1. Hépatite BHépatite B
Fabien ZoulimFabien Zoulim
Département d’hépatologieDépartement d’hépatologie
& INSERM U871, Lyon& INSERM U871, Lyon

2. VHBVHB HCAHCA cirrhosecirrhose CHCCHC
VaccinVaccin ANTIVIRAUXANTIVIRAUX
IFNIFN
Antiviraux/IFN?Antiviraux/IFN?
NiederauNiederau 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 ansGuérisonGuérison
Lee, N Engl J Med 1997; Lok, Hepatology 2001Lee, N Engl J Med 1997; Lok, Hepatology 2001

3. EPIDEMIOLOGIE DE L’HÉPATITE BEPIDEMIOLOGIE DE L’HÉPATITE B

5. EPIDEMIOLOGIE DE L'INFECTION A VHBEPIDEMIOLOGIE DE L'INFECTION A VHB
• Hépatites aiguesHépatites 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

6. MODES DE TRANSMISSION DU VIRUS DE L'HÉPATITE B EN EUROPEMODES DE TRANSMISSION DU VIRUS DE L'HÉPATITE B EN EUROPE
sexuellesexuelle
34%34%
hétérohétéro
23%23%
homohomo
11%11%
drogue IVdrogue IV
26%26%
inconnueinconnue
31%31%
hémodialyséshémodialysés
8%8%
transfusionstransfusions
2%2%
personnels de santépersonnels de santé
2%2%
contact aveccontact avec
porteur du VHBporteur du VHB
4%4%
AsieAsie
Transmission verticaleTransmission verticale

7. 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

8. 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"

9. Circuit de l’information
Biologiste
InVS
MISP de DDASS du
département
d’exercice
Médecin
prescripteur
Fiche de notification autocopiante à 4 feuillets
Partie 1 : code d’anonymat irréversible, caractéristiques du patient
Partie 2 : information biologique
Parties 3-4-5 : information clinique et épidémiologique
Parties 6-7 : identification du médecin prescripteur et du biologiste déclarants
Feuillet 1 :
parties 1-2 et
6-7 renseignées
Feuillets 2 et 3
à compléter
Feuillet 2 :
parties 3-4-5
complétées
Feuillets 1 et 2 complétés et validés
Relance

10. Results
158 acute hepatitis cases158 acute hepatitis cases
• Hospital doctor in 64% casesHospital 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 femalesMedian 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)

11. 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
Classes d'âge
% de cas
Réseau "Sentinelles" Déclarations obligatoires
years 1991- 94
n= 151
March 03- February 04
n= 158

12. 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 treatmentInvasive treatment 1515 10,3%10,3%
– Tatoo, piercingTatoo, piercing 55 3,4%3,4%
– FamilialFamilial 1414 9,7%9,7%
– Perinatal 2Perinatal 2 1,4%1,4%
– Live in instiutionLive 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 91-96Sentinel networks 91-96
N=195N=195
– sexualsexual 35%35%
– IVDUIVDU 19%19%
– « percutaneous »« percutaneous » 15%15%
– No factorNo factor 35%35%

13. 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

14. VIROLOGIEVIROLOGIE

15. • 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

17. LE GÉNOME DU VIRUS DE L’HÉPATITE BLE GÉNOME DU VIRUS DE L’HÉPATITE B
déterminant adéterminant a
vaccin/IgHBsvaccin/IgHBs
Gène polGène pol
antivirauxantiviraux
Mt pre-coreMt pre-core
Réponse anti-HBeRéponse anti-HBe ??
Mt du coreMt du core
Réponse CTLRéponse CTL
8 génotypes8 génotypes
A to HA to H
Tiollais Nature 1985Tiollais Nature 1985
Günther Adv Virus Res 1999Günther Adv Virus Res 1999
Norder J Gen Virol 2003Norder J Gen Virol 2003

