Eradication of HBV: can we eliminate cccDNA

Massimo Levrero

Dipartimento di Medicina Interna e Specialita’ Mediche

Laboratorio Life-Nanoscience

EAL INSERM U795

Sapienza Universita’ di Roma

Grand Hyatt Kauai

Koloa, Kauai, Hawaii

December 4-8, 2011

Outline

1. Treating CHB: where we are ..

2. cccDNA in patients

3. cccDNA in HBV life cycle

4. Developing a cccDNA functional assay

5. Epigenetic control of cccDNA function

6. Can we modulate cccDNA function

7. Targeting cccDNA stability: are we there?

Where we are?

• Sustained suppression of HBV replication with reduction in

histological activity of chronic hepatitis lessening the risk

of cirrhosis and decreasing (but not eliminating) the risk of

HCC can be reached and maintained in the vast majority of

patients

• Clinical impact of HBV resistance is reduced (more drugs,

better drugs)

Treatment endpoints

in HIV, HBV and HCV infections

H

HBV1,2

Host cell

cccDNA

Host DNA

Integrated DNA

Nucleus

H

HIV1

Host cell

Host DNA

Proviral DNA

Nucleus

H

HCV1,3

Host cell

Host DNA

Nucleus

HCV RNA

Life-long suppression

of viral replication

Definitive viral clearance

and SVR

Long-term suppression

of viral replication

Adapted from 1. Sorriano V, et al. J Antimicrob Chemother 2008;62:1-4. 2. Locarnini S and Zoulim F.

Antiviral Therapy 2010;15 (suppl 3):3-14. 3. Sarrazin C and Zeuzem S. Gastroenterology 2010;138:447-462.

Treatment can be stopped:

• Stable HBeAg to anti-HBe seroconversion (up to 50% of HBeAg +ve patients)

• HBsAg to anti HBsAg seronversion:

- 10-15% of HBeAg+ve patients treated long term with NUCs

- 10-15% of anti-HBe+ve patients after PEH-IFNa treatment

cccDNA life cicle 1

from Nassal et al, Virus Research 2008

HBV replication: 2 arms

- nuclear transcription of cccDNA (from cccDNA to viral mRNAs)

- cytoplasmic RT/Pol (core particles) (from pgRNA to HBV-DNA)

HBV morphogenesis:

- Endoplasmic reticulum and Golgi

cccDNA life cicle 2

Adapted from Nassal et al, Virus Research 2008

TDP2/TTRAP

5'-tyrosyl DNA phosphodiesterase 2

TRAF and TNF receptor-associated protein

cccDNA formation requires

cellular proteins

cccDNA intracellular

recycling controlled in a

virus/cell specific manner

Koch et al Plos Path 2010

cccDNA in the livers of HBV patients

Werle, Petersen, Locarnini, Zoulim, Gastroenterology 2004

Laras, Hepatology 2006

cccDNA ~ 2 Logs < HBV-DNA

n. of positive hepatocytes

cccD

NA

(co

pie

s/c

ell)

DAYS

cccD

NA

(co

pie

s/c

ell)

Variability of cccDNA levels

Zhang et al, PNAS USA 2003

Duckling with chronic DHBV infection

Limiting dilution single cell cccDNA analysis

Persistence of cccDNA

Belloni, Levrero, Gaeta HBV meeting 2010

B2B1 B3 B4 C2C1 N1 N2

pg

RN

Ac

p/n

g c

DN

A

0

0.02

0.04

0.06

0.08

0.10

0

0.25

0.50

0.75

1.0

cc

cD

NA

co

pie

s/c

ell

1149.4neg>250

HBsAg

Persistence of cccDNA in 3 out of 4 patients with long

term HBV suppression under lamivudine

Maynard et al. J Hepatol 2005

Persistence of cccDNA after HBs seroconversion

In vitro studies on cccDNA stability during ADV

treatment using WHV-infected hepatocytes

WC-hepatocyte culture

ccc

4 weeks of Adefovir treatment strongly inhibited

viral replication without reducing cccDNA levels

Dandri et al., Hepatology 2000

Antivirals do not directly target cccDNA

Modified from Nassal et al, Virus Research 2008

?

