Hépatite B.pdf

Natural history of hepatitis B Acute infection

Chronic infection: 400 million carriers !

Immune tolerance

Chronic hepatitis Inactive carrier

Resolved infection 5% neonates 90% adults

Wild type virus HBeAg+ Pre-core mutant HBeAg-

Cirrhosis

Hepatocellular carcinoma

Reactivation

30-50 years Seeger, Zoulim, Mason; Fields Virology; 2007

Déclaration obligatoire de l’hépatite B en France :

résultats des 12 premiers mois de notification

Denise Antona, E Delarocque-Astagneau, D Lévy-Bruhl département des maladies infectieuses

Incidence of acute hepatitis B in France Sentinel networks 1991-1996 et Lyon (COURLY) 1983-1997

0

5

10

15

20

25

1983 1985 1987 1989 1991 1993 1995 1997

Tau

x /100 0

00

COURLY Réseau "Sentinelles"

Circuit de l’information Biologiste

InVS

MISP de DDASS du département

d’exercice

Médecin prescripteur

Fiche de notification autocopiante à 4 feuillets Partie 1 : code d’anonymat irréversible, caractéristiques du patient Partie 2 : information biologique Parties 3-4-5 : information clinique et épidémiologique Parties 6-7 : identification du médecin prescripteur et du biologiste déclarants

Feuillet 1 : parties 1-2 et 6-7 renseignées

Feuillets 2 et 3 à compléter

Feuillet 2 : parties 3-4-5 complétées

Feuillets 1 et 2 complétés et validés

Relance

Results

Age distribution: comparison of the different periods 1991-94 versus 03/2003 - 02/2004

0%

10%

20%

30%

40%

0-9 ans 10-19 ans 20-29 ans 30-39 ans 40-49 ans 50-59 ans

! 60 ans

Classes d'âge

% de cas

Réseau "Sentinelles" Déclarations obligatoires

years 1991- 94 n= 151

March 03- February 04 n= 158

Risk exposure within 6 months preceding the acute case Source : obligatory declaration 2003-04

Hépatites virales B: épidémiologie

- Vaccin mais 400 millions de porteurs chroniques dans le monde -  280 000 porteurs chroniques en France (INVS) -  45% ignorent leur statut -  1 300 décès par an en France -  60 000 avec hépatite chronique active -  Environ 15 000 patients traités

•  FAMILLE : Hepadnaviridae, seul représentant humain

•VIRUS RESISTANT : - 7 jours dans l’environnement - pendant 5 mn à 100°C, 10 h à 60°C - à la congélation.

LE VIRUS DE L ’HEPATITE B

S small surface protein

M middle surface protein

L large surface protein

core capsid protein

HBeAg secreted e antigen

pol polymerase

HBx X protein (non-secreted)

v

v

v

sphere filament

Dane particle

HBeAg

HBsAg

The HBV genome

Tiollais, Nature 1985

The viral replication cycle

Zoulim & Locarnini, Gastroenterology 2009

ARN pg

ss DNA

RC DNA cccDNA

intégration

virion

virion 10%

90%

ds DNA

cccDNA illégitime

noyau

Réplication du génome viral. Implication pour la persistance virale et l’intégration du génome viral

Membrane cellulaire

Transgenic mice Humanized mice

Human Chimpanzee Gibbon baboons

Tupaïa

Woolley monkey

Ground squirrel

American woodchuck

Pekin Duck Grey Heron

The animal models of HBV infection

HIV (Ritonavir )

HCV (IFN- )

HBV (Lamivudine)

Plasma virus

Half-life 5.8 h 2.7 - 7.2 h 24 h Mean viral generation time

2.7 d 3.8 - 7.3 d 24.7 d

Daily turnover 95% 94% - 99.8% 50% Daily production (plasma)

1010 (1.1 - 12.7)*1011

1011

Total load 1.2*109 (3.8 - 5.6)*1010 2*1011 Infected cells

Half-life 1.6 d 2.4 - 4.9 d 10 - 100 d Mean lifespan 2.3 d 3.5 - 7.1 d 23.3 d Daily turnover 38% 13% - 25% 1% - 7%

(Tsiang et al. Hepatology 1999)

HBV replication and its role in HCC development

Wands, NEJM 2004

Role du VHB dans l’oncogénèse hépatique!

VHB!INFECTION CHRONIQUE!

CARCINOGENES!CO-FACTEURS!

REACTION INFLAMMATOIRE CHRONIQUE!REGENERATION HEPATIQUE!

MUTAGENESE INSERTIONNELE!TRANSACTIVATION DE GENES CELLULAIRES!

INTERACTIONS PROTEIQUES!INACTIVATION DE GENES SUPPRESSEURS DE TUMEUR!

CHC!

Immunopathology

The level of viral antigen presented by hepatocytes influences CD8 T-cell function

•  Hepatocyte antigen presentation was generally inefficient, and the quantity of

viral antigen strongly influenced CD8 T-cell antiviral function.

•  High levels of hepatitis B virus production induced robust IFN-gamma and TNF-

alpha production in virus-specific CD8 T cells,

•  while limiting amounts of viral antigen, both in hepatocyte-like cells and

naturally infected human hepatocytes, preferentially stimulated CD8 T-cell

degranulation. •  Virus-specific CD8 T-cell function is influenced by the quantity of virus

produced within hepatocytes Gehring AJ, et al . J Virol 2007;81:2940-9.

