Revisiting HBV biology: perspective for cure

Fabien ZoulimHepatology Department, Hospices Civils de LyonINSERM U1052, Cancer Research Center of Lyon

Lyon University, France

What do we want to achieve ?

Therapy

HB

V D

NA

chan

ge fr

om b

asel

ine

(log

10c/

mL)

0.0

-1.0

-2.0

-3.0

-4.0

+1.0

Time

HBsAg

HBVDNA

cccDNA

SERUM

LIVER

+/- Anti-HBsAb

Lok et al, Hepatitis B Cure: From Discovery to Regulatory Approval; Hepatology / J Hepatol joint publication; 2017

Partial Cure

FunctionalCure

Complete Cure

SterilizingCure

Zoulim & Locarnini, Gastroenterology 2009; Zoulim Antiviral Research 2012; Mico et al J Hepatol 2013; Boni et al Hepatology 2015

NK cells

Innate responses

CD8+ cells

B cells

CD4+ cells

Adaptive immune responses Interferon

alphahNTCP

The HBV life cycle

Nucleos(t)ide analogues

Barriers to eradicating HBV

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.

cccDNA reservoirLong t1/2Continuous replenishmentNot affected by NAs and IFN

Integrated forms

HBV persistence

Maynard et al, J Hepatol 2005

cccDNA persistence after HBs seroconversion

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

Boyd et al, J Hepatol 2016

New round of infection and/or replenishment of the cccDNA pool occurdespite « viral suppression »

Persistence of intrahepatic viral DNA synthesis during long Tenofovir therapy

(HIV-HBV cohort)

Zoulim, et al, Clin GastroenterolHepatol 2013Lucifora et al, Science 2014Belloni et al, JCI 2012Koeniger etal, PNAS 2014Durantel&Zoulim, J Hepatol 2016

cccDNA loss

cccDNA silencing

cccDNAdegradation

cccDNAformation

Targeting cccDNA, the viral minichromosome

cccDNArepleneshiment

cccDNArepleneshiment

Li et al, elife 2012; Urban et al, Gastroenterology 2014

Model for HBV entry in hepatocytes and development of entry inhibitors

Entry inhibitorsMyrcludex(pre-S1 peptide)Blank et al, J Hepatol 2016Bogomolov et al, J Hepatol 2016

EzetimibeLucifora, Antiviral Res 2013ProanthocyanidinTsukuda, Hepatology 2017Cyclosporin analogues Shimura, J Hepatol 2017

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

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

NUCs“Polymerase inhibitors”

plasma

membrane

CpAMs“Capsid inhibitors”

CpAMs“Capsid inhibitors”

Lucifora et al, Science 2014; Shlomai & Rice, Science 2014

Model for cccDNA degradationIFNalpha /Lymphotoxin beta can induce APOBEC3A/B dependent

degradation of HBV cccDNA

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. 2014 & 2016

In vivo proliferation of hepadnavirus‐infected hepatocytes induces loss of cccDNA in mice

Dandri et al, Hepatology 2010

Li & Seeger, J Virol 2017

Reduction of nuclear DNA consistent with symetrical distribution to daughter cells

CCC DNA formation and candidate targets

Nassal Gut 2015

Formation of cccCNARelaxed circular DNA mimics damaged cellular DNA

Nassal, Gut 2015Koeniger et al, PNAS 2014

Cui X, et al. PlosOne, 2015

HBV infection in HepG2-NTCP Tdp2 knockout cells

Relaxedcircular

DNA

HBV minichromoso

me

Covalentlyclosed circular

DNA

HBx protein

HBc protein

DNA repair Histone deposition

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

cccDNA formation: a multistep process

Locatelli et al, HBV conference 2017

Chromatin structure

• 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

Histone VariantsH1 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, transcription)

• 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.

HIRA : An histone chaperone involved in DNA repair

Transcription recovery after DNA damage requires chromatin priming by the H3.3 histone chaperone HIRA. Adam S, Polo SE, Almouzni G. Cell, 2013.

Relaxedcircular

DNA

HBV minichromosome

Covalentlyclosed circular

DNA

HBx protein

HBc protein

DNA repair Histone deposition

Is HIRA involved in cccDNA establishment?

Locatelli et al, HBV conference 2017

Does HIRA interact with cccDNA?

HBx protein

HBc protein?

