Hepatitis B: New TreatmentTargets and Possible Cure

Fabien Zoulim

Hepatology Department, Hospices Civils de Lyon

INSERM U1052, Cancer Research Center of Lyon

Lyon University, France

Why a need for new antiviral targets for hepatitis B ?

Current antivirals achieve viral suppression in the majority of patients (in western countries)

Issues with antiviral drug resistance in developing countries (use of low barrier to resistance antivirals)

The cure rate (cccDNA / HBsAg loss) remains very low

Life-long therapy is needed in the majority of cases

Treatment with finite duration if:

� cccDNA control or loss

� HBsAg loss

HBsAg clearance is associated with a lower risk of HCC development

Zoulim, Antiviral Research 2012

Definition of HBV cure

Virologic definition

- Functional cure

- Situation where antiviral therapy could be stopped

with a minimal risk of viral reactivation

- HBsAg loss with anti-HBsAb seroconversion

- cccDNA inactivation and/or control by host

mechanisms

- Complete cure

- HBsAg clearance and cccDNA eradication

Clinical definition

- Functional cure associated with a regression in the

risk of progression of fibrosis and HCCZeisel, Lucifora et al, Gut 2015

Results of current antivirals

Zoulim & Locarnini, Gastroenterology 2009; Zoulim Antiviral Research 2012; Mico et al J Hepatol 2013; Lucifora et al Science 2014

NK

cells

Innate responses

cells

CD8

+

cells

B

cells

cells

CD4

+

cells

Adaptive immune

responses

Nucleos(t)ide analogues

Interferon

alphaNTCP

Mode of action of antivirals for CHB

Current treatments: virus suppression and sustained disease control

Entecavir1,2 Tenofovir3 PEG-IFN α-2a4,5

HBeAg positive n = 354 n = 176 n = 271

HBV DNA undetectable 67% 76% 25%a

HBeAg seroconversion 21% 21% 27%

ALT normalisation 68% 68% 39%

HBsAg loss 2% 3.2% 2.9%b

HBeAg negative n = 325 n = 250 n = 177

HBV DNA undetectable 90% 93% 63%a

ALT normalisation 78% 76% 38%

HBsAg loss 0.3% 0% 0.6%b

1. Chang T-T, et al. N Engl J Med 2006;354:1001–10.

2. Lai C-L, et al. N Engl J Med 2006;354:1011–20.

3. Marcellin P, et al. N Engl J Med 2008;359:2442–55.

4. Lau GKK, et al. N Engl J Med 2005;352:2682–95.

5. Marcellin P, et al. N Engl J Med 2004;351:1206–17.

Results at 48 weeks a HBV DNA < 400 copies/mL; b At 72 weeks

Persistence of intrahepatic viral DNA synthesis during Tenofovir therapy

(HIV-HBV cohort)

Boyd et al, in revision

New round of infection and/or replenishment of the cccDNA pool occur

despite « viral suppression »

NUC Therapy

HB

V D

NA

ch

an

ge f

rom

baselin

e (

log

10

c/m

L)

0.0

-1.0

-2.0

-3.0

-4.0

+1.0

Time

Long-term therapy is required to maintain viral suppression

HBsAg

HBVDNA

cccDNA

SERUM

LIVER

New treatment concepts for a functional cure of HBV infection

Antiviral

s

Therapy0.0

-1.0

-2.0

-3.0

-4.0

+1.0

Time

HBsAg

HBVDNA

cccDNA

Immune

restoration

SERUM

LIVER Decay or epigenetic control

0 4 8 12 16 20 24

0

2

4

6

8

10

Stopping TDF therapy after long-term viral suppressionHigh rates of viral replapse & ALT elevations3 patients with HBsAg loss out of 41

24-week TFFU Completers (n=41)

HBeAg positive (n=4)

HB

V D

NA

(L

og10

IU/m

L)

HBeAg negative (n=37)

Follow-up Week

Buti et al AASLD 2015

AL

T,

Mu

ltip

le o

f U

LN

Follow-up Week

Characterisation of the immune profile in Chronic Hepatitis B, with CyTOF, to identify biomarkers of

immune control following NUC therapy discontinuationExpanded global CD4+ T cell memory populations in ‘non-flare’ vs flare patients