1 yr of monotherapy with nucleos(t)ide analogues (ADV, LAM,

ETV) reduced median intrahepatic cccDNA amounts by 1 log

Zoulim,Petersen,Locarnini, Gastroenterology 2004,

Wong, Antivir Ther 2006, Sung, Gastroenterology 2005

HBV DNA

Virions

replication

Virions + defective particles(exceeding virions by a factor of 103 - 105)

qHBsAg

replication cccDNA

transcription mRNAs

translation

cccDNA, Serum HBsAg and HBV DNA levels in CHB

Serum HBV DNA: marker of virus replication

Serum HBsAg marker of transcriptionally active cccDNA in infected cells

more than the overall amount of cccDNA

cccDNA:- Template for transcription

- “Archive” for mutations

viral mRNAs:- translated into

viral proteins

(HBsAg etc)

Confoundings: Integrated HBV DNA (source of HBsAg)

Secretion rate (proportion of LHBs, mutants)

Irrevevant: HBV DNA (viremia) (NOT a source of HBsAg)

HBsAg

Modeling of cccDNA

Cell division in the setting of liver regenerationinduced cccDNA destabilization and formation ofcccDNA-free cells

cccDNA loss

cccDNA dilution

without loss

Implications

Not only viral suppression but also some cell injury and compensatory cell growth

may be necessary to significantly reduce cccDNA loads in vivo and possibly to

achieve control of HBV infection with consecutive reduction or loss of HBsAg

2.5 cccDNA copies /PTH before Tx

cccDNA copies after TX

Days after transplantation

0.01

10

0.1

1

0 10 20 40 80

Lo

g c

ccD

NA

co

pie

s /

PT

H

cccDNA decline per infected cell

Lütgehetmann et al., Hepatology 2010

Courtesy of J. Petersen

cccDNA in chronic HBV infection open issues

• mechanisms regulating maintenance of the

cccDNA pool

• “archiving” mutations

• molecular basis of cccDNA stability

• viral and cellular factors regulating transcriptional

activity of the cccDNA minichromosome in vivo

(cccDNA epigenetics)Modified from J Petersen

Studying cccDNA function in vivo

cccDNA

AAAAAA

AAA

liver tissue

Huh7 or HepG2 cells

transient transfection of

linear full-length HBV monomers

HBV

cccDNA ChIP assaycrosslink

sonicate

reverse crosslink

purify DNA

Reference

input DNA

immunoprecipitate

with specific antibodies

PCR or real time PCR with cccDNA

specific primers

Pollicino et al. Gastroenteroplogy 2006

Levrero et al. J Hepatol, 2009

Belloni et al, PNAS 2009

1) cccDNA chromatin Immuno

Precipitation assay (ChIP)

2) PCR-based method that

allows cccDNA quantitation

3) transient transfection of

linear HBV full-length genomes

into HuH7 hepatoma cells

Bock, T. et al 1994. Virus Genes;8:215

Bock, T. et al 2001. JMB;307:183

The cccDNA is a minichromosome

Epigenetic marks

of open and condensed chromatin

cccDNA-bound H3 and H4 histones

are acetylated in HuH7 cells replicating HBV

Pollicino et al., Gastroenterology, 2006

HBV replication parallels the acetylation status

of HBV cccDNA-bound H3 and H4 histones

Histones

HBV infection of PHH or HepaRG cells

cccDNA detected

H3 and AcH3 ChIP performed

level of infection infection (5 to 10 %) as limitation

cccDNA ChIP assay performance in in vitro and in vivo

HBV replication models

HepG2,

HepaRG

PHHwt HBV

cccDNA

Bac-HBV-1.1

Bac-HBV transduction of HepaRG cells

cccDNA formed from nucleocapsid recycling

AcH3/H4 ChIP positive

Bac-HBV HepG2 cells

Lucifora et al., J Gen Virol. 2008 Lucifora et al., J Hepatol 2011

HBV infected HepaRG cells

Human

Hepatocytes in uPA mice

Inp

ut

Ac

h4

IgG

cccDNA primers

Belloni et al., JCI 2011

uPA/SCID chimera mice

in vivoHBV infection

PPARa/RXRa, FXR,

HNF4a, HNF3, HNF1,

C/EBPa, FoxO1,

SP1 , NFkB

PPARa/RXRa, HNF4a, HNF3,

C/EBPa, CREB

cABL, RFX1, AP1, p53

HNF1, Oct1, NFkB

STATs

CREB, NF-Y

Transcriptional coactivators and repressors

are recruited onto cccDNA

Belloni et al. PNAS 2009

Guerrieri, Belloni unpublished

HBV preC/C promoter (cccDNA specific primers)

