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

Lim, C. K.; Romeo, O.; Chilver, A. P.; Tran, B. M.; Flanagan, D.; Kirby, E. N.; Breen, J.; Vincan, E.; Warner, N.; McCartney, E. M.; Van der Hoek, M.; Ruszkiewicz, A.; Tse, E.; Beard, M. R.

doi:10.1101/2022.10.20.513112

bioRxiv · 10.1101/2022.10.20.513112

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

Lim, C. K.·Romeo, O.·Chilver, A. P.·Tran, B. M.·Flanagan, D.·Kirby, E. N.·Breen, J.·Vincan, E.·Warner, N.·McCartney, E. M.·Van der Hoek, M.·Ruszkiewicz, A.·Tse, E.·Beard, M. R.

View original source ↗Export metadata ↓

Abstract

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.

Explore related subjects

microbiology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Lim, C. K., Romeo, O., Chilver, A. P., Tran, B. M., Flanagan, D., Kirby, E. N., Breen, J., Vincan, E., Warner, N., McCartney, E. M., Van der Hoek, M., Ruszkiewicz, A., Tse, E., Beard, M. R.. 2022-10-21. Human Liver Organoids as a Patient-derived Model for HBV Infection and Cellular Response. https://doi.org/10.1101/2022.10.20.513112

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

A conserved cysteine-histidine-glutamate metal site identifies DUF501 (Rv1025), an essential uncharacterised protein family of Mycobacterium tuberculosis, as a candidate metalloenzyme and drug target

A substantial fraction of the Mycobacterium tuberculosis proteome remains functionally uncharacterised. Rv1025, a 155-residue protein carrying the domain of unknown function DUF501 (Pfam PF04417), is essential by transposon mutagenesis and vulnerable by CRISPR interference, an attractive but neglected drug target, yet has never been functionally described. The family (4,370 proteins, no Gene Ontology term, no solved structure) is uncharacterised across all organisms and essential in three Actinobacterial genera. A Foldseek search of the AlphaFold model against complete structural databases finds no significant homolog, indicating a novel fold. The operon eno-divIC-Rv1025-ppx2 is conserved across the Actinobacteria phylum, yet AlphaFold-Multimer finds no direct complex between Rv1025 and its neighbour DivIC. Instead, conservation across 8,700 homologous sequences reveals a near-invariant Cys113-His115-Glu59 cluster forming a pocket. Holo AlphaFold3 predictions with Zn, Fe and Mn confidently place a divalent metal on this triad at 2.25-2.47 A; mutating the triad relocates the metal, and an independent backbone-geometry predictor recovers the same site, confirming specificity. The triad is universal across the family: present in all 1,472 near-complete bacterial sequences of the Pfam alignment, with no non-conservative substitution among the 2,228 sequences examined, a defining feature of bacterial DUF501 rather than a mycobacterial peculiarity. We propose that DUF501 is a metal-binding protein and candidate metalloenzyme, the first functional hypothesis for this family, whose conserved, essential metal pocket is a promising drug target. As the predictions build on a conservation-defined site within a fully computational study, they are supportive rather than proof of metal occupancy and warrant experimental validation.

microbiology↗

bioRxiv2026

Mycoplasmal endosymbionts of Trichomonas vaginalis are associated with reduced risk for Chlamydia trachomatis endometrial infection in asymptomatic, coinfected, women.

Trichomonas vaginalis is a protozoan parasite that causes trichomoniasis, the most common curable non-viral sexually transmitted infection, and Chlamydia trachomatis is a bacterial pathogen that can ascend to the upper genital tract and cause pelvic inflammatory disease, infertility, and ectopic pregnancy. T. vaginalis harbors bacterial endosymbionts, including Candidatus Malacoplasma girerdii, an obligate symbiont, and Metamycoplasma hominis, which can live freely or symbiotically. In a 16S rRNA sequencing study of the cervicovaginal microbiome of women at high risk for chlamydial infection, Ca. M. girerdii abundance was one of 13 features predicting lack of chlamydial spread to the endometrium, despite no direct association between T. vaginalis infection and reduced chlamydial ascension. Investigating the relationship between these microorganisms further, we found that T. vaginalis vaginal abundance correlated positively with chlamydial burden in women whose infection was confined to the cervix, while a nonsignificant inverse relationship was seen in women with endometrial spread. Among participants with high chlamydial burden, Ca. M. girerdii was detected exclusively in women without endometrial infection. Both endosymbionts trended toward more frequent detection, and higher abundance, in coinfected women without endometrial spread, while M. hominis abundance correlated strongly with T. vaginalis burden in this group. These findings suggest that mycoplasmal endosymbionts of T. vaginalis, rather than T. vaginalis itself, are microbial factors limiting chlamydial ascension, and point to a three-way interaction between parasite, endosymbiont, and bacterial pathogen that shapes upper genital tract C. trachomatis infection risk.

microbiology↗

bioRxiv2026

Understanding the physiological alterations of Vibrio cholerae upon exposure to L-ascorbic acid

The scourge of cholera remains a major global public health threat. It affects up to 4 million people worldwide and causes tens of thousands of deaths each year. The disease is experiencing a concerning resurgence in many parts of Africa, the Middle East, and Asia. To effectively tackle cholera and circumvent rising antimicrobial resistance, targeted biological and preventive approaches, complementing traditional rehydration, are urgently needed. In this regard, our group has demonstrated the efficacy of L-ascorbic acid in controlling the growth and pathogenesis of Vibrio cholerae in vitro. The present work further provides a mechanistic elucidation of the L-ascorbic acid-mediated physiological changes in V. cholerae and also bolsters such a non-antibiotic approach to control cholera.

microbiology↗