Zoulim

Hépatite B

Fabien Zoulim

Département d’hépatologie

& INSERM U1052, Lyon

Natural history of hepatitis B

Acute infection

Chronic infection: 400 million carriers !

Immune tolerance

Chronic hepatitis

Inactive carrier

Resolved infection5% neonates

90% adults

Wild type virus HBeAg+ Pre-core mutant HBeAg-

Cirrhosis

Hepatocellular carcinoma

Reactivation

30-50 yearsSeeger, Zoulim, Mason; Fields Virology; 2007

EPIDEMIOLOGIE DE L’HÉPATITE B

EPIDEMIOLOGIE DE L'INFECTION A VHB

• Hépatites aigues – VHA : 40%

– VHB : 30%

– VHC : 20%

• incidence : 300 000 infections à VHB / an

• 30 000 nouveaux porteurs chroniques / an

• 3 000 décès / an

AUX USA

MODES DE TRANSMISSION DU VIRUS DE L'HÉPATITE B EN EUROPE

sexuelle34%

hétéro23%

homo11%

drogue IV26%

inconnue31%

hémodialysés8%

transfusions2%

personnels de santé 2%

contact avecporteur du VHB

4%

AsieTransmission verticale

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

Results

158 acute hepatitis cases

• Hospital doctor in 64% cases

• Sex ratio M/F : 2,95 (118/40)

• Median age: 37 yrs for males, 36yrs for females

• Jaundice : 69%

• Hospitalisation : 46%

• Fulminant hepatitis : 3 (2 death)

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

• Source: obligatory declaration march 03- february 2004 N=145

– Sexual 59 40,6% No factor43 29,6%

– IVDU 9 6,2% >1 factor 3826,3%

– Invasive treatment 15 10,3%

– Tatoo, piercing 5 3,4%– Familial 14 9,7%– Perinatal 2 1,4%– Live in instiution 11 7,6%– Travel in endemic 21 14,5% areas91/145 patients (63 %) had a vaccine indication (2 vaccinated ≥

3 doses)

• Sentinel networks 91-96 N=195 – sexual

35% – IVDU

19%– « percutaneous »

15%– No factor

35%

Surveillance épidémiologique de l’infection HBV

• 14 446 adultes testés• Prévalence de l’AgHBs 0,65% (280 000 porteurs

chroniques du VHB)• Homme 1,1% versus 0,2% femme• Naissance en zone d’endémie 4% versus 0,5%• Précarité, séjour en institution, homosexualité,

usage de drogues

Meffre et al, J. Med Virol 2004

Hépatites virales B: épidémiologie

- Vaccin mais 250 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

VIROLOGIE

• 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

spherefilament

Dane particle

HBeAg

HBsAg

The HBV genome

Tiollais, Nature 1985

déterminant avaccin/IgHBs

Gène pol antiviraux

Mt pre-coreRéponse anti-HBe ?

Mt du coreRéponse CTL

8 génotypesA to H

The viral replication cycle

Zoulim & Locarnini, Gastroenterology 2009

Model for sodium-dependent taurocholic cotransporting polypeptide (NTCP) binding to preS1

Seeger C , and Mason W S Gut 2013 in press; Yan H, et al. eLife 2012;1:e00049; Hu NJ, et al. Nature 2011;478:408–11.

• Sodium-dependent transporter for taurocholic acid • Expressed at the basolateral membrane of hepatocytes• Mediates the transport of conjugated bile acids • 349 amino acid-long glycosylated transmembrane protein. • Expression controlled by hepatocyte-specific transcription

factors, including HNF3 and C/EBP

Model for sodium-dependent taurocholic cotransporting polypeptide (NTCP) binding to preS1

Seeger C , and Mason W S Gut 2013 in press; Yan H, et al. eLife 2012;1:e00049; Hu NJ, et al. Nature 2011;478:408–11.

Transgenic miceHumanized mice

HumanChimpanzeeGibbonbaboons

Tupaïa

Woolley monkey

Ground squirrel

American woodchuck

Pekin DuckGrey Heron

Summers PNAS 1978, Mason J Virol 1981, Chisari Science 1985, Petersen PNAS 1998, Lanford PNAS 1998

The animal models of HBV infection

• Polymerase virale

– DHBV : lysat réticulocytaire

– HBV : baculovirus

Modèles in vitro

UPolymerase VHB

DNA(-)

ELONGATIONCCC -

RC -L -

SS -

• Culture cellulaire

– Transfection : lignées d’hépatome

– Infection : hépatocytes primaires, HepaRG

– Baculovirus ou adenovirus recombinant

Sells PNAS 1987, Wang Cell 1992, Zoulim J Virol 1994, Lanford J Virol 1995, Gripon PNAS 2002, Sprinzl J Virol 2001

Infection à VHB et risque de CHC

• Etude de Beasley à Taiwan– risque relatif = 100 chez les porteurs de l'AgHBs

• Etude de Tsukuma– risque cumumatif de CHC à 3 ans

• 12,5% chez 240 patients avec cirrhose

• 3,8% chez 677 patients avec hépatite chronique

– risque x 7 si AgHBs +

– risque X 4 si anti-HCV +

• Facteurs associés : alcool, tabac, aflatoxine

• Diminution incidence avec la vaccination de masse (Chen,

NEJM 1995)

CARCINOME HEPATOCELLULAIRE ET VIRUS DE L'HEPATITE B

• Co-incidence de répartition géographique

VHB / CHC

• Porteurs AgHBs : RR x 100 pour le CHC

• CHC dans les modèles animaux de l'hépatite B :

– marmotte

– écureuil

• Présence d'ADN viral intégré dans les tumeurs

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

PHYSIOPATHOLOGIE / IMMUNOPATHOLOGIE

Ganem and Prince, NEJM 2004

HÉPATOCYTE INFECTÉ

VHB

CTL

Fasperforine

HÉPATOCYTENON INFECTÉ

IMMUNOPATHOGÉNIE DES HÉPATITES B CHRONIQUES

AgHBc/e

HLAI

cytokines

RÉPONSE IMMUNITAIRECYTOKINES

ANTIVIRAUX

ANTICORPS NEUTRALISANTS

IMMUNOPATHOLOGY OF HBV INFECTION

Immune tolerance

Clearance phaseChronic hepatitis

SeroconversionRemission

CD8+

HBV

CD8+ HBV

CD8+HBV

Immunopathology

Fulminant hepatitis

CD8+

HBV

Hépatocyte infecté

HBV

Hépatocyte non infecté

Phase de tolérance immunitaire

MarqueursAgHBe +HBV DNA +++ALAT = NFoie = N

HBc/e Ag


HBV

CD8

Fasperforine


Phase de clairance immune(hépatite chronique)

