Zoulim Du 2009

Hépatite BHépatite B

Fabien ZoulimFabien Zoulim

Département d’hépatologie Département d’hépatologie

& INSERM U871, Lyon& INSERM U871, Lyon

VHBVHB HCAHCA cirrhose cirrhose CHCCHC

VaccinVaccin ANTIVIRAUXANTIVIRAUXIFNIFN

Antiviraux/IFN?Antiviraux/IFN?Niederau Niederau N Engl J Med 1996N Engl J Med 1996 & Liaw & Liaw N Engl J Med 2004N Engl J Med 2004

RESISTANCE VIRALERESISTANCE VIRALE

30-50 ans30-50 ansGuérison Guérison

Lee, N Engl J Med 1997; Lok, Hepatology 2001Lee, N Engl J Med 1997; Lok, Hepatology 2001

EPIDEMIOLOGIE DE L’HÉPATITE BEPIDEMIOLOGIE DE L’HÉPATITE B

EPIDEMIOLOGIE DE L'INFECTION A VHBEPIDEMIOLOGIE DE L'INFECTION A VHB

• Hépatites aigues Hépatites aigues

– VHA : 40%VHA : 40%

– VHB : 30%VHB : 30%

– VHC : 20%VHC : 20%

• incidence : 300 000 infections à VHB / anincidence : 300 000 infections à VHB / an

• 30 000 nouveaux porteurs chroniques / an30 000 nouveaux porteurs chroniques / an

• 3 000 décès / an3 000 décès / an

AUX USAAUX USA

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

sexuellesexuelle34%34%

hétérohétéro23%23%

homohomo11%11%

drogue IVdrogue IV26%26%

inconnueinconnue31%31%

hémodialyséshémodialysés8%8%

transfusionstransfusions2%2%

personnels de santé personnels de santé 2%2%

contact aveccontact avecporteur du VHBporteur du VHB

4%4%

AsieAsieTransmission verticaleTransmission 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

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

Taux /100 000

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 patientPartie 2 : information biologiqueParties 3-4-5 : information clinique et épidémiologiqueParties 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

158 acute hepatitis cases158 acute hepatitis cases

• Hospital doctor in 64% cases Hospital doctor in 64% cases

• Sex ratio M/F : 2,95 (118/40)Sex ratio M/F : 2,95 (118/40)

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

• Jaundice : 69%Jaundice : 69%

• Hospitalisation : 46%Hospitalisation : 46%

• Fulminant hepatitis : 3 (2 death)Fulminant hepatitis : 3 (2 death)

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- 94n= 151

March 03- February 04n= 158

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

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

– SexualSexual 5959 40,6%40,6% No factorNo factor 4343 29,6%29,6%– IVDUIVDU 99 6,2% 6,2% >1 factor>1 factor 3838 26,3%26,3%– Invasive treatment Invasive treatment 1515 10,3%10,3% – Tatoo, piercing Tatoo, piercing 55 3,4% 3,4%– FamilialFamilial 1414 9,7% 9,7%– Perinatal 2Perinatal 2 1,4% 1,4%– Live in instiution Live in instiution 1111 7,6% 7,6%– Travel in endemic 21Travel in endemic 21 14,5% 14,5%

areasareas

91/145 patients (63 %) had a vaccine indication (2 vaccinated ≥ 3 doses) 91/145 patients (63 %) had a vaccine indication (2 vaccinated ≥ 3 doses)

• Sentinel networks 91-96 Sentinel networks 91-96 N=195 N=195 – sexualsexual 35% 35% – IVDUIVDU 19%19%– « percutaneous » « percutaneous » 15%15%– No factor No factor 35% 35%

Hépatites virales B: épidémiologie

- Vaccin mais 400 millions de porteurs chroniques dans le monde

- 300 000 porteurs chroniques en France (INVS)

- 1 300 décès par an en France

- 60 000 avec hépatite chronique active

- Seulement 13 000 patients traités

VIROLOGIEVIROLOGIE

• 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

LE GÉNOME DU VIRUS DE L’HÉPATITE BLE GÉNOME DU VIRUS DE L’HÉPATITE B

déterminant adéterminant avaccin/IgHBsvaccin/IgHBs

Gène pol Gène pol antivirauxantiviraux

Mt pre-coreMt pre-coreRéponse anti-HBeRéponse anti-HBe ??

Mt du coreMt du coreRéponse CTLRéponse CTL

8 génotypes8 génotypesA to HA to H

Tiollais Nature 1985Tiollais Nature 1985Günther Adv Virus Res 1999Günther Adv Virus Res 1999Norder J Gen Virol 2003Norder J Gen Virol 2003

Ganem & Prince, NEJM 2004Ganem & Prince, NEJM 2004

ARN pg

ss DNA

RC DNA

cccDNA

intégration

virion

virion10%

90%

ds DNA

cccDNAillégitime

noyau

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

Membrane cellulaire

VHB HUMAINVHB HUMAIN

MARMOTTE (WHV)MARMOTTE (WHV) CANARD (DHBV)CANARD (DHBV)

QuickTime™ et un décompresseurPhoto - JPEG sont requis pour visualiser

cette image.

Modèles AnimauxModèles Animaux

Souris TransgéniquesSouris TransgéniquesSouris SCID uPaSouris SCID uPa

ChimpanzéChimpanzé

TupaiaTupaia

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

• Polymerase viralePolymerase virale

– DHBV : lysat réticulocytaireDHBV : lysat réticulocytaire

– HBV : baculovirusHBV : baculovirus

Modèles Modèles in vitroin vitro

UUPolymerase VHBPolymerase VHB

DNA(-)DNA(-)

ELONGATIONELONGATIONCCC -CCC -

RC -RC -L -L -

SS -SS -

• Culture cellulaireCulture cellulaire

– Transfection : lignées d’hépatomeTransfection : lignées d’hépatome

– Infection : hépatocytes primaires, HepaRGInfection : hépatocytes primaires, HepaRG

– Baculovirus ou adenovirus recombinantBaculovirus ou adenovirus recombinant

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

interaction

virion

noyau

hépatocyte

traduction

encapsidation transcription inverse

ARNpgADN (-)

amplificationde l’ADNccc

transcription

ARNm

ARNpgAAA

AAAAAA

AAAADNcccADNccc

formation d’ADNccc

ADN RC

entrée

polymérase

ADN (+)

Synthèse du brin (+)

sécrétion des virions

RE

Ag HBe

Ag HBs

récepteur ?

RE

sécrétion desprotéines virales

Cycle de réplication du HBV

Analogues de nucléosides

ADN RC

Comparative dynamics among three viruses Comparative dynamics among three viruses

(Tsiang et al. Hepatology 1999)

Infection à VHB et risque de CHCInfection à VHB et risque de CHC

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

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

• 12,5% chez 240 patients avec cirrhose12,5% chez 240 patients avec cirrhose

• 3,8% chez 677 patients avec hépatite chronique3,8% chez 677 patients avec hépatite chronique

– risque x 7 si AgHBs +risque x 7 si AgHBs +

– risque X 4 si anti-HCV +risque X 4 si anti-HCV +

• Facteurs associés : alcool, tabac, aflatoxineFacteurs associés : alcool, tabac, aflatoxine

• Diminution incidence avec la vaccination de masse Diminution incidence avec la vaccination de masse (Chen, (Chen,

NEJM 1995)NEJM 1995)

CARCINOME HEPATOCELLULAIRE ET VIRUS CARCINOME HEPATOCELLULAIRE ET VIRUS DE L'HEPATITE B DE L'HEPATITE B

• Co-incidence de répartition géographique Co-incidence de répartition géographique

VHB / CHCVHB / CHC

• Porteurs AgHBs : RR x 100 pour le CHCPorteurs AgHBs : RR x 100 pour le CHC

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

– marmottemarmotte

– écureuilécureuil

• Présence d'ADN viral intégré dans les tumeursPrésence d'ADN viral intégré dans les tumeurs

