses. These features can be utilised for development of personalised drug testing platform for antivirals. ### Competing Interest Statement The authors have declared no competing interest.