48 hours

96 hours

Histones

CBP

p300

PCAF Sirt1

HDAC1Ezh2

DNMT3a

Most evidence from in vitro study or

in non replicative cell culture models

classical transactivation studies

Acetylation of cccDNA-Bound H3 and H4 Correlates to

HBV Viremia Levels in Chronic Hepatitis B Patients

ChIP of liver nuclear extracts from

10 HBsAg-posi tive CH pts

using specific antibodies

to AcH3, AcH4, HDAc1 or control IgG

A. B.

Serum HBV DNA quantification in HBsAg-positive pts with

- active (AcH3 - AcH4 positive/HDAc1 negative, 4 cases)

(AcH3-AcH4 positive/HDAc1 positive, 2 cases)

- suppressed (AcH3-AcH4 negative/ HDAc1positive, 4 cases)

HBV replication. P value: Wilcoxon rank sum test.

Pollicino et al., Gastroenterology, 2006

Inactive HBV carrier

LOW-REPLICATIVE STATE HIGH-REPLICATIVE STATE

– spontaneously

– during immunosuppression

Low-replicative or latent infectionEpigenetic control

Histones

PCAFp300 PCAF

p300Sirt1

Sirt1HDAC1HDAC1

Histones

Pollicino et al. Gastroenteroplogy 2006

Levrero et al. J Hepatol, 2009

Occult HBV Infection Is Associated to Hypermethylated and

Deacetylated HBV cccDNA-bound histones

Input 1 2 3 4 5 6 IgG

cccDNA-ChIP

Occult HBV

Overt HBV

1:

2:

3:

4:

5:

6:

HP1

MECP2

SUV39

HDAC1

Ac.H3

Ac.H4

Pollicino, et al, unpublished

Definition: Presence of HBV DNA in the liver (± serum) of individuals with

undetectable HBsAg by the use of currently available tests.

Taormina statements on occult HBV infection. J Hepatol 2008

Class I/II and class III histone deacetylase inhibitors increase HBV

replication and acetylation of cccDNA-bound H3 and H4 histones

Pollicino et al., Gastroenterology 2006;

Belloni et al., HBV meeting 2006

B.

Input

IgG

AcH4

A.

cytoplasmic HBV

replicative intermediates

op

tical d

en

sit

y

fold

in

cre

ase

1

10

5

NT VPA TSA NAM

NT: untreated

VPA: treated for 16 hrs with 5 mM VPA

TSA: treated for 16 hrs with 300 nM TSA

NAM: treated for 16 hrs with 25 mM NAM

ChIP (cccDNA specific primers)

OC

DS

SS

HBV viral particles secreted

into the medium

NT VPA TSANAM1

10

5

op

tical d

en

sit

y

fold

in

cre

ase

OC

Low Replication

Sirt1

TF TFEzh2

TF TF

HDA

C1TF

Sirt1Ezh2

TFPCAF

HDAC1

tolerance chronic hepatitis inactive carrier pre-core mt occult HBV

0,001

0,01

0,1

1

10

100

1000DNA

cccDNA status in HBV patients

liver tissue

Huh7 or HepG2 cells

transient transfection of

linear full-length HBV monomers

HBV

cccDNA ChIP assay

Pollicino et al. Gastroenteroplogy 2006

Levrero et al. J Hepatol, 2009

Belloni, PNAS 2009

A methodology to study cccDNA function

in vitro,

in animal models

ex vivo (liver samples/biopsies)

