Revisiting HBV biology: perspective for cure

Fabien Zoulim Hepatology Department, Hospices Civils de Lyon INSERM U1052, Cancer Research Center of Lyon Lyon University, France What do we want to achieve ?

SERUM +1.0 c/mL) 10 Therapy 0.0 HBsAg Partial Cure -1.0 +/- Anti-HBsAb

-2.0 Functional Cure -3.0

HBVDNA Sterilizing -4.0 Cure HBV DNA HBV changeDNA from baseline(log Time

LIVER cccDNA Complete Cure

Lok et al, Cure: From Discovery to Regulatory Approval; Hepatology / J Hepatol joint publication; 2017 The HBV life cycle Innate responses Adaptive immune responses NK Interferon cells alpha CD8+ hNTCP cells 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 Barriers to eradicating HBV

Defective CD8+ responses cccDNA reservoir Long t1/2 Defective B cell responses Continuous replenishment Not affected by NAs and IFN Inefficient innate response Integrated forms HBV persistence 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. cccDNA persistence after HBs seroconversion

Maynard et al, J Hepatol 2005 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 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 Targeting cccDNA, the viral minichromosome

cccDNA loss cccDNA repleneshiment

cccDNA formation

cccDNA degradation cccDNA repleneshiment

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 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 Core inhibitors inhibit viral genome replication and prevent cccDNA formation when administered prior to HBV inoculation

Berke et al. Antimicrob. Agents Chemother. 2017;61:e00560-17 plasma

membrane

CpAMs “ inhibitors”

CpAMs “Capsid inhibitors”

NUCs “ inhibitors” 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 Targeting Hepatitis B 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. 2014 & 2016 13 In vivo proliferation of hepadnavirus‐infected hepatocytes induces loss of cccDNA in mice

Dandri et al, Hepatology 2010 Reduction of nuclear DNA consistent with symetrical distribution to daughter cells

Li & Seeger, J Virol 2017 CCC DNA formation and candidate targets

Nassal Gut 2015 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 cccDNA formation: a multistep process

HBx protein

HBc protein

DNA repair deposition

Relaxed Covalently HBV circular closed circular minichromoso DNA DNA me

Are DNA repair and chromatinization coupled in the formation of cccDNA ?

Locatelli et al, HBV conference 2017 structure

Histone Variants H1 H1.1 ; H1.5 ; H1° H3 H3.1 ; H3.2 ; H3.3 ; CENP-A H2A H2A.X ; H2A.Z ; macroH2A H2B H2B1 ; H2BW H4

• Specific dynamics . Genomic regions (centromere, telomere, genes) . DNA processes (replication, repair, ) • H3.3 is a DNA replication- independent 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 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. Is HIRA involved in cccDNA establishment?

HBx protein

HBc protein

DNA repair Histone deposition

Relaxed Covalently HBV circular closed circular minichromosome DNA DNA

Locatelli et al, HBV conference 2017 Does HIRA interact with cccDNA?

HBx protein

? HBc protein

Experimental approach: Chromatin Immunoprecipitation on infected cells

cccDNA copy number/nucleus

4 3

1.5 rcDNA

1.0 Linear HBV (3,2 kb)

0.5

cccDNA copies / nucleus cccDNA / copies cccDNA 0.0

30 2h 4 h 8 h 12h 24h 48h 72h

Time

Locatelli et al, HBV conference 2017 HIRA and H3.3 bind to cccDNA H3.3 binding is concomitant with the beginning of transcription

cccDNA copy number/nucleus

4 ChIP experiment – HepG2-NTCP (n=4) 3

1.5

HIRA binding to cccDNA H3.3 binding on cccDNA 1.0

128 32

64 16 0.5

32 8 cccDNA copies / nucleus cccDNA / copies

16 4 0.0

30 2h 4 h 8 h 8 2 12h 24h 48h 72h

4 1 Time

2 0.5 % Input cccDNA % Input % Input cccDNA % Input

1 0.25 3.5 kb HBV RNA

2h 4 h 8 h 2 h 4h 8h 4 12h 24h 48h 72h 12h 24h 48h 72h

Time Time 3

2

H3.3 1 normalized on HKG normalized

H3.3 R e la tiv e fo ld in d u ctio n HIRA HIRA H3.3 0

2 h 4h 8h 12h 24h 48h 72h H3.3 H3.3 HIRA Infection course Time rcDNA enters 2 hours P.I 24h P.I the nucleus Initiation of the transcription

Locatelli et al, HBV conference 2017 Silencing of HIRA decreases cccDNA levels

2nd siRNA 1st siRNA transfection transfection HBV infection Cell havrvesting HepG2-NTCP D -4 D -2 D 0 72h post-infection

Hira expression post-silencing

150 CTL S iH ira 100 24h PI 72h PI

50 to C TL cells Percentage normalyzed Percentage 0

CTL SiH ira

rcDNA

Viral parameters post silencing 4,9 kb

CTL Linear DNA 2,8 kb 100 S iH ira

2,3 kb 50

to C TL cells cccDNA Percentage normalyzed Percentage 0 TRA* : Transfection

pgRNA cccDNA reagent Total RNA

HBV total DNA

Locatelli et al, HBV conference 2017 cccDNA chromatinisation involves HIRA recruitment and deposition of H3.3

Hepatitis B virus

HBV

Translation

Reverse transcription pgRNA

Hepatocyte Secretion

Cytoplasm e

Locatelli et al, HBV conference 2017 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 Modulation of cccDNA epigenetic control

In vivo DHBV HBV-infected HBV infection Tet-on cell line HepG2-NTCP

Belloni, JCI 2012

Liu, Plos Path 2013 Tropberger, PNAS 2015

Alpha-IFN: • inhibits cccDNA transcription (no reduction in cccDNA levels) • reduces the of cccDNA-bound histones 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