Experimental approach: ChromatinImmunoprecipitation on infected cells

30 2h 4 h 8 h1 2 h

2 4 h4 8 h

7 2 h0 .0

0 .5

1 .0

1 .5

3

4

c c c D N A c o p y n u m b e r /n u c le u s

T im e

cc

cD

NA

co

pie

s /

nu

cle

us

rcDNA

Linear HBV (3,2 kb)

cccDNA

Locatelli et al, HBV conference 2017

2 h 4 h 8 h1 2 h

2 4 h4 8 h

7 2 h0

1

2

3

4

3 .5 k b H B V R N A

T im e

Re

lati

ve

fo

ld i

nd

uc

tio

nn

orm

ali

zed

on

HK

G

ChIP experiment – HepG2-NTCP (n=4)

30 2h 4 h 8 h1 2 h

2 4 h4 8 h

7 2 h0 .0

0 .5

1 .0

1 .5

3

4

c c c D N A c o p y n u m b e r /n u c le u s

T im e

cccD

NA

co

pie

s / n

ucl

eus

2 h 4 h 8 h1 2 h

2 4 h4 8 h

7 2 h1

2

4

8

1 6

3 2

6 4

1 2 8

H IR A b in d in g to c c c D N A

T im e

% In

pu

t cc

cDN

A

2 h 4 h 8 h1 2 h

2 4 h4 8 h

7 2 h0 .2 5

0 .5

1

2

4

8

1 6

3 2

H 3 .3 b in d in g o n c c c D N A

T im e

% In

pu

t cc

cDN

A

rcDNA enters the nucleus

24h P.IInitiation of the transcription

HIRA

2 hours P.I

Infection course

HIRAH3.3

HIRAH3.3H3.3

H3.3

H3.3

HIRA and H3.3 bind to cccDNAH3.3 binding is concomitant with the beginning of transcription

Locatelli et al, HBV conference 2017

D -2 72h post-infectionD 0

Cell havrvesting

D -4

1st siRNA transfection HBV infection2nd siRNA

transfection

c c cD N A

H B V tota

l DN A

p g R N A

T o tal R

N A

0

5 0

1 0 0

Pe

rce

nta

ge

no

rma

lyze

dto

CT

L c

ell

s

C TL

S iH ira

Silencing of HIRA decreases cccDNA levels

Viral parameters post silencing

C T L

S iHira

0

5 0

1 0 0

1 5 0

H ira e x p re s s io n p o s t-s ile n c in g

Pe

rce

nta

ge

no

rma

lyze

dto

CT

L c

ell

s

C TL

S iH ira

HepG2-NTCP

rcDNA

Linear DNA

cccDNA

24h PI 72h PI

4,9 kb

2,8 kb

2,3 kb

TRA* : Transfection reagent

Locatelli et al, HBV conference 2017

Cytoplasme

Hep

atoc

yte

Hepatitis B virus

Secretion

Reversetranscription

Translation

HBV antigens

pgRNA

cccDNA chromatinisation involves HIRA recruitment and deposition of H3.3 histones

Locatelli et al, HBV conference 2017

Epigenetics of covalently closed circular (ccc)DNA(Regulation by viral proteins HBc and HBx)

Levrero et al, J Hepatol 2009; Nassal, Gut 2015,

Guo et al Hepatology 2017

SilencingEpigenome modifyers,Interferon alpha, Capsid inhibitors,HBx inhibitors

Tropberger et al, PNAS 2015; Decorsière et al. Nature 2016; Liang JT, Nature 2016

Belloni, JCI 2012

Modulation of cccDNA epigenetic control

In vivoHBV infection

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

Liu, Plos Path 2013

DHBV Tet-on cell line

HBV-infectedHepG2-NTCP

Tropberger, PNAS 2015

Chromatin proteomics of HBV minichromosome

HepG2-NTCP

PHH

Common

Gene Set Enrichment Analysis

GO

slim

cat

egor

ies

Significant differences between transformed and primary human hepatocytes

363 proteins were differentiallyidentified in either infected HepG2 or PHH respect to mock-infected cells

Experimental design

Testoni et al, HBV conference 2017

Gene ontology analysis of cccDNA-associated proteins

Transcription/mRNA processing

Ubiquitin/Proteasome Translation initiation

Kinases/Chromatin

modification

Signaling flux in the cell

Chromatinstructure

*363 proteins identified in either HepG2 or PHH Testoni et al, HBV conference 2017

Functional investigation of selected cccDNA partners

Knock-down of selected cccDNA-associated proteins in infected PHHs had a strong impact on HBV replication

Total HBV DNA

cccDNA-associated proteinssiRNA

Total HBV RNA

cccDNA-associated proteinssiRNA

HBV pregenomic (pg)RNA

cccDNA-associated proteinssiRNA

cccDNA-associated proteinssiRNA

cccDNA

mRNA knock-down

Testoni et al, HBV conference 2017

Further knowledge required

Partial effect?Efficacy in vivo?

Off-target effect?Delivery?

Specificity for cccDNA?Delivery?