Epigenetic control of cccDNA transcription

1. HBx modulates HBV transcription by affecting cccDNA-bound

histone acetylation

2. HBx repression of miR224 expression relieves the negative

effects of miR-224 on HBV replication

3. IL6: modulate cccDNA transcription by targeting liver enriched

transcription factors required for cccDNA transcription

4. IFNa: represses HBV transcription by recruiting the PRC2

repressor polycomb complex on the cccDNA

HBx impacts on the epigenetic control of HBV cccDNA

Belloni et al., PNAS 2009

Input

aHBx

IgG

wt

mt

HB

x

aHBx

5

10

15

20

25

30

RT

-PC

R

Arb

itra

ry U

nit

s

5

10

15

20

25

p300 HDAC Sirt1 E2F1

RT

-PC

R

Arb

itra

ry U

nit

s

Input

aAcH4

IgG

wt

mt

HB

x

1

2

3

4

aAcH4

RT

-PC

R

Arb

itra

ry U

nit

s

p_ 0.02<

Input IgG

Sirt1 HDAC1

p300 E2F1

wt

mt

HB

x

wt

mt

HB

x

HBx mutant recruits histone deacetylases and

transcribes less pgRNA

PCAF

Sirt1 Sirt1

HDAC1HDAC1

Histones

TFTF TF TF

PCAF p300

TF TF

HBx mutant HBV

HBV wild type

Histones

TF

PCAFp300

TF

HBx

TF TF

PCAF p300

TF

HBx

TF

IFNa and HBV

Interferon-a (IFNa) is an effective treatment for hepatitis B

virus (HBV) infection.

Class I IFNs inhibit HBV replication in vitro and in vivo but the

mechanism of action has not been identified.

An “interferon stimulated responsive element” (ISRE) is present

in the enhancer 1/X gene promoter region of the HBV genome

(Tur Kaspa, 1990) but the effect of IFNa and the role of STATs

protein on HBV transcription is not established (Alcantara,

2002).

Post-translational mechanisms and degradation of HBV

transcripts might also be involved:

- IFNa accelerates decay of replication-competent core particles (Xu,

2010)

- IFNa-induced MyD88 accelerates degradation of pgRNA (Li, 2010)

Inhibition of Intrahepatic viral productivity

by combination therapy with IFNa & ADV

Baseline (n=24)

1100

0

100

200

(503)

(735)

(861)

(1088)

300

W48(n=19)

W144(n=16)

-99%

-76%

p=0,001p=0,001

rcDNA/cccDNA

Lütgehetmann, Antiviral Therapy 2008

Effects of IFNa treatment on HBV replication and

transcription in humanized uPA/SCID mice

Petersen, PNAS 1998, Dandri, Hepatology 2001, 2002, 2008, J Hepatol 2005, Petersen, Nature Biotech.2008

woodchuck,

tupaia, human

hepatocytes

In vivo

HBV infection

Belloni et al., JCI 2011

The HBV ISRE mediates IFNa transcriptional repression

- ISREmt HBV transcribes less pgRNA but

its transcription is not repressed by IFN

ISRE

Belloni et al., JCI 2011

IFNAR1 IFNAR2

ISRE

Jak1Tyk2

Stat1 Stat2

IRF-9

Type I IFN signalingSTATs as “latent cytoplasmic factors”

ISGs

P

IFN-a

IFNAR1 IFNAR2

Dimerization

Jak1Tyk2

Stat1

Stat2Stat1

P

P P

Stat2

ISRE

Stat2Stat1

PP

IRF-9

ISRE

Different classes of IFNa activated and repressed genes

Testoni et al, Oncogene 2011; Testoni et al., JBC 2011

IFNα inhibits cccDNA transcription by recruiting the

Polycomb Repressor Complex 2 on the cccDNA

these results provide a molecular mechanism for IFNa repression of HBV transcritpion

In response to IFNa HDAC1 and Polycomb

corepressors are recruited on to the cccDNA

**

HBVtot

Arb

itra

ry U

nit

s

***

96t

0.2

0.4

0.6

0.8

1.0

48t +

48nt

96nt 48t +

48nt

96nt

pgRNA

******

96t

0.2

0.4

0.6

0.8

1.0

48t +

48nt

96nt

**

96t

1

2

3

a-Ezh2 RBAP48

Su

z12

Ezh

1

Me Me

YY1

PRC2 complex

EED

Ezh

2

Ac

Ac

no treatment IFNa off IFNa

TFn1TFn2

Ac Ac

H3K27H4K5/8/12/16

RBAP48

Su

z12

Ezh

1

Me Me

YY1

PRC2 complex

EED

Ezh

2

Ac

Ac

RBAP48

Su

z12

Ezh

1

Me Me

YY1

PRC2 complex

EEDE

zh

2

Ac

Ac

ACTIVE TRANSCRIPTION

(pgRNA; sub-genomic RNAs)

HIGH REPLICATION

TRANSCRIPTION REPRESSION

( pgRNA; sub-genomic RNAs)

LOW REPLICATION

TRANSCRIPTION REPRESSION

( pgRNA; sub-genomic RNAs)

LOW REPLICATION

• ACTIVE HBV CARRIER

• ACTIVE LIVER DISEASE

PROGRESSION

• HBsAG TITER DECLINE

• INACTIVE CARRIER

• INACTIVE LIVER DISEASE

REMISSION

• HBsAG TITER DECLINE

• INACTIVE CARRIER

• INACTIVE LIVER DISEASE

“OFF THERAPY” REMISSION

Model of IFNα activity on cccDNA transcription

Sustained virological suppression achieved by IFNα treatment (30-35% of HBeAg+ patients and 20-25% of HBeAg-

patients, is commonly thought to reflect the transition to the “immune-control” phase that characterize the inactive

HBsAg carrier state.

