Grand Hyatt Kauai

Koloa, Kauai, Hawaii

December 4-8, 2011

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

Outline
1.
2.
3.
4.
5.
6.
7.

Treating CHB: where we are ..

cccDNA in patients
cccDNA in HBV life cycle
Developing a cccDNA functional assay
Epigenetic control of cccDNA function
Can we modulate cccDNA function
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
HBV1,2

HIV1
Host cell

Host cell

Host cell
cccDNA

Host DNA

H
Proviral DNA

Host DNA

HCV1,3

Host DNA

HCV RNA

Integrated DNA

Nucleus

Nucleus

Life-long suppression
of viral replication

Long-term suppression
of viral replication

Nucleus

Definitive viral clearance

and SVR

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

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.

cccDNA life cicle 1

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
from Nassal et al, Virus Research 2008

cccDNA life cicle 2

cccDNA formation requires
cellular proteins

cccDNA intracellular
recycling controlled in a
virus/cell specific manner

TDP2/TTRAP
5'-tyrosyl DNA phosphodiesterase 2
TRAF and TNF receptor-associated protein
Adapted from Nassal et al, Virus Research 2008
Koch et al Plos Path 2010

cccDNA in the livers of HBV patients

Laras, Hepatology 2006

Werle, Petersen, Locarnini, Zoulim, Gastroenterology 2004

cccDNA ~ 2 Logs < HBV-DNA

cccDNA (copies/cell)

cccDNA (copies/cell)

Variability of cccDNA levels

DAYS

n. of positive hepatocytes

Duckling with chronic DHBV infection

Limiting dilution single cell cccDNA analysis

Zhang et al, PNAS USA 2003

Persistence of cccDNA

pgRNA cp/ng cDNA

>250 neg 9.4 114

1.0

0.10
0.08

0.75

0.06
0.50

0.04
0.02

0.25
0

0
B1 B2 B3

B4

C1 C2

N1

cccDNA copies/cell

HBsAg

N2

Persistence of cccDNA in 3 out of 4 patients with long

term HBV suppression under lamivudine
Belloni, Levrero, Gaeta HBV meeting 2010

Persistence of cccDNA after HBs seroconversion

Maynard et al. J Hepatol 2005

In vitro studies on cccDNA stability during ADV

treatment using WHV-infected hepatocytes

ccc

WC-hepatocyte culture

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

cccDNA, Serum HBsAg and HBV DNA levels in CHB

qHBsAg

HBV DNA

Virions + defective particles

Virions

(exceeding virions by a factor of 103 - 105)

replication

replication
transcription
translation

cccDNA
mRNAs

Serum HBV DNA: marker of virus replication

Serum HBsAg marker of transcriptionally active cccDNA in infected cells
more than the overall amount of cccDNA

viral mRNAs:
cccDNA:
- Template for transcription
- Archive for mutations

- 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

Log cccDNA copies /PTH

cccDNA decline per infected cell

cccDNA loss

cccDNA dilution
without loss

2.5 cccDNA copies /PTH before Tx

cccDNA copies after TX

10

0.1

0.01
0

10 20
40
80
Days after transplantation

Cell division in the setting of liver regeneration

induced cccDNA destabilization and formation of
cccDNA-free cells
Courtesy of J. Petersen

Ltgehetmann et al., Hepatology 2010

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

cccDNA in chronic HBV infection open issues

mechanisms regulating maintenance of the

cccDNA pool

cccDNA
AAA

archiving mutations
molecular basis of cccDNA stability

AAA
AAA

Modified from J Petersen

viral and cellular factors regulating transcriptional

activity of the cccDNA minichromosome in vivo
(cccDNA epigenetics)

Studying cccDNA function in vivo

The cccDNA is a minichromosome

Huh7 or HepG2 cells
HBV

liver tissue

transient transfection of
linear full-length HBV monomers

cccDNA ChIP assay

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

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

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

crosslink

sonicate

immunoprecipitate
with specific antibodies

reverse crosslink
purify DNA
Reference
input DNA
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

Epigenetic marks
of open and condensed chromatin

HBV replication parallels the acetylation status

of HBV cccDNA-bound H3 and H4 histones
cccDNA-bound H3 and H4 histones
are acetylated in HuH7 cells replicating HBV

Histones

Pollicino et al., Gastroenterology, 2006

cccDNA ChIP assay performance in in vitro and in vivo

HBV replication models
wt HBV

HepG2,
HepaRG
PHH

cccDNA

Bac-HBV transduction of HepaRG cells

cccDNA formed from nucleocapsid recycling
AcH3/H4 ChIP positive

Bac-HBV-1.1

HBV infection of PHH or HepaRG cells

cccDNA detected
H3 and AcH3 ChIP performed
level of infection infection (5 to 10 %) as limitation