HBV replication and its role in HCC development

Wands, NEJM 2004

PATHOGENIE DU CARCINOME PATHOGENIE DU CARCINOME HEPATOCELLULAIREHEPATOCELLULAIRE

VHBVHB ALCOOLALCOOLVHCVHC

LESIONS HEPATIQUES CHRONIQUESLESIONS HEPATIQUES CHRONIQUES

ACTIVATION FACTEURS ACTIVATION FACTEURS DE CROISSANCEDE CROISSANCE

REGENERATIONREGENERATION

ALTERATIONS GENETIQUESALTERATIONS GENETIQUES

CARCINOME HEPATOCELLULAIRECARCINOME HEPATOCELLULAIRE

DESORDRES DESORDRES METABOLIQUESMETABOLIQUES

FACTEURSFACTEURSENVIRONNEMENTAUXENVIRONNEMENTAUX

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 / PHYSIOPATHOLOGIE / IMMUNOPATHOLOGIEIMMUNOPATHOLOGIE

Ganem and Prince, NEJM 2004Ganem and Prince, NEJM 2004

HÉPATOCYTE INFECTÉHÉPATOCYTE INFECTÉ

VHBVHB

CTLCTL

FasFasperforineperforine

HÉPATOCYTEHÉPATOCYTENON INFECTÉ NON INFECTÉ

IMMUNOPATHOGÉNIE IMMUNOPATHOGÉNIE DES HÉPATITES B CHRONIQUESDES HÉPATITES B CHRONIQUES

AgHBc/eAgHBc/e

HLAIHLAI

cytokinescytokines

RÉPONSE IMMUNITAIRERÉPONSE IMMUNITAIRECYTOKINESCYTOKINES

ANTIVIRAUXANTIVIRAUX

ANTICORPS NEUTRALISANTSANTICORPS NEUTRALISANTS

IMMUNOPATHOLOGY OF HBV INFECTIONIMMUNOPATHOLOGY OF HBV INFECTION

Immune toleranceImmune tolerance

Clairance phaseClairance phaseChronic hepatitisChronic hepatitis

SeroconversionSeroconversionRemission Remission

CD8+CD8+

HBVHBV

CD8+CD8+ HBVHBV

CD8+CD8+HBVHBV

Immunopathology

Fulminant hepatitisFulminant hepatitis

CD8+CD8+

HBVHBV

Non cytolytic processesNon cytolytic processesTH1 cytokines with direct antiviral TH1 cytokines with direct antiviral

effecteffect

Turn-over of infected cellsTurn-over of infected cellsImmune mediated lysis of infected cellsImmune mediated lysis of infected cells

DucksDucks

WoodchucksWoodchucks(Guo J Virol 1999(Guo J Virol 1999

Summers PNAS 2003&2004)Summers PNAS 2003&2004)

Transgenic miceTransgenic miceChimpanzeesChimpanzees

(Guidotti Science 1999, (Guidotti Science 1999, Thimme J Virol 2003)Thimme J Virol 2003)

AntiviralsAntiviralsInhibit ion of viral DNA synthesisInhibit ion of viral DNA synthesis-> inhibit ion of intracellular recycling of -> inhibit ion of intracellular recycling of cccDNAcccDNA(Werle Gastroenterology 2004)(Werle Gastroenterology 2004)Restoration of anti-HBV immune responseRestoration of anti-HBV immune response(Boni Hepatology 2000)(Boni Hepatology 2000)

MECHANISMS OF VIRAL CLEARANCEMECHANISMS OF VIRAL CLEARANCE

Non cytolytic clearance of acute Non cytolytic clearance of acute HBV infection in chimpanzeeHBV infection in chimpanzee

Wieland S et al, PNAS 2004

Hepatocyte turn-over is required for clearance of Hepatocyte turn-over is required for clearance of viral infection in acute infectionviral infection in acute infection

Summers et al, PNAS 2003 & 2004Summers et al, PNAS 2003 & 2004

Hépatocyte infectéHépatocyte infecté

HBVHBV

Hépatocyte Hépatocyte non infecté non infecté

Phase de tolérance immunitairePhase de tolérance immunitaire

MarqueursMarqueursAgHBe +AgHBe +HBV DNA +++HBV DNA +++ALAT = NALAT = NFoie = NFoie = N

HBc/e AgHBc/e Ag


HBVHBV

CTLCTL

FasFasperforineperforine


Phase de clairance immunePhase de clairance immune(hépatite chronique)(hépatite chronique)

MarqueursMarqueursAgHBe+AgHBe+HBV DNA +HBV DNA +ALAT +++ALAT +++Foie: Foie: Hépatite Hépatite chroniquechronique

HBc/e AgHBc/e Ag

HLAIHLAI

cytokinescytokines


HBs AgHBs Ag


MarqueursMarqueursAgHBe-AgHBe-anti-HBe +anti-HBe +HBV DNA < 10HBV DNA < 1044 /mL /mLALAT = NALAT = NFoie = rémissionFoie = rémission

Phase de rémissionPhase de rémissionportage inactif de l’AgHBs portage inactif de l’AgHBs

RéactivationRéactivationVirus sauvage Virus sauvage ou mt pre-coreou mt pre-coreOncogénèseOncogénèse

Hépatocytes infectésHépatocytes infectés

Hépatocytes Hépatocytes non infectésnon infectés

MarqueursMarqueursHBsAg -HBsAg -

anti-HBc +anti-HBc +Anti-HBs +/-Anti-HBs +/-

HBV DNA - mais PCR +HBV DNA - mais PCR +

Clairance de l’AgHBsClairance de l’AgHBs

Mutants d’échappementMutants d’échappementInfections occultesInfections occultes

OncogénèseOncogénèse

cccD

NA

cc

cDN

A (c

op

ies/

cell)

(co

pie

s/ce

ll)

To

tal

HB

V D

NA

T

ota

l H

BV

DN

A

(co

pie

s/ce

ll)(c

op

ies/

cell)

cccDNA levels in the different phases of cccDNA levels in the different phases of chronic HBV infectionchronic HBV infection

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

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

HBeAg+ (63)

HBeAg+ (63)

Inact.

Carriers

(10)

Inact.

Carriers

(10)

HBSAg- (7)

HBSAg- (7)

HBeAg- (18)

HBeAg- (18)

HBeAg+ (63)

HBeAg+ (63)

Inact.

Carriers

(10)

Inact.

Carriers

(10)

HBSAg- (7

HBSAg- (7)

HBeAg- (18)

HBeAg- (18)

Werle et al, Gastroenterology 2004

HISTOIRE NATURELLE ET VIROLOGIE CLINIQUE

Histoire Naturelle de l’hépatite BHistoire Naturelle de l’hépatite BInfection aigueInfection aigue

Infection chroniqueInfection chronique

Tolérance immunitaireTolérance immunitaire

Hépatite chroniqueHépatite chronique

Portage inactifPortage inactif

GuérisonGuérison5% nx-nés5% nx-nés90% adultes 90% adultes

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

Cirrhose Cirrhose

Carcinome hépatocellulaireCarcinome hépatocellulaire

Réactivation Réactivation

30-50 ans30-50 ans

Seeger, Zoulim, Mason; Fields Virology; 2007

MARQUEURS SEROLOGIQUESMARQUEURS SEROLOGIQUES

• Système AgHBe /anti-HBeSystème AgHBe /anti-HBe

– distinction virus sauvage / virus muté AgHBe distinction virus sauvage / virus muté AgHBe

négatifnégatif

• VirémieVirémie

– détection quantitative de l'ADN viraldétection quantitative de l'ADN viral

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

– Évolution plus fréquente vers la chronicitéÉvolution plus fréquente vers la chronicité

• Prodromes:Prodromes:– Maladie sérique : arthralgies, urticaire, Maladie sérique : arthralgies, urticaire,

acrodermatite etc. ..acrodermatite etc. ..