MarqueursAgHBe+HBV DNA > 2000 IU/mLALAT +++Foie: Hépatite chronique

HBc/e Ag

HLAI

cytokines


HBs Ag


MarqueursAgHBe-anti-HBe +HBV DNA < 2000 IU/mLALAT = NFoie = rémission

Phase de rémissionportage inactif de l’AgHBs

RéactivationVirus sauvage ou mt pre-coreOncogénèse

CD8CD4

Hépatocytes infectés

Hépatocytes non infectés

MarqueursHBsAg -

anti-HBc +Anti-HBs +/-

PCR sérum (-) / foie (+)

Clairance de l’AgHBs

Mutants d’échappementInfections occultes

Oncogénèse

CD8CD4

B

cccD

NA

(co

pie

s/ce

ll)

Tota

l H

BV

DN

A

(co

pie

s/ce

ll)

cccDNA levels in the different phases of chronic HBV infection

• HBeAg+ patients had significantly higher cccDNA (90-fold) and total HBV DNA (147- fold) levels compared to HBeAg- patients. (p<0.001, Wilcoxon tests)

HBeAg+ (63)

Inact.

Carriers

(10)

HBSAg- (7)

HBeAg- (18)

HBeAg+ (63)

Inact.

Carriers

(10)

HBSAg- (7)

HBeAg- (18)

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 infectionEpigenetic control

Histones

CBPPCAFp300

CBPPCAF

p300Sirt1

Sirt1HDAC1HDAC1

Histones

Pollicino et al., Gastroenterology 2006

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

HISTOIRE NATURELLE ET VIROLOGIE CLINIQUE

Histoire Naturelle de l’hépatite BInfection aigue

Infection chronique

Tolérance immunitaire

Hépatite chronique

Portage inactif

Guérison5% nx-nés90% adultes

Virus sauvage (HBeAg+) Mutant pre-core (HBeAg-)

Cirrhose

Carcinome hépatocellulaire

Réactivation

30-50 ans

Seeger, Zoulim, Mason; Fields Virology; 2007

Seeger, Zoulim, Mason – Fields Virology 2007

HEPATITE B AIGUE• Incubation 1 à 6 mois• Le plus souvent asymptomatique

– Évolution plus fréquente vers la chronicité• Prodromes:

– Maladie sérique : arthralgies, urticaire, acrodermatite etc. ..

• Formes ictériques : + graves que VHA et VHC– Durée de l’ictère : jusqu’à 4 mois

• Evolution : chronicité 5 à 10%• Hépatites fulminantes

Laboratory Diagnosis of Acute Hepatitis B

ALT

HBsAg

HBeAg

HBV DNA

Normal

Months After Exposure

ALT

an

d H

BV

DN

A

IU/L

an

d m

illio

n c

op

ies/

ml

Symptoms

Anti-HBs Ab

Anti-HBe Ab

IgM anti-HBc

Total anti-HBc

Seeger, Zoulim, Mason, Fields Virology 2007

HEPATITE B PROLONGEE

• Définition–Persistance réplication virale à la 8ème

semaine d’évolution :–AgHBe + ou ADN-VHB +

• Evolution–Chronicité : 8 cas / 10

• Traitement : IFN –Guérison : 7 à 8 cas / 10

INFECTIONS CHRONIQUES A VHBFORMES CLINIQUES

• virus sauvage– tolérance immunitaire– rupture de tolérance -> lésions hépatocytaires : HCA– séroconversion anti-HBe spontanée (portage inactif) :

5-10% /an– > diminution significative réplication virale– > amélioration signes histologiques

• virus muté pré-C (-)– sélection au moment de la séroconversion anti-HBe– dépend du génotype viral– immunopathologie ?– sévérité de l'hépatopathie : controversée– association au CHC

ALT

HBsAg

HBeAg

HBV DNA

Normal


ALT

an

d H

BV

DN

A

IU/L

or

mill

ion

co

pie

s/m

l

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


ALT

``HBsAg

HBeAg

HBV DNA

Normal


ALT

an

d H

BV

DN

A

IU/L

an

d m

illio

n c

op

ies/

ml

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


ALT

HBsAg

HBV DNA

Normal ALT levels

Months

ALT

an

d H

BV

DN

A

IU/L

an

d m

illio

n c

op

ies/

ml Anti-HBeHBeAg

Laboratory Diagnosis of HBeAg negative Chronic Hepatitis B


0,001

0,01

0,1

1

10

100

1000 ALAT

ADN-VHB

AgHBe + anti-HBe +UI/mlpg/ml

AgHBs

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

hybridation

PCR

9 log

8 log

7 log

6 log

5 log

4 log

3 log

2 log

1 log

Dynamic ranges of quantificationof HBV DNA assays

Amplicor HBV Monitor v2.0 (Roche)

HBV Hybrid-Capture II (Digene)

Ultra-sensitive HBVHybrid-Capture II

Versant HBV DNA3.0 (bDNA, Siemens)

Cobas Taqman HBV(Roche)

Abbot Real-time HBV(Abbott)

Versant HBV DNA 1.0(kPCR, Siemens)*

*in development

10 102 103 104 105 106 107 108 109

RealArt HBV LC PCR(Artus Biotech)

Formes cliniques

MANIFESTATIONS EXTRAHEPATIQUES DU VHB

• PAN– Complexes immuns circulants HBs/anti-HBs– Dépots artères moyens et petit calibre– Traitement : plasmaphéreses, corticoides, antiviraux

(vidarabine / IFN / famciclovir / lamivudine)• Glomérulonéphrites• Cryoglobulinémies• Guillain-Barré• Myocardite