Wieland S et al, PNAS 2004

10 -3

10 -2

10 -1

10 0

10 1

10 2

10 3

10 4

10 -3

10 -2

10 -1

10 0

10 1

10 2

10 3

Werle et al, Gastroenterology 2004

Inactive HBV carrier ●  Not virologically inactive:

–  low levels of viremia –  episomal HBV DNA in the liver

LOW-REPLICATIVE STATE HIGH-REPLICATIVE STATE –  spontaneously

–  during immunosuppression

Low-replicative or latent infection Epigenetic control

Histones

CBP PCAF p300

CBP PCAF

p300 Sirt1

Sirt1 HDAC1 HDAC1

Histones

Pollicino et al., Gastroenterology 2006

Pollicino et al. Gastroenteroplogy 2006 Levrero et al. J Hepatol, 2009

HISTOIRE NATURELLE ET VIROLOGIE CLINIQUE

Seeger, Zoulim, Mason; Fields Virology; 2007

HEPATITE B AIGUE

Laboratory Diagnosis of Acute Hepatitis B

01002003004005006007008009001000

0 1 2 3 4 5 6 12 24 36 48 60

ALT

HBsAg

HBeAg

HBV DNA

Normal

Months After Exposure

ALT

and

HB

V D

NA

IU/L

and

mill

ion

copi

es/m

l

Symptoms

Anti-HBs Ab Anti-HBe Ab

IgM anti-HBc

Total anti-HBc

Seeger, Zoulim, Mason, Fields Virology 2007

HEPATITE B PROLONGEE

0

100

200

300

400

500

600

700

800

0 1 2 3 4 5 6 12 24 36 48 60

ALT

HBsAg

HBeAg

HBV DNA

Normal


ALT

and

HB

V D

NA

IU

/L o

r mill

ion

copi

es/m

l

Laboratory Diagnosis of Chronic Hepatitis B associated with wild type virus infection


ALT

`` HBsAg

HBeAg

HBV DNA

Normal


ALT

and

HB

V D

NA

IU/L

and

mill

ion

copi

es/m

l

Anti-HBe

Laboratory Diagnosis of Transition of Chronic Hepatitis B to The inactive Carrier State

0 100 200 300 400 500 600 700 800

0 1 2 3 4 5 6 12 24 36 48 60 72 80 92 104


050100150200250300350400450

0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48

ALT

HBsAg

HBV DNA

Normal ALT levels

Months

ALT

and

HB

V D

NA

IU/L

and

mill

ion

copi

es/m

l Anti-HBe HBeAg

Laboratory Diagnosis of HBeAg negative Chronic Hepatitis B


0,001

0,01

0,1

1

10

100

1000 ALAT ADN- VHB

Tolérance hép chronique p. inactif mt pré-core VHB occulte

9 log

8 log

7 log

6 log

5 log

4 log

3 log

2 log

1 log

Dynamic ranges of quantification of HBV DNA assays

Amplicor HBV Monitor v2.0 (Roche)

HBV Hybrid-Capture II (Digene)

Ultra-sensitive HBV Hybrid-Capture II

Versant HBV DNA 3.0 (bDNA, Siemens)

Cobas Taqman HBV (Roche)

Abbot Real-time HBV (Abbott)

Versant HBV DNA 1.0 (kPCR, Siemens)*

*in development

RealArt HBV LC PCR (Artus Biotech)

Formes cliniques

Pathophysiologic Cascade of Chronic HBV Infection

Adapted from: Lavanchy D. Journal of Viral Hepatitis, 2004, 11, 97–107. Chen JC, et al. JAMA. 2006;295:65-73. Iloeje U. H, et al. Gastroenterology. 2006;130:678-86.

Charge virale et incidence de la cirrhose

R.E.V.E.A.L. – HBV Study Année de suivi

Inci

denc

e cu

mul

ativ

e de

cir

rhos

e

.2

.1

0 1 2 3 4 5 6 7 8 9 10 11 12 13

0

.4

.3

P <0.001

n=3774 1.0 x 106 n=627 1.0-9.9x105 n=344 1.0-9.9x104 n=649 300-9.9x103 n=1210 <300 n=944

5.2% 6.3%

10.0%

23.0%

37.1%

Iloeje UH et al. Gastroenterology 2006; 130: 678-686

Survie chez les patients au stade cirrhose

1. Weissberg et al. Ann Intern Med. 1984;101:613. 2. De Jongh et al. Gastroenterology. 1992;103:1630.

1 32 4 50

20

40

60

100

80

Cirrhosis1

(n = 130)

Decompensated cirrhosis2

(n = 21) 14%

55%

Patie

nts

Surv

ivin

g, %

Years

0

AgHBeAg et risque de CHC

Yang et al. N Engl J Med. 2002;347:168-174.

Cum

ulat

ive

inci

denc

e (%

)

Year

HBsAg+ HBeAg+

HBsAg+, HBeAg -

HBsAg -, HBeAg -

62 10 0

4

6

8

12

10

2

0 4 8

•  11,893 Taiwanese men; 92,359 person-years follow-up

Charge virale et incidence du CHC

Chen et al; JAMA 2006

REVEAL-Incidence of HCC Increases with Increasing HBV DNA

Baseline Viral Level

Chen JC, et al. JAMA. 2006;295:65-73.

14.9%

12.2%

3.6% 1.4% 1.3%

0%

5%

10%

15%

20%

<300 >300 - 103

Baseline HBV DNA (copies/mL)

% c

umul

ativ

e in

cide

nce

of H

CC

> 103 - 104 >104 - 106 ≥106

High Baseline Serum HBV DNA Levels are Associated with Increased Risk of HCC Mortality

in HBsAg-Positive Patients

80%

84%

88%

92%

96%

100%

0 1 2 3 4 5 6 7 8 9 10 11 12

Survival time (Years)

Su

rviv

al d

istr

ibu

tio

n f

un

cti

on

HBV DNA Negative

p < 0.001 across viral categories

http://www.fccc.edu/docs/sci_report/Evans.pdf#search=%22haimen. Accessed 1/23/07. Chen G, et al. J Hepatology 2005; 42 (suppl 2):477A. Chen G, et al. Hepatology 2005; 40 (suppl 1):594A.