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

• Evolution : chronicité 5 à 10%Evolution : chronicité 5 à 10%• Hépatites fulminantesHépatites fulminantes

Laboratory Diagnosis of Acute Hepatitis B

0

100200

300

400

500600

700

800900

1000

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

ALT

HBsAg

HBeAg

HBV DNA

Normal

Months After Exposure

AL

T a

nd

HB

V D

NA

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

• DéfinitionDéfinition– Persistance réplication virale à la 8ème Persistance réplication virale à la 8ème

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

• EvolutionEvolution– Chronicité : 8 cas / 10Chronicité : 8 cas / 10

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

INFECTIONS CHRONIQUES A VHBINFECTIONS CHRONIQUES A VHBFORMES CLINIQUESFORMES CLINIQUES

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

5-10% /an5-10% /an– > diminution significative réplication virale> diminution significative réplication virale– > amélioration signes histologiques> amélioration signes histologiques

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

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


AL

T a

nd

HB

V D

NA

IU/ L

or

mil l

ion

co

pi e

s/m

l

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

Diapositive 25


ALT

``HBsAg

HBeAg

HBV DNA

Normal


AL

T a

nd

HB

V D

NA

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


0

50

100

150

200

250

300

350

400

450

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

AL

T a

nd

HB

V D

NA

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 +AgHBe + anti-HBe +anti-HBe +UI/mlUI/mlpg/mlpg/ml

AgHBsAgHBs

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

hybridationhybridation

PCRPCR

9 log

8 log

7 log

6 log

5 log

4 log

3 log

2 log

1 log

HBV DNA concentration in log IU/mL

Ranges of quantitative HBV DNA assays

0 2 4 6 8 10

COBAS Amplicor HBV Monitor

COBAS Taqman 48 HBV

Amplicor HBV MonitorRoche Molecular Systems

Bayer Corp.

Versant HBV DNA 3.0

Versant HBV DNA 1.0

Digene Corp.

HBV Digene Hybrid Capture I

Artus Biotech

HBV Digene Hybrid Capture II

Ultra-Sensitive Digene Hybrid Capture II

Real Art HBV PCR AssayAbbott Molecular

ABBOTT real time HBV DNA assay

Formes cliniques

MANIFESTATIONS MANIFESTATIONS EXTRAHEPATIQUES DU VHBEXTRAHEPATIQUES DU VHB

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

(vidarabine / IFN / famciclovir / lamivudine)(vidarabine / IFN / famciclovir / lamivudine)

• GlomérulonéphritesGlomérulonéphrites• CryoglobulinémiesCryoglobulinémies• Guillain-BarréGuillain-Barré• MyocarditeMyocardite

TRANSMISSION VERTICALE DU VHBTRANSMISSION VERTICALE DU VHB

• mère AgHBe +mère AgHBe +

– transmission : 90%transmission : 90%

• mère anti-HBe +mère anti-HBe +

– transmission : 10-20%transmission : 10-20%

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

• chronicité chez l’enfant : 90%chronicité chez l’enfant : 90%

PRESENTATION CLINIQUEPRESENTATION CLINIQUE• INFECTION PERI-NATALEINFECTION PERI-NATALE

– ALT normales ou subnormalesALT normales ou subnormales

– ADN-VHB > 1000 pg/mlADN-VHB > 1000 pg/ml

– histologie : lésions minimeshistologie : lésions minimes

• INFECTION POST-NATALEINFECTION POST-NATALE

– ALT élevéesALT élevées

– ADN-VHB < 1000 pg/mlADN-VHB < 1000 pg/ml

– histologie : hépatite modérée à sévèrehistologie : hépatite modérée à sévère

• CARCINOME HEPATOCELLULAIRE : 30 ANSCARCINOME HEPATOCELLULAIRE : 30 ANS

Histoire naturelle de l’infection chronique par le virus de l’hépatite B

en Alaska• McMahon BJ, Ann Intern Med 2001;135(9):759-68McMahon BJ, Ann Intern Med 2001;135(9):759-68• 1536 natifs d’Alaska : 641 AgHBe+, 83 anti-HBe+1536 natifs d’Alaska : 641 AgHBe+, 83 anti-HBe+• Probabilité d’éliminer l’Ag HBe à 10 ans : 72,5 %.Probabilité d’éliminer l’Ag HBe à 10 ans : 72,5 %.• Elimination de l’Ag HBs chez 106 porteurs Elimination de l’Ag HBs chez 106 porteurs

chroniques du VHB (7 %) chroniques du VHB (7 %) • Incidence des événements cliniques: 2,3/1000 Incidence des événements cliniques: 2,3/1000

porteurs/année porteurs/année • Incidence du CHC: 1,9/1000 porteurs/année (2,3 chez Incidence du CHC: 1,9/1000 porteurs/année (2,3 chez

l’homme; 1,2 chez la femme). l’homme; 1,2 chez la femme).

Pathophysiologic Cascade of Chronic HBV Infection

HBV ReplicationHBV Replication(Measured by (Measured by

Serum HBV DNA)Serum HBV DNA)

HBV ReplicationHBV Replication(Measured by (Measured by

Serum HBV DNA)Serum HBV DNA)

Liver Liver InflammationInflammation

Liver Liver InflammationInflammation

Worsening HistologyWorsening Histology• NecroinflammationNecroinflammation• FibrosisFibrosis• CirrhosisCirrhosis

Worsening HistologyWorsening Histology• NecroinflammationNecroinflammation• FibrosisFibrosis• CirrhosisCirrhosis

Disease ProgressionDisease Progression• Liver FailureLiver Failure• Liver CancerLiver Cancer• TransplantTransplant• DeathDeath

Disease ProgressionDisease Progression• Liver FailureLiver Failure• Liver CancerLiver Cancer• TransplantTransplant• DeathDeath

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 ALT ElevationElevation

ALT ALT ElevationElevation

Normal Aminotransferase Levels and Risk of Mortality from Liver

Diseases

Kim HC et al. Kim HC et al. BMJBMJ 2004; 328:983 2004; 328:983

0 10 20 30 40 50 60 70 80 90

Risk ratio (95% CI)

<20

20-29

30-39

40-49

50-99

>100

ALT

1.01.0

2.92.9

9.59.5

19.219.2

30.030.0

59.059.0

NormalNormal

ElevatedElevated

• Korea Medical Insurance Corporation– 94,533 men; 47,522 women

– 35-59 yrs old

– Relative risk for liver mortality compared with AST and ALT <20 IU/l

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

AgHBeAg et risque de CHC

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

Cu

mu

lati

ve i

nci

den

ce (

%)

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

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

80%

84%

88%

92%

96%

100%

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

Survival time (Years)

Survival distribution function

HBV DNA Negative

HBV DNA LowHBV DNA Low< 10< 1055 copies/mL copies/mL RR = 1.7 (0.5-5.7)RR = 1.7 (0.5-5.7)

HBV DNA HighHBV DNA High≥ ≥ 101055 copies/mL copies/mL

RR = 11.2 (3.6-35.0)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

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 VHBVARIABILITE GENETIQUE DU VHB

• Multiplication viraleMultiplication virale

» taux d'erreur de la transcriptase inversetaux d'erreur de la transcriptase inverse

• Pression de sélectionPression de sélection

» réponse immunitaire cellulaire / humoraleréponse immunitaire cellulaire / humorale

» antivirauxantiviraux

-> possibilité de variants d'échappement-> possibilité de variants d'échappement

• Conséquences cliniquesConséquences cliniques

» diagnostic sérologiquediagnostic sérologique

» traitements antivirauxtraitements antiviraux

• SOUS-TYPES : acides aminés et déterminants HBsSOUS-TYPES : acides aminés et déterminants HBs

– boucle 139-147 -> det aboucle 139-147 -> det a

– 122 -> det d ou y122 -> det d ou y

– 127 -> det w1-4127 -> det w1-4

– 160 -> det w ou r160 -> det w ou r

• GENOTYPES : variabilité de séquence génomique GENOTYPES : variabilité de séquence génomique

– du génome complet : 8%du génome complet : 8%

– du gène S : 4%du gène S : 4%

– 8 génotypes A à H8 génotypes A à H

• MUTANTS DU VHBMUTANTS DU VHB

– mutations ponctuelles / délétions / insertionsmutations ponctuelles / délétions / insertions

VARIABILITE GENETIQUE DU VHBVARIABILITE GENETIQUE DU VHB

8 genotypes, numerous sub-genotypes, and recombinant forms

World J Gastroenterol 2007; 13: 14-21

D/E

B6

D1

Génotypes VHB chez les patients atteints Génotypes VHB chez les patients atteints d’hépatite chronique en Franced’hépatite chronique en FranceN

umbe

r of

sub

ject

sN

umbe

r of

sub

ject

s

FF GGAA BB CC DD EE00

1010

2020

3030

4040

5050

6060

7070

8080

9090

100100

30.2%30.2%

7.9%7.9%

12.5%12.5%

37.4%37.4%

11.3%11.3%

0.4 %0.4 % 1.1%1.1%

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%

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 VHBLES MUTANTS DU GÉNOME DU VHB

déterminant adéterminant avaccin/HBIgvaccin/HBIg

polymérasepolyméraseantivirauxantiviraux

Mt pré-coreMt pré-coreRéponse anti-eRéponse anti-e ??

Mt coreMt coreRéponse CTLRéponse CTL

ROLE DE LA RÉGION PRÉ-C ET DE L’AgHBeROLE DE LA RÉGION PRÉ-C ET DE L’AgHBe

• Non nécessaire à la réplication du VHBNon nécessaire à la réplication du VHB– Culture cellulaireCulture cellulaire

– Modèles in vivo Modèles in vivo • MarmotteMarmotte• CanardCanard

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

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

LES MUTANTS PRÉ-C (-)LES MUTANTS PRÉ-C (-)

• codon stop / région pré-Ccodon stop / région pré-C

TGG -> TTGG -> TAAG en pos. 1896G en pos. 1896

– génotypes B à E (A : exceptionnel)génotypes B à E (A : exceptionnel)

– arrêt traduction protéine pré-C/C arrêt traduction protéine pré-C/C

– AgHBe négatifAgHBe négatif

• mutation dans promoteur pré-Cmutation dans promoteur pré-C

TTAAAGG -> TTAATTAAAGG -> TTAATTGGAA en pos. 1762 /1764 en pos. 1762 /1764

– génotypes A à Egénotypes A à E

– transcrits pré-C/C :transcrits pré-C/C :

– synthèse d'AgHBe :synthèse d'AgHBe :

Carman et al Lancet 1989, Okamoto et al J Virol 1990/1994, Tong et al Virology 1990Carman 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 ’AgHBeVARIANTS NÉGATIFS POUR L ’AgHBe

mRNAmRNA

ProtéineProtéinepré-C/Cpré-C/C

PRE-CPRE-C CCPROMOTEURPROMOTEUR

TAGTAG******

1762-17641762-1764 18961896

arrêt des synthèses protéiquesarrêt des synthèses protéiques

Diminution de l’expression de l ’AgHBeDiminution de l’expression de l ’AgHBe

Main pre-c/core promoter mutations observed in vivo

GGGGGAGGAGATTAGGTTAAAGGTCTTTGTATTAGGAGGCTGTAGGCATAAATT

Pre-C mRNAPre-C mRNA

Basic core promoter

17621762 6464

TTGGAA

LEF

HNF1GGTTAATNATTA

HNF4AGGTCA

TTTTAA

6666 6868

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

Mutation AGG to TCA and insertion TA at 65/66

WTRTTKRY

Insertion (TTG) at 66/67

TTTTGGHNF3

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

0500

1000150020002500

temps

ALATALAT

ADN-VHBADN-VHB

AgHBeAgHBe Anti-HBeAnti-HBe

0

20

40

60

80

100

temps

sauvagesauvage

Mt pré-CMt pré-C

Outcome of Chronic Anti-HBe Positive Hepatitis B

0

100

200

300

400

0

100

200

300

400

0

100

200

300

400

Biochemical patterns in 164 untreated patientsBiochemical patterns in 164 untreated patientsafter 23 months (range 12-36) monthly monitoringafter 23 months (range 12-36) monthly monitoring

00 1212 2424monthsmonths

With flares and normalizationWith flares and normalization

Without flaresWithout flares

With flares and without normalizationWith flares and without normalization

73 pts 73 pts ( 44.5% )( 44.5% )

59 pts 59 pts ( 36.0% )( 36.0% )

32 pts 32 pts ( 19.5% )( 19.5% )

Asymptomatic Asymptomatic flare-up: flare-up:

90% of cases90% of cases

AALLTT Flare-up yearlyFlare-up yearly

frequency:frequency:once 57.1%once 57.1%twice 20%twice 20%

< once 22.8%< once 22.8%

Brunetto MR et al, J Hepatol 2002Brunetto MR et al, J Hepatol 2002

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

No pre-core mutationNo pre-core mutation (n = 42; 14.8%) (n = 42; 14.8%)

Both mutationsBoth mutations (n = 95; 33.6%) (n = 95; 33.6%)

Promoter mutation Promoter mutation (n = 99; 27.9%) (n = 99; 27.9%)

Stop codon mutation Stop codon mutation (n = 55; 19.4%) (n = 55; 19.4%)

Data unavailableData unavailable (n = 12; 4.2%) (n = 12; 4.2%)

Pre-core mutationsPre-core mutations

Lamivir cohort, Zoulim et al, J Viral Hepatitis 2006

HBe serotype and pre-core mutationsHBe serotype and pre-core mutations

00

1010

2020

3030

4040

5050

6060

7070

8080

9090

No pre-coreNo pre-coremutationmutation

Stop codonStop codonmutationmutation

Promoter Promoter mutationmutation

BothBothmutationsmutations

Num

ber

of s

ubje

cts

Num

ber

of s

ubje

cts

HBe-positiveHBe-positive

HBe-negativeHBe-negative


MUTANTS PRÉ-C ET SÉVÉRITÉ HISTOLOGIQUEMUTANTS PRÉ-C ET SÉVÉRITÉ HISTOLOGIQUELA CONTROVERSELA CONTROVERSE

• ItalieItalie– Cirrhose plus fréquenteCirrhose plus fréquente

• Bonino Gastroenterology 1986, Fattovich Hepatology 1988Bonino Gastroenterology 1986, Fattovich Hepatology 1988

• FranceFrance– Activité idem / cirrhose plus fréquenteActivité idem / cirrhose plus fréquente

• Zarski et al, J Hepatol 1993Zarski et al, J Hepatol 1993• Grandjacques et al, J Hepatol 2000Grandjacques et al, J Hepatol 2000• Zoulim et al, J Viral Hepatitis 2006Zoulim et al, J Viral Hepatitis 2006

• AsieAsie– Mt promoteur : activité histologique et fibrose plus importanteMt promoteur : activité histologique et fibrose plus importante– Mt pré-C : activité histologique moins importanteMt pré-C : activité histologique moins importante

• Lindh et al, J Infect Dis 1999Lindh et al, J Infect Dis 1999– Rémission histologiqueRémission histologique

• Chan et al, Hepatology 1999Chan et al, Hepatology 1999

• AfriqueAfrique– Mt promoteur : plus fréquents dans le CHCMt promoteur : plus fréquents dans le CHC

• Baptista et al, Hepatology 1999Baptista et al, Hepatology 1999

HBe serotype and liver pathologyHBe serotype and liver pathology

0-40-4 5-95-9 10-1410-14 15-2215-2200

1010

2020

3030

4040

5050

6060

7070

Knodell scoreKnodell score

Num

ber

of s

ubje

cts

Num

ber

of s

ubje

cts

Metavir scoreMetavir score

≤≤ F2F2 F3F3 F4F400

1010

2020

3030

4040

5050

6060

7070

HBe-positiveHBe-positive

HBe-negativeHBe-negative


HÉPATITES FULMINANTES ET MUTANTS PRE-CHÉPATITES FULMINANTES ET MUTANTS PRE-C

• Lien de causalité :Lien de causalité :

– Épidémies hépatites fulminantesÉpidémies hépatites fulminantes

– Transmission souche mutée pré-C (-)Transmission souche mutée pré-C (-)

– Rôle immunomodulateur de l ’AgHBeRôle immunomodulateur de l ’AgHBe

• Pas de lien de causalitéPas de lien de causalité

– Séquençage génome completSéquençage génome complet

– Pas de profil commun de mutationPas de profil commun de mutation

• Sélection des mutants par la réponse immunitaire cytotoxique Sélection des mutants par la réponse immunitaire cytotoxique

dirigée contre la souche à l ’origine de l ’HFdirigée contre la souche à l ’origine de l ’HF

Stuyver et al, Hepatology 1999, Sternbeck et al Hepatology 1996, Liang et al, NEJM 1991Stuyver et al, Hepatology 1999, Sternbeck et al Hepatology 1996, Liang et al, NEJM 1991

DIAGNOSTICS DIFFICILESDIAGNOSTICS DIFFICILES

I. Porteur inactifI. Porteur inactifII. ExacerbationII. Exacerbation

Diagnosis of inactive carrier versus Diagnosis of inactive carrier versus HBeAg negative chronic hepatitisHBeAg negative chronic hepatitis

• Inactive CarrierInactive Carrier– Persistently normal ALT levelsPersistently normal ALT levels

– Persistently low levels of serum HBV DNAPersistently low levels of serum HBV DNA• Threshold : 10Threshold : 1033 copies / mL ? copies / mL ?