18. Ganem & Prince, NEJM 2004Ganem & Prince, NEJM 2004

19. ARN pg
ss DNA
RC DNA
cccDNA
intégration
virion10%
90%
ds DNA
cccDNA
illégitime
noyau
Réplication du génome viral. Implication pour la
persistance virale et l’intégration du génome viral
Membrane cellulaire

20. VHB HUMAINVHB HUMAIN
MARMOTTE (WHV)MARMOTTE (WHV) CANARD (DHBV)CANARD (DHBV)
QuickTime™ et un décompres seur
Photo - JPEG sont requis pour visualiser
cette image.
Modèles AnimauxModèles Animaux
Souris TransgéniquesSouris Transgéniques
Souris 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

21. • Polymerase viralePolymerase virale
– DHBV : lysat réticulocytaireDHBV : lysat réticulocytaire
– HBV : baculovirusHBV : baculovirus
ModèlesModèles in vitroin vitro
UU
Polymerase VHBPolymerase VHB
DNA(-)DNA(-)
ELONGATIONELONGATION
CCC -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

22. interaction
virion
noyau
hépatocyte
traduction
encapsidation
transcription inverse
ARNpgADN (-)
amplification
de l’ADNccc
transcription
ARNm
ARNpg
AAA
AAA
AAA
AAA
ADNcccADNccc
formation d’ADNccc
ADN RC
entrée
polymérase
ADN (+)
Synthèse du brin
(+)
sécrétion des virions
RE
Ag HBe
Ag HBs
récepteur ?
RE
sécrétion des
protéines virales
Cycle de réplication du HBV
Analogues de
nucléosides
ADN RC

23. Comparative dynamics among three virusesComparative dynamics among three viruses
(Tsiang et al. Hepatology 1999)

24. 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 masseDiminution incidence avec la vaccination de masse (Chen,(Chen,
NEJM 1995)NEJM 1995)

25. CARCINOME HEPATOCELLULAIRE ET VIRUSCARCINOME HEPATOCELLULAIRE ET VIRUS
DE L'HEPATITE BDE L'HEPATITE B
• Co-incidence de répartition géographiqueCo-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

26. HBV replication and its role in HCC development
Wands, NEJM 2004

27. PATHOGENIE DU CARCINOMEPATHOGENIE DU CARCINOME
HEPATOCELLULAIREHEPATOCELLULAIRE
VHBVHB
ALCOOLALCOOL
VHCVHC
LESIONS HEPATIQUES CHRONIQUESLESIONS HEPATIQUES CHRONIQUES
ACTIVATION FACTEURSACTIVATION FACTEURS
DE CROISSANCEDE CROISSANCE
REGENERATIONREGENERATION
ALTERATIONS GENETIQUESALTERATIONS GENETIQUES
CARCINOME HEPATOCELLULAIRECARCINOME HEPATOCELLULAIRE
DESORDRESDESORDRES
METABOLIQUESMETABOLIQUES
FACTEURSFACTEURS
ENVIRONNEMENTAUXENVIRONNEMENTAUX

28. 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

29. PHYSIOPATHOLOGIE /PHYSIOPATHOLOGIE /
IMMUNOPATHOLOGIEIMMUNOPATHOLOGIE

30. Ganem and Prince, NEJM 2004Ganem and Prince, NEJM 2004

31. HÉPATOCYTE INFECTÉHÉPATOCYTE INFECTÉ
VHBVHB
CTLCTL
FasFas
perforineperforine
HÉPATOCYTEHÉPATOCYTE
NON INFECTÉNON INFECTÉ
IMMUNOPATHOGÉNIEIMMUNOPATHOGÉNIE
DES HÉPATITES B CHRONIQUESDES HÉPATITES B CHRONIQUES
AgHBc/eAgHBc/e
HLAIHLAI
cytokinescytokines
RÉPONSE IMMUNITAIRERÉPONSE IMMUNITAIRE
CYTOKINESCYTOKINES
ANTIVIRAUXANTIVIRAUX
ANTICORPS NEUTRALISANTSANTICORPS NEUTRALISANTS