Modified from Nassal, Gut 2015FORMATION

DESTRUCTION

TRANSCRIPTIONAL CONTROL

minichromosome

Challenges in targeting cccDNA

Testoni et al, Sem Liver Dis 2017; Hong et al, Hepatology 2017

pgRNA Encapsidated pgRNA

Rc-DNA cccDNA SubgenomicRNAs Exosomes

Nuc

leus

Cyt

opla

smEx

trac

ellu

lar /

ser

um

sphere

filament

qHBsAgDane particles cir HBV-RNAs HBcrAg

HBeAgDNA free particles

Dane particles

HBV RNA particles

Subgenomic RNAspgRNA

Testoni et al, Sem Liver Dis, 2017

Standardization of cccDNA assaysL Allweiss et al, ANRS/DZIF/ICE-HBV concerted action

Tracking cccDNA by FISHZhang et al, JCI 2016 ; Li et al J Virol 2017

Biomarkers, surrogatesvan Bommel et al, Hepatology 2015Chen et al, Sci Rep 2017

Novel biomarkers to assist drug development

Therapy

HB

V D

NA

chan

ge fr

om b

asel

ine

(log

10c/

mL)

0.0

-1.0

-2.0

-3.0

-4.0

+1.0

Time

HBsAg

HBVDNA

cccDNA

SERUM

LIVER

+/- Anti-HBsAb

Lok et al, Hepatitis B Cure: From Discovery to Regulatory Approval; Hepatology / J Hepatol joint publication; 2017

Partial Cure

FunctionalCure

Complete Cure

SterilizingCure

Can we cure both the infection and the diseased liver ?

Slower HBsAg decline in HBeAg negative patients: is it linked to HBV integration ?

0

0,2

0,4

0,6

0,8

1

1,20 12 24 36 48 60 72 84 96

ETV HBeAg(-)ETV+TDF HBeAg(-)ETV HBeAg(+)ETV+TDF HBeAg(+)

ETV vs ETV+TDF By HBeAg status

Duration of treatment (weeks)

Mea

n H

BsA

gde

clin

e fr

om

base

line,

log 1

0IU

/mL

(SE)

Zoulim et al, J Hepatol 2015

Week

HB

sAg

[IU/m

L]

0 20 40 60 8010

100

1000

10000

10000001-7981 E Pos01-7982 E Pos01-7985 E Pos

First dose ofextension

Last dose ofextension

Single doseCohort 7

ExtensionCohort 10

01-7988 E Neg

Week

HB

sAg

[IU/m

L]

0 20 40 60 800.1

1

10

100

1000

10000

First dose ofextensionLast dose ofextension

Single doseCohort 7

ExtensionCohort 10

01-7973 E neg01-7974 E Neg

01-7983 E Neg01-7986 E Neg

SiRNABy HBeAg status

Wooddell et al., Sci Transl Med 2017

pgRNARc-DNA cccDNA SubgenomicRNAs

Nuc

leus

Cyt

opla

smEx

trac

ellu

lar/

ser

um

sphere

filament

qHBsAgDane particles

Subgenomic RNAspgRNA

Testoni et al, Sem Liver Dis 2017

Both cccDNA and viral integrants are the template for HBsAg expression

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 cirrhosis

• Liver damage results of immune killing of hepatocytes

• Clonal expansion of hepatocytes not supporting HBV replicationoccurs 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 ?

UNTREATED

Liver

Blood

Rc-DNA

HBsAg

ccc-DNA

HBV-DNA

Normal Cirrhosis Hepatocellular carcinoma

Liver

Blood

Rc-DNA

HBsAg

ccc-DNA

NUCs

Normal Cirrhosis Hepatocellular carcinoma

Risk of HCC reduced (after 5 yrs)

but not eliminated

Liver

Blood

ccc-DNA Liver

Blood

NMEs NMEs“Cure”

Direct Antiviral Agents

Immunomodulatory strategies

Normal Cirrhosis Hepatocellular carcinoma

Zoulim & Levrero

Acknowledgements

Hepatology Unit INSERM U1052 Collaborations

Barbara TestoniJulie LuciforaDavid DurantelBernd StadelmeyerGuada MartinezMaelle LocatelliFleur ChapusAurore InchauspéMaud MicheletJudith Fresquet

C. Caux, Lyon CRCLFL. Cosset, Lyon CIRIA. Boyd, ParisF Carrat, ParisC Ferrari, ParmaP Lampertico, MilanA Craxi, PalermoJP Quivy, Institut CurieG Almouzni, Institut CurieM Dandri, HamburgXX Zhang, Shanghai

Marc BoninThomas LahlaliLucyna CovaRomain ParentAnna SalvettiBirke BartoschEve PecheurBoyan GrigorovChristophe Combet

François BaillySamir BenmakloufMarie EcochardKerstin HartigFanny LebosséMassimo LevreroMarianne MaynardChristian Trépo