• HBeAg negative chronic hepatitisHBeAg negative chronic hepatitis– Fluctuation / exacerbation of ALTFluctuation / exacerbation of ALT– Fluctuations of HBV DNA levels usually below 10Fluctuations of HBV DNA levels usually below 1066

copies / mLcopies / mL– Presence of pre-core / core promoter mutationsPresence of pre-core / core promoter mutations

DIAGNOSTIC D'UNE EXACERBATION AIGUE DIAGNOSTIC D'UNE EXACERBATION AIGUE SUR HEPATITE B CHRONIQUESUR HEPATITE B CHRONIQUE

• Définition : poussée cytolytiqueDéfinition : poussée cytolytique≠ réactivation viraleréactivation virale

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

• Anti-HBe + initialementAnti-HBe + initialement– réactivation virus sauvage : -> AgHBe +réactivation virus sauvage : -> AgHBe +– réactivation virus muté pré-C (-)réactivation virus muté pré-C (-)– corticothérapiecorticothérapie– surinfection delta / VHCsurinfection delta / VHC

0

5

10

15

20

25

0 1 2 5 9 12 13 16months

1

10

100

1000

10000

100000

1000000

10000000

100000000

1000000000

10000000000

ALT

pre-S1

bDNA

PCR

case#6case#6Genotype AGenotype A

pre-C promoterpre-C promoterWTWTMTMTpre-C regionpre-C regionWTWTM2M2M4M4M2+M4M2+M4

--++

--++

++++

++--

--++

++--

++------

++------

++------

++------

++------

++------

HBeAgHBeAgAnti-HBe AbAnti-HBe Ab

--++

++++

++--

--++

--++

++--

--++

--++

interferoninterferon

Pichoud et al, J hepatol 2000

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 neutral izing antibodies

sG145R

sP120T

sD144H/A/E

PreS1PreS2

SPol

Pré-C

C

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

X

TATAAU5-like

DR1

DR2Enh1Enh2

GRE0/3221

SHBs (S)MHBs (preS2+S)

LHBs (preS2+preS2+S)

Variants de l'Ag HBsVariants de l'Ag HBs

• échappement à la réponse humorale anti-HBséchappement à la réponse humorale anti-HBs

– naturellenaturelle

– vaccination (transmission mère-enfant)vaccination (transmission mère-enfant)

– immunoprophylaxie (transplantation hépatique)immunoprophylaxie (transplantation hépatique)

• infection active malgré Ac anti-HBsinfection active malgré Ac anti-HBs

• sérologie AgHBs faussement négativesérologie AgHBs faussement négative

�� Risques : transmission virale + infections occultesRisques : transmission virale + infections occultes

VARIANTS DE L'AgHBsVARIANTS DE L'AgHBs

• Mutations ponctuelles dans le déterminant a de Mutations ponctuelles dans le déterminant a de

l'AgHBs (124-147)l'AgHBs (124-147)

– aa 145 : Gly -> Arg aa 145 : Gly -> Arg

– aa 126 : Ile -> Ser / Thr -> Asnaa 126 : Ile -> Ser / Thr -> Asn

• transmission mère-enfant malgré la serovaccination transmission mère-enfant malgré la serovaccination

(3%)(3%)

• infection du greffon hépatique malgré infection du greffon hépatique malgré

Immunoglobulines anti-HBsImmunoglobulines anti-HBs

• hépatites chroniques avec anti-HBc et 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

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

ROLEin

HCC

Diagnostic

Tools ?

Worsen HCVinfection ?

Co-infections ?Therapy?

To beimproved

Specific treatments ?

Not fully understood ?

Occult HBV infections: unresolved issues

AntivirauxAntivirauxPersistance viralePersistance virale

Resistance aux antivirauxResistance aux antivirauxMonitoring des traitementsMonitoring des traitements

Goals and types of responseGoals and types of response

Biochemical responseBiochemical response- normalization of ALT levels- normalization of ALT levels

Virological responseVirological response- HBV DNA < 10- HBV DNA < 1044 or 10 or 1033 copies/mL copies/mL

Histological responseHistological response- improvement in HAI or Metavir score- improvement in HAI or Metavir score

Combined responseCombined responseComplete responseComplete response

-> decrease in viral load-> decrease in viral load-> normalization of ALT levels-> normalization of ALT levels-> HBe/HBs seroconversion-> HBe/HBs seroconversion-> improvement of liver disease-> improvement of liver disease

Hoofnagle, J Hepatol 2003Hoofnagle, J Hepatol 2003

Mommeja-Marin H et al. Hepatology 2003 Mommeja-Marin H et al. Hepatology 2003

Median logMedian log1010 HBV DNA level decrease from Baseline HBV DNA level decrease from Baseline

Efficacy : Correlation between HBV DNA Efficacy : Correlation between HBV DNA response and histologic benefitresponse and histologic benefit

Me

dia

n H

isto

log

ic A

cti

vity

In

de

x

Me

dia

n H

isto

log

ic A

cti

vity

In

de

x

Imp

rove

me

nt

fro

m B

as

eli

ne

Imp

rove

me

nt

fro

m B

as

eli

ne

11 22 33 44 55

IFNIFN

Definition of response to therapyDefinition of response to therapy

Initial responseInitial response- decrease in viral load by at least one log10- decrease in viral load by at least one log10

Maintained responseMaintained response- viral load below 12IU /mL- viral load below 12IU /mL

End of treatment response End of treatment response Sustained reponseSustained reponse

-> can we stop therapy ?-> can we stop therapy ?

Pyrimidine dideoxynucleoside analogues

2 ’ Fluoro-substituted

arabinosylpyrimidines

Lamivudine Emtricitabine ElvucitabineClevudine

OH

O OHN

HN

O

OCH3

β-L-2’-deoxythymidine

telbivudine

Purine dideoxynucleoside analogues

acyclic nucleoside phosphonates carbocyclic guanosine analogue

entecaviradefovir

N

N

N

N

NH2

P OHO

HO

O

CH3

tenofovirPMPA

Anti-HBV Active Compounds

Drug type Approved Phase 3 Phase 2

Nucleoside analogs

LamivudineEntecavir

TelbivudineEmtricitabine*

Clevudine**Elvucitabine

ValtorcitabineAmdoxovir

RacivirLB80380

Nucleotide analogs

Adefovir Tenofovir

AlamifovirPradefovir

CytokinesInterferon alfaPeg-interferon

alfa-2a* Currently approved for HIV** Approved in South Korea

L(-)-SddC, 3TCL(-)-SddC, 3TCLamivudineLamivudineL(-)-SddCL(-)-SddC

mitochondriamitochondria

nucleusnucleus

L(-)-SddC-TPL(-)-SddC-TP HBV DNAHBV DNA

Nuclear DNANuclear DNA

Mt DNAMt DNA

L(-)-SddC-TPL(-)-SddC-TP

L(-)-SddC-TPL(-)-SddC-TP

cytoplasmcytoplasm

kinasekinase

L(-)-SddUL(-)-SddU

deaminasedeaminase

Bridges; Progress in Liver Disease 1995

interaction

Virion

Nucleus

Hepatocyte

translation

encapsidation reverse transcription

pgRNADNA (-)

cccDNA amplification

transcription

mRNA

pgRNAAAAAAA

AAAAAA

cccDNAcccDNA

cccDNA formation

RC DNA

entry

polymerase

DNA (+)

(+) strand synthesis

virion secretion

ER

HBeAg

HBsAg

receptor ?