32. IMMUNOPATHOLOGY OF HBV INFECTIONIMMUNOPATHOLOGY OF HBV INFECTION
Immune toleranceImmune tolerance
Clairance phaseClairance phase
Chronic hepatitisChronic hepatitis
SeroconversionSeroconversion
RemissionRemission
CD8+CD8+
HBVHBV
CD8+CD8+ HBVHBV
CD8+CD8+
HBVHBV

33. Immunopathology
Fulminant hepatitisFulminant hepatitis
CD8+CD8+
HBVHBV

34. Non cytolytic processesNon cytolytic processes
TH1 cytokines with direct antiviralTH1 cytokines with direct antiviral
effecteffect
Turn-over of infected cellsTurn-over of infected cells
Immune 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 mice
ChimpanzeesChimpanzees
(Guidotti Science 1999,(Guidotti Science 1999,
Thimme J Virol 2003)Thimme J Virol 2003)
AntiviralsAntivirals
Inhibition of viral DNA synthesisInhibition of viral DNA synthesis
-> inhibition of intracellular recycling of-> inhibition of intracellular recycling of
cccDNAcccDNA
(Werle Gastroenterology 2004)(Werle Gastroenterology 2004)
Restoration of anti-HBV immune responseRestoration of anti-HBV immune response
MECHANISMS OF VIRAL CLEARANCEMECHANISMS OF VIRAL CLEARANCE

35. Non cytolytic clearance of acuteNon cytolytic clearance of acute
HBV infection in chimpanzeeHBV infection in chimpanzee
Wieland S et al, PNAS 2004

36. Hepatocyte turn-over is required for clearance ofHepatocyte 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

37. Hépatocyte infectéHépatocyte infecté
HBVHBV
HépatocyteHépatocyte
non infecténon infecté
Phase de tolérance immunitairePhase de tolérance immunitaire
MarqueursMarqueurs
AgHBe +AgHBe +
HBV DNA +++HBV DNA +++
ALAT = NALAT = N
Foie = NFoie = N
HBc/e AgHBc/e Ag

38. Hépatocyte infectéHépatocyte infecté
HBVHBV
CTLCTL
FasFas
perforineperforine
HépatocyteHépatocyte
non infecténon infecté
Phase de clairance immunePhase de clairance immune
(hépatite chronique)(hépatite chronique)
MarqueursMarqueurs
AgHBe+AgHBe+
HBV DNA +HBV DNA +
ALAT +++ALAT +++
Foie:Foie: HépatiteHépatite
chroniquechronique
HBc/e AgHBc/e Ag
HLAIHLAI
cytokinescytokines

39. Hépatocyte infectéHépatocyte infecté
HBs AgHBs Ag
HépatocyteHépatocyte
non infecténon infecté
MarqueursMarqueurs
AgHBe-AgHBe-
anti-HBe +anti-HBe +
HBV DNA < 10HBV DNA < 1044
/mL/mL
ALAT = NALAT = N
Foie = rémissionFoie = rémission
Phase de rémissionPhase de rémission
portage inactif de l’AgHBsportage inactif de l’AgHBs
RéactivationRéactivation
Virus sauvageVirus sauvage
ou mt pre-coreOncogénèseOncogénèse

40. Hépatocytes infectésHépatocytes infectés
HépatocytesHépatocytes
non infectésnon infectés
MarqueursMarqueurs
HBsAg -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’échappement
Infections occultesInfections occultes
OncogénèseOncogénèse

41. cccDNAcccDNA(copies/cell)(copies/cell)
TotalHBVDNATotalHBVDNA
(copies/cell)(copies/cell)
cccDNA levels in the different phases ofcccDNA 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) levelsHBeAg+ 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
100
101
102
103
104
10-3
10-2
10-1
100
101
102
103
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