Relationship Between Persistent Viremia and HCC: Argument For Antiviral Therapy

•  Persistent replication associated with greater risk of HCC •  Decreased risk when viral replication declines

Chen, et al. JAMA 2006

Baseline HBV DNA, (copies/mL) < 104 ≥105 ≥105 ≥105

Follow-up HBVDNA, copies/mL --- < 104 104 to <105 ≥105

Adjusted RR (95% CI)

1.0 (ref)

3.6 (1.7-7.6)

6.9 (3.4-13.8)

9.1 (5.8-14.1)

P Value -- < 0.001 < 0.001 < .001

HC

C In

cide

nce

Rat

e Pe

r 100

,000

0

1473

5882

8730 10,108

2.0x103

4.0x103 6.0x103 8.0x103 1.0x104

1.2x104

Impact Clinique de la Variabilité du Génome Viral

World J Gastroenterol 2007; 13: 14-21

B6

D1

Zoulim et al J Viral Hepatitis 2006

Impact du génotype sur la séroconversion

1 Janssen, Lancet 2005; 2 Flink, Am J Gastro 2006

PEG-IFN a-2b

HBeAg Loss 1

0

10

20

30

40

50

A n=90

28%

47% 44%

25%

B n=23

C n=39

D n=103

Perc

enta

ge o

f pat

ient

s (%

)

HBV genotype

0

3

6

9

12

15

A n=90

5%

8%

0%

B n=23

C n=39

D n=103

18 15%

Perc

enta

ge o

f pat

ient

s (%

) 21

HBV genotype

PEG-IFN a-2b

HBsAg Loss 2

HBeAg and Precore Mutation G 1896A = stop codon, TAG

ATG ATG Core gene

HBeAg and Precore Mutation

ATG ATG Core gene

Main pre-c/core promoter mutations observed in vivo

GGGGGAGGAGATTAGGTTAAAGGTCTTTGTATTAGGAGGCTGTAGGCATAAATT

Basic core promoter LEF

HNF1 GGTTAATNATTA

HNF4 AGGTCA

Deletion 63-70 Insertion (RGTTAATYATTA) at 74/75

Mutation AGG to TCA and insertion TA at 65/66

WTRTTKRY

Insertion (TTG) at 66/67

HNF3

05001000150020002500

temps

020406080100

temps

Outcome of Chronic Anti-HBe Positive Hepatitis B

0

100

200

300

4000

100

200

300

400

0

100

200

300

400

Augmentation de prévalence des hépatites chroniques avec AgHBe négatif en France

HBeAg(+)HBeAg(-)

48% N=119

62% N=164

Zoulim et al, J Viral Hepatitis 2006

Lamivir cohort, Zoulim et al, J Viral Hepatitis 2006



0

5

10

15

20

25

0 1 2 5 9 12 13 16months1

10

100

1000

10000

100000

1000000

10000000

100000000

1000000000

10000000000

ALT

pre-S1

bDNA

PCR

Pichoud et al, J hepatol 2000

COOH

137 149

107

99 NH2

S - S

S - S S - S

S- S

S-S

138

139 147

Tiollais P. et al., Nature 1985. Torresi J., J. Clin Virol 2002; Dryden KA. et al., Mol Cell 2006

« a » determinant

HBs Ag

« a » determinant induces the synthesis of anti-HBs neutralizing antibodies

sG145R

sP120T

sD144H/A/E

PreS1 PreS2

S Pol

Pré-C

C

Brin(+) 2,4kb Brin(-) 3,2kb

X

TATAA U5-like

DR1

DR2 Enh1 Enh2

0/3221

SHBs (S) MHBs (preS2+S)

LHBs (preS2+preS2+S)

Presence of HBV DNA in the liver (± serum) of

individuals testing HBsAg negative by currently

available assays

Occult HBV Infection (OBI)

Raimondo et al, J Hepatol 2008

How to Detect Occult HBV Infection

Currently there is no standardized

diagnostic assay for occult HBV infection

Reported Prevalence of Occult HBV Infection in HIV Positive Patients

Study Country N° of patients

Occult HBV

N° (%) Methods

Hofer, 1998 Switzerland 57 51 (89%) “nested” PCR (serial evaluation)

Torres-Baranda, 2006 Mexico 35 7 (20%) “nested” PCR

Filippini, 2006 Italy 86 17 (20%) single step PCR

Mphahlele, 2006 South Africa 140 31 (22.%) “nested” PCR

Pogany, 2005 Netherlands 93 4 (4%) single step PCR

Neau, 2005 France 160 1 (0.6%)

Santos, 2003 Brazil 101 16 (16%) single step PCR

Wagner, 2004 France 30 11 (37%) “nested” PCR

Goncales, 2003 Brazil 159 8 (5%) “nested” PCR

Nunez, 2002 Spain 85 0 Cobas Amplicor HBV Monitor (Roche)

Piroth, 2000 France 37 13 (35%) single step PCR

Raffa, 2007 Italy “nested” PCR (liver)

Cobas Amplicor HBV Monitor (Roche)

101 42 (41%)

Raimondo et al, J Hepaol 2007, modified

OBI

Cause(s) for the failure of HBsAg detection

Suppression of HBV replication and

gene expression

Infection by S gene Variants

“false” OBI

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

H3/H4

AcH3/AcH4 Core

Methylated H3/H4

and DNA

Sirt1 Sirt1

HDAC1 HDAC1

Histones TF

MeCP2

TF TF TF

HP1 Suv39

TF TF

Pollicino et al., unpublished

Occult HBV infection

HBV cccDNA Integrated HBV DNA

HBV mutants Epigenetic control

HBV replication

Immune surveillance Viral co-infections

OBI

Seropositive Seronegative

HBsAg lost during CH

HBsAg lost after AH

Progressive antibody disappearence

Primary occult

Schematic representation of HBV serum marker profile in OBI and “false” OBI

„false“ OBI

S gene escape mutants

HBV DNA levels comparable to overt infection

HBV DNA levels < 200 UI/ml

Occult hepatitis B Torbenson M. & Thomas D.L., Lancet Inf Dis, 2002

High prevalence

ROLE in

HCC

Diagnostic

Tools ?

Worsen HCV infection ?

Co-infections ? Therapy?

To be improved

Specific treatments ?

Not fully understood ?

Occult HBV infections: unresolved issues

HBeAg(+) HBeAg(-) / anti-HBe(+)

ALAT

HBV DNA

Minimal CH Moderate to severe CH Moderate to severe CH Remission

Cirrhosis

Immunotolerant phase

Immuno-active phase

Inactive phase Low replication

Reactivation phase

Cirrhosis

109-1012 IU/mL >2000-<109 IU/mL <2000 IU/mL >2000 IU/mL

Inactive cirrhosis

Adapted from Fattovich G. Sem Liver Dis. 2003

Treatment indicated Treatment indicated

HBsAg Occult infection

Endpoints of therapy

Persistence of high viral load is associated with a significant risk of progression of the liver disease and of HCC

Aim of antiviral therapy:

HBV DNA < 10-15 IU/mL by real-time PCR assays

No replication =

No resistance

Viral suppression

Histological and clinical improvement

Chen CJ, et al. JAMA 2006. Iloeje UH, et al. Gastroenterology 2006. Chen C, et al. Am J Gastroenterol 2006. Zoulim & Perrillo J Hepatol 2008. Zoulim & Locarnini Gastroenterology 2009

Antivirals approved for hepatitis B

*Currently approved for HIV **development on hold

Drug Type Approved Phase 3 Phase 2 Nucleoside analogs •  Lamivudine*

•  Entecavir •  Telbivudine

•  Emtricitabine* •  Clevudine**

•  Elvucitabine •  Valtorcitabine •  Amdoxovir •  Racivir •  LB80380

Nucleotide analogs •  Adefovir dipivoxil •  Tenofovir*

•  Alamifovir •  Pradefovir

Cytokines •  Interferon alfa •  Pegylated Interferon alfa-2a

•  IL7 • IFN Lambda • Vaccine therapy

Treatment failure

Primary non response Partial response

Secondary treatment failure Antiviral drug resistance

Host factors Drug metabolism Patient’s compliance

Drug factors Antiviral potency

Drug factors Barrier to resistance

Viral factors Resistant mutants

Zoulim & Perrillo J Hepatol 2008; EASL CPG J Hepatol 2009

Clinical definition of resistance

•  Virologic Breakthrough: Rebound in serum HBV DNA levels (e.g. 1 log10 above nadir)

•  Genotypic Resistance: Detection of mutations known to confer resistance while on therapy

•  Virologic Breakthrough with Genotypic Resistance: Viral rebound associated with a mutation(s) known to cause resistance.

•  Primary non response: <1log10 decrease of viral load after 3 months

•  Partial response: detectable HBV DNA levels during therapy

Zoulim & Perrillo, J Hepatol 2008; EASL CPG, J Hepatol 2009

Laboratory Definition of HBV Resistance to Antivirals

Laboratory Investigations •  Phenotypic Resistance: Decreased susceptibility (in vitro

testing) to inhibition by anti-viral drugs associated with genotypic resistance.

•  Cross Resistance: Mutants selected by one agent that also confer resistance to other antiviral agents

Zoulim et al; Future Virology 2006

The main differences between HIV, HBV and HCV

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.

Si Ahmed et al. Hepatology. 2000; Yuen et al Hepatology 2001; Locarnini et al Antiviral Therapy 2004; Villet et al Gastroenterology 2006 J Hepatol 2007 & 2008; Pallier et al J Virol 2007; Yim et al Hepatology 2006.

Kinetics of emergence of HBV drug resistant mutants

Lamivudine Resistance Accelerates Progression of Liver Disease

0

5

10

15

20

25

0 6 12 18 24 30 36Time after randomization (Months)

% W

ith d

isea

se p

rogr

essi

on Placebo (N=215)

YMDDm (N=209) (49%)Wild Type (N=221)

YMDDm

WT

Placebo

5%

13%

21%

Liaw YF et al. N Engl J Med. 2004;351:1521-1531

Biochemical and Histologic Correlates of HBV Resistance

•  Rise in ALT levels – Mild ALT elevations in most cases – ALT flares with acute exacerbations and liver failure:

especially patients with liver cirrhosis and/or pre-core mutant infection

•  Progression of liver disease – Progressive worsening of liver histology – Clinical deterioration, liver decompensation, HCC

development Lai et al Clin Infect Dis 2003; 36: 687-696; Dienstag et al Gastroenterology 2003;124:105-117 ; Lok et al Gastroenterology 2003; 125 : 1714-1722; Hadziyannis et al Hepatology 2000;32:847-851; Si Ahmed et al Hepatology 2000; Zoulim et al J Viral Hepatitis 2006;13:278-288 ; Fung et al J Hepatol 2005;43:937-943; Liaw et al NEJM 2004;351:1521-1531.

ALT flares in patients with lamivudine resistance over time

Lok et al Gastroenterology 2003; 125 : 1714-1722

Drug and patient population

Resistance at year of therapy expressed as percentage of patients

1 2 3 4 5 6 Lamivudine 23 46 55 71 80 - Telbivudine HBeAg-Pos 4.4 21 - - - - Telbivudine HBeAg-Neg 2.7 8.6 - - - - Adefovir HBeAg-Neg 0 3 6 18 29 - Adefovir (LAM-resistant) Up to 20% - - - - - Tenofovir 0 0 0 0 - - Entecavir (naïve) 0.2 0.5 1.2 1.2 1.2 1.2 Entecavir (LAM resistant) 6 15 36 46 51 57

Incidence of drug resistance over time

CL Lai Clin Infect Dis 2003; CL Lai NEJM 2007; Hadzyiannis Gastroenterology 2006; Marcellin NEJM 2008; CL Lai & Chang NEJM 2006; Zoulim & Locarnini Gastroenterology 2009

Zoulim & Locarnini, Gastroenterology, 2009

Zoulim & Locarnini, Gastroenterology, 2009

Determinants of viral persistence

Bridges; Progress in Liver Disease 1995

Nucleos(t)ide analogs

The HBV life cycle

Zoulim & Locarnini, Gastroenterology 2009

uncoating! CCC DNA!

removal of protein primer!removal of RNA primer!completion of viral (+) strand DNA!ligation of DNA strands extremities!

supercoiled DNA!minichromosome!

viral polymerase?!DNA repair protein?!other cellular enzymes?!

Topoisomerase (TDP2) ?!Acetyl transferase ?!Histones!

Tuttleman et al Cell 1986 Le Guerhier et al AAC 2000 Delmas et al AAC 2002 Kock et al Hepatology 2003 Cortes Ledesma et al Nature 2009 Boeck et al Plos Pathogen 2010

Antivirals ?

Can we prevent cccDNA formation ? Nucleoside analogs in monotherapy or combination therapy cannot prevent the de novo formation of cccDNA in hepatocyte culture and in vivo in animal experiments (Delmas et al AAC 2000; Seigneres et al AAC 2002)

Can we clear cccDNA from a chronically infected cell ? The decrease of intrahepatic cccDNA during nucleoside analog requires hepatocyte turn over in animal experiments (Zhu et al J Virol 2001; Litwin et al J Clin Virol 2005)

Zhu et al, J Virol 2001

ADV Associated Serum HBsAg Reductions are Similar in Magnitude to cccDNA Reductions

-6

-5

-4

-3

-2

-1

0

Cha

nges

in H

BV

Mar

kers

from

Bas

elin

e(lo

g10

copi

es/c

ell(m

l))Serum HBV DNA

Total Intracellular

DNA cccDNA Serum

HBsAg

Werle et al, Gastroenterology 2004

Kinetics of spread and emergence of drug resistant virus during antiviral therapy

Zhou T, et al. Antimicrobial Agents and Chemotherapy 1999; 43: 1947-1954.

antiviral

wt

ni

Free liver space

Mutant fitness

I II III IV INHIBITION OF WILD TYPE VIRUS REPLICATIONS DELAYED EMERGENCE OF

DRUG RESISTANT VIRUS

ni = non-infected wt = wild type

mt = mutant type

mt

Kinetics of HBV drug resistance emergence

Si Ahmed et al. Hepatology. 2000; Yuen et al Hepatology 2001; Locarnini et al Antiviral Therapy 2004; Villet et al Gastroenterology 2006 J Hepatol 2007 & 2008; Pallier et al J Virol 2007; Yim et al Hepatology 2006.

Treatment begins

Drug-resistant variant

Drug-susceptible virus

Naturally—occurring viral variants

Time

HB

V re

plic

atio

n

Primary resistance mutations

Secondary resistance mutations / compensatory resistance mutations

Partial response to adefovir dipivoxil is not due to the selection of DR mutants

•  The top 25% patients (quartile 1): > 4.91 log10 reduction in serum HBV DNA at week 48. •  In Q2: 3.52 to 4.90 log10 reduction of viral load. •  In Q3: 2.22 to 3.51 log10 reduction in viral load. •  The bottom 25% of patients (Q4):< 2.22 log10 reduction in HBV DNA levels at week

48. •  Phenotypic analysis of viral strains: Q4 as sensitive to ADV as Q1 strains •  Documented Drug Compliance (% of days without taking ADV)

•  Wilcoxon rank sum test, P=0.01 Durantel et al, Antiviral Therapy, 2008

Virological Response Q1 (best response)

(n=38)

Virological Response Q2

(n=38)

Virological Response Q3

(n=38)

Virological Response Q4 (worse response)

(n=38)

Median 99% 99% 99% 97% a

range 86-100% 41*-100% 91-100% 70-100%

M204V reduces pocket size

Steric clash between lamivudine and V204

Wild-type M204/L180

L180

M204

LVD-TP

LVDr M204V/L180M

L180M

M204V

LVD-TP

LVDr M204V/L180M

L180M

M204V

ETV-TP

Langley DR, et al. J Virol. 2007;81:3992-4001.

Amino acid substitutions result in conformation changes of the polymerase catalytic site

Minimal steric clash between entecavir and V204

Definition of fitness

•  A parameter that quantifies the adaptation of an organism or a virus to a given environment

•  For a virus, ability to produce infectious progeny relative to a reference viral clone, in a defined environment

Esteban Domingo, In Fields Virology 2007

Cross-resistance data for the main mutants and the commercially available drugs

Pathway Amino acid substitutions in the rt domain

Lamivudine Telbivudine Entecavir Adefovir Tenofovir

Wild type S S S S S L-nucleoside M204I R R I S S L-nucleoside L180M+M204V R R I S S

Alkyl phosphonate

N236T S S S R I

Shared A181T/V I/R I/R S R I D-Cyclopentane

(ETV) L180M+M204V/I ±I169T±V173L

±M250V

R R R S S

D-Cyclopentane (ETV)

L180M+M204V/I±T184G±S202I/G

R R R S S

MDR V173L+L180M+A181V+N36T

R R S R S

Zoulim & Locarnini Gastroenterology 2009

•  cccDNA in the liver:

–  Is propagated during the normal replication cycle of HBV

–  Can serve as a template for the production of new virus

Archiving of viral variants Viral quasispecies

cccDNA variants

Liver Majority population Minority variants Resistant variants

Blood circulation

Zhou et al, AAC 1999; Zoulim F. Antivir Res. 2004. Zoulim F & Perillo R. J Hepatol. 2008




•  It is believed that viral variants with antiviral resistance may be archived in this way


cccDNA variants

Blood circulation






•  It is believed that viral variants with antiviral resistance may be archived in this way


cccDNA variants


Blood circulation


Phenotyping of HBV clinical isolates

1. Durantel D, et al., Hepatology, 2004;40:855-64. 2. Yang H, et al., Antiv Ther, 2005;10:625-33.

Southern blot analysis

Patient serum

PCR cloning

Whole genome HBV clones

Transfection

HepG2 Huh7

IC50 reference strain

IC50 mutant Fold resistance =

Wild-type virus

Increasing antiviral concentration

Cell culture plate

Patient’s virus

SS -

RC -

lamivudine adefovir

ADV rtN236T +/or rtA181V

Wild-type virus

ADV-resistant virus

LAM-resistant virus LAM rtM204V/I ± rtL180M

ETV-resistant virus

rtT184 or rtS202 or rtM250 ETV

rtM204V/I rtL180M +/-

TDF

TDF: what can we expect?

rtM204V/I +/- rtL180M

LAM then ETV

rtT184 or rtS202 or rtM250

LAM + TDF – what do we see?

Maximising the barrier to resistance

Can we detect low frequency mutants prior to or during therapy ?

Use of pyrosequencing to detect low frequency mutants

• May detect mutants representing as low as 0.1% of the viral population

• The clinical significance for treatment choice or adaptation needs to be determined by prospective studies

Important factors involved in selection of MDR mutants

•  Use of inadequate sequential monotherapies and inadequate treatment adaptation

•  Incomplete viral suppression –  > Persistent replication in the presence of antiviral pressure

•  Use of drugs sharing cross-resistance characteristics –  One mutation may confer resistance to several drugs

–  > Persistent replication

•  Accumulation of mutations

•  Wide replication space (liver transplantation)

?

Multiple drug resistant mutants with complex pattern of mutations

+ one mutation + one mutation

Drug A Drug B

Risk of selection of MDR mutants by sequential therapy -  drugs sharing cross-resistance characteristics -  incomplete viral suppression -  liver transplantation

The problem of sequential therapy with nucleoside analogues

Zoulim F, et al. J Hepatol. 2008;48:S2-19. Yim et al, Hepatology 2006; Villet et al Gastroenterology 2006 & 2009

103

104

105

106

107

108

109

0 20 40 60 80 100 120 Treatment (months)

HB

V D

NA

(cop

ies/

ml)

entecavir IFN adefovir

lamivudine Genotype H

lamivudine

Drugs sharing cross-resistance characteristics: Switching strategy emergence of MDR mutant

L180M+S202G+M204V

L180M+M204V

Villet et al, J Hepatol 2007

Trea

tmen

t (m

onth

s)

lam

ivud

ine

ente

cavi

r

0 20 40 60 80 100

1

L180M+M204V M204V wt

V173L+L180M+M204V

L180M+M204V V173L+L180M+M204V

L180M+S202G+M204V I169L+L180M+S202G+M204V V173L+P177S+L180M+S202G+M204V

V173L+P177S+L180M+S202G+M204V L180M+A181G+S202G+M204V

L180M+S202G+M204V

L180M+A181G+S202G+M204V

wt

% clones in the quasi-species

27/0

0

11

34

36

-  Lamivudine therapy: Selection of a main population harboring the V173L+L180M+M204V mutations = primary resistance mutations

-  Entecavir therapy: Selection of three populations, all harboring the L180M+S202G+M204V mutations = secondary resistance mutations

Genotypic analysis of the viral quasi-species during lamivudine and entecavir therapy

Lamivudine rebound

Entecavir rebound

Villet et al, J Hepatol 2007

Role of cross-resistance, inefficacy of viral load suppression, and replication space, in MDR mutant selection

Villet et al Gastroenterology 2006

Genotype E

102

103

104

105

106

107

108

0 500 1000 1500 2000 2500 3000 3500

days of treatment

HB

V D

NA

(Meq

/ml)

lamivudine

adefovir

HBIg tenofovir

V173L+L180M+A181V+N236T L180M+M204V

Liver transplantation

0 10 20 30 40 50 60 70 80 90 100

1

Lam

ivud

ine+

adef

ovir

treat

men

t (m

onth

s)

1

8

24

34

38

40

42 to 50

Viral rebound

0

16

26

30

32

34 to 42

Time post-transplantation (m

onths)

% of variants in the viral quasi-species

Accumulation of mutations and selection of a complex mutant

Terminal Protein spacer Pol/RT RNaseH

V173L L180M A181V N236T

Pre-S/S gene

P120S

dominant HBV mutant

L180M+M204I

wt

V173L+L180M+A181V+M204V

M204I

V173L+L180M+A181V V173L+L180M+A181V+M204V+N236T

V173L+L180M+A181V+N236T

V173L+L180M+A181V+N236T

V173L+L180M+A181V+N236T

V173L+L180M+A181V+M204V+N236T

V173L+L180M+A181V+M204V

V173L+L180M+A181V+M204V

V173L+L180M+A181V+M204V

V173L+L180M+A181V+M204V

L180M+M204I

V173L+L180M+A181V+M204I

I169V+L180M+T184I+M204V

V173L+L180M+A181V+N236T

A single a.a. substitution at position rt181 may be responsible for multidrug resistance

Villet S, et al. J Hepatol. 2008;48:747-55.

wt A181V A181T A181V + N236T A181T + N236T N236T

N236T + N238T M204V M204I L80V L80V + M204I

LVD

LVD+TDF LVD+ADV+TDF

Patient #1 (67 months)










LVD+ADV ADV

Warner et al Hepatology 2009 Kamili et al Hepatology 2009 Villet et al Gastroenterology 2009

Impact on virus infectivity and fitness

Impact on virion release (intracellular retention) and virologic monitoring of breakthrough

Impact on vaccine prophylaxis efficacy

Potential risk of transmission of HBV DR mutants

Clements et al, Bull WHO 2009

Conclusions – Main issues regarding viral resistance and persistence

1) Persistence of cccDNA A major driver of viral persistence Can we quantify HBsAg as a surrogate / non invasive marker ?

2) Viral quasi-species Role of more sophisticated methodologies to detect HBV variants, i.e. Ultradeep sequencing ?

3) Viral fitness A major determinant in the barrier to resistance Is monotherapy sufficient on the long-term to maintain a high barrier to resistance ?

4) New treatment targets in combination with NUCs for finite duration

Management algorithm Antiviral treatment

Treatment failure

Viral load asssessment

Add-on therapy based on cross-resistance data

Check compliance Primary non response

Switch to more potent drug Zoulim and Perrillo, J Hepatol, 2008; EASL CPG J Hepatol 2009

Viral genome sequence analysis

Wild type virus HBV drug resistant mutant

Check compliance

Management algorithm

Antiviral treatment

Treatment response

Viral load asssessment

Zoulim and Perrillo, J Hepatol, 2008; EASL CPG J Hepatol 2009

Check for HBe/HBs seroconversion on a regular basis (6 monthly)

Virologic Consequences of Persistent Viremia

  Infection of new hepatocytes slower kinetics of clearance infected cells and cccDNA

  Increases the risk of occurrence and subsequent selection of HBV mutations responsible for drug resistance

  On-treatment prediction of HBV drug resistance

Le Guerhier et al Antimicrob Agents Chemoter 2000;44:111-122; Delmas et al Antimicrob Agents Chemother 2002; 46:425-433; Kock et al Hepatology2003; 38:1410-1418; Richman Hepatology 2000;32:866-867

Comment adapter le traitement ?

Zoulim Antivir Res 2004; 64: 1-15. Villeneuve et al J Hepatol 2003. Lampertico et al Gastroenterology 2007

Wild type

LAM-R

ADV-R

ADV

+LAM

ADV

LAM

Months

ADV mono

Pat

ient

s w

ith v

irolo

gica

l bre

akth

roug

h

273 268 256 225 201 158 61

30%

6%

P<0.001

ADV+LAM

255 238 223 213 200 177 103 P

atie

nts

with

AD

V-R

229 225 217 194 179 146 57

16%

0%

P<0.001

ADV mono ADV+LAM

242 227 214 205 200 174 92

3-yr cumulative probability

* > 1 log rebound of HBV DNA compared to on-treatment nadir ** N236T or A181T-V in patients with a virological breakthrough

Patients still at risk

Virologic breakthrough* Virologic breakthrough* and ADV resistance**

Lampertico P for the AISF ADV Study Group, 57th AASLD Meeting, October 27-31, 2006, Boston, USA. Oral presentation LB5. Hepatology. 2006;44(4, suppl 1):229A-30 (Abstract 110).

0

20

40

60

80

100

0 3 6 9 12 15 18 21 24 27 30 33 36 0

20

40

60

80

100

0 3 6 9 12 15 18 21 24 27 30 33 36

Patients with lamivudine resistance: adefovir add-on strategy

HBV DNA Δ ALT

The problem of sequential therapy and switching strategy

Villeneuve et al, J Hepatol 2003

N236T

Ser

um H

BV

DN

A (L

og10

cop

ies/

mL) A

LT (IU/L)

300

250

200

150

100

50

L180M+M204V

LAM ADV

Reverted to wild type

2

3

4

5

6

7

8

9

10

janv-98 janv-99 janv-00 janv-01 janv-02 janv-03 janv-04 janv-05

LAM

M0

M6

M12

M18

M24

M30

M36

ALT

0

2

4

6

8

ALT HBV DNA

Month of therapy

Rescue therapy in patients with clinical breakthrough

Drug A

Drug B

Seru

m H

BV D

NA

(Log

10 c

opie

s/m

L)

and

ALT

(x U

LN)

M0

M6

M12

M18

M24

M30

M36

ALT

0

2

4

6

8

ALT

HBV DNA

Month of therapy

Rescue therapy in patients at the time of virologic breakthrough

Drug A

Drug B

Seru

m H

BV D

NA

(Log

10 c

opie

s/m

L)

and

ALT

(x U

LN)

M0

M6

M12

M18

M24

M30

M36

ALT

0

2

4

6

8

ALT

HBV DNA

Month of therapy

Early add-on therapy to prevent drug resistance

Drug A Drug B

Seru

m H

BV D

NA

(Log

10 c

opie

s/m

L)

and

ALT

(x U

LN)

Very Early Add-on Therapy to Keep Viral Load as Low as Possible

2

3

4

5

6

7

8

M0 M3 M6 M9 M12 M15 M18 M21 M24

Seru

m H

BV

DN

A (L

og10

cop

ies/

mL)

Month of therapy

1. Start with a drug having a high genetic barrier for resistance 2. Add a drug with a different cross-resistance profile

outgrowth of drug resistant mutant ?

MDR ?