Our results indicate that IFNα induces a condition of “active epigenetic control” of HBV cccDNA transcription that likely

contributes to the persistent, yet reversible, “off therapy” inhibition of HBV replication

Low Replication / HBx mt

Si

rt

1TF TF

E

z

h

2

TF TF

HDA

C1TF

Si

rt

1E

z

h

2

TFP

C

AF

HD

AC

1

tolerance chronic hepatitis inactive carrier pre-core mt occult HBV

0,001

0,01

0,1

1

10

100

1000DNA

If we cannot destroy it .... let’s make it “locked” (“occult”)

… in all patients

-hSirt1 agonists

-EZH2 agonists

-IFN mimics (i.e. non IFN inducers of ISGs)

IFNa

Control vs eradication

Low Replication / HBx mt

Si

rt

1TF TF

E

z

h

2

TF TF

HDA

C1TF

Si

rt

1E

z

h

2

TFP

C

AF

HD

AC

1

tolerance chronic hepatitis inactive carrier pre-core mt occult HBV

0,001

0,01

0,1

1

10

100

1000DNA

HBV “Sleeping beauty”

IFNa

If we cannot destroy it .... let’s make it

“locked” (“occult”) … in all patients

-hSirt1 agonists

-EZH2 agonists

Low Replication / HBx mt

Si

rt

1TF TF

E

z

h

2

TF TF

HDA

C1TF

Si

rt

1E

z

h

2

TFP

C

AF

HD

AC

1

tolerance chronic hepatitis inactive carrier pre-core mt occult HBV

0,001

0,01

0,1

1

10

100

1000DNA

IFNa

HBV “Sleeping beauty”

If we cannot destroy it .... let’s make it

“locked” (“occult”) … in all patients

-hSirt1 agonists

-EZH2 agonists

What we want, what we can...

HBV suppression block HBV DNA synthesis (RT-DNA Pol inhibitors)

inhibit cccDNA transcription

target morphogenesis (?)

……

HBV eradication target cccDNA stability/formation

viral entry inhibitors

……

Make all active carriers „true“ inactive

and, eventually, over time „occult“carriers

What we are trying to do ...

HBV suppression block HBV DNA synthesis (RT-DNA Pol inhibitors)

inhibit cccDNA transcription

target morphogenesis (?)

……

HBV eradication target cccDNA stability/formation

HBc as a target

Chromatin assembly/reassembly

viral entry inhibitors

……

Make all active carriers „true“ inactive

and, eventually, over time „occult“carriers

Open issues and new perspectives

cccDNA assay

1. (further) standardization of the assay

2. improve sensitivity (less chromatin …. infection models)

Clinical / translational implications

1. Eradication vs control: make it “locked” (“occult”) or destroy it

2. Finite therapy: identify stable cccDNA inactivition

3. Should we ChIP it, measure it or look for surrogate markers

(pgRNA, qHBsAg, ….. )

Laura Belloni

Francesca Guerrieri

Natalia Pediconi

Cecilia Scisciani

Letizia Cimino

Rossana De Iaco

Valeria Schinzari

Barbara Testoni

Massimo Levrero

Collaborations:

Dept. of Internal Medicine

University of Messina

Giovanni Raimondo

Teresa PollicinoGiuseppina Raffa

Giovanni Squadrito

INSERM U761 -Lyon

Fabien ZoulimJulie Lucifora

David Durantel

University Medical Hospital Hamburg

Jorg Petersen

Maura DandriLena Allweiiss

Tassilo Voltz

Technische Universität Munchen

Helmholtz Zentrum Munchen

Ulrike Protzer

Julie Lucifora

Laboratory of Gene Expression

With the support of Fondazione

Andrea

Cesalpino

Anna TramontanoAndrea Sbardellati,

Daniel D’Andrea

Francesco Cicconardi

Collaborations: