1

v

Professor Massimo Levrero

Sapienza University of Rome, Italy

18 th Annual Resistance and

Antiviral Therapy Meeting

Thursday 18 September 2014, Royal College of Physicians, London

Massimo LevreroDipartimento di Medicina Interna e Specialita’ Mediche - DMISM

IIT - Center for Life-Nanoscience

Sapienza Universita’ di Roma

Can we eradicate chronic hepatitis B infection?

Dipartimento dimedicina interna e specialitÀ mediche

2

Disclosures

● BMS: Advisory Board and invited speaker; investigator

● Gilead: Advisory Board and invited speaker; investigator

● Janssen: Advisory Board and invited speaker, investigator

● MSD: Advisory Board and invited speaker; investigator

● Roche: Consultancy; nvited speaker; investigator

● Tekmira: Advisory Board

Outline

1. Concepts on HBV cure

2. HBV cccDNA as a minichromosome: HBV replication is

controlled by epigenetic mechanisms

1. modulation of cccDNA function by epigenetic drugs

2. anti-capsid drugs also target cccDNA

3

H

Host cell

cccDNA

Host DNA

Integrated DNA

Nucleus

Long-term suppression

of viral replication

(DAA)

Adapted from 1. Soriano 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.

HBV the concept of « cure »

Viral Suppression

Functional cure (very few)

Eradication

�. anti-HBV therapies achieve ~ 100 % long term on

treatment suppression but only 20 to 15 % off treatment

control with IFN (12 months) or NUCs (3 to 5 years) off

treatment sustained viral suppression .. adherence

Suppression

of viral replication

Immune control

(IFNα)

• High rates of regression or

halting of fibrosis/cirrhosis with

TDF/ETV

• Established safety profile up to

Year 7 of treatment

• Resistance with some

antivirals (LAM, ADV)

• Risk of HCC persists

• Persistence of cccDNA (even

after therapy or spontaneous

recovery)

- Viral reactivation

- Hepatocarcinogenesis

• Integrated viral genome

- Hepatocarcinogenesis

HBV1,2

HBV challenges

• Treat more effectively: HBV and HCC

• Develop/use tools to Predict Treatment Outcomes– qHBsAg

– qHBeAg

• Aim for cure

– Functional cure

• off-therapy persistent HBV suppression

– cccDNA eradication

• surrogate endpoint HBsAg loss and anti-HBs seroconversion

• Strategies

– Immune system

– Viral targets

4

HBV life cicle

from Nassal et al, Virus Research 2008

• HBV hepatic “latent” reservoires (non integrated functionally

competent HBV genomes) is now established – OBI and more

• extra-hepatic reservoires described & but never formally

established &. cccDNA presence unproven

cccDNA half-life: supposedly long, not established

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

No data on IFN-a monotherapy

5

Persistence of cccDNA

Belloni, Levrero, Gaeta HBV meeting 2010

B2B1 B3 B4 C2C1 N1 N2

pg

RN

Acp

/ng

cD

NA

0

0.02

0.04

0.06

0.08

0.10

0

0.25

0.50

0.75

1.0

cccD

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

In 2 out 3 patients cccDNA is inactive (no pgRNA)

• Detected in the liver of NUCs long-term suppressed patients after HBsAg to anti-HBs

seroconversion [Maynard, 2005; Belloni unpublished]

• Detected in the liver of HBsAg negative patients (occult HBV infection)

[Werle-Lapostolle, 2004; Pollicino unpublished]

• Present in 30 /30 patients with occult HBV infection and HCC [Pollicino, 2004]

liver tissue

Huh7 or HepG2 cells

transient transfection of

linear full-length HBV monomers

HBV

cccDNA as a minichromosome: the cccDNA ChIP assays

Pollicino et al. Gastroenteroplogy 2006

Levrero et al. J Hepatol, 2009

Belloni, PNAS 2009

Belloni, JCI 2012

A methodology to study cccDNA function

[ HBV minichromosome structure ]

[ modifications of cccDNA bound histones ]

[ binding of TF and coregulators ]

in vitro,

in animal models

ex vivo (liver samples/biopsies)

PCR or real time PCR

with cccDNA

specific primers

crosslink

sonicate

reverse crosslink

purify DNA

Reference

input DNA

immunoprecipitate

with specific antibodies

Massive Parallel

Sequencing

(ChIP-Seq)

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.

Bock, T. et al 2001.

6

Studying cccDNA back in 2004

from Nassal et al, Virus Research 2008

- No reliable infection system

- HBV transgenic mice: cccDNA (-)

- HBV stable cell lines (2.2.15) cccDNA +/-

- HBV Transfection:

• 1.2-1.3 HBV constructs: cccDNA +/- or (-)

• 1.0 HBV linear genome: cccDNA ++

1.0 HBV1.2-1.3 HBV or

HBV DNA transfection

cccDNA

formation

cccDNA

transcription

Epigenetic marks

of open and condensed chromatin

Arrowsmith CH et al., Nat Rev Drug Discov. 2012

7

HBV cccDNA ChIP assay

• HBV replication parallels the acetylation status of HBV

cccDNA-bound H3 and H4 histones in HBV replicating cells

and in vivo in patients

• Transcription factors and transcriptional coactivators and

repressors are recruited onto cccDNA

– Stat1/2, Stat3, HNF1a, HNF4a, p65, YY1, FXR

– HATs (p300, PCAF, CBP)

– HDACs (HDAC1, hSirt1)

– HMTs (Ezh2); PRMTs (PMRT1; PMRT5) DNA-MTs (DNMT3a)

• Viral proteins bind to and modulate cccDNA functions

– HBx (increases/required for transcription; increase p300 and

prevents HDAC1 binding)

– HBc

Pollicino, Gastroenterology 2006; Belloni, PNAS 2009; Levrero, J Hepatol 2009; Belloni, JCI 2012

PCAF

Sirt1 Sirt1

HDAC1HDAC1

HistonesTFTF

TF TF

PCAF p300

TF TF

HistonesTF

PCAFp300

TF

HBx

TF TF

PCAF p300

TF

HBx

TF

Active cccDNA

Inactive cccDNA

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

8

spontaneously

iatrogenic

immunosuppression

iatrogenic

immunosuppression

tolerance chronic hepatitis inactive carrier pre-core mt occult HBV

0,001

0,01

0,1

1

10

100

1000

DNA

cccDNA status in HBV patients

Histones

PCAFp300 PCAF

p300

High Replication Low Replication

Sirt1

Sirt1HDAC1

HDAC1

Histones

Ezh2

Sirt1

Sirt1HDAC1

HDAC1

Histones

HP1MeCP2

Suv39

Occult HBV

Input 1 2 3 4 5 6 IgG

ChIP

Low-replicative to latent infectionEpigenetic control

Pollicino et al., Gastroenterology 2006;

Belloni et al., HBV meeting 2006

Pollicino, unpublished

cccDNA ChIP assay in vitro and in vivo

HBV replication models

HepG2,

HepaRG

PHH

wt HBV

cccDNA

Bac-HBV-1.1

uPA/SCID mice

in vivo

HBV infection

Human Hepatocytes in uPA mice[Belloni, 2012; Belloni & Allweiss, unpublished]

• cccDNA detected

• H3 and AcH3 ChIP performed

• infection efficiency: cccDNA levels required 0.5-1 cp/cell

Bac-HBV transduction of HepaRG cells[Lucifora, 2008]

• cccDNA formed from nucleocapsid recycling

• AcH3/H4 ChIP positive

HBV infection of PHH or HepaRG cells[Lucifora, 2011; Sonnabend & Belloni, unpublished]

• cccDNA detected

• H3 and AcH3 ChIP performed

• infection efficiency as limitation

0.2 cccDNA/cell = cccDNA ChIP LOD

0.5-1.0 cccDNA/cell = multiple parameters cccDNA ChIP

wt HBV

cccDNANTCP-HepG2

HBV infection of NTCP-HepG2 cells[Sonnabend & Belloni, unpublished]

• cccDNA detected

• H3 and AcH3 ChIP performed

Liver biopsies

Human Hepatocytes[Pollicino, 2006; Belloni, Pollicino, Guerrieri, unpublished]

• cccDNA detected

• H3, AcH3, HDACs, HMTs ChIP performed

9

cccDNA

formation

cccDNA Y Y Y N N N Y/N

cccDNA

chromatin

assembly

cccDNA

ChIPY/N * Y/N* Y/N* N Y N Y

cccDNA

chromatin

function

cccDNA

ChIPY/N* Y/N* Y/N* Y/N* Y Y/N Y

cccDNA

stability

cccDNA Y/N Y/N Y Y/N Y/N Y N

cccDNA

transcription

pgRNA Y Y Y Y Y Y/N Y/N

Core particles

recyclingY Y Y/N Y/N N Y Y

HBV

replication

Cp

HBV-

DNA

Y Y Y Y Y Y Y

Chronic

infectionN Y/N Y Y N Y/N Y/N

HepaRG

PHH

[infection]

NTCP

HepG2

HepaRG

[infection]

uPA/SCID

Mice

[infection]

HBV

isolated

hepatocytes

uPA mice

[ex vivo]

HepG2

HBV 1.0

[transfection]

HepG2

HBV H1.3

[stable]

HepG2

AD38

[inducible]

* inferred; direct study limited by cccDNA levels

HBV models for cccDNA

HBV cure

• Aim for cure

– Functional cure

Off-therapy persistent HBV suppression

– cccDNA eradication

• Strategies

- Immune system

- Viral targets

cccDNA degradation

cccDNA silencing

cccDNA dilution

10

Adapted from Nassal M et al. Virus Res 2008;134:235–249;

Grimm D et al. Hepatol Int 2011;5:644–653.

cccDNA formation

inhibitors

Nucleocapsid assembly

inhibitors

cccDNA transcription

Inhibitors

Emerging antiviral approaches

• cccDNA - silencing

- degradation

• pgRNA - siRNAs

• sub-genomic RNAs - siRNAs

• RNAse H inhibitors

• “capsid”- Core protein Assembly

Modulators (CpAMs)

• Entry inhibitors (HBV and HDV)

• Prenylation inhibition (HDV)

• HBsAg release inhibitors

• Cyclophilin inhibitors

• TLR agonists

• Therapeutic vaccination

anti-HBV siRNAs

cccDNA

destabilization/degradation

Adapted from Petersen J et al. Hepatol Rev 2007;4:9–13 and Levrero M et al. J Hepatol 2009;51:581–592.

Silencing cccDNA

“functional cure” – off therapy control

11

IFNα targets the HBV cccDNA

Interferon-α (IFNα) inhibits HBV transcription and

replication in vitro and in vivo by favoring the long

term recruitment to the nuclear cccDNA mini-

chromosome of the class III HDAC hSirt1 and of

the PRC2 repressive complex, including the

transcriptional co-repressors HDAC1 and Ezh2

RBAP48

Suz12

Ezh1

Me Me

YY1

PRC2 complex

EED

Ezh2

Ac

Ac

IFNa (1000 UI/ml)

NT

Belloni et al., JCI 2012

Effects of IFNα treatment on HBV

replication and transcription confirmed

in humanized uPA/SCID mice

The HBV ISRE mediates IFNα transcriptional

repression

Pollicino T et al. Gastroenterology 2006;130:823–837; Adapted from Levrero M et al. J Hepatol 2009;51:581–592;Belloni L et al. J Clin Invest 2012;122:529–537.

Histone acetylation/methylation

affects the regulation of gene expressionIFNα treatment is accompanied by a

decrease in the acetylation of cccDNA

bound H4 histones in vitro

PCAFPCAF

p300p300

Sirt1Ezh2YY1HDAC1

acetylated histones

deacetylated histones

+ IFNα

HBx mt

Transcription of the HBV cccDNA mini-

chromosome can be regulated epigenetically

12

IFNα targets the HBV cccDNA

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 (30-

35% of HBeAg+ patients and 20-25% of

HBeAg- patients) and suggest that HBV

cccDNA function can be modulated by

epigenetic drugs

RBAP48

Suz12

Ezh1

Me Me

YY1

PRC2 complex

EED

Ezh2

Ac

Ac

HBV-specific CD8

CMV-specific CD8

Micco L et al J. Hepatol. 2012Belloni et al., JCI 2012

HepAD38 cells

HBV stable clone

Ladner 1997cccDNA

- Tet

days0 36

Cp-HBV-DNA

pgRNA

Drug

Ezh2JMD3

MC3119

Me Me

Ezh2

Ac

Ac

Palumbo GA et al. AASLD 2013 Abs 928

Ladner SK et al. Antimicrob Agents and Chemo1997;41:1715-20.

Modulation of Ezh2 histone methyltransferase

activity mimics IFNα-induced repression of

cccDNA transcription

0

0.2

0.4

0.6

0.8

1

1.2

2^

-(CtpgRNA

-Ctb

eta

ctin

a)

pgRNAChIP

α-Me3K27H3

Fold

in

du

ctio

n

0

1

2

3

4

0

0.2

0.4

0.6

0.8

1

1.2

HB

V D

NA

co

pie

s/ce

ll

Cp HBV-DNA

13

• Modulation of Ezh2 activity by MC3119 mimics IFNa-induced

transcriptional repression of the cccDNA.

• PERSPECTIVE:

- explore sequential treatments as a model for IFN sparing regimens

- increase the subset of IFN SVRs

Make active carriers „true“ inactive

and, eventually, over time „occult“

carriers by „locking“ the cccDNA

Proof of concept of modulation of cccDNA function

by “epigenetic” compounds

Palumbo GA et al. AASLD 2013 Abs 928

Proof of concept of modulation of cccDNA function

by “epigenetic” compounds

TFn1TFn2

Ac AcAc

Ac

• Inhibitors of PCAF/p300 and activators of

hSirt1/2 display the most significant

effects on cccDNA transcription and HBV

replication

• Inhibition of cccDNA bound HATs activity

leads to detachment of (PCAF) and p300

• The hSirt1/2 agonist induce a shift in

hSirt1/ hSirt2 occupancy and global

cccDNA-bound H4 deacetylation

• These results provide a proof of concept

that small molecules / drugs that affect

cccDNA bound chromatin modifying

enzymes can modulate HBV transcription

and replication

• PERSPECTIVE

• - possible synergisms ??

TFn1TFn2

Ac Ac

Ac

Ac

Active cccDNA

Suppressed cccDNA

Palumbo GA et al. AASLD 2013 Abs 928

14

HBc protein / capsid

HBV capsid

(120 HBc dimers)

Bock, 2001

HBc dimer

Input

aHBc

IgG

wt

mock

aHBc

5

10

15

20

Arb

itra

ry U

nit

s

Belloni 2009

� HBc binds the cccDNA and regulates its function

HBc protein / capsid

HBV capsid

(120 HBc dimers)

Bock, 2001

HBc dimer

Input

aHBc

IgG

wt

mock

aHBc

5

10

15

20

Arb

itra

ry U

nit

s

Belloni 2009

ChIP aHBc

Lupacchini (unpublished)

� HBc binds the cccDNA and regulates its function

� HBc binds to cellular promoters and regulates gene expression

15

HBV capsid

(120 HBc dimers)

HBc dimer

HAP12

� The Hap12 Core Protein Assembly Modulator (CpAM)

• inhibit HBV replication

CpAMs

HBV capsid

(120 HBc dimers)

HBc dimer

HAP12

� The Hap12 Core Protein Assembly Modulator (CpAM)

• inhibit HBV replication

• target the cccDNA

NT

1µM HAP

Palumbo (unpublished)

CpAMs

16

HBV capsid

(120 HBc dimers)

HBc dimer

HAP12

� The Hap12 Core Protein Assembly Modulator (CpAM)

• inhibit HBV replication

• target the cccDNA

• affect HBc occupancy on both the cccDNA and cellular promoters

NT

1µM HAP

Palumbo (unpublished)

CpAMs

HEPATITIS B CORE (HBC) PROTEIN IS A KEY AND VERY EARLY

NEGATIVE REGULATOR OF THE INTERFERON RESPONSE

HBc affects the epigenetic state of the host genome to control innate immunity- very early inhibition of the hepatocyte IFN response by core protein through the recruitment of

epigenome-modifying enzymes (Ezh2 and G9 HMTs) that leads to repressive marks (H3K27me3

and H3K9me3)

Gruffaz M, Testoni B et al AASLD 2013 Abs 136

17

HBV capsid

(120 HBc dimers)

HBc dimer

HAP12

� The Hap12 Core Protein Assembly Modulator (CpAM)

• inhibit HBV replication

• target the cccDNA

• affect HBc occupancy on both the cccDNA and cellular promoters

NT

1µM HAP

Palumbo (unpublished)

CpAMs

More assembly

Products are normal.ish

more free dimers

More assembly

Larger products

Different CpAMs – Different Results ?

Adapted from Zlotnick, ICAR 2014

18

CpAMs Conclusions

● HAP12 “early treatment” inhibits cccDNA accumulation and

HBV replication

[direct effect on cccDNA or NUC-like effect on capsid recycling ?]

● HAP12 treatment affects significantly an existing cccDNA pool

[experiments in HepG2-NTCP stable infection needed]

● HAP12 treatment apparently does not change HBc nuclear

levels

[how it works ?]

● HAP12 treatment effect on cccDNA transcription “possible”

but not “proven”

Lucifora et al. Science 343, 1221-8, 2014

- Interferon-α and lymphotoxin-β-receptor

activation up-regulated APOBEC3A and 3B

cytidine-deaminases, respectively, in HBV-

infected cells, primary hepatocytes and

human liver-needle biopsies.

- HBV-core protein mediates the interaction

with nuclear cccDNA resulting in cytidine-

deamination, apurinic/apyrimidinic site

formation and finally cccDNA degradation

19

Lucifora et al. Science 343, 1221-8, 2014

- Interferon-α and lymphotoxin-β-receptor

activation up-regulated APOBEC3A and 3B

cytidine-deaminases, respectively, in HBV-

infected cells, primary hepatocytes and

human liver-needle biopsies.

- HBV-core protein mediates the interaction

with nuclear cccDNA resulting in cytidine-

deamination, apurinic/apyrimidinic site

formation and finally cccDNA degradation

Targeted gene disruption strategies

Recognized challenge: delivery vehicle

� Zinc Finger

� Talen

� CRISP / Cas

20

Targeted gene disruption strategies

Recognized challenge: delivery vehicle

� Zinc Finger

� Talen

� CRISP / Cas

� TALENs treatment

reduces cccDNA level in

vitro and and HBV pgRNA

in vivo

Chen, Mol Therapies, 2014

� Inhibition of HBV replicazion with ZNF in Hep AD38 cells Weber, Plos1, 2014

Conclusions

● Still striving for finite therapy (“functional cure”)

�. “where the old and the new ways meet”;

● In theory, the ideal goal of antiviral therapy for CHB

would be complete HBV elimination including complete

eradication of cccDNA from infected hepatocytes

.� “a new beginning” � and keep open mind and acombinatorial attitude (HAPs+NUCs; HAPs+Mycludex �.)

● Lack of adequate in vitro replication models to study

cccDNA

�. “one model does not fit all �. yet”

21

Future directions: target & drug discovery to cure HBV infection

Immune modulation

• Toll-like receptors

agonists, Gilead,

Roche

• Anti-PD-1 mAb,

BMS, Merck

• Vaccine therapy

Transgene, Gilead,

Roche Innovio,

Medimmune, ITS

Development stage: preclinical, clinical

Zoulim F, et al. Antiviral Res 2012;96(2):256–9; HBF Drug Watch, Available at:

http://www.hepb.org/professionals/hbf_drug_watch.htm.

HBx

Endosome

rcDNA

cccDNA

Polymerase

pgRNA

Core

Surface

proteins

Entry inhibitors

• Lipopeptides, e.g.

Myrcludex-B

Targeting

cccDNA

Inhibition of nucleocapsid assembly,

Novira, Assemblyparm, Gilead, Janssen

Polymerase inhibitors

• Nucleoside

analogues, e.g.

Gilead, BMS

• Non-nucleoside,

e.g. LB80380

Inhibitors of HBsAg

release, ReplicorRNA interference,

Arrowhead, Tekmira,

Alnylam, GSK

What Might HBV Cure Will Look Like?

Potent NA

cccDNA

Inhibitor

Immune

Activator

HBV

Antigen

Inhibitor

⊕

⊕

⊕

to prevent viral spread and cccDNA re-amplification

safe and selective agent to reduce or silence cccDNA

agent(s) to activate specific antiviral immune

responses or relieve repression/exhaustion of the

immune system

agent(s) to block/inhibit the HBV life-cycle [entry,

cell-spread, capsid assembly, HBx, HBeAg, HBsAg]

Modified from S. Locarnini 6.2014

22

Laura Belloni Natalia Pediconi

Francesca Guerrieri Cecilia Scisciani

Aurora Palumbo Ludovica Calvo

Letizia Cimino Barbara Testoni

Rossana DeIaco Valeria Schinzari

Massimo Levrero

Collaborations:

Dept. of Internal Medicine - University of Messina

Giovanni Raimondo

Teresa Pollicino

INSERM U761 -Lyon

Fabien Zoulim

Barbara Testoni

University Medical Hospital Hamburg

Jorg Petersen

Maura Dandri

TUM - Helmholtz Zentrum München

Ulrike Protzer

Julie Lucifora

Laboratory of Gene Expression

With the support of Fondazione

Andrea

Cesalpino

Anna TramontanoAndrea Sbardellati,

Daniel D’Andrea

Francesco Cicconardi

Collaborations at Sapienza:

Dept of Molecular & Cellular Biochemistry

Indiana University

Adam Zlotnick

Antonello MaiDaniela Secci

Dante Rotili

Sergio Valente

Dept of Molecular Virology – Heidelberg Univ Hospital

Jessika Sonnabend

Stephan Urban

Assembly Pharma

Uri Lopatin