Input

HBV infected HepaRG cells

Human
Hepatocytes in uPA mice
IgG

Bac-HBV HepG2 cells

Ach4

uPA/SCID chimera mice

in vivoHBV infection

cccDNA primers
Lucifora et al., J Gen Virol. 2008

Lucifora et al., J Hepatol 2011

Belloni et al., JCI 2011

Transcriptional coactivators and repressors

are recruited onto cccDNA
CREB, NF-Y

HNF1, Oct1, NFkB

48 hours

STATs

96 hours
PPARa/RXRa, HNF4a, HNF3,
C/EBPa, CREB
cABL, RFX1, AP1, p53

HBV preC/C promoter

(cccDNA specific primers)

PPARa/RXRa, FXR,
HNF4a, HNF3, HNF1,
C/EBPa, FoxO1,
SP1 , NFkB

Histones
Most evidence from in vitro study or
in non replicative cell culture models
classical transactivation studies

Belloni et al. PNAS 2009

Guerrieri, Belloni unpublished

Ezh2
DNMT3a

PCAF
CBP
p300

Sirt1
HDAC1

Acetylation of cccDNA-Bound H3 and H4 Correlates to

HBV Viremia Levels in Chronic Hepatitis B Patients
A.

B.

ChIP of liver nuclear extracts from

10 HBsAg-posi
tive CH pts
using specific antibodies
Ac
to H3, AcH4, HDAc1 or control IgG
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 or latent infection

Epigenetic control

HDAC1HDAC1 Sirt1

Sirt1

PCAF

p300
p300

PCAF

Histones

Histones

LOW-REPLICATIVE STATE

HIGH-REPLICATIVE STATE

spontaneously
during immunosuppression

Pollicino et al. Gastroenteroplogy 2006

Levrero et al. J Hepatol, 2009

Occult HBV Infection Is Associated to Hypermethylated and

Deacetylated HBV cccDNA-bound histones
cccDNA-ChIP

Input 1

2 3

4:
5:
6:

HDAC1

IgG

Occult HBV

Overt HBV
1:
2:
3:

HP1
SUV39
MECP2

Ac.H3
Ac.H4

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

Pollicino, et al, unpublished

Class I/II and class III histone deacetylase inhibitors increase HBV
replication and acetylation of cccDNA-bound H3 and H4 histones
B.

A.

Input
OC
DS

OC

AcH4
IgG

SS

ChIP (cccDNA specific primers)

10
optical density
fold increase

optical density
fold increase

10

NT

VPA TSA NAM

cytoplasmic HBV
replicative intermediates

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

NT VPA TSA NAM

HBV viral particles secreted

into the medium

Pollicino et al., Gastroenterology 2006;

Belloni et al., HBV meeting 2006

cccDNA status in HBV patients

1000
100
10
1
0,1
0,01
0,001

DNA

tolerance chronic hepatitis inactive carrier pre-core mt

Sirt1
Sirt1
HDAC1 Ezh2
Ezh2
HDA
PCAF
TF TF TF
TF TF TF
C1

Low Replication

occult HBV

cccDNA ChIP assay

Huh7 or HepG2 cells

HBV

liver tissue

transient transfection of
linear full-length HBV monomers

A methodology to study cccDNA function

in vitro,
in animal models
ex vivo (liver samples/biopsies)

Pollicino et al. Gastroenteroplogy 2006

Levrero et al. J Hepatol, 2009
Belloni, PNAS 2009

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

HBx

HBx

PCAF

PCAF p300

p300

Input

Input

IgG

aHBx

aAcH4

p300

E2F1

IgG

IgG

Sirt1

HDAC1

RT-PCR
Arbitrary Units

RT-PCR
Arbitrary Units

< 0.02
p_
30
25
20
15
10
5
aHBx

4
3
2
1

RT-PCR
Arbitrary Units

Input

TF

TF

TF

TF
Histones

HBV wild type

25
20
15
10

Sirt1

Sirt1

HDAC1
PCAF HDAC1
PCAF

TF

aAcH4

TF

mt HBx

wt

mt HBx

wt

wt
mt HBx

wt
mt HBx

TF

p300 HDAC Sirt1

TF

TF

TF

p300

TF

TF
Histones

E2F1

HBx mutant HBV

HBx mutant recruits histone deacetylases and

transcribes less pgRNA
Belloni et al., PNAS 2009

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.

Inhibition of Intrahepatic viral productivity

by combination therapy with IFNa & ADV

rcDNA/cccDNA
1100
(1088)

p=0,001

p=0,001

(861)
(735)

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)

300

(503)

200
-76%

100
-99%
0
Baseline

W48

W144

(n=24)

(n=19)

(n=16)

