Beard, M. R. | bioRxiv Science

bioRxiv2022

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

Background & AimsCurrent 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. MethodsDuctal 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- and viral mimic (PolyI:C). ResultsFollowing 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- and PolyI:C stimulation, HBV infection in liver organoids did not reveal innate immune activation. ConclusionsAdSC 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. Lay SummaryHuman liver organoid culture provides a personalised assessment of HBV infection, replication and responsiveness to antiviral therapy. This model system has a robust innate immune response and could be used to assess novel immune-modulating curative therapy.

microbiology↗

bioRxiv2022

Constitutive expression and distinct properties of IFN-epsilon protect the female reproductive tract from Zika virus infection

The immunological surveillance factors controlling vulnerability of the female reproductive tract (FRT) to sexually transmitted viral infections are not well understood. Interferon-epsilon (IFN{varepsilon}) is a distinct, immunoregulatory type-I IFN that is constitutively expressed by FRT epithelium and is not induced by pathogens like other antiviral IFNs , {beta} and {lambda}. We show the necessity of IFN{varepsilon} for Zika Virus (ZIKV) protection by: increased susceptibility of IFN{varepsilon}-/- mice; their "rescue" by intravaginal recombinant IFN{varepsilon} treatment and blockade of protective endogenous IFN{varepsilon} by neutralising antibody. Complementary studies in human FRT cell lines showed IFN{varepsilon} had potent anti-ZIKV activity, associated with transcriptome responses similar to IFN{lambda} but lacking the proinflammatory gene signature of IFN. IFN{varepsilon} activated STAT1/2 pathways similar to IFN and {lambda} that were inhibited by ZIKV-encoded non-structural (NS) proteins, but not if IFN{varepsilon} exposure preceded infection. This scenario is provided by the constitutive expression of endogenous IFN{varepsilon}. However, the IFN{varepsilon} expression was not inhibited by ZIKV NS proteins despite their ability to antagonise the expression of IFN{beta} or {lambda}. Thus, the constitutive expression of IFN{varepsilon} provides cellular resistance to viral strategies of antagonism and maximises the antiviral activity of the FRT. These results show that the unique spatiotemporal properties of IFN{varepsilon} provides an innate immune surveillance network in the FRT that is a significant barrier to viral infection with important implications for prevention and therapy. Author SummaryThe female reproductive tract (FRT) is vulnerable to sexually transmitted infections and therefore a well-tuned immune surveillance system is crucial for maintaining a healthy FRT. However, our understanding of the factors that impact viral infection of the FRT and the host response are not well understood. In this work we investigate the role of a hormonally regulated type I interferon, IFN epsilon (IFN{varepsilon}) in control of Zika virus (ZIKV) infection of the FRT. IFN{varepsilon} is unique compared to other canonical type-I IFNs in that it is constitutively expressed by epithelial cells of the FRT with expression levels controlled by progesterone and not in response to viral infection. We demonstrate that IFN{varepsilon} has anti-ZIKV properties using a combination of IFN{varepsilon} KO mice, blockade of endogenous IFN{varepsilon} by neutralising Abs and rescue of IFN{varepsilon} KO mice by recombinant IFN{varepsilon} administered directly to the FRT. Furthermore, we complemented our in vivo studies using human FRT derived cell lines. Importantly, ZIKV NS proteins did not block IFN{varepsilon} expression despite their ability to antagonise the expression of IFN{beta} or {lambda}. Collectively this work implicates IFN{varepsilon} as a key type-I IFN that provides a distinct homeostatic antiviral environment in the FRT.

immunology↗