TRANSMISSION VERTICALE DU VHB

• mère AgHBe +– transmission : 90%

• mère anti-HBe +– transmission : 10-20%

– VHB muté pré-C (-) : hépatites fulminantes

• chronicité chez l’enfant : 90%

PRESENTATION CLINIQUE• INFECTION PERI-NATALE

– ALT normales ou subnormales

– ADN-VHB > 1000 pg/ml

– histologie : lésions minimes

• INFECTION POST-NATALE

– ALT élevées

– ADN-VHB < 1000 pg/ml

– histologie : hépatite modérée à sévère

• CARCINOME HEPATOCELLULAIRE : 30 ANS

Pathophysiologic Cascade of Chronic HBV Infection

HBV Replication(Measured by

Serum HBV DNA)

Liver Inflammation

Worsening Histology• Necroinflammation• Fibrosis• Cirrhosis

Disease Progression• Liver Failure• Liver Cancer• Transplant• Death

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.

ALT Elevation

Charge virale et incidence de la cirrhose

R.E.V.E.A.L. – HBV Study

Année de suivi

Incid

en

ce c

um

ula

tive d

e

cir

rhose

.2

.1

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

0

.4

.3

P <0.001

n=37741.0 x 106 n=6271.0-9.9x105 n=3441.0-9.9x104 n=649300-9.9x103 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%

Pa

tie

nts

Su

rviv

ing

, %

Years

0

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

um

ula

tive

in

cid

ence

of

HC

C

> 103 - 104 >104 - 106 ≥106

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

in HBsAg-Positive Patients HBV DNA Negative

HBV DNA Low< 105 copies/mL RR = 1.7 (0.5-5.7)

HBV DNA High≥ 105 copies/mL

RR = 11.2 (3.6-35.0)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.

http://www.fccc.edu/docs/sci_report/Evans.pdf%23search=%22haimen

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

cid

ence

Rat

e P

er 1

00,0

00

0

1473

5882

873010,108

2.0x103

4.0x103

6.0x103

8.0x103

1.0x104

1.2x104

Impact Clinique de la Variabilité du Génome Viral

VARIABILITE GENETIQUE DU VHB

• Multiplication virale» taux d'erreur de la transcriptase inverse

• Pression de sélection» réponse immunitaire cellulaire / humorale» antiviraux

-> possibilité de variants d'échappement• Conséquences cliniques

» diagnostic sérologique» traitements antiviraux

8 genotypes, numerous sub-genotypes, and recombinant forms

World J Gastroenterol 2007; 13: 14-21

B6

D1

Génotypes VHB chez les patients atteints d’hépatite chronique en France

Nu

mb

er

of

sub

ject

s

F GA B C D E0

10

20

30

40

50

60

70

80

90

100

30.2%

7.9%

12.5%

37.4%

11.3%

0.4 % 1.1%

Zoulim et al J Viral Hepatitis 2006

Impact du génotype sur la séroconversion Hbe/HBs

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%

Bn=23

C n=39

D n=103

Per

cen

tag

e o

f p

atie

nts

(%

)

HBV genotype

0

3

6

9

12

15

A n=90

5%

8%

0%

Bn=23

C n=39

D n=103

1815%

Per

cen

tag

e o

f p

atie

nts

(%

) 21

HBV genotype

PEG-IFN a-2b

HBsAg Loss 2

LES MUTANTS DU GÉNOME DU VHB

déterminant avaccin/HBIg

polyméraseantiviraux

Mt pré-coreRéponse anti-e ?

Mt coreRéponse CTL

ROLE DE LA RÉGION PRÉ-C ET DE L’AgHBe

• Non nécessaire à la réplication du VHB– Culture cellulaire– Modèles in vivo

• Marmotte• Canard

• Modulation de la réponse immune– Tolérogène : souris transgéniques– Cible de la réponse anti-capside

Chang et al, J. Virol 1987; Schlicht et al J. Virol 1987; Chen J. Virol 1992; Millich et al PNAS

LES MUTANTS PRÉ-C (-)

• codon stop / région pré-C

TGG -> TAG en pos. 1896

– génotypes B à E (A : exceptionnel)

– arrêt traduction protéine pré-C/C

– AgHBe négatif

• mutation dans promoteur pré-C

TTAAAGG -> TTAATGA en pos. 1762 /1764

– génotypes A à E

– transcrits pré-C/C :

– synthèse d'AgHBe :

Carman et al Lancet 1989, Okamoto et al J Virol 1990/1994, Tong et al Virology 1990

HBeAg and Precore Mutation

1814 1901

Precore Coreregion region

HBcAg

HBeAg

G 1896A = stop codon, TAG

ATG ATG

Virion

Serum

Core gene

HBeAg and Precore Mutation

1814 1901

Precore Coreregion region

HBcAg

HBeAg

ATG ATG

Virion

Serum

Core gene

VARIANTS NÉGATIFS POUR L ’AgHBe

mRNA

Protéinepré-C/C

PRE-C CPROMOTEUR

TAG***

1762-1764 1896

arrêt des synthèses protéiques

Diminution de l’expression de l ’AgHBe

Sélection des mutants pré-core au cours de l’histoire naturelle de l’hépatite B chronique

ALAT

ADN-VHB

AgHBe Anti-HBe

sauvage

Mt pré-C

Outcome of Chronic Anti-HBe Positive Hepatitis BBiochemical patterns in 164 untreated patients

after 23 months (range 12-36) monthly monitoring

0 12 24months

With flares and normalization

Without flares

With flares and without normalization

73 pts ( 44.5% )

59 pts ( 36.0% )

32 pts ( 19.5% )

Asymptomatic flare-up:

90% of cases

ALT Flare-up yearly

frequency:once 57.1%twice 20%

< once 22.8%

Brunetto MR et al, J Hepatol 2002

Augmentation de prévalence des hépatites chroniques avec AgHBe négatif en France

42%N=119

58 %N=164

Zoulim et al, J Viral Hepatitis 2006

DIAGNOSTICS DIFFICILES

I. Porteur inactifII. Exacerbation

Diagnosis of inactive carrier versus HBeAg negative chronic hepatitis

• Inactive Carrier– Persistently normal ALT levels– Persistently low levels of serum HBV DNA

Threshold : 2,000 IU/ mL (see EASL CPG J Hepatol 2009/2012)

• HBeAg negative chronic hepatitis– Fluctuation / exacerbation of ALT– Fluctuations of HBV DNA levels usually > 2000 IU/ mL– Presence of pre-core / core promoter mutations

DIAGNOSTIC D'UNE EXACERBATION AIGUE SUR HEPATITE B CHRONIQUE

• Définition : poussée cytolytique≠ réactivation virale

• Ag HBe + initialement– rupture de tolérance immunitaire– séroconversion anti-HBe– très fréquent chez patients asiatiques

• Anti-HBe + initialement– réactivation virus sauvage : -> AgHBe +– réactivation virus muté pré-C (-)– Corticothérapie, biothérapie, chimiothérapie– surinfection delta / VHC

COOH

137149

107

99 NH2

S - S

S - S S - S

S- S

S-S

138

139147

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

PreS1PreS2

SPol

Pré-C

C

Brin(+) 2,4kb

Brin(-) 3,2kb

X

TATAAU5-like

DR1

DR2

Enh1Enh2

GRE0/322

1

SHBs (S)MHBs (preS2+S)

LHBs (preS2+preS2+S)

Variants de l'Ag HBs

• échappement à la réponse humorale anti-

HBs

– naturelle

– vaccination (transmission mère-enfant)

– immunoprophylaxie (transplantation

hépatique)

• infection active malgré Ac anti-HBs

• sérologie AgHBs faussement négative

Risques : transmission virale + infections

occultes

VARIANTS DE L'AgHBs

• Mutations ponctuelles dans le déterminant

a de l'AgHBs (124-147)

– aa 145 : Gly -> Arg

– aa 126 : Ile -> Ser / Thr -> Asn

• transmission mère-enfant malgré la

serovaccination (3%)

• infection du greffon hépatique malgré

Immunoglobulines anti-HBs

• hépatites chroniques avec anti-HBc et anti-

HBs +

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° ofpatients

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 ofHBV replication and

gene expression

Infection byS gene Variants

“false” OBI

Occult HBV infection

HBV cccDNA Integrated HBV DNA

HBV mutants Epigenetic control

HBV replication

Immune surveillanceViral co-infections

OBI

Seropositive Seropositive SeronegativeSeronegative

HBsAg lost during CH

HBsAg lost during CH

HBsAg lost after AH

HBsAg lost after AH

Progressive antibody disappearence

Progressive antibody disappearence

Primary occult 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

High prevalence

ROLEin

HCC

Diagnostic

Tools ?

Worsen HCVinfection ?

Co-infections ?Therapy?

To beimproved

Specific treatments ?

Not fully understood ?

Occult HBV infections: unresolved issues

AntivirauxPersistance virale

Resistance aux antivirauxMonitoring des traitements

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

ALAT

HBV DNA

Minimal CH Moderate to severe CH Moderate to severe CHRemission

Cirrhosis

Immunotolerantphase

Immuno-activephase

Inactive phaseLow 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

HBsAgOccult infection

Antivirals approved for hepatitis B

*Currently approved for HIV**development on hold

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

• Entecavir • Telbivudine

• Emtricitabine*• Clevudine**

Nucleotide analogs • Adefovir dipivoxil• Tenofovir*

Cytokines • Interferon alfa• Pegylated Interferon alfa-2a

• TLR7 agonists•IL7•IFN Lambda•Vaccine therapy

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

Treatment failure

Primary non responsePartial response

Secondary treatment failureAntiviral drug resistance

Host factorsDrug metabolismPatient’s compliance

Drug factorsAntiviral potency

Drug factorsBarrier to resistance

Viral factorsResistant mutants

Zoulim et al Hepatol 2008; EASL CPG J Hepatol 2009; Lancet Infect Dis 2012

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

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

developmentLai 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

6

3

LVD ADV LdT ETV TDF

0

10

20

30

40

50

60

70

80

23

Pro

port

ion

of p

atie

nts

(%)

46

55

71

80

0

11

18

29

5

25

0.2 0.51.2

0

1 2 3 4 5 1 2 3 4 5 1 2 1 2 3 4 5 1 2 3

0 0

Option to add emtricitabine at

week 72*

*Patients confirmed to be viraemic at Week 72 or beyond could add emtricitabine to TDF at the discretion of the investigator. Clinical data on the safety and efficacy of emtricitabine and TDF in CHB are pending

Rates of resistance with lamivudine (LVD), adefovir (ADV), telbivudine (LdT), entecavir (ETV) and tenofovir (TDF) among NA-naïve patients

4

0

High barrier to resistance

Adapted from Gish, Jia, Locarnini & Zoulim, Lancet Infect Dis 2012

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 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

Terminal protein Spacer POL/RT RNaseH

1 183 349 (rt) 692 (rt 344) 845 a.a.

I(G) II(F) A B C D E

F_V_LLAQ_YMDD

*rtA181T/V and/or rtN236T cause reduced sensitivity*rtA194T association with rtL180M+rtM204V (to be confirmed)

LMV resistance/ rtL80IrtL180M

rtM204V/ILdT resistance

rtA181T/V

ADV resistance rtA181T/V rtN236TTDF resistance* ?ETV resistance rtL180M rtM204I/V

rtT184*** rtS202**** rtM250I/Vrtl169T

***S/A/I/L/G/C/M****C/G/I

Zoulim F & Locarnini Gastroenterology 2009;137:1593-1608.

rtV173L

* Role of complex mutants: rtA181T+rtN236T ?

Zoulim & Locarnini, Gastroenterology, 2009

Multiple factors are associated with the barrier of resistance & drug efficacy

•Adherence• Immune status•Prior antiviral exposure•Metabolism•Body mass

Patient

Antiviral Drug

•Antiviral potency•Number of mutations needed to overcome drug suppression

•Level of exposure to drug•Chemical structure Virus

Locarnini S, et al. Antivir Ther. 2004;9:679–93. Locarnini S, et al. Antivir Ther. 2007;12:H15-H23. 3. Ghany M & Liang TJ. Gastroenterology 2007;132:1574-85. Zoulim F, et al. Antiviral Res. 2004;64:1-15. Locarnini S, et al. J Hepatol. 2003;39:S124-S132.; Zoulim & Locarnini Gastroenterology 2009

•Replication fitness and space•Persistence of archived mutations as cccDNA

•Pre-existing mutations

Adherence to nucleos(t)ide analogues for chronic hepatitis B in clinical practice and correlation with virological breakthroughs

W. Chotiyaputta et al, Journal of Viral Hepatitis, Volume 19, Issue 3, pages 205-212, 14 JUL 2011

http://onlinelibrary.wiley.com/doi/10.1111/jvh.2012.19.issue-3/issuetoc

http://onlinelibrary.wiley.com/doi/10.1111/jvh.2012.19.issue-3/issuetoc

L(-)-SddC, 3TCLamivudineL(-)-SddC

mitochondria

nucleus

L(-)-SddC-TP HBV DNA

Nuclear DNA

Mt DNA

L(-)-SddC-TP

L(-)-SddC-TP

cytoplasm

kinase

L(-)-SddU

deaminase

Bridges; Progress in Liver Disease 1995

Nucleos(t)ide analogs

The HBV life cycle

Zoulim & Locarnini, Gastroenterology 2009

uncoating CCC DNA

removal of protein primerremoval of RNA primercompletion of viral (+) strand DNAligation of DNA strands extremities

supercoiled DNAminichromosome

viral polymerase?DNA repair protein?other cellular enzymes?

Topoisomerase (TDP2) ?Acetyl transferase ?Histones

Formation of the recalcitrant cccDNA: a difficult target for antiviral therapy

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

Kinetics of Viral Loss During Antiviral Therapy with L-FMAU (clevudine) in the woodchuck model

Zhu et al, J Virol 2001

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

SerumHBVDNA

TotalIntracellular

DNAcccDNA Serum

HBsAg

48 weeks of ADV resulted in significant reductions in : serum HBV DNA > total intrahepatic HBV DNA > cccDNA

Changes in HBsAg levels correlated with cccDNA changes-> 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 that cell turn-over occurred but was associated with infection of new cells during therapy

Immunohistochemical Staining of Patient Biopsies at Baseline and After 48 Weeks ADV Therapy

Baseline Week 48

Maynard et al, J Hepatol 2005

Persistence of cccDNA after HBs seroconversion

Clearance of viral infection versus selection of escape mutants

The most important factors to consider:

§ The rate of immune killing of infected hepatocytes

§ The rate of replication and spread of mutant virus in the

chronically infected liver (I.e. fitness of the virus: the rate of

spread to uninfected hepatocytes)

§ Small changes in these factors may have profound effect on

whether treatment response is durable or subject to rapid

rebound (Litwin et al J Clin Virol 2005)

§ These factors may be subject to therapeutic intervention

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 IVINHIBITION OF WILD TYPE VIRUS REPLICATIONS DELAYED EMERGENCE OF

DRUG RESISTANT VIRUS

ni = non-infectedwt = 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 r

eplic

atio

n

Primary resistance mutations

Secondary resistance mutations/ compensatory resistance mutations

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

Zoulim & Locarnini Gastroenterology 2009; Liver Int 2013

Pathway Amino Acid Substitutions in the rt Domain

LMV LdT ETV ADV TFV

Wild-type S S S S S

L-Nucleoside (LMV/LdT)

M204I/V R R I S S

Acyclic phosphonate (ADV)

N236T S S S R I

Shared (LMV, LdT, ADV)

A181T/V R R S R I

Double (ADV, TFV) A181T/V + N236T R R S R R

D-Cyclopentane (ETV)

L180M+M204V/I± I169 ± T184± S202 ± M250

R R R S S

Multi-Drug Resistance

A181T+N236T+ M250V

R R R R R

• 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 variantsViral quasispecies

cccDNA variants

LiverMajority population

Minority variants

Resistant variants

Blood circulation

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

• cccDNA in the liver:– Is propagated during the normal replication

cycle of HBV– Can serve as a template for the production of

new virus

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

Archiving of viral variants

Viral quasispecies

cccDNA variants

Blood circulation


Minority variants

Resistant variants


• cccDNA in the liver:– Is propagated during the normal replication

cycle of HBV– Can serve as a template for the production of

new virus

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

Archiving of viral variants

Viral quasispecies

cccDNA variants


Minority variants

Resistant 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.

Lab S

train

Clone

A

Clone

A

Clone

CClo

ne D

Clone

ESouthern blotanalysis

Patient serum

PCR cloning

Whole genomeHBV clones

Transfection

HepG2Huh7

IC50 reference strain

IC50 mutantFold 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 virusLAM rtM204V/I ± rtL180M

ETV-resistant virus

rtT184 or rtS202 or rtM250ETV

rtM204V/I rtL180M+/-

TDF

TDF: what can we expect?

rtM204V/I +/- rtL180M

LAMthen 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

(co

pie

s/m

l)

entecavirIFNadefovir

lamivudineGenotype H

lamivudine

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

L180M+S202G+M204V

L180M+M204V

Villet et al, J Hepatol 2007

Warner et al Hepatology 2009Kamili et al Hepatology 2009Villet 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

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

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

Prevention of resistanceImpact of first line therapy

• Choose an antiviral drug with

1. A potent antiviral activity

2. A high barrier to resistance

6

3

LVD ADV LdT ETV TDF0

10

20

30

40

50

60

70

80

23

Pro

port

ion

of p

atie

nts

(%)

46

55

71

80

0

11

18

29

5

25

0.2 0.51.2 0

1 2 3 4 5 1 2 3 4 5 1 2 1 2 3 4 5 1 2 3

0 0

Option to add emtricitabine at

week 72*

*Patients confirmed to be viraemic at Week 72 or beyond could add emtricitabine to TDF at the discretion of the investigator. Clinical data on the safety and efficacy of emtricitabine and TDF in CHB are pending

Rates of resistance with lamivudine (LVD), adefovir (ADV), telbivudine (LdT), entecavir (ETV) and tenofovir (TDF) among NA-naïve patients

4

0

High barrier to resistance

5

0

Gish, Jia, Locarnini, Zoulim, Lancet Infect Dis 2012

Management of partial response – The case of Entecavir

Zoutendijk et al, HepatologyVolume 54, Issue 2, pages 443-451, 25 JUL 2011

Kaplan-Meier curve for the probability of achieving virological response for 243 NA-naïve patients according to HBeAg status at baseline. P value was determined using log-rank testing.

http://onlinelibrary.wiley.com/doi/10.1002/hep.v54.2/issuetoc



Entecavir treatment for chronic hepatitis B: Adaptation is not needed for the majority of naïve patients with a partial virological response

Zoutendijk et al Hepatology Volume 54, Issue 2, pages 443-451, 25 JUL 2011

.Kaplan-Meier curve for the probability of achieving a VR for NA-naïve patients with a PVR according to HBV DNA at week 48. Three patients were switched to TDF plus emtricitabine, and one patient received TDF add-on therapy. P value was determined using log-rank testing.



Zoulim & Locarnini, Gastroenterology 2009; EASL CPG J Hepatol 2009 & 2012

Mangement of antiviral drug resistance• Impact of second line therapy

– Early treatment adaptation to prevent accumulation of mutations

– Choice always based on cross-resistance data

– Add-on strategy versus switch ?

• Good results with TDF switch

• Some cases of suboptimal responses

• Combination to increase the barrier to resistance

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

Zoulim & Locarnini Gastroenterology 2009; Liver Int 2013

Pathway Amino Acid Substitutions in the rt Domain

LMV LdT ETV ADV TFV

Wild-type S S S S S

L-Nucleoside (LMV/LdT)

M204I/V R R I S S

Acyclic phosphonate (ADV)

N236T S S S R I

Shared (LMV, LdT, ADV)

A181T/V R R S R I

Double (ADV, TFV) A181T/V + N236T R R S R R

D-Cyclopentane (ETV)

L180M+M204V/I± I169 ± T184± S202 ± M250

R R R S S

Multi-Drug Resistance

A181T+N236T+ M250V

R R R R R

Manns M, et al., EASL 2008; Oral # 1587.

Tenofovir efficacy in LAM Experienced vs. Naïve

Study 103:

N=176Study 102:

N=250 Total

LAM-Naïve, nLAM-Experienced, n

1688

20941

37749

• Study 102 actively enrolled both LAM experienced and LAM-naïve patients • Study 103 enrolled eight LAM experienced patients despite LAM-naïve inclusion criteria

P=0.718

ITT Missing=Failure

Combined data includes both HBeAg +/- patients

Reijnders, JGP et al. J Hepatol 2010

Virologic response to Entecavir according to Lamivudine exposure

% C

umul

ated

resp

onse

2 80 10 124 60

20

60

80

40

100LVD-naïve (N=118)

LVD-experienced without development of LVD-resistance (N=20)

LVD-experienced with a prior history of LVD-resistance (N=14)

LVD-experienced with LVD-resistant mutations at baseline (N=9)

P = 0.007

2 80 10 124 60

100

20

60

80

40

Reijnders, JGP et al.. J Hepatol. 2010

Virologic response to Entecavir according to Adefovir exposure

ADV-naïve (N=119)

ADV-experienced without development of ADV-resistance (N=30)ADV-experienced with ADV-resistant mutations at baseline (N=12)

% C

umul

ated

resp

onse

P = NS

29 29 29 29 27 26 24 24 33 33 33 31 30 29 27 2614 14 14 14 14 14 14 1411 11 11 11 10 10 10 1017 16 16 16 16 16 16 1612 12 12 12 12 11 10 10

n =n =n =n =n =n =

Response by Baseline Resistance at Week 168TDF vs. FTC/TDF for Treatment-Experienced Patients:

Weeks on Study

Berg et al, Gastroenterology 2010; Ms submitted

Patients heavily exposed to NUCs with low barrier to resistance – Risk of MDR selection

• Risk of multidrug resistance by sequential accumulation of resistance mutations

• Risk of partial response, even with the newest NUCs -> long-term impact ?

?

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

Liu et al, Antivir Ther. 2010;15(8):1185-90.

Sequential therapy with NUCs and the risk of MDR

Accumulation of multiple mutations on the same viral genome

Complete change of the viral quasi-species

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

L80VL80V + M204I

LVD

LVD+TDF LVD+ADV+TDF

Patient #1(67 months)










LVD+ADVADV

Impact of rtA181 and rtN236 mutations on antiviral drug efficacy and cross-resistance


BL W4 W12 W24 W36 W480%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

N236T

A181V + N236T

A181V

A181S + N236T

A181T + N236T

A181T

wt

#1051 (L180L/M,A181A/V/T,A194A/T,S202S/I,N236N/T)

Week

0 4 8 12 16 20 24 28 32 36 40 44 48

HB

V D

NA

(lo

g 10

cp

/mL

)

0

1

2

3

4

5

6

7

8

9

10

Viral load

BL viral load = 8.75logTreatment: TDF

Adherence : 95.2%

Patient 1051 data:

LLOD

Evolution of viral genome during Tenofovir therapy in patients who previously failed ADV

Impact of persisting low viremia levels on treatment outcome ?Impact of persisting resistant mutants ?

Lavocat et al, AASLD 2010 & Ms submitted

Virologic response to TDF according to ADV resistance mutations at baseline

The Australian Experience

Patterson S J et al. Gut 2011;60:247-254

Tenofovir + Emtricitabine in patients with treatment failure – treatment intensification

0

1

2

3

4

5

6

7

Baseline M3 M6 M12

Time after TDF+FTC initiation (months)

HBV

DN

A (lo

g10

IU/m

L)

HBV DNA kinetics after TDF+FTC initiation in 59 patients with treatment intensification

Si-Ahmed et al, Antiviral Research 2011Time to undetectable DNA (<50 IU/mL)

14121086420

Pro

babili

ty

1,0

,8

,6

,4

,2

0,0

≤ 4 logs

> 4 logs

Viral load initiation

Rescue therapy with ETV + TDF in CHB patients with advanced liver disease and complex viral resistance patterns or showing partial antiviral responses to preceeding therapies (Virgil network)

ETV + TDF combination in patients with treatment failure

Petersen J, et al. J Hepatol 2012.

HBV DNA Viremia

1002100310041005100610071008100910101011

Baseline 3 6 9 12 15 18 21 24

10 6

Δ 3 log10 c/mL reductionP=0.0001

LLoD

HB

V D

NA

[IU

/ml]

Months

Management algorithm

Antiviral treatment

Treatment failure

Viral load asssessment

Second line therapybased on cross-resistance data(Add-on or switch…)

Check compliance Primary non response

Switch to more potent drug

Viral genome sequence analysis

Wild type virus HBV drug resistant mutant

Check compliance

Zoulim and Perrillo, J Hepatol, 2008; EASL CPG J Hepatol 2012

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)

Suggested treatment adpatation in patients with treatment failure

Type of failure Treatment adaptation

Lamivudine resistance 1) add TFV (add ADV if TFV not available)2) a switch to TFV is also advised by some guidelines

Adefovir resistance 1) switch to TFV (if available) and a 2nd drug2) if no history of LMV, switching to ETV is also effective. 3) If rtN236T substitution, consider adding LMV, ETV, or LdT to the TFV or switch to TFV plus FTC4) If rtA181V/T substitution, alone or in combination with rtN236T, switch to TFV plus ETV

Telbivudine resistance 1) add TFV2) a switch to TFV has been considered in some guidelines

3) a switch to ADV is not recommended

Entecavir resistance add TFV

Tenofovir resistance 1) not been confirmed so far 2) genotyping and phenotyping required3) may add ETV

EASL CPG, J Hepatol 2009 & 2012; Zoulim & Locarnini Liver Int 2013

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 circulationviral load

No

v.

99

Ja

n.

00

Se

pt.

00

Ma

r. 0

1

Ju

ly 0

2

De

c.

02

De

c.

03

2.00E+02

3.00E+02

4.00E+02

5.00E+02

6.00E+02

7.00E+02

8.00E+02

9.00E+02

0.01

0.1

1

10

100

Viral load

HBs Ag

Lamivudine 100 mg/day

Clinical example of HBsAg clearance

HBs AgPositivity cut off: 0.05

Viral loadDetection treshold

Viral loadLog Copies/ml

HBs Ag IU/ml

* *

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

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

Cumulative Probability of HBsAg Loss During TDF Administration

Cum

ula

tive P

robability F

unction E

stim

ate

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10

0.11

0.12

Weeks on Study

0 12 24 36 48 64 80 96 108 120 132 144 156 168 180 192

10.8%

8.5%

• TDF-TDF• ADV-TDF

Switch to Open Label TDF

Cumulative probability of seroconversion to anti-HBs: 7.7% TDF-TDF 7.3% ADV-TDF

*Kaplan-Meier Heathcote E-J, et al., AASLD 2010; Poster #477.

• TDF-TDF• ADV-TDF

0 12 24 36 48 64 80 96 108 120 132 144 156 168 180 192

Weeks

Cu

mu

lati

ve P

rob

abil

ity

Fu

nct

ion

Est

imat

e 0.12

0.11

0.10

0.09

0.08

0.07

0.06

0.05

0.04

0.03

0.02

0.01

0.00

Percentage of TDF-TDF Patients with HBsAg Loss

Key Characteristic HBsAg Clearance by Year 4 n/N (%)

Genotype A or D 14/95 (15%)

HBV DNA ≥ 9 log10 copies/mL 12/75 (16%)

HBsAg ≥ 4.5 log10 IU/mL 14/90 (16%)

Knodell Necroinflammatory Score ≥ 9 13/114 (11%)

Heathcote E-J, et al., AASLD 2010; Poster #477.

No HBsAg loss in : Asian patientsHBeAg negative patientsGenotype B or C

High rate of HBsAg clearance among sustained responders to PEG-IFN-2a ± LAM

Marcellin et al. APASL 2009* Modified ITT analysis (missing = non response); § last observation carried forward

5 years post-treatment with PEG-IFN-2a ± LAM (N=230)

<10,000 cp/mL* <400 cp/mL* Cleared HBsAg§

Pat

ien

ts

(%)

21%

17%

12%

64%

0

5

10

15

20

25

30

Mason, W. S. et al. 2009 / 2010. J. Virol

Devons nous redéfinir la tolérance immunitaire et repenser les indications thérapeutiques ?

Observation d’une expansion clonale des hépatocytes

- Cellules qui n’expriment pas les antigènes viraux

- Diminution de la charge virale malgré l’absence de lésion hépatique mesurable

- L’une des premières étapes du CHC

Tolérance Immunitaire

- Presque tous les hepatocytes sont infectés - Viremies > 10E9 copies/mL - Devrions nous réaliser une biopsie lorsque la charge virale diminue sans élévation des ALAT ? Et penser à un traitement antiviral ?

Zoulim & Mason, W. S. Gut 2012

Why a need for new antiviral targets ?

• Current antivirals achieve viral suppression in the majority of patients

• But the rate of cccDNA / HBsAg loss is very low• Life-long therapy needed• HBsAg clearance is associated with a lower risk of

HCC development• Treatment with finite duration if:

– cccDNA control or loss– HBsAg loss

Zoulim, Antiviral Research 2012

Current treatment: sustained disease control achieved with NUCs/IFN in majority of patients

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

ALT, alanine aminotransferase; INF, interferon; NUCs, nucleos(t)ide analogues; PEG-INF, peginterferon α-2a;

Evolution of viral markers during NUC therapy

Wong et al, Clin Gastroenterol Hepatol 2013

New targets

Immune system

Zoulim, Antiviral Research, 2012

Zoulim, et al, Clinical Gastroenterology and Hepatology 2013

Hepatocyte turn-over

cccDNA silencing

cccDNA destruction

Zoulim et al, Gastroenterology 2013

Antiviral activity of a TLR7 agonist in HBV infected chimpanzees

Lanford et al, Gastroenterology 2013

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

CD8 T cells

Infected hepatocytes Infected hepatocytes

INF-gTNF-aIL-2

GranzymePerforin

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;143:1576–85Figure 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.

Perspectives of anti-HBV immune therapy

The concept of combination therapy

Entry inhibition

cccDNA- formation- stability / destruction- epigenetic regulation

Viral core functions

Other viral targets

Stimulating innate responsesSpecific ligands

Stimulating adpative responsesCo-inhibitory signals

Co-stimulatory signals

Therapeutic vaccinationFunctional cure / controlReal cure ?

Viral targets Immune modulation

Conclusions 1• Maladie fréquente et grave

– 300 000 porteurs chroniques en france– 1ère cause de cancer du foie dans le monde– 1300 décès par an en France

• Maladie méconnue– Souvent asymptomatique, ou symptomes non spécifiques– Seulement 60 000 personnes connaissent leur maladie– 15 000 sont traitées

• Persistance virale– Pas d’éradication du génome viral– Surveillance prolongée, possibilité de réactivations

Conclusions 2

• Différentes formes d’hépatites en fonction de l’interaction virus / réponse immunitaire– Portage asymptomatique / hépatite chronique / cirrhose /

cancer du foie

• Impact de la variabilité du génome viral- Role dans la persistance virale et la résistance aux antiviraux

- Echappement diagnostique

• Nécessité d’un dépistage et traitement précoce des formes chroniques

• Prévention par la vaccination !!!

Acknowledgements

Hepatology Unit INSERM U1052 Collaborations

David DurantelBarbara TestoniMalika Ait-GoughoulteSouphalone LuangsayMarion GruffazNathalie IsorceFanny LebosséMaelenn FournierJulie LuciforaMaud MicheletJudith Fresquet

LabEx

C. Caux, Lyon CRCLU. Hasan, Lyon CIRIT. Henry, Lyon CIRIFL. Cosset, Lyon CIRIM. Levrero, RomeM. Tommasino, IARC

IHU

Potential risk of transmission of HBV DR mutants

Clements et al, Bull WHO 2009

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

L80VL80V + M204I

LVD

LVD+TDF LVD+ADV+TDF











LVD+ADVADV

Impact of rtA181 and rtN236 mutations on antiviral drug efficacy and cross-resistance


Month of therapy

Rescue therapy in patients with clinical breakthrough

Drug A

Drug B

Seru

m H

BV

DN

A (

L og1

0 c

opi e

s /m

L )

an

d A

LT (

x U

L N)

Month of therapy

Rescue therapy in patients at the time of virologic breakthrough

Drug A

Drug B

Seru

m H

BV

DN

A (

L og1

0

c op

i es/

mL )

an

d A

LT (

x U

L N)

Month of therapy

Early add-on therapy to prevent drug resistance

Drug A

Drug B

Seru

m H

BV

DN

A (

L og1

0 c

opi e

s /m

L )

an

d A

LT (

x U

LN)

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

Drug ADrug A

+Drug B

Month of therapy

1. Start with a drug having a high genetic barrier for resistance2. Add a drug with a different cross-resistance profile

outgrowth of drug resistant mutant ?

MDR ?

RA

ND

OM

IZAT

ION

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

P

e

r

c

en

t

age

(

%

)

0

10

20

30

40

50

60

70

80

90

100

Weeks on Study

0 4 8 12 24 36 48 60 72 84 96 108 120 132 144 156 168

TreatmentTDFFTC/TDF

P

e

r

c

en

t

age

(

%

)

0

10

20

30

40

50

60

70

80

90

100

Weeks on Study

0 4 8 12 24 36 48 60 72 84 96 108 120 132 144 156 168

P

e

r

c

en

t

age

(

%

)

0

10

20

30

40

50

60

70

80

90

100

Weeks on Study

0 4 8 12 24 36 48 60 72 84 96 108 120 132 144 156 168

TreatmentTDFF

TreatmentTDFFTC/TDF

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

‡

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

Per

cen

tage

(%

)

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

Study 106 – Treatment-Experienced Patients

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

Virologic response to TDF according to ADV resistance mutations at baseline - The Australian Experience

Patterson S J et al. Gut 2011;60:247-254

BL W4 W12 W24 W480%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

A181T

A181T + N236T

wt

#1046 (A181A/T/V,N236N/T)

Week

0 4 8 12 16 20 24 28 32 36 40 44 48

HB

V D

NA

(lo

g 10

cp

/mL

)

0

1

2

3

4

5

6

7

8

9

10

Viral load

BL viral load = 6.85logTreatment: TDFAdherence : 68%

Patient 1046 data:

LLOD

Evolution of viral genome during Tenofovir therapy in patients who prevously failed ADV

Patient #1046

Lavocat & Zoulim, AASLD 2010.

BL W4 W12 W24 W36 W480%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

N236T

A181V + N236T

A181V

A181S + N236T

A181T + N236T

A181T

wt

#1051 (L180L/M,A181A/V/T,A194A/T,S202S/I,N236N/T)

Week

0 4 8 12 16 20 24 28 32 36 40 44 48

HB

V D

NA

(lo

g 10

cp

/mL

)

0

1

2

3

4

5

6

7

8

9

10

Viral load

BL viral load = 8.75logTreatment: TDFAdherence : 95.2%

Patient 1051 data:

LLOD

Evolution of viral genome during Tenofovir therapy in patients who prevously failed ADV

Patient #1051

Impact of persisting low viremia levels on treatment outcome ?

Lavocat & Zoulim, AASLD 2010.

nucleus

cccDNA

PreC/pgRNA

AAAAAA

AAAAAA

Pre-S1Pre-S2

X

core

polymerase

pgRNA

surface proteins

rcDNA

HBeAg

HBsAg

HBx

1. entry

2. trafficking

3. cccDNA formation

4. transcriptionviral RNA

5. translation

6. nucleocapsid formation pgRNA packaging

8a. morphogenesis and secretion

7. DNA synthesis

8b. trafficking

endosome

9. integration into the host DNA

NK cells

PRRs

Type I IFN induction

Innate responses

CD8+ cells

B cells

CD4+ cells

Adaptive immune responses

Zoulim, Antiviral Research, 2012

Zoulim

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