42. HISTOIRE NATURELLE ET
VIROLOGIE CLINIQUE

43. Histoire Naturelle de l’hépatite BHistoire Naturelle de l’hépatite B
Infection aigueInfection aigue
Infection chroniqueInfection chronique
Tolérance immunitaireTolérance immunitaire
Hépatite chroniqueHépatite chronique
Portage inactifPortage inactif
GuérisonGuérison
5% nx-nés5% nx-nés
90% adultes90% adultes
Virus sauvage (HBeAg+)Virus sauvage (HBeAg+)
Mutant pre-core (HBeAg-)Mutant pre-core (HBeAg-)
CirrhoseCirrhose
Carcinome hépatocellulaireCarcinome hépatocellulaire
RéactivationRéactivation
30-50 ans30-50 ans
Seeger, Zoulim, Mason;
Fields Virology; 2007

44. MARQUEURS SEROLOGIQUESMARQUEURS SEROLOGIQUES
• Système AgHBe /anti-HBeSystème AgHBe /anti-HBe
– distinction virus sauvage / virus muté AgHBedistinction virus sauvage / virus muté AgHBe
négatifnégatif
• VirémieVirémie
– détection quantitative de l'ADN viraldétection quantitative de l'ADN viral

45. 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

46. Laboratory Diagnosis of Acute Hepatitis B
0
100
200
300
400
500
600
700
800
900
1000
0 1 2 3 4 5 6 12 24 36 48 60
ALT
HBsAg
HBeAg
HBV DNA
Normal
Months After Exposure
ALTandHBVDNA
IU/Landmillioncopies/ml
Symptoms
Anti-HBs Ab
Anti-HBe Ab
IgM anti-HBc
Total anti-HBc
Seeger, Zoulim, Mason, Fields Virology 2007

47. HEPATITE B PROLONGEE
• DéfinitionDéfinition
– Persistance réplication virale à la 8èmePersistance 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 : IFNTraitement : IFN
– Guérison : 7 à 8 cas / 10Guérison : 7 à 8 cas / 10

48. INFECTIONS CHRONIQUES A VHBINFECTIONS CHRONIQUES A VHB
FORMES 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

49. 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
ALTandHBVDNA
IU/Lormillioncopies/ml
Laboratory Diagnosis of Chronic Hepatitis B
associated with wild type virus infection
Diapositive 25
Seeger, Zoulim, Mason, Fields Virology 2007

50. ALT
``HBsAg
HBeAg
HBV DNA
Normal
Months After Exposure
ALTandHBVDNA
IU/Landmillioncopies/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

51. 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
ALTandHBVDNA
IU/Landmillioncopies/ml
Anti-HBeHBeAg
Laboratory Diagnosis of HBeAg negative
Chronic Hepatitis B
Seeger, Zoulim, Mason, Fields Virology 2007

52. 0,001
0,01
0,1
1
10
100
1000 ALAT
ADN-
VHB
AgHBe +AgHBe + anti-HBe +anti-HBe +
UI/mlUI/ml
pg/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

53. 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 Monitor
Roche 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 Assay
Abbott Molecular
ABBOTT real time HBV DNA assay

54. Formes cliniques

55. MANIFESTATIONSMANIFESTATIONS
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, antivirauxTraitement : plasmaphéreses, corticoides, antiviraux
(vidarabine / IFN / famciclovir / lamivudine)(vidarabine / IFN / famciclovir / lamivudine)
• GlomérulonéphritesGlomérulonéphrites
• CryoglobulinémiesCryoglobulinémies
• Guillain-BarréGuillain-Barré
• MyocarditeMyocardite

56. 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%

57. 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

58. 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 porteursElimination de l’Ag HBs chez 106 porteurs
chroniques du VHB (7 %)chroniques du VHB (7 %)
• Incidence des événements cliniques: 2,3/1000Incidence des événements cliniques: 2,3/1000
porteurs/annéeporteurs/année
• Incidence du CHC: 1,9/1000 porteurs/année (2,3 chezIncidence du CHC: 1,9/1000 porteurs/année (2,3 chez
l’homme; 1,2 chez la femme).l’homme; 1,2 chez la femme).