CD4+ T cells CD127+ (IL7R) CD4+ T cells

Non flare

Flare

CD

4+

T c

ells

(%)

0

20

Naive

*80

40

60

TCM TEM TEMRA

*

CD

127 +

CD

4+

T c

ells

(%)

20

40

Naive

*100

60

80

TCM TEM TEMRA

HBV polymerase specific T cell responses on NUC therapy are predictive of hepatic flares with

NUC discontinuationOn NUC therapy NUC discontinuation

0

100

X

300

200

Core Env Pol

*250

150

50

*** *

SF

C/1

05

cells

0

100

X

1000

200

300

Core Env Pol

** *

� Patients with detectable frequencies of circulating HBV-specific T cells controlled

HBV replication / no hepatic flare following NUC discontinuation

SF

C/1

05

cells

Gill US, et al. AASLD 2015, San Francisco. #167

Improvement of existing drugsExample of TAF for tenofovir

N

N

N

N

NH2

OP

O

OO

O

O

O

OO

O

N

N

N

N

NH2

OP

O

HOOH

N

N

N

N

NH2

OP

O

NH O

O

O

Tenofovir

Disoproxil FumarateTenofovir

Tenofovir

Alafenamide

LYMPHOID CELLS/

HEPATOCYTESPLASMAGUT

TFV

TFV

TFV-MP

TFV-DP

TDF/TFVTDF

TFV TDF TAF

TAF

Cathepsin A

CES1

TAF TAF

♦ Improved stability in

plasma:

– Enhanced delivery of

active form (TFV-DP) to

hepatocytes

– Lower doses are used;

systemic exposures of

TFV reduced

Agarwal K et al. AASLD 2013, Poster # 973

Murakami E et al. HepDART 2013, Abstract 104CES1 = carboxylesterase 1; DP= di-phosphate; MP= mono-phosphate.

Phase 1B results: HBV DNA kinetics on 28 days

♦No differences in viral declines over range of TAF 8 mg to 120 mg

♦Viral suppression over 4 weeks with TAF was similar to TDF

Study Day

TAF 8 mg (n= 10)

TAF 40 mg (n=10)

TAF 120 mg (n=11)

TDF 300 mg (n=10)

TAF 25 mg (n=10)

Study Day

Me

an

HB

V D

NA

(L

og10

IU/m

L)

Agarwal K et al. AASLD 2013, Poster # 973

GS-US-320-0101 - Clinicaltrials.gov NCT01671787

New targets for HBV therapy

Vaccine therapy

Check-point

inhibitors

TLR agonistsBlockade

of immune-

suppressive

cytokines

Chimeric antigen

Receptors (CAR)

Antiviral cytokines

Entry inhibitors

Core modulators

Targeting cccDNA

Polymerase

inhibitors

RNA

interference

Egress Inhibitors

Core modulators

Targeting

HBx

Testoni and Zoulim, Hepatology 2015

Li et al, elife 2012

Urban et al, Gastroenterology 2014

Model for HBV entry in hepatocytes and

development of entry inhibitors

Myrcludex B, a peptidic inhibitor of

NTCP-mediated entry of HBV and HDV

• Myrcludex B is an HBV preS-derived lipopetide binding sodium-taurocholate co-transporting polypeptide

(NTCP).

• Myrcludex B inhibits HBV and HDV receptor function of NTCP in vitro and in animal models (IC50∼ 80 pM).

• Myrcludex B inhibits NTCP-mediated bile salt uptake into hepatocytes (IC50∼ 100 nM).

• Myrcludex B specifically targets liver hepatocytes after subcutanous administration.

• Myrcludex B showed safety in Phase I clinical trials.

⇒ (A) Proof of safety and efficacy in chronically HBV infected individuals.

⇒ (B) Proof of safety and efficacy in chronically HBV/HDV co-infected individuals.

AASLD 2014 Boston, SIG HBV, Nov. 10thS Urban Heidelberg U & MyrGmbH

HBV Serum DNA-levels decline

during Myrcludex B treatment

0,5 mg/d 1 mg/d 2 mg/d

5 mg/d 10 mg/d ETV 0,5 mg/d

⇒ HBV DNA levels decline significantly during Myrcludex B treatment in all groups.

⇒ Pronounced effects by > 1log in 6/8 patients were observed in the 10 mg dosing group.

⇒ 7/40 showed > 1log HBV reduction in lower dosing groups.

AASLD 2014 Boston, SIG HBV, Nov. 10thS Urban Heidelberg U & MyrGmbH

Lucifora et al, Science 2014

Zoulim, et al, Clin

Gastroenterol Hepatol 2013

Belloni et al, JCI 2012

Koeniger etal, PNAS 2014

Tropberger et al, PNAS 2015

Hepatocyte turn-over

cccDNA silencing

cccDNA

degradation

cccDNA

formation

Targeting cccDNA

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

Model for cccDNA degradation

IFNalpha /Lymphotoxin beta can induce APOBEC3A/B dependent

degradation of HBV cccDNA

Similar observation with IFNγ and TNFα – Xia et al, Gastroenterology 2015

Epigenetics of covalently closed circular (ccc)DNA

Regulation by viral proteins (HBc and HBx)

Michael Nassal Gut doi:10.1136/gutjnl-2015-309809

Pollicino et al. Gastroenteroplogy 2006

Levrero et al. J Hepatol, 2009

Lucifora et al, J Hepatol 2012

Belloni et al, PNAS 20O9

Belloni et al, J Clin Invest 2012

Strubin et al, HBV conference 2015

Silencing

Interferon alpha,

Capsid inhibitors,

Epigenome modifyers

Challenges in targeting cccDNA

• cccDNA formation: involves nuclear enzyme / DNA repair machinery

• cccDNA degradation: is the whole pool of cccDNA susceptible to

degradation ? will all infected cells be susceptible ?

• cccDNA damage: CRISPR/cas9 technologies and others. Issues

with delivery ?

• cccDNA silencing: targeting virus-specific mechanisms to avoid

safety issues

• Hepatocyte turn-over: may trigger the clonal selection of

hepatocytes in the context of an oncogenic virus

• Small molecules needed !

Targeting the HBV capsid

Targeting HBV nucleocapsids

Deres et al, Science 2003

Klumpp et al, PNAS 2015

Heteroaryldihydropyrimidines

Destabilization of nucleocapsids

Phenylpropenamide derivatives

Prevent pgRNA encapsidation

Antimicrob Agents Chemother. 2002.

Novel classes of capsid inhibitors based on the 3D structure of HBc

Novira, Assembly Biosciences, Janssen, Roche, and others

Phase 1 studies with Novira completed

Effective reduction of serum HBV DNA by NVR 3-778(Novira)

All treatments reduced HBV DNA

ETV and NVR 3-778

monotherapies show similar

antiviral activity (p>0.05)

PEG-IFN + NVR 3-778

combination provides

highest antiviral efficacy

5/5 mice achieve serum HBV

DNA BLQ

25

Lam A, et al. AASLD 2015, San Francisco. #33

Phase 1b clinical trial: NVR 3-778 reduces serum HBV DNA and RNA (Novira)

Serum HBV DNA: mean 1.7 log reduction (600 mg BID)

Serum HBV RNA: mean 0.86 log reduction (600 mg BID)

26

Cohort I: 600 mg BID

mean serum HBV RNA change from baseline

Yuen M-F, et al. AASLD 2015, San Francisco. #LB-10

mRNA

mRNAdegradation

dsRNAdicer

Cleavage

Strand separation

Complementary pairing

Cleavage

(A)n

(A)n

Natural Process of RNAi

Synthetic siRNA

Targeted Gene

Silencing

RISC

RNA Interference

Reductions in cccDNA under NUC and ARC-520 therapy in

chimpanzees with chronic hepatitis B virus infection implicate

integrated DNA in maintaining circulating HBsAg

Wooddell CI, et al. AASLD 2015, San Francisco.

#32

Total Liver HBV DNA cccDNA

Pre-

study

After NUC

lead-in

Pre-

study

After NUC

lead-in

108

107

106

105

104

103

102

HBeAg

+

HBeAg-

LLOQ

Liv

er

HB

V D

NA

(G

E/µ

g)

� ARC-520 reduced total liver DNA and

cccDNA beyond levels achieved in

HBeAg-pos NUC treatment during lead-in

� ARC-520 and NOT NUC reduced

intrahepatic HBV RNA and antigens

109

108

107

106

105

104

HBeAg+ HBeAg-

HB

V m

RN

A in liv

er

(copy/µ

g)

Total HBV RNAPre-core/Core RNA1000

Weig

hte

d R

NA

reads

HBeAg+ HBeAg-

100

10

1

Total HBV RNAPre-core/Core RNA

50%

5%

49%

5%

ARC-520 produces deep and durable knockdown of viral antigens and DNA in a phase II study in patients with chronic hepatitis B

Yuen M-F, et al. AASLD 2015, San Francisco. #LB-9

Small dose-related reduction in HBsAg

Maximum effective dose not reached

HBV DNA results pending in ETV naïve patients

-1.4

-1.2

-1

-0.8

-0.6

-0.4

-0.2

0

0.2

-10.00 10.00 30.00 50.00 70.00 90.00

Log

Re

du

ctio

n i

n v

ira

l a

nti

ge

n

Days

HBsAg PBO HBsAg ARC-520 HBeAg PBO

HBeAg ARC-520 HBcrAg PBO HBcrAg ARC-520

HBsAg reduction in ETV naïve

patients with a single 4 mg dose

(cohort 7)

HBV antigen reduction in ETV

experienced HBeAg-positive

patients with a single 4 mg dose

(cohort 5)

Direct antiviral effect lasted up to 57 days after

a single dose of ARC-520, delayed response

duration >85 days

Restoration of antiviral immunity

Bertoletti A, Gehring AJ (2013) Immune Therapeutic Strategies in Chronic Hepatitis B Virus Infection: Virus or Inflammation

Control?. PLoS Pathog 9(12): e1003784. doi:10.1371/journal.ppat.1003784

http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003784

Antiviral activity of a TLR7 agonist

in HBV infected chimpanzees

Lanford et al, Gastroenterology 2013

The Oral Toll-Like Receptor-7 Agonist GS-9620 in

Patients with Chronic Hepatitis B Virus Infection

Gane et al, Journal of Hepatology, 2015

Effective T-cells control virus Exhausted T-cells lose control of

virus

CD8 T cells

Infected hepatocytes Infected hepatocytes

INF-γ

TNF-α

IL-2

Granzyme

Perforin

Can an effective antiviral T-cell response be recovered?

Patients who have resolved HBV Patients with chronic HBV

Restoration of defective T-cell immune control

Figure adapted from Nebbia G, et al. Q J Med 2012;105:109–13 and Freeman G, et.al. J Exp Med 2006;203(10):2223–7.

Effective T-cells control virus Exhausted T-cells lose control of

virus

CD8 T cells

Infected hepatocytes Infected hepatocytes

INF-γ

TNF-α

IL-2

Granzyme

Perforin

Prolonged nucleotide therapy in a subset of patients1

Patients who have resolved HBV Patients with chronic HBV

Restoration of defective T-cell immune control

1. Boni C, et al. Gastroenterology 2012;143:963–73

Figure adapted from Nebbia G, et al. Q J Med 2012;105:109–13 and Freeman G, et.al. J Exp Med 2006;203(10):2223–7.

Effective T-cells control virus Exhausted T-cells lose control of

virus

CD8 T cells

Infected hepatocytes Infected hepatocytes

INF-γ

TNF-α

IL-2

Granzyme

Perforin

Specific immunomodulation of existing T-cells e.g. PD-1 blockade1,2

Patients who have resolved HBV Patients with chronic HBV

Restoration of defective T-cell immune control

1. Fisicaro P, et al. Gastroenterology 2010;138:682–93. 2. Fisicaro P, et al. Gastroenterology 2012;143:1576–85

Figure adapted from Nebbia G, et al. Q J Med 2012;105:109–13 and Freeman G, et.al. J Exp Med 2006;203(10):2223–7.

Triple combination therapy of ETV treatment, DNA

vaccination and in vivo PD-L1 blockade in WHV infected

woodchucks

Liu J, Zhang E, Ma Z, Wu W, et al. (2014) Enhancing Virus-Specific Immunity In Vivo by Combining Therapeutic Vaccination and

PD-L1 Blockade in Chronic Hepadnaviral Infection. PLoS Pathog 10(1): e1003856. doi:10.1371/journal.ppat.1003856

In vivo PD-L1 blockade synergizes with therapeutic

vaccination to control WHV replication

Liu J, Zhang E, Ma Z, Wu W, et al. (2014) Enhancing Virus-Specific Immunity In Vivo by Combining Therapeutic Vaccination and

PD-L1 Blockade in Chronic Hepadnaviral Infection. PLoS Pathog 10(1): e1003856. doi:10.1371/journal.ppat.1003856

Antiviral

s

Therapy

HB

V D

NA

ch

an

ge

fro

m b

as

elin

e (

log

10

c/m

L)

0.0

-1.0

-2.0

-3.0

-4.0

+1.0

Time

New treatment concepts for a functional cure of HBV infection

HBsAg

HBVDNA

cccDNA

Immune

restoration

Target & drug discovery to cure HBV infection

Immune modulation

• Toll-like receptors

agonists, Gilead,

Roche

• PD1 blockade,

BMS, Merck etc.

• Vaccine therapy

Transgene, Gilead,

Roche Innovio,

Medimmune, ITS

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, AssemblyBiosc,

Gilead, Janssen, Roche

Polymerase

inhibitors

• Nucleoside

analogues, e.g.

Gilead, BMS

• Non-nucleoside,

e.g. LB80380

• RNAseH inhibitors

Targeting HBsAg

Mab, Gilead

Release, Replicor

RNA interference,

Arrowhead, Arbutus,

Alnylam, GSK

Cyclophilin

inhibitors

Arbutus

Can we cure the liver disease ?

• Mechanisms of HBV-induced HCC involve several

factors: inflammation, fibrosis, chromosomal integration,

HBx etc.

• Improvement of liver inflammation & fibrosis with long-

term NUC

• HCC not always seen on a background of cirrhosis

• Clonal expansion of hepatocytes not supporting HBV

replication occurs even before cirrhosis

• NUCs decrease, but don’t eliminate, the risk of HCC

• Early treatment intervention & better antivirals needed

Zoulim & Mason, Gut 2012; 61 : 333-336

Acknowledgements

Hepatology Unit INSERM U1052 Collaborations

David Durantel

Barbara Testoni

Julie Lucifora

Malika Ait-Goughoulte

Souphalone Luangsay

Marion Gruffaz

Nathalie Isorce

Fanny Lebossé

Maelenn Fournier

Maud Michelet

Judith FresquetLabEx

C. Caux, Lyon CRCL

FL. Cosset, Lyon CIRI

K. Lacombe, Paris

M. Levrero, Rome/Lyon

JP Quivy, Institut Curie

IHU

Maelle Locatelli

Valentina d’Arienza

Pascal Jalaguier

Thomas Lahlali

Dulce Alafaiate

Lucyna Cova

Romain Parent

Anna Salvetti

Birke Bartosch

Eve Pecheur

Boyan Grigorov

Christophe Combet

Third ANRS “HBV cure” Workshop HBV pathobiology and target discovery

Scientific coordination: Fabien Zoulim

Tuesday, May 31st, 2016

Union internationale des chemins de fer (UIC) 16, rue Jean Rey - 75015 PARIS

HBV cure 2014: Zeisel, M. B. et al. Towards an HBV cure: state-of-the-art and unresolved

questions-report of the ANRS workshop on HBV cure. Gut, doi:10.1136/gutjnl-2014-308943 (2015).

HBV cure 2015: http://www.anrs-hbvcure2015.com/

15/12/2015 43

348 HBeAg(+) and HBeAg(-) CHB patients from phase 3 studies who enrolled in a

long-term rollover study were evaluated for long-term liver histology outcomes51% of

patients had regression of fibrosis, including 71/96 patients with cirrhosis

(Ishak score ≥ 5) at phase 3 study baseline

Regression of fibrosis and cirrhosis during long-term Tenofovir therapy

Marcellin P, et al. Lancet 2013;381:468–75.