Rationale for de novo Combination Therapy

Drug A

Drug B

Wild type

Drug B resistant mutant

Drug A resistant mutant

wt

Clavel et al NEJM 2004;350:1023-35 ; Zoulim Antiviral Res 2004;64: 1-15

M0

M6

M12

M18

M24

M30

M36

ALT

0

2

4

6

8

ALT

HBV DNA

Month of therapy

De novo combination therapy to prevent drug resistance

Drug A

Drug B

Seru

m H

BV D

NA

(Log

10 c

opie

s/m

L)

and

ALT

(x U

LN)

Preventing L-Nucleosides Resistance with de novo Combination Therapy

1 Marcellin et al. N Engl J Med 2004; 351: 1206-17 2 Lau et al. Hepatology 2004;40:171A

3 Lai et al. Hepatology 2003;38:262A 4 Sung et al. J Hepatol 2003 ;38 (suppl 2):25-26 5 Lau et al. Hepatology 2004:40:666A

* After 1- year therapy

20% 18%

34%

21%

2% 1% 11% 12%

5%

0

20

40

60

80

100

Sung 4 Marcellin 1 Lau 2 Lai 3

LAM LAM LAM LAM LAM

+ADV

LAM

+Peg

LAM

+Peg

LAM

+LdT

Inci

denc

e of

resi

stan

ce* (

%)

LdT FTC FTC

+ADV

0% 0%

Lau 5

RAND

OMIZ

ATIO

N 1:

1

Tenofovir DF 300 mg (TDF)

FTC 200 mg / Tenofovir DF 300 mg (FTC/TDF)

Study 106: TDF Versus FTC/TDF for Treatment of CHB in Patients with Persistent Viral Replication Receiving ADV

Double Blind

End of Study

Week 24*

Blinded TDF or

OL FTC/TDF

Blinded FTC/TDF or

OL FTC/TDF

Final Study Results (AASLD 2010)

‡

Berg T, et al., AASLD 2010; Oral# 136.

Week 48 Week 96

*From Week 24 on, patients with confirmed (within 4 weeks) plasma HBV DNA ≥ 69 IU/mL had the option to add FTC (as fixed dose FTC/TDF) or discontinue from the trial and initiate commercially available therapy

Week 168

Blinded TDF or

OL FTC/TDF

Blinded TDF or

OL FTC/TDF

Blinded FTC/TDF or

OL FTC/TDF

Blinded FTC/TDF or

OL FTC/TDF

* From Week 24 on, patients with confirmed HBV DNA ≥ 400 copies/mL (69 IU/mL) could switch to open label (OL) FTC/TDF or discontinue from the trial and initiate commercially available therapy

Study 106 – Treatment-Experienced Patients

Primary Efficacy Analysis: Comparison of the Two Treatment Strategies

82% FTC/TDF

82% TDF

ITT: NC=F*

Two patients on study at Week 168 had HBV DNA ≥400 copies/mL

‡


Perc

enta

ge (%

)

*NC=F, Non-completer counted as failure in this ITT analysis, including patients who switched to open-label FTC/TDF fixed-dose combination


% of Patients with HBV DNA < 400 copies/mL (69 IU/mL)

Mean HBV DNA (log10c/mL) by Study Visit

* Includes patients who switched to open-label FTC/TDF fixed-dose combination

2.26 TDF 2.24 FTC/TDF

Bert T, et al., AASLD 2010; Oral# 136.

‡ M

ean

(95%

CI)

HB

V D

NA

(log

10 c

opie

s/m

L)


Mean HBV DNA by Baseline LAM-R and Treatment


‡ Study 106 – Treatment-Experienced Patients

Mean HBV DNA by Baseline ADV-R and Treatment

‡



0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

BL W4 W12 W24 W48

A181T

A181T + N236T

wt

Viral load

BL viral load = 6.85log Treatment: TDF Adherence : 68%

Patient 1046 data:

LLOD

Lavocat & Zoulim, AASLD 2010.

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

BL W4 W12 W24 W36 W48

N236T

A181V + N236T

A181V

A181S + N236T

A181T + N236T

A181T

wt

Viral load

BL viral load = 8.75log Treatment: TDF Adherence : 95.2%

Patient 1051 data:

LLOD

Impact of persisting low viremia levels on treatment outcome ? Lavocat & Zoulim, AASLD 2010.

Perspectives / Prevention of drug resistance

•  First line therapy –  Use of antivirals with high antiviral potency and high barrier to

resistance –  Combination therapy with complementary drugs to increase the

barrier to resistance •  Second line treatment

–  Add-on strategies with complementary drugs preferred to sequential monotherapies

–  Early treatment adaptation to prevent accumulation of mutations

–  Choice always based on cross-resistance data

Perspectives beyond the guidelines

•  Early treatment intervention to prevent disease progression ? screening program non invasive evaluation of liver disease / biomarkers

•  Can we prevent prevent HCC development ? decreased risk of HCC if HBsg clearance <50 yrs (Yuen et al, Gastroenterology 2008)

•  Can we clear cccDNA and/or HBsAg ? new treatment strategies new treatment targets

HBsAg clearance

Werle-Lapostolle B et al., Gastroenterology 2004;126: 1750-58.

Infected hepatocytes!

Infected liver

CD8

NKT

CD4

B

cccDNA

Antivirals

Clearance of HBsAg?

Blood circulation viral load

0,01

0,1

1

10

2,00E+02

3,00E+02

4,00E+02

5,00E+02

6,00E+02

7,00E+02

8,00E+02

9,00E+02

Nov

. 99

Jan.

00

Sept

. 00

Mar

. 01

July

02

Dec

. 02

Dec

. 03

Viral load

HBs Ag

Lamivudine 100 mg/day

Clinical example of HBsAg clearance

HBs Ag Positivity cut off: 0.05

Viral load Detection treshold

Viral load Log Copies/ml

HBs Ag IU/ml

* *

*: Anti-HBs antibody Negativation of HBe Ag May 1987 HBe Seroconversion between June 87 and November 1996?

Borgniet O et al., J Med Virol , 2009;81:1336-42.

New targets

Immune system

Conclusions 1

Conclusions 2

Documents

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