ER

viral proteins secretion

The HBV life cycle

Zoulim et al Future Virology 2006

Nucleoside analogs

Blood streamviral load

Infected hepatocytes

Infected liver

CD8

NKT

CD4

B

Blood StreamViral Load

Infected hepatocytes

Infected Liver

Antiviral

CD8

NKT

CD4

B

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?Acetyl transferase ?Histones

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

Tuttleman et al Cell 1986Le Guerhier et al AAC 2000Delmas et al AAC 2002Kock et al Hepatology 2003

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)

QuickTime™ et undécompresseur TIFF (non compressé)

sont requis pour visionner cette image.

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

Zhu et al, J Virol 2001

M0 M2 M6

L-FMAU + FTC + Ad-IFNL-FMAU + FTC + Ad-IFNγγ Untreated Untreated

M0 M2 M6

M0 M2M1

Failure to eradicate cccDNA with a combination of Failure to eradicate cccDNA with a combination of nucleoside analogs and IFN gammanucleoside analogs and IFN gamma

Jacquard et al AAC 2004Jacquard et al AAC 2004

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

-6

-5

-4

-3

-2

-1

0

Changes in HBV Markers

from Baseline

(log

10

copies/cell(ml))

SerumHBVDNA

TotalIntracellular

DNAcccDNA Serum

HBsAg

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

Changes in HBsAg levels correlated with cccDNA changesChanges in HBsAg levels correlated with cccDNA changes-> 14 years of therapy to clear completely viral cccDNA-> 14 years of therapy to clear completely viral cccDNA

Werle et al, Gastroenterology 2004

• 0.8 log10 (84%) decline in cccDNA, not paralleled by a similar decline in the number of HBcAg+ cells

• Suggests cccDNA depleted primarily by non-cytopathic mechanisms or that cell turn-over Suggests cccDNA depleted primarily by non-cytopathic mechanisms or that cell turn-over occurred but was associated with infection of new cells during therapyoccurred but was associated with infection of new cells during therapy

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

BaselineBaseline Week 48Week 48

Maynard et al, J Hepatol 2005Maynard et al, J Hepatol 2005

Persistence of cccDNA after HBs seroconversionPersistence of cccDNA after HBs seroconversion

Clearance of viral infection versus selection of Clearance of viral infection versus selection of escape mutantsescape mutants

The most important factors to consider: The most important factors to consider:

§ The rate of immune killing of infected hepatocytesThe rate of immune killing of infected hepatocytes

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

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

spread to uninfected hepatocytes)spread to uninfected hepatocytes)

§ Small changes in these factors may have profound effect on Small changes in these factors may have profound effect on

whether treatment response is durable or subject to rapid whether treatment response is durable or subject to rapid

rebound rebound (Litwin et al J Clin Virol 2005)(Litwin et al J Clin Virol 2005)

§ These factors may be subject to therapeutic interventionThese factors may be subject to therapeutic intervention

LamivudineLamivudine

II IIII IIIIII IVIV

wtwt mtmt

nini

INHIBITION OF WILD TYPE VIRUS REPLICATIONINHIBITION OF WILD TYPE VIRUS REPLICATION DELAYED EMERGENCE OF DELAYED EMERGENCE OF DRUG RESISTANT VIRUSDRUG RESISTANT VIRUS

XX

XX

XX

XX

XX

XXXX

Zhou et al AAC 1999

Kinetics of emergence of drug resistant Kinetics of emergence of drug resistant virus during antiviral therapyvirus during antiviral therapy

• Free l iver space

• Mutant f i tness

1,E+01

1,E+03

1,E+05

1,E+07

1,E+09

-3 1 5 9 13 17 21 25 29 33

Vir

al l

oa

d

LamivudineLamivudine

Changement de la quasi-espèce virale et de la sensibilité aux Changement de la quasi-espèce virale et de la sensibilité aux drogues pendant le traitement antiviraldrogues pendant le traitement antiviral

0 0,1 0,2 0,4 0,8 1,6 3,2 6,3 12,5 mM

Lamivudine

3

21.51.2

1

Lamivudine

0 1,6 3,2 6,3 12,5 25 50 100 mM

3

2

1.51.2

Lineards HBV

1

IC50» 0.1 µM IC90» 2 µM

IC50> 50 µM IC90>>100 µM

Quasi-espèce

Phenotype

Quasi-espèce

Durantel, Hepatology 2004

Mechanisms of HBV Drug Resistance

Viral persistence

cccDNA Long half-life

Infected cells Long half-life

Defective Defective immune immune

responseresponse

VirusVirus HepatocytesHepatocytes

Impairment of Impairment of innate responseinnate response

HostHost

Selective pressure Antivirals or others

Viral polymerasespontaneous error rate

VirusVirus

Selection of escape Selection of escape mutantsmutants

Treatment failureTreatment failure

Replication fitnessReplication space

Viral quasi-species

Immune responseDrug PK

Zoulim Antivir Res 2004;64:1–15

Polymerase gene mutations reponsible for drug resistance

Allen et al. Hepatology 1998; Gish et al. J Hepatol 2005; Qi et al. J Hepatol 2004; Tenney et al. AAC 2004 & 2007; Lai et al. Gastroenterology 2005; Sheldon et al. Antivir Ther 2005; Delaney et al. AAC 2006 ; Schildgen et al NEJM 2006 ; Borroto-Esoda JID 2007; Durantel et al Antiviral Therapy 2008; Villet et al Gastroenterology 2006, J Hepatol 2007 & 2008; Warner et al Hepatology 2008

RNaseH

845 a.a.

Terminal protein Spacer Pol/RTPol/RT

AA BB CC EED D

1 183 349 692

YMDD

V173L

L180M M204I/V

GVGLSPFLLA

I(G)I(G) II(F) II(F)

(rt1) (rt 344)

LAM / FTC

ETV I169T T184G S202G/I M250V

ADV A181V/T N236TI233V ?

LdT M204I

TDF A194T ?

M204I/V

Mechanisms of resistance

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

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 ResponseQ1 (best response)

(n=38)

Virological ResponseQ2

(n=38)

Virological ResponseQ3

(n=38)

Virological ResponseQ4 (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

Polymerase gene mutations may result in decreased inhibitory activity of antivirals

Jacquard et al, Antimicrob Agents Chemother 2006

wt polymerase 3TC-R polymerase PMEA-R polymerase 3TC+PMEA-R polymerase

Drug IC50 (µM) P IC50 (µM) P IC50 (µM) P IC50 (µM) P

Elongation

FLG-TP 4 ± 0.9 5.43 ± 0.6 7.8 ± 1.9 6.33 ± 1.3

3TC-TP 10.75 ± 4.8 <0.05 >100 <0.05 14 ± 5.7 <0.05 >100 <0.05

PMEA-DP 2.8 ± 0.3 >0.05 0.9 ± 0.1 <0.05 49.5 ± 3.4 <0.05 16.5 ± 7.2 <0.05

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

Polymerase gene mutations Surface gene mutations

wt none none

mutant #1 T128I; V173L; L180M; A181V; N236T F20S; P120S; E164D; L173F

mutant #2 T128I; V173L; L180M; A181V; M204V R79H; P120S; E164D; L173F; I195M; Y206F

mutant #3 ∆111-120; T128I; V173L; L180M; A181V F20S; ∆102-111; P120S; E164D; L173F

mutant #4 T128I; V173L; L180M; A181V; M204V; L220I; N236T P120S; E164D; L173F; I195M

Polymerase clonal genetic analysis

lamivudine

adefovir

HBIg

wt

Mutant #1

Mutant #2

Mutant #3

Mutant #4

Villet et al, Gastroenterology 2006

Villet, Billioud et al, Gastroenterology 2008

0

50

100

150

200

250

300

350

400

wt #1 #2 #3 #4 Mutant

Mu

tan

t re

plic

ati

on

cap

aci

ty /

wt

(%)

Viral replication capacity in the presence of both antivirals (LAM + ADV)

Mutant

wt #1 #2 #3 #4

1,7 kb

A

B

wt #1 #2 #3 #4 Mutant

Mu

tan

t in

fect

ivit

y / w

t (%

)

0

20

40

60

80

100

120

Villet, Billioud et al, Gastroenterology 2008

Infectivity of the mutants in HepaRG cellsImpact of mutations in the overlapping S gene

HDV hybrids with HBV mutant envelopesHDV replication in HepaRG cells as a reporter of infection

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

Model for the selection of drug resistant mutants

Villet et al, J Hepatol 2007; Villet et al Gastroenterology 2006; Yim et al Hepatology 2006; Palier et al J Virol 2006; Durantel et al Hepatology 2004; Tenney et al AAC 2004 & 2007

LAM-R

204+ 180

184 / 202 / 250

ETV

236 and/or 181ADV

204 ± 180

Wild-type

204 + 180LAM

Wild-type

Wild-type

ETV 184 / 202 /250

ADV-R

ETV-R

ETV-R

LAM-R

TDF ?

Archiving of resistant mutants

• 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


Multidrug resistance

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)Zoulim & Perrillo, J Hepatol 2008

The Problem of Sequential Therapywith Nucleoside Analogs

+ one mutation + one mutation

?

Multiple drug resistant mutants with complex pattern of mutations

Drug ADrug B

� Risk of selection of MDR mutants by sequential therapy, especially when using drugs sharing cross-resistance characteristics

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

Tre

atm

ent (

mon

ths)

lam

ivud

ine

ente

cavi

r

0 20 40 60 80 100

1

L180M+M204VM204Vwt

V173L+L180M+M204V

L180M+M204VV173L+L180M+M204V

L180M+S202G+M204VI169L+L180M+S202G+M204VV173L+P177S+L180M+S202G+M204V

V173L+P177S+L180M+S202G+M204VL180M+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

Lamivudinerebound

Entecavirrebound

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

(M

eq

/ml)

lamivudine

adefovir

HBIg tenofovir

V173L+L180M+A181V+N236TL180M+M204V

Liver transplantation

0 10 20 30 40 50 60 70 80 90 100

1

Lam

ivud

ine+

adef

ovir

trea

tmen

t (m

onth

s)

1

8

24

34

38

40

42 to 50

Viral rebound

0

16

26

30

32

34 to 42

Tim

e po st-transp lantation (month s)

% of variants in the viral quasi-species

Accumulation of mutations and selection of a complex mutant

YMDDYMDDTerminal

Protein

spacer Pol/RT RNaseH

V173L L180M A181V N236T

Pre-S/S gene

P120S

dominant HBVmutant

L180M+M204I

wt

V173L+L180M+A181V+M204V

M204I

V173L+L180M+A181VV173L+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

Conclusions

• Resistant mutants do pre-exist prior to therapy

• Their selection depends on:

– their intrinsic fitness

• infectivity

• replication capacity

• level of resistance

– replication space in the liver

• Resistant mutants are archived in cccDNA

• Complex mutants can be selected leading to multidrug resistance

PerspectivesPrevention of drug resistance

• First line therapy

– Use of antivirals with high antiviral potency and high barrier to resistance

– Combination therapy with complementary drugs

• 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

AntivirauxMonitoring et resistance

Aspects cliniques

Clinical Definition of HBV Resistance to AntiviralsClinical• Genotypic Resistance: Detection of mutations in the HBV genome,

known to confer resistance, which develop during anti-viral therapy

• Virologic Breakthrough: Rebound in serum HBV DNA levels following the development of genotypic resistance

• Clinical Breakthrough: Virologic breakthrough with increased ALT levels or worsening histology

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

Nafa et al Hepatology 2000; Lok et al. J Clin Microbiol. 2002Nafa et al Hepatology 2000; Lok et al. J Clin Microbiol. 2002

Line Probe Assay Versus Sequencing for the Detection of Line Probe Assay Versus Sequencing for the Detection of HBV Drug ResistanceHBV Drug Resistance

Can detect any new mutation

Very sensitive (minor species and low viremia)

Line probe assayLine probe assay

Sequencing of PCR productsSequencing of PCR products

0

20

40

60

80

100

HB

V D

NA

(10E

+6

gen

ome

eq/

ml)

AL

T(U

/L)

020

60

100

140

180

1 595100 200 300 400

Codon 528 LiPASeq

Codon 552 LiPASeq

Codon 555 LiPASeq

1

LL

MM

VV

39

LL

MM

VV

290

L/ML/M

M/VM

VV

400

MM

VV

VV

595

MM

VV

VV

Day

T A T A T G

C T C M T G

G A T

G C T

600

500

200

150

100

50

0

8

7

6

5

4

3

0 6 12 18 24 30 36

Lamivudine

Months42

AL

T (

U/L

)

HB

V D

NA

log

co

pie

s/m

L

MMV

MM/V

V

LMV

L/MMV

MM/VV

LMV

MVV

Codon 180Codon 204Codon 207

MVV

Genotypic resistance

Dynamics of Resistance EmergenceGenotypic Resistance

Si Ahmed et al. Hepatology 2000;32:1078–88

600

500

200

150

100

50

0

8

7

6

5

4

3

0 6 12 18 24 30 36

Lamivudine

Months42

PCR assay

AL

T (

U/L

)

MMV

MM/V

V

LMV

L/MMV

MM/VV

LMV

MVV


MVV

HB

V D

NA

log

co

pie

s/m

L

Rebound of serum HBV DNA*

> 1 log10 copies/mL

Dynamics of Resistance EmergenceVirologic Breakthrough


600

500

200

150

100

50

0

8

7

6

5

4

3

0 6 12 18 24 30 36

Lamivudine

Months42

Rise in serum transaminases

PCR assay

MMV

MM/V

V

LMV

L/MMV

MM/VV

LMV

MVV


MVV

AL

T (

U/L

)

HB

V D

NA

log

co

pie

s/m

L

Worsening of liver disease

Dynamics of Resistance EmergenceClinical Breakthrough


Sequence of Events in Resistance to antiviral Therapy

Time

Antiviral drug

HB

V D

NA

(lo

g1

0 I

U/m

L)

AL

T (IU

/L)

4

6

5

3

2 ULN

1

0

Detection of Genotypic Resistance

Nadir1 log10

VirologicBreakthrough

BiochemicalBreakthrough

Genotypic resistance

VirologicalBreakthrough

BiochemicalBreakthrough

Incidence of HBV Drug Resistance• Definitions of antiviral drug resistance vary across the

clinical trials

• Few studies report on primary non response

• Clinical impact on treatment management

– Incidence of resistance in nucleoside naive patients• Choice of drug as a first line treatment

– Incidence of resistance in patients who are in previous treatment failure (lamivudine resistance as a current problem)

• Choice of drug as a second line treatment

Incidence of Resistance in Nucleoside Naive Patients

% o

f pa

tien

ts w

ith

resi

stan

ce m

utat

ions

Lai et al CID 2003; Hadzyiannis et al Gastroenterology 2006; Colonno et al AASLD 2006; Di Bisceglie et al AASLD 2006

0

10

20

30

40

50

60

70

80

Lamivudine Adefovir Entecavir Telbivudine Tenofovir

year 1year 2

year 3year 4

year 5

Incidence of Resistance in Lamivudine Refractory Patients

% o

f pa

tien

ts w

ith

resi

stan

ce m

utat

ions

0

10

20

30

40

Adefovirswitch

Adefoviradd-on

Entecavirswitch

Tenofovir +FTC/3TC

baseline

Year 1

Year 2

Year 3

Year 4

Lampertico et al Hepatology 2005 & Gastroenterology 2007; Colonno et al AASLD 2007; Lacombe et al AIDS 2006

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

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sont requis pour visionner cette image.

Lok et al Gastroenterology 2003; 125 : 1714-1722

Lamivudine Resistance Accelerates Progression of Liver Disease

0

5

10

15

20

25

0 6 12 18 24 30 36

Time after randomization (Months)

% With disease progression

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

Impact of Adefovir Resistance on Virologic and Biochemical Correlates

0% 3%

11%18%

29%

0% 3%8%

13% 16%

0% 2%6%

10% 11%

0%

20%

40%

60%

80%

100%

Year 1 Year 2 Year 3 Year 4 Year 5

Years of lamivudine therapy

% of patients

Hadzyiannis et al, Gastroenterology 2006

Cumulative probabilities calculated by Life-Table analysis

* ALT = >1X ULN

M: detection of mutationsVR: virologic breakthroughALT: biochemical breakthrough*

M M + VR M + VR + ALT

Baseline Predictive Factors of Resistance• Lamivudine trials

• Positive correlation with emergence of Lamivudine resistant mutants– Baseline virus levels– Disease severity assessed by H.A.I. score– Increased body mass index

• Lack of emergence of lamivudine resistant strains– Asian ethnicity– Female sex

Lai et al Clin Infect Dis 2003; 36: 687-696; Zoulim et al, J Viral Hepatitis, 2006; 13:278-288

• Adefovir, entecavir: ?

Age: ≥ 50 years old

Alcohol consumption

Place of birth: Asia

HBV DNA: > 5 x 106 IU/ml

Metavir score: ≥ F3

LiPA genotype: C

0.1110Odds ratio

Zoulim et al, J Viral Hepatitis, 2006

Multivariate logistic analysis: VIRAL LOAD was the only parameter associated Multivariate logistic analysis: VIRAL LOAD was the only parameter associated with the emergence of YMDD mutationswith the emergence of YMDD mutations

Predictive Factors of Lamivudine Resistance Pre-treatment Factors

(Cohort study of 295 patients undergoing lamivudine therapy)

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

Viral Load at Week 24 is a Predictor of Resistance at Week 104 of Therapy (Telbivudine vs. Lamivudine trial)

4%

25%29%

30%

9%

24%

41%45%

0%

20%

40%

60%

80%

100%

< QL,n=203,146

QL - 3,n=57,63

3 to 4,n=83,79

> 4,n=115,175

% of patients with resistance

2%

12%

20%

60%

5% 6%

50%

56%

0%

20%

40%

60%

80%

100%

< QL,n=178,157

QL - 3,n=18,20

3 to 4,n=16,24

> 4,n=10,23

% of patients with resistance

Telbivudine Lamivudine

HBeAg Positive, n=921HBeAg Positive, n=921 HBeAg Negative, n=446HBeAg Negative, n=446

Lai et al , NEJM, 2007

HBeAg Seroconversion at 2 Years vs. Antiviral Effect at Week 24

PercentHBeAg

Seroconversion

Serum HBV DNA Level at Week 24

HBeAg Positive Patients, Combined Treatment GroupsHBeAg Positive Patients, Combined Treatment Groups

39%

46%

19%

6%

0%

20%

40%

60%

Below QL QL to 3 log 3 to 4 log > 4 log

Lai et al , NEJM, 2007

Secondary Treatment Preferences Based on Virologic Monitoring

Partial virologic response Virologic breakthrough

Nucleoside analog treatment

Add a more potent agent* or switch to a combination of

emtricitabine/tenofovir*

* Choice based on cross-resistance data

Monitorat 12-24 weeks

Early non reponse

Monitorevery 12 weeks

Switch to morepotent agent*

Zoulim & Perrillo, J Hepatol in press

Lamivudine Telbivudine Entecavir Adefovir Tenofovir

Wild-type S S S S S

M204l R R I/R S S

L180M + M204V

R R I S S

A181 T/V I S S R S

N236T S S S R I

I169T + V173L + M250V*

R R R S S

T184G + S202lI/G * R R R S S

*(+ L180M + M204I/V).

Treatment adaptation should be based on cross-resistance data

Durantel et al Hepatology 2004; Brunelle et al Hepatology 2005; Yang et al Antiviral Therapy 2005; Villet et al Gastroenterology 2006; Delaney et al AAC 2006; Villet et al J Hepatol 2007; Brunelle et al AAC 2007;

Qi et al Antiviral therapy 2007; Tenney et al AAC 2004 & 2007 ; Villet et al J Hepatol 2008

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

ien t

s w

ith v

ir olo

gic a

l bre

a kth

rou g

h

273 268 256 225 201 158 61

30%

6%

P<0.001

ADV+LAM

255 238 223 213 200 177 103P

atie

n ts

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 ∆ ALT

The problem of sequential therapy and switching strategy

Villeneuve et al, J Hepatol 2003

N236T

Ser

um H

BV

DN

A

(Log

10 c

opie

s /m

L)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

Resistance to Lamivudine / Telbivudine

Add:• ADV • TDFSwitch to TDF+FTC*

Switch to ETVNot valid• LAM• FTC• LdT

Zoulim and Perrillo, J Hepatol, 2008

Management of HBV resistance

*The association of FTC/TDF is not yet approved in the treatment of chronic hepatitis B

Resistance to adefovir

Add :• Lamivudine• ETV• Telbivudine

Switch to TDF+FTC*Switch to :• TDF• TVD• ETV• LdT

Non response to adefovir




Resistance to Entecavir

Add•ADV•TDF

Switch to TDF+FTCNot valid •LAM•LdT




M0 M6M12M18M24M30 M36

ALT0

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

AL T

(x U

LN)

M0 M6M12 M18 M24 M30 M36

ALT0

2

4

6

8

ALTHBV 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

AL T

(x U

LN)

M0 M6M12 M18 M24 M30 M36

ALT0

2

4

6

8

ALTHBV DNA

Month of therapyMonth 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

AL T

(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

Serum HBV DNA (Log10 copies/mL)

Drug ADrug ADrug ADrug A

++Drug BDrug 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 ?

Rationale for de novo Combination Therapy

Drug A

Drug B

Wild type

Drug B resistant mutant

Drug A resistant mutant

�� Combination of drugs without cross-resistanceCombination of drugs without cross-resistance

wt

Low risk of Low risk of selection of MDRselection of MDR

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

M0 M6M12 M18 M24 M30 M36

ALT0

2

4

6

8

ALTHBV DNA

Month of therapyMonth 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

AL T

(x U

LN)

Preventing L-Nucleosides Resistance with de novo Combination Therapy

1 Marcellin et al. N Engl J Med 2004; 351: 1206-172 Lau et al. Hepatology 2004;40:171A

3 Lai et al. Hepatology 2003;38:262A4 Sung et al. J Hepatol 2003 ;38 (suppl 2):25-265 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 4Marcellin 1 Lau 2 Lai 3

LAMLAM LAM LAM LAM

+ADV

LAM

+Peg

LAM

+Peg

LAM

+LdT

Inci

den

ce

of

resi

stan

ce*

(%)

LdT FTC FTC

+ADV

0% 0%

Lau 5

Future Needs for the Management of

HBV Drug Resistance• Algorithm for the use of viral load & genomic assays in

the monitoring of antiviral therapy

• Management of drug resistance: – Best strategies to rescue drug resistance with long-term

treatment end-points

• Prevention of drug resistance: – Treatment strategy trials: de novo combination versus early

add-on therapy– Drugs without cross-resistance– Long-term endpoints

Conclusions 1

• Maladie fréquente et graveMaladie fréquente et grave– 300 000 porteurs chroniques en france300 000 porteurs chroniques en france– 1ère cause de cancer du foie dans le monde1ère cause de cancer du foie dans le monde

– 1300 décès par an en France1300 décès par an en France

• Maladie méconnueMaladie méconnue– Souvent asymptomatique, ou symptomes non spécifiquesSouvent asymptomatique, ou symptomes non spécifiques– Seulement 60 000 personnes connaissent leur maladieSeulement 60 000 personnes connaissent leur maladie– 13 000 sont traitées13 000 sont traitées

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

Conclusions 2

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

cancer du foiecancer du foie

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

- Echappement diagnostique- Echappement diagnostique

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

• Prévention par la vaccination !!!Prévention par la vaccination !!!

Health & Medicine

Zoulim Du 2009