Ishak Score

0

20

40

60

80

100

Baseline Year 1 Year 5

6

5

4

3

2

1

0

Japanese cohorts: Entecavir reduced HCC incidence, compared with controls

PS-matched cohort multivariate cox regression analysis:*

HR 0.37 (95% CI 0.15–0.91) p = 0.030

*Adjusted for age, sex, alcohol, smoking, cirrhosis, HBV genotype, HBeAg status, HBV-DNA, ALT, albumin,

γGTP, total bilirubin and platelet count.

Cum

ula

tive H

CC

rate

s (

%)

Log-rank test: p<0.001

Treatment duration (years)

0

10

20

30

7.2%

13.7%

3.7%

1.2%

0 1 3 5 72 4 6

No. at risk

ETV

Control316

316

316

316

264

277

185

246

101

223

44

200

2

187

2

170

Adapted from Hosaka T, et al. Hepatology 2013 [Epub ahead of print]. doi: 10.1002/hep.26180.

HR, hazard ratio; PS, propensity score

Control

ETV

HCC occurence in HBsAg loss patients

Simonetti et al, Hepatology 2010

The incidence of HCC after clearance of HBsAg was 36.8 per 100,000

person-year (95% CI 13.5-80.0) which was significantly lower than the rate

in those who remained HBsAg-positive (195.7 cases per 100,000 person-

years of follow-up [95% CI 141.1-264.5; P < 0.001])

Liver Damage and HBV infection

HCC not always seen on a background of cirrhosis

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

Zoulim & Mason, Gut 2012; 61 : 333-336

Mechanisms of HBV-related hepatocarcinogenesis

1. Lupberger J, Hildt E. World J Gastroenterol. 2007;13:74–81; 2. But DYK, et al. World J Gastroenterol. 2008;14:1652–6; 3. Neuvert C, et al. J

Hepatol. 2010;52:594–604; 4. Tan YJ. World J Gastroenterol. 2011;17:4853–7; 5. Wong DKH, et al. J Hepatol. 2006;45:553–9;

6. Friedman SL. Gastroenterol. 2008;134:1655–69.

Chronic liver

inflammation,

hepatocyte injury,

proliferating fibroblasts,

fibrosis/cirrhosis

Hepatocellular carcinoma

Chronic HBV Infection

Viral replication

Host DNA mutations

due to high hepatocyte

turnover, cytokine and

growth factor release

Chromosomal

integrationHBV protein X

Chromosomal

instability, altered gene

expression or function

Interference with cellular

transcription and

signaling pathways

Mason W S et al. Proc. Natl. Acad. Sci. USA 2005; J Virol 2009 & 2010

Analysis of liver tissue for variability in levels of HBV infection - Chimpanzee

Analysis of liver fragments for clonal expansion of cells by serial dilution - woodchucks

Similar observations in HBV infected

chimpanzees and patients

Clonal expansion of hepatocytes not supporting

active viral replication

� 48 weeks of ADV resulted in significant reductions in :

serum HBV DNA > total intrahepatic HBV DNA > cccDNA

> 14 years of therapy to clear completely viral cccDNA

Werle et al, Gastroenterology 2004

� 0.8 log10 (84%) decline in cccDNA, not paralleled by a similar decline in the

number of HBcAg+ cells

� Suggests cccDNA depleted primarily by non-cytopathic mechanisms or new

rounds of hepatocyte infection occurred during therapy

Baseline Week 48

Werle et al, Gastroenterology 2004

Koeniger et al, PNAS 2014

cccDNA formation: identification of TDP2

TDP2: Tyrosyl DNA Phosphodiesterase

Cortes Ledesma et al, Nature 2009

53

Targeting Hepatitis B Virus With CRISPR/Cas9

Mol Ther Nucleic Acids. 2014; Seeger et al

Induction of deletions in cccDNA

Decreased number of cells expressing viral antigens

Histone modifications, Pol2 binding and transcriptional profile of cccDNA

Tropberger et al, PNAS 2015

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

Dandri et al, Hepatology 2010