Ltgehetmann, Antiviral Therapy 2008

Effects of IFNa treatment on HBV replication and

transcription in humanized uPA/SCID mice
woodchuck,
tupaia, human
hepatocytes

In vivo
HBV infection

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

Belloni et al., JCI 2011

The HBV ISRE mediates IFNa transcriptional repression

ISRE

- ISREmt HBV transcribes less pgRNA but

its transcription is not repressed by IFN
Belloni et al., JCI 2011

Type I IFN signaling

STATs as latent cytoplasmic factors

IFN-a
IFNAR1
Tyk2

Stat1

IFNAR2

IFNAR1
Tyk2
P

Jak1

Stat2

IFNAR2
Jak1
P

Stat2

Stat1

Stat1

Dimerization

Stat2

IRF-9

Stat1Stat2
IRF-9
ISRE
ISRE

ISRE

ISGs

Different classes of IFNa activated and repressed genes

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

IFN inhibits cccDNA transcription by recruiting the

Polycomb Repressor Complex 2 on the cccDNA

PRC2 complex

Arbitrary Units

1.0
0.8
0.6
0.4
0.2

0.8
0.6
0.4
0.2
96nt 96t 48t +
48nt

Suz12

***
**1.0

EED

a-Ezh2
*
*

pgRNA
***
***

Ezh2

Ezh1

HBVtot

RBAP48 YY1

Me

Me

Ac

3
Ac

2
1
96nt 96t 48t +
48nt

96nt 96t 48t +

48nt

In response to IFNa HDAC1 and Polycomb

corepressors are recruited on to the cccDNA

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

Model of IFN activity on cccDNA transcription

no treatment

H4K5/8/12/16

IFNa

off IFNa

PRC2 complex

H3K27

PRC2 complex
Suz12

EED

RBAP48 YY1

RBAP48 YY1

Me

Ac Ac

Ezh2

Ezh1

Suz12

Ezh2

Ezh1

EED

TFn2 TFn1

Me

Me

Ac
Ac

Me

Ac
Ac

ACTIVE TRANSCRIPTION
(pgRNA; sub-genomic RNAs)

TRANSCRIPTION REPRESSION
( pgRNA; sub-genomic RNAs)

TRANSCRIPTION REPRESSION
( pgRNA; sub-genomic RNAs)

HIGH REPLICATION

LOW REPLICATION

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

Sustained virological suppression achieved by IFN treatment (30-35% of HBeAg+ patients and 20-25% of HBeAgpatients, is commonly thought to reflect the transition to the immune-control phase that characterize the inactive
HBsAg carrier state.
Our results indicate that IFN 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

Control vs eradication
1000
100
10
1
0,1
0,01
0,001

DNA

tolerance chronic hepatitis inactive carrier pre-core mt

occult HBV

Si
Si
HD
E rt
HDA
P rt E
AC
zTF 1
TF
z
C1
C 1 TF
TF 1 TF
TF
h
AF h
2
2

Low Replication / HBx mt

IFNa

If we cannot destroy it .... lets make it locked (occult)

in all patients

-hSirt1 agonists
-EZH2 agonists
-IFN mimics (i.e. non IFN inducers of ISGs)

HBV Sleeping beauty

1000
100
10
1
0,1
0,01
0,001

DNA

tolerance chronic hepatitis inactive carrier pre-core mt

occult HBV

Si
Si
HD
E rt
HDA
P rt E
AC
zTF 1
TF
z
C1
C 1 TF
TF 1 TF
TF
h
AF h
2
2

Low Replication / HBx mt

IFNa

If we cannot destroy it .... lets make it

locked (occult) in all patients
-hSirt1 agonists
-EZH2 agonists

HBV Sleeping beauty

1000
100
10
1
0,1
0,01
0,001

DNA

tolerance chronic hepatitis inactive carrier pre-core mt

occult HBV

Si
Si
HD
E rt
HDA
P rt E
AC
zTF 1
TF
z
C1
C 1 TF
TF 1 TF
TF
h
AF h
2
2

Low Replication / HBx mt

IFNa

If we cannot destroy it .... lets 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 (?)

Make all active carriers true inactive
and, eventually, over time occultcarriers

HBV eradication
target cccDNA stability/formation
viral entry inhibitors

What we are trying to do ...

HBV suppression
block HBV DNA synthesis (RT-DNA Pol inhibitors)
inhibit cccDNA transcription
target morphogenesis (?)

Make all active carriers true inactive

and, eventually, over time occultcarriers

HBV eradication
target cccDNA stability/formation
HBc as a target
Chromatin assembly/reassembly
viral entry inhibitors

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, .. )

Collaborations:

Laboratory of Gene Expression

Laura Belloni
Francesca Guerrieri
Natalia Pediconi
Cecilia Scisciani
Letizia Cimino
Rossana De Iaco
Valeria Schinzari
Barbara Testoni

Lena Allweiiss
Tassilo Voltz

Dept. of Internal Medicine

University of Messina
Giovanni Raimondo
Teresa Pollicino
Giuseppina Raffa
Giovanni Squadrito

Technische Universitt Munchen

Helmholtz Zentrum Munchen
Ulrike Protzer
Julie Lucifora

Massimo Levrero
Collaborations:

INSERM U761 -Lyon

Fabien Zoulim

Anna Tramontano

Julie Lucifora
David Durantel

Andrea Sbardellati,
Daniel DAndrea
Francesco Cicconardi

With the support of

University Medical Hospital Hamburg

Jorg Petersen
Maura Dandri

Fondazione
Andrea
Cesalpino