Revisiting HBV biology: perspective for cure

Transcription

1 Revisiting HBV biology: perspective for cure Fabien Zoulim Hepatology Department, Hospices Civils de Lyon INSERM U1052, Cancer Research Center of Lyon Lyon University, France

2 What do we want to achieve? SERUM HBV DNA change from baseline (log 10 c/ml) Therapy HBsAg HBVDNA Partial Cure Functional Cure +/- Anti-HBsAb Time Sterilizing Cure LIVER cccdna Complete Cure Lok et al, Hepatitis B Cure: From Discovery to Regulatory Approval; Hepatology / J Hepatol joint publication; 2017

3 Adaptive immune responses CD8+ cells The HBV life cycle Innate responses hntcp NK cells Interferon alpha CD4+ cells B cells Nucleos(t)ide analogues Zoulim & Locarnini, Gastroenterology 2009; Zoulim Antiviral Research 2012; Mico et al J Hepatol 2013; Boni et al Hepatology 2015

4 Barriers to eradicating HBV cccdna reservoir Long t1/2 Continuous replenishment Not affected by NAs and IFN Integrated forms HBV persistence Defective CD8+ responses Defective B cell responses Inefficient innate response Defective immune responses Revill, Testoni, Locarnini, S. & Zoulim, F. et al. (2016) Global strategies are required to cure and eliminate HBV infectionnat. Rev. Gastroenterol. Hepatol.

5 cccdna persistence after HBs seroconversion Maynard et al, J Hepatol 2005

6 Late hepatitis B reactivation following DAA-based treatment of recurrent hepatitis C in an anti-hbc-positive liver transplant recipient Vionnet et al, Hepatology 2017

7 Persistence of intrahepatic viral DNA synthesis during long Tenofovir therapy (HIV-HBV cohort) New round of infection and/or replenishment of the cccdna pool occur despite «viral suppression» Boyd et al, J Hepatol 2016

8 Targeting cccdna, the viral minichromosome cccdna repleneshiment cccdna loss cccdna formation cccdna repleneshiment cccdna degradation cccdna silencing Zoulim, et al, Clin Gastroenterol Hepatol 2013 Lucifora et al, Science 2014 Belloni et al, JCI 2012 Koeniger etal, PNAS 2014 Durantel&Zoulim, J Hepatol 2016

9 Model for HBV entry in hepatocytes and development of entry inhibitors Entry inhibitors Myrcludex (pre-s1 peptide) Blank et al, J Hepatol 2016 Bogomolov et al, J Hepatol 2016 Ezetimibe Lucifora, Antiviral Res 2013 Proanthocyanidin Tsukuda, Hepatology 2017 Cyclosporin analogues Shimura, J Hepatol 2017 Li et al, elife 2012; Urban et al, Gastroenterology 2014

10 Core inhibitors inhibit viral genome replication and prevent cccdna formation when administered prior to HBV inoculation Berke et al. Antimicrob. Agents Chemother. 2017;61:e

11 plasma membrane CpAMs Capsid inhibitors CpAMs Capsid inhibitors NUCs Polymerase inhibitors

12 Model for cccdna degradation IFNalpha /Lymphotoxin beta can induce APOBEC3A/B dependent degradation of HBV cccdna Lucifora et al, Science 2014; Shlomai & Rice, Science 2014 Similar observation with IFNγ and TNFα Xia et al, Gastroenterology 2015

13 Targeting Hepatitis B Virus With CRISPR/Cas9 Mutations and deletions with functional inactivation of cccdna Over 90% of HBV DNA cleaved by Cas9 Cas9 cleavage 15,000 times more efficient than APOBEC-mediated cytosine deamination following IFNα treatment Seeger et al, Mol Ther Nucleic Acids &

14 In vivo proliferation of hepadnavirus infected hepatocytes induces loss of cccdna in mice Dandri et al, Hepatology 2010

15 Reduction of nuclear DNA consistent with symetrical distribution to daughter cells Li & Seeger, J Virol 2017

16 CCC DNA formation and candidate targets Nassal Gut 2015

17 Formation of ccccna Relaxed circular DNA mimics damaged cellular DNA Nassal, Gut 2015 Koeniger et al, PNAS 2014 HBV infection in HepG2-NTCP Tdp2 knockout cells Cui X, et al. PlosOne, 2015

18 cccdna formation: a multistep process HBx protein HBc protein DNA repair Histone deposition Relaxed circular DNA Covalently closed circular DNA HBV minichromoso me Are DNA repair and chromatinization coupled in the formation of cccdna? Locatelli et al, HBV conference 2017

19 Chromatin structure Histone Variants H1 H1.1 ; H1.5 ; H1 H3 H2A H2B H3.1 ; H3.2 ; H3.3 ; CENP-A H2A.X ; H2A.Z ; macroh2a H2B1 ; H2BW H4 Specific dynamics Genomic regions (centromere, telomere, genes) DNA processes (replication, repair, transcription) H3.3 is a DNA replicationindependent histone variant Replaces conventional H3 in a wide range of nucleosomes in active genes Role of histone chaperones Two modes of mitochondrial dysfunction lead independently to lifespan extension in Caenorhabditis elegans. Yang W., Hekimi S. Aging Cell. 2010

20 HIRA : An histone chaperone involved in DNA repair HIRA is known to recognize naked DNA to deposit histone H3.3. HIRA is responsible for chromatin priming in response to DNA damage allowing transcription recovery after repair. Transcription recovery after DNA damage requires chromatin priming by the H3.3 histone chaperone HIRA. Adam S, Polo SE, Almouzni G. Cell, 2013.

21 Is HIRA involved in cccdna establishment? HBx protein HBc protein DNA repair Histone deposition Relaxed circular DNA Covalently closed circular DNA HBV minichromosome Locatelli et al, HBV conference 2017

22 Does HIRA interact with cccdna?? HBx protein HBc protein Experimental approach: Chromatin Immunoprecipitation on infected cells cccdna copy number/nucleus 4 cccdna copies / nucleus rcdna Linear HBV (3,2 kb) cccdna 30 2h 4 h 8 h 12h 24h 48h 72h Time Locatelli et al, HBV conference 2017

23 HIRA and H3.3 bind to cccdna H3.3 binding is concomitant with the beginning of transcription cccdna copy number/nucleus ChIP experiment HepG2-NTCP (n=4) % Input cccdna HIRA binding to cccdna % Input cccdna H3.3 binding on cccdna cccdna copies / nucleus h 4 h 8 h 12h 24h Time 3.5 kb HBV RNA 48h 72h 2h 4 h 8 h 12h 24h 48h 72h 2 h 4h 8h 12h 24h 48h 72h 4 Time HIRA H3.3 HIRA H3.3 Time H3.3 R e la tiv e fo ld in d u ctio n normalized on HKG H3.3 HIRA H3.3 2 h 4h 8h 12h 24h 48h 72h Infection course Time rcdna enters the nucleus 2 hours P.I 24h P.I Initiation of the transcription Locatelli et al, HBV conference 2017

24 Silencing of HIRA decreases cccdna levels HepG2-NTCP 1st sirna transfection 2 nd sirna transfection HBV infection Cell havrvesting D -4 D -2 D 0 72h post-infection Hira expression post-silencing 150 Percentage normalyzed to C TL cells CTL S ih ira 24h PI 72h PI CTL S ih ira Viral parameters post silencing 4,9 kb rcdna Percentage normalyzed to C TL cells cccdna HBV total DNA pgrna Total RNA CTL S ih ira TRA* : Transfection reagent 2,8 kb 2,3 kb Linear DNA cccdna Locatelli et al, HBV conference 2017

25 cccdna chromatinisation involves HIRA recruitment and deposition of H3.3 histones Hepatitis B virus HBV antigens Translation Hepatocyte pgrna Reverse transcription Secretion Cytoplasm e Locatelli et al, HBV conference 2017

26 Epigenetics of covalently closed circular (ccc)dna (Regulation by viral proteins HBc and HBx) Silencing Epigenome modifyers, Interferon alpha, Capsid inhibitors, HBx inhibitors Levrero et al, J Hepatol 2009; Nassal, Gut 2015, Guo et al Hepatology 2017 Tropberger et al, PNAS 2015; Decorsière et al. Nature 2016; Liang JT, Nature 2016

27 Modulation of cccdna epigenetic control In vivo HBV infection DHBV Tet-on cell line HBV-infected HepG2-NTCP Belloni, JCI 2012 Liu, Plos Path 2013 Tropberger, PNAS 2015 Alpha-IFN: inhibits cccdna transcription (no reduction in cccdna levels) reduces the acetylation of cccdna-bound histones

28 Chromatin proteomics of HBV minichromosome Experimental design 363 proteins were differentially identified in either infected HepG2 or PHH respect to mock-infected cells Gene Set Enrichment Analysis GOslim categories HepG2-NTCP PHH Common Significant differences between transformed and primary human hepatocytes Testoni et al, HBV conference 2017

29 Gene ontology analysis of cccdna-associated proteins Transcription/ mrna processing Ubiquitin/ Proteasome Translation initiation Kinases/ Chromatin modification Chromatin structure Signaling flux in the cell *363 proteins identified in either HepG2 or PHH Testoni et al, HBV conference 2017

30 Functional investigation of selected cccdna partners mrna knock-down cccdna Total HBV DNA sirna cccdna-associated proteins sirna cccdna-associated proteins Total HBV RNA HBV pregenomic (pg)rna sirna cccdna-associated proteins sirna cccdna-associated proteins Knock-down of selected cccdna-associated proteins in infected PHHs had a strong impact on HBV replication Testoni et al, HBV conference 2017

31 Challenges in targeting cccdna TRANSCRIPTIONAL CONTROL Specificity for cccdna? Delivery? Further knowledge required minichromosome Partial effect? Efficacy in vivo? Off-target effect? Delivery? DESTRUCTION FORMATION Modified from Nassal, Gut 2015 Testoni et al, Sem Liver Dis 2017; Hong et al, Hepatology 2017

32 Novel biomarkers to assist drug development Dane particles cir HBV-RNAs qhbsag HBcrAg Nucleus Cytoplasm Extracellular / serum Biomarkers, surrogates van Bommel et al, Hepatology 2015 Chen et al, Sci Rep 2017 pgrna Tracking cccdna by FISH Zhang et al, JCI 2016 ; Li et al J Virol 2017 Standardization of cccdna assays L Allweiss et al, ANRS/DZIF/ICE-HBV concerted action Subgenomic RNAs filament sphere HBeAg HBV RNA particles DNA free particles Dane particles Rc-DNA cccdna pgrna Encapsidated pgrna Subgenomic RNAs Exosomes Testoni et al, Sem Liver Dis, 2017

33 Can we cure both the infection and the diseased liver? SERUM HBV DNA change from baseline (log 10 c/ml) Therapy HBsAg HBVDNA Partial Cure Functional Cure +/- Anti-HBsAb Time Sterilizing Cure LIVER cccdna Complete Cure Lok et al, Hepatitis B Cure: From Discovery to Regulatory Approval; Hepatology / J Hepatol joint publication; 2017

34 Slower HBsAg decline in HBeAg negative patients: is it linked to HBV integration? ETV vs ETV+TDF By HBeAg status SiRNA By HBeAg status Mean HBsAg decline from baseline, log 10 IU/mL (SE) 0 0,2 0,4 0,6 0,8 1 1, Duration of treatment (weeks) ETV HBeAg(-) ETV+TDF HBeAg(-) ETV HBeAg(+) ETV+TDF HBeAg(+) HBsAg [IU/mL] HBsAg [IU/mL] Single dose Cohort 7 Extension Cohort Week Single dose Cohort 7 Extension Cohort Week E Pos E Pos E Pos First dose of extension Last dose of extension E Neg E Neg E Neg E neg E Neg First dose of extension Last dose of extension Zoulim et al, J Hepatol 2015 Wooddell et al., Sci Transl Med 2017

35 Both cccdna and viral integrants are the template for HBsAg expression Nucleus Cytoplasm Extracellular / serum Dane particles pgrna qhbsag filament sphere Subgenomic RNAs Rc-DNA cccdna pgrna Subgenomic RNAs Testoni et al, Sem Liver Dis 2017 Wooddell et al., Sci Transl Med 2017

36 Integration in host chromosomes and clonal expansion of hepatocytes in all disease phases Mason et al, Gastroenterology 2016

37 Liver Damage and HBV infection Early occurrence of HBV integration HCC not always seen on a background of cirrhosis Liver damage results of immune killing of hepatocytes Clonal expansion of hepatocytes not supporting HBV replication occurs even before cirrhosis Experimental models show that clonal hepatocyte repopulation is a major risk factor for HCC Integration contributes to T cell exhaustion via HBsAg expression Strong arguments for early treatment intervention Zoulim & Mason, Gut 2012; Mason et al, Gastroenterology 2016

38 Can we cure both the infection and the diseased liver? ccc-dna Liver Normal Cirrhosis Hepatocellular carcinoma Rc-DNA HBV-DNA HBsAg UNTREATED Blood Normal Cirrhosis Hepatocellular carcinoma ccc-dna Liver Rc-DNA NUCs HBsAg Blood Risk of HCC reduced (after 5 yrs) but not eliminated ccc-dna Liver Liver NMEs Cure NMEs Normal Cirrhosis Hepatocellular carcinoma Blood Blood Direct Antiviral Agents Immunomodulatory strategies Zoulim & Levrero

39 Acknowledgements Hepatology Unit INSERM U1052 Collaborations François Bailly Samir Benmaklouf Marie Ecochard Kerstin Hartig Fanny Lebossé Massimo Levrero Marianne Maynard Christian Trépo Barbara Testoni Julie Lucifora David Durantel Bernd Stadelmeyer Guada Martinez Maelle Locatelli Fleur Chapus Aurore Inchauspé Maud Michelet Judith Fresquet Marc Bonin Thomas Lahlali Lucyna Cova Romain Parent Anna Salvetti Birke Bartosch Eve Pecheur Boyan Grigorov Christophe Combet C. Caux, Lyon CRCL FL. Cosset, Lyon CIRI A. Boyd, Paris F Carrat, Paris C Ferrari, Parma P Lampertico, Milan A Craxi, Palermo JP Quivy, Institut Curie G Almouzni, Institut Curie M Dandri, Hamburg XX Zhang, Shanghai