SPONSOR & COPYRIGHT NOTICE

Transcription

1 SPONSOR & COPYRIGHT NOTICE THIS PRESENTATION WAS GIVEN AT THE LIVER MEETING 2014 (AASLD), DURING AN INVESTIGATOR MEETING SPONSORED BY CN BIO INNOVATIONS LIMITED (UK REGISTERED COMPANY: ) This presentation is copyright of Marcus Dorner. Approval to reproduce this work has been granted to CN Bio Innovations by M. Dorner No permission or licence, express or implied, is granted to copy, reproduce, distribute, transmit, broadcast, display, sell, or otherwise exploit this presentation for any other purposes without the prior written consent of Marcus Dorner 1

2 Limitations and Innovations in model systems for the study of hepatitis B virus in vitro and in vivo Marcus Dorner, Ph.D. Imperial College London, UK AALSD 2014 Boston, USA

3 Tools for unraveling the HBV life cycle In vitro: Overlength HBV genomes Reporter HBV genomes HepG cells stably replicating HBV The HepaRG cell system The HepG2/hNTCP system Primary human hepatocyte-based systems In vivo: The HBV-transgenic mouse The AAV8/HBV mouse Human liver-chimeric mouse Human immune system (HIS) and liver chimeric mice

4 The HBV life cycle and associated problems for research

5 The HBV life cycle and associated problems for research

6 Overlength genomes for initiating HBV replication Lucifora J. et al J. Gen. Virol.

7 Overlength genomes for initiating HBV replication HBV cccdna is organized on host cellular histones Without selection the transient nature enables only studies up to 6 days Stable cell lines expressing tetracycline-inducible versions of the 1.3x HBV genome are available (HepAD38 cells) (Ladner et al Antimicrob. Agents Chemother.) Lucifora J. et al J. Gen. Virol.

8 HBV genomes encoding reporter genes Fluorescent reporter gene expression via insertion into HBsAg No evidence for cccdna maintenance or genetic stability Hong et al J. Virol.

9 HBV genomes encoding reporter genes Fluorescent reporter gene expression via IRES-mediated translation No evidence for cccdna maintenance or genetic stability Wang et al PLoS One

10 The HepG cell system Stable expression of two head-to-tail HBV dimers resulting in replicationcompetent HBV Contains subtype ayw genomes both as cccdna as well as integrated copies Genetic heterogeneity and genetic differences to HepG2 cells make direct comparisons impossible Bock et al Virus Genes; Zhao et al World J. Gastroenterol.

11 The HepaRG cell system Gripon et al Proc. Natl. Acad. Sci. USA

12 The HepaRG cell system Human hepatoma cell line recapitulating the HBV life-cycle Permissive to HBV infection at high multiplicities of infection in the presence of PEG Requires addition of DMSO for extensive periods of time Maintenance of cccdna Gripon et al Proc. Natl. Acad. Sci. USA

13 The HepG2/NTCP system Human hepatoma cell line recapitulating the HBV life-cycle following stable overexpression of human NTCP Permissive to HBV infection at high multiplicities of infection Requires addition of DMSO and PEG for extensive periods of time Maintenance of cccdna Yan et al elife

14 Primary human hepatocytes 2D vs. 3D models vge per cell needed for infection PHH cultured in presence of 0.5% DMSO Infection in presence of 4% PEG Konig et al J. Hepatol. Schulze et al Hepatol.

15 Pha se Micropattern co-culture models for HBV infection Primary human hepatocyte / mouse embryonic fibroblast (NIH3T3-J2) co-cultures recapitulate the HBV life-cycle at low levels Requirement of innate immune blunting via JAK/STAT pathway inhibition Shlomai et al Proc. Natl. Acad. Sci. USA

16 3D microfluidic hepatocyte cultures for HBV infection Tight junctions Microvili Lumen of bile canaliculus 500nm Long-term culture (>20 days) of primary human hepatocytes All HBV life-cycle steps including cccdna maintenance are recapitulated Mono-cultures as well as co-cultures with immune cells (i.e. NK cells) is possible and demonstrates HBVspecific immune activation Wai et al. AASLD 2014 & manuscript in preparation Quantum-B and LiverChip are trademarks of CN Bio Innovations Ltd.

17 3D microfluidic hepatocyte cultures for HBV infection Hepatocytes have an intact innate immune response to HBV Monoculture of hepatocytes solely activates IL28b and induces a selective ISG profile Co-culture of hepatocytes and Kupffer cells results in more physiological innate immune responses Wai et al. AASLD 2014 & manuscript in preparation Quantum-B and LiverChip are trademarks of CN Bio Innovations Ltd.

18 Comparison of in vitro HBV models HepG HepaRG HepG2/NTCP PHH Micropattern co-cultures Recapitulation of the complete HBV life-cycle No, entry not recapitulated Yes, but high moi needed Yes, but high moi needed Yes, but only short-term cccdna maintenance Throughput Physiological host cell profile Yes High No, HepG2 cell background Yes High No, DMSO/PEG addition needed Yes High No, DMSO/PEG addition needed Yes Medium Yes, short-term (3-5 days) Yes Yes Medium Yes, but immune blunting required Theoretical ability to cure HBV No, integrated HBV present Quantum-B Yes Yes Medium Yes Yes Yes Yes Yes Yes

19 Studying HBV in vivo HBV transgenic mice Stable transgenic expression of HBV pgrna (1.3xHBV, subtype ayw) No cccdna maintenance No HBV-specific immune responses Guidotti et al J. Virol.

20 Studying HBV in vivo AAV8/HBV mice Long-term persistence of AAV8/HBV-infected hepatocytes in vivo Ability to study human immune responses in HLA-A2-transgenic mice cccdna maintenance not confirmed Dion et al J. Virol

21 Studying HBV in vivo Human liver-chimeric mice Highly immunodeficient mice lacking T, B and NK cells serve as xenorecipients Liver injury in murine hepatocytes (i.e. upa tg or Fah -/- ) gives human hepatocytes a growth advantage Yan et al elife

22 Studying HBV in vivo Human liver-chimeric mice human serum albumin (mg/ml, log 10 ) FRG upa-scid TK-NOG AFC8 * HCV HBV H&E FAH chimerism (% human albumin-positive cells) FRG upa-scid TK-NOG AFC8 de Jong et al Science Transl. Med.

23 Studying HBV in vivo Human liver/immune system mice Wilson et al Stem Cell Res

24 Studying HBV in vivo Human liver/immune system mice de Jong et al. EASL 2014; AASLD Poster #1709

25 Studying HBV in vivo Human liver-chimeric mice Permissive to HBeAg + and HBeAg - patient isoates upa-scid Alb-uPa FRG or FNRG TK-NOG AFC8 Yes Yes Yes Not shown Inducible liver injury model No Yes Yes Yes Ability to repopulate with human liver and immune system No Yes No Yes Repopulation efficiency High High Medium Low

26 Comparison of in vivo HBV models HBV transgenic mice AAV8/HBV mice Liver-chimeric mice Liver/Immune systemchimeric mice Recapitulation of the complete HBV life-cycle No, entry not recapitulated No, entry not recapitulated cccdna maintenance Throughput Host cell background Theoretical ability to cure HBV No High Mouse No, integrated HBV present No Medium Mouse Yes, but no cccdna Yes Yes Low Human Yes Yes Yes Low Human Yes

27 Take home lessons Achievements: Identification of NTCP as entry factor Stable and inducible HBV systems Primary hepatocyte culture models recapitulating the HBV life cycle Human xenograft models allowing the study of HBV in human hepatocytes Challenges: No equivalent to the HCV replicon system No cell-based high throughput reporters Limited virally encoded reporter strains No mouse model recapitulating all HBV life cycle steps with fully functional immune system Southern blot for the detection of HBV cccdna is still gold standard

28 Acknowledgements Imperial College London Sandi Wai Antonia Evripioti Jordan Roberts Mindy Gore Christine Styles Mark Thursz Jacob Baum Fiona Angrisano Andrew Blagborough CN Bio Innovations Emma Large Terri Cornforth Cliff Rowe David Hughes Emma Sceats University College London Laura Pallett Kerstin Stegmann Mala Maini King s College London Marion Lussignol Maria Teresa Catanese Roland Fleck Rockefeller University Ype de Jong Eva Billerbeck Michiel Mommersteeg Jing Xiao Charles Rice