HepG2-NTCP

PHH GOslim categories Common

Significant differences between transformed and primary human hepatocytes

Testoni et al, HBV conference 2017 Gene ontology analysis of cccDNA-associated proteins

Ubiquitin/ Proteasome Translation initiation Transcription/ mRNA processing

Kinases/ Chromatin modification

Chromatin structure

Signaling flux in the cell

*363 proteins identified in either HepG2 or PHH Testoni et al, HBV conference 2017 Functional investigation of selected cccDNA partners

mRNA knock-down

cccDNA Total HBV DNA

siRNA siRNA cccDNA-associated proteins cccDNA-associated proteins

Total HBV RNA HBV pregenomic (pg)RNA

siRNA siRNA cccDNA-associated proteins 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 Challenges in targeting cccDNA

TRANSCRIPTIONAL CONTROL

Specificity for cccDNA? Delivery?

Partial effect? Efficacy in vivo? minichromosome Off-target effect? Delivery?

DESTRUCTION Further knowledge required

FORMATION Modified from Nassal, Gut 2015

Testoni et al, Sem Liver Dis 2017; Hong et al, Hepatology 2017 Novel biomarkers to assist drug development Dane particles cir HBV-RNAs qHBsAg HBcrAg

DNA free filament HBeAg particles sphere Biomarkers, surrogates Dane van Bommel et al, Hepatology 2015 particles Chen et al, Sci Rep 2017

HBV RNA particles Extracellular / serum

Subgenomic RNAs pgRNA

Cytoplasm Tracking cccDNA by FISH Zhang et al, JCI 2016 ; Li et al J Virol 2017 Nucleus Standardization of cccDNA assays L Allweiss et al, ANRS/DZIF/ICE-HBV concerted action

Subgenomic Rc-DNA cccDNA pgRNA Encapsidated Exosomes pgRNA RNAs

Testoni et al, Sem Liver Dis, 2017 Can we cure both the infection and the diseased liver ?

SERUM +1.0 c/mL) 10 Therapy 0.0 HBsAg Partial Cure -1.0 +/- Anti-HBsAb

-2.0 Functional Cure -3.0

HBVDNA Sterilizing -4.0 Cure HBV DNA HBV changeDNA from baseline(log Time

LIVER cccDNA Complete Cure

Lok et al, Hepatitis B Cure: From Discovery to Regulatory Approval; Hepatology / J Hepatol joint publication; 2017 Slower HBsAg decline in HBeAg negative patients: is it linked to HBV integration ?

ETV vs ETV+TDF SiRNA By HBeAg status By HBeAg status Single dose Extension Cohort 7 Cohort 10 0 100000 01-7981 E Pos (SE) 0,2 01-7982 E Pos 10000 01-7985 E Pos 0,4

IU/mL IU/mL 1000 First dose of

decline from extension 10 0,6

HBsAg [IU/mL] HBsAg 100 Last dose of 0,8 extension 10 HBsAg 1 0 20 40 60 80 Week 1,2 baseline, log Mean Mean 0 12 24 36 48 60 72 84 96 Single dose Extension Cohort 7 Cohort 10 Duration of treatment (weeks) 10000 01-7988 E Neg ETV HBeAg(-) 1000 01-7986 E Neg ETV+TDF HBeAg(-) 01-7983 E Neg ETV HBeAg(+) 100 01-7973 E neg 01-7974 E Neg ETV+TDF HBeAg(+) 10 First dose of HBsAg [IU/mL] HBsAg 1 extension Last dose of 0.1 extension 0 20 40 60 80 Week

Zoulim et al, J Hepatol 2015 Wooddell et al., Sci Transl Med 2017 Both cccDNA and viral integrants are the template for HBsAg expression

Dane particles qHBsAg filament serum

/ sphere Extracellular

Subgenomic RNAs pgRNA Cytoplasm Nucleus

Rc-DNA cccDNA pgRNA Subgenomic RNAs

Testoni et al, Sem Liver Dis 2017 Wooddell et al., Sci Transl Med 2017 Integration in host chromosomes and clonal expansion of hepatocytes in all disease phases

Mason et al, Gastroenterology 2016 Liver Damage and HBV infection

• Early occurrence of HBV integration

• HCC not always seen on a background of

• 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 Can we cure both the infection and the diseased liver ?

Normal Cirrhosis ccc-DNA Liver

Rc-DNA UNTREATED HBsAg HBV-DNA Blood

Normal Cirrhosis Hepatocellular carcinoma

ccc-DNA Liver

Rc-DNA NUCs HBsAg Risk of HCC reduced Blood (after 5 yrs) but not eliminated

Normal Cirrhosis Hepatocellular carcinoma ccc-DNA Liver Liver NMEs “CNMEsure”

Direct Antiviral Agents

Blood Blood Immunomodulatory strategies

Zoulim & Levrero Acknowledgements

Hepatology Unit INSERM U1052 Collaborations

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 François Bailly Barbara Testoni Marc Bonin G Almouzni, Institut Curie Samir Benmaklouf Julie Lucifora Thomas Lahlali M Dandri, Hamburg Marie Ecochard David Durantel Lucyna Cova XX Zhang, Shanghai Kerstin Hartig Bernd Stadelmeyer Romain Parent Fanny Lebossé Guada Martinez Anna Salvetti Massimo Levrero Maelle Locatelli Birke Bartosch Marianne Maynard Fleur Chapus Eve Pecheur Christian Trépo Aurore Inchauspé Boyan Grigorov Maud Michelet Christophe Combet Judith Fresquet