Hépatite B Hépatite B Fabien Zoulim Fabien Zoulim Département d’hépatologie Département d’hépatologie & INSERM U871, Lyon & INSERM U871, Lyon
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'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
• 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
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
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
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
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
Hépatocyte infectéHépatocyte infecté
HBVHBV
CTLCTL
FasFasperforineperforine
Hépatocyte Hépatocyte non infecté non infecté
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
Hépatocyte infectéHépatocyte infecté
HBs AgHBs Ag
Hépatocyte Hépatocyte non infecté non infecté
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 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
Months After Exposure
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
Seeger, Zoulim, Mason, Fields Virology 2007
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
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
Seeger, Zoulim, Mason, Fields Virology 2007
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
Seeger, Zoulim, Mason, Fields Virology 2007
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
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
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.
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
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
Lamivir cohort, Zoulim et al, J Viral Hepatitis 2006
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
Lamivir cohort, Zoulim et al, J Viral Hepatitis 2006
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
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
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
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 ?
• 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
LiverMajority population
Minority variants
Resistant variants
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
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
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
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
Codon 180Codon 204Codon 207
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
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
Rise in serum transaminases
PCR assay
MMV
MM/V
V
LMV
L/MMV
MM/VV
LMV
MVV
Codon 180Codon 204Codon 207
MVV
AL
T (
U/L
)
HB
V D
NA
log
co
pie
s/m
L
Worsening of liver disease
Dynamics of Resistance EmergenceClinical Breakthrough
Si Ahmed et al. Hepatology 2000;32:1078–88
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
Zoulim and Perrillo, J Hepatol, 2008
*The association of FTC/TDF is not yet approved in the treatment of chronic hepatitis B
Management of HBV resistance
Resistance to Entecavir
Add•ADV•TDF
Switch to TDF+FTCNot valid •LAM•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
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 !!!