59. 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)
LiverLiver
InflammationInflammation
LiverLiver
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.
ALTALT
ElevationElevation
ALTALT
ElevationElevation

60. Normal Aminotransferase Levels and
Risk of Mortality from Liver
Diseases
Kim HC et al.Kim HC et al. BMJBMJ 2004; 328:9832004; 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

61. 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%
PatientsSurviving,%
Years
0

62. AgHBeAg et risque de CHC
Yang et al. N Engl J Med. 2002;347:168-174.
Cumulativeincidence(%)
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

63. Charge virale et incidence du CHC
Chen et al; JAMA 2006

64. 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)
%cumulativeincidenceofHCC
> 103
- 104
>104
- 106
≥106

65. 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/mLcopies/mL
RR = 1.7 (0.5-5.7)RR = 1.7 (0.5-5.7)
HBV DNA HighHBV DNA High
≥≥ 101055
copies/mLcopies/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.

66. 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
HCCIncidence
RatePer100,000
0
1473
5882
8730
10,108
2.0x103
4.0x103
6.0x103
8.0x103
1.0x104
1.2x104

67. Impact Clinique de la Variabilité
du Génome Viral

68. 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

69. • 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 génomiqueGENOTYPES : 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

70. 8 genotypes, numerous sub-genotypes, and
recombinant forms
World J Gastroenterol 2007; 13: 14-21
D/E
B6
D1

71. Génotypes VHB chez les patients atteintsGénotypes VHB chez les patients atteints
d’hépatite chronique en Franced’hépatite chronique en FranceNumberofsubjectsNumberofsubjects
FF GGAA BB CC DD EE
00
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

72. Impact du génotype sur la
séroconversion
1
Janssen, Lancet 2005; 2
Flink, Am J Gastro 2006
PEG-IFN a-2b
HBeAg Loss 1
0
10
20
30
40
50
A
n=90
28%
47%
44%
25%
B
n=23
C
n=39
D
n=103
Percentageofpatients(%)
HBV genotype
0
3
6
9
12
15
A
n=90
5%
8%
0%
B
n=23
C
n=39
D
n=103
18
15%
Percentageofpatients(%)
21
HBV genotype
PEG-IFN a-2b
HBsAg Loss 2

73. LES MUTANTS DU GÉNOME DU VHBLES MUTANTS DU GÉNOME DU VHB
déterminant adéterminant a
vaccin/HBIgvaccin/HBIg
polymérasepolymérase
antivirauxantiviraux
Mt pré-coreMt pré-core
Réponse anti-eRéponse anti-e ??
Mt coreMt core
Réponse CTLRéponse CTL

74. 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 vivoModèles 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

75. 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/Carrêt traduction protéine pré-C/C
– AgHBe négatifAgHBe négatif
• mutation dans promoteur pré-Cmutation dans promoteur pré-C
TTAAAGG -> TTAATTAAAGG -> TTAATTGGAA en pos. 1762 /1764en 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

76. HBeAg and Precore Mutation
1814 1901
Precore Core
region region
HBcAg
HBeAg
G 1896A = stop codon, TAG
ATG ATG
Virion
Serum
Core gene

77. HBeAg and Precore Mutation
1814 1901
Precore Core
region region
HBcAg
HBeAg
ATG ATG
Virion
Serum
Core gene

78. VARIANTS NÉGATIFS POUR L ’AgHBeVARIANTS NÉGATIFS POUR L ’AgHBe
mRNAmRNA
ProtéineProtéine
pré-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

79. 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 63-70
Insertion (RGTTAATYATTA) at 74/75
Mutation AGG to TCA and insertion TA at 65/66
WTRTTKRY
Insertion (TTG) at 66/67
TTTTGG
HNF3

80. Sélection des mutants pré-core au cours deSélection des mutants pré-core au cours de
l’histoire naturelle de l’hépatite B chroniquel’histoire naturelle de l’hépatite B chronique
0
500
1000
1500
2000
2500
temps
ALATALAT
ADN-VHBADN-VHB
AgHBeAgHBe Anti-HBeAnti-HBe
0
20
40
60
80
100
temps
sauvagesauvage
Mt pré-CMt pré-C

81. 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 patients
after 23 months (range 12-36) monthly monitoringafter 23 months (range 12-36) monthly monitoring
00 1212 2424
monthsmonths
With flares and normalizationWith flares and normalization
Without flaresWithout flares
With flares and without normalizationWith flares and without normalization
73 pts73 pts
( 44.5% )( 44.5% )
59 pts59 pts
( 36.0% )( 36.0% )
32 pts32 pts
( 19.5% )( 19.5% )
AsymptomaticAsymptomatic
flare-up:flare-up:
90% of cases90% of cases
AA
LL
TT 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

82. 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

83. 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 mutationPromoter mutation
(n = 99; 27.9%)(n = 99; 27.9%)
Stop codon mutationStop 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

84. 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-core
mutationmutation
Stop codonStop codon
mutationmutation
PromoterPromoter
mutationmutation
BothBoth
mutationsmutations
NumberofsubjectsNumberofsubjects
HBe-positiveHBe-positive
HBe-negativeHBe-negative
Lamivir cohort, Zoulim et al, J Viral Hepatitis 2006

85. MUTANTS PRÉ-C ET SÉVÉRITÉ HISTOLOGIQUEMUTANTS PRÉ-C ET SÉVÉRITÉ HISTOLOGIQUE
LA 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

86. HBe serotype and liver pathologyHBe serotype and liver pathology
0-40-4 5-95-9 10-1410-14 15-2215-22
00
1010
2020
3030
4040
5050
6060
7070
Knodell scoreKnodell score
NumberofsubjectsNumberofsubjects
Metavir scoreMetavir score
≤≤ F2F2 F3F3 F4F4
00
1010
2020
3030
4040
5050
6060
7070
HBe-positiveHBe-positive
HBe-negativeHBe-negative
Lamivir cohort, Zoulim et al, J Viral Hepatitis 2006

87. 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 cytotoxiqueSélection 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

88. DIAGNOSTICS DIFFICILESDIAGNOSTICS DIFFICILES
I. Porteur inactifI. Porteur inactif
II. ExacerbationII. Exacerbation

89. Diagnosis of inactive carrier versusDiagnosis 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

90. DIAGNOSTIC D'UNE EXACERBATION AIGUEDIAGNOSTIC 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

91. 0
5
10
15
20
25
0 1 2 5 9 12 13 16
months
1
10
100
1000
10000
100000
1000000
10000000
100000000
1000000000
10000000000
ALT
pre-S1
bDNA
PCR
case#6case#6
Genotype AGenotype A
pre-C promoterpre-C promoter
WTWT
MTMT
pre-C regionpre-C region
WTWT
M2M2
M4M4
M2+M4M2+M4
--
++
--
++
++
++
++
--
--
++
++
--
++
--
--
--
++
--
--
--
++
--
--
--
++
--
--
--
++
--
--
--
++
--
--
--
HBeAgHBeAg
Anti-HBe AbAnti-HBe Ab
--
++
++
++
++
--
--
++
--
++
++
--
--
++
--
++
interferoninterferon
Pichoud et al, J hepatol 2000

92. COOH
137
149
107
99 NH2
S - S
S - S S - S
S- S
S-S
138
139
147
Tiollais P. et al., Nature 1985. Torresi J., J. Clin Virol 2002; Dryden KA. et al., Mol Cell 2006
« a » determinant
HBs Ag
« a » determinant induces the synthesis of
anti-HBs neutralizing antibodies
sG145R
sP120T
sD144H/A/E
PreS1
PreS2
SPol
Pré-C
C
Brin(+) 2,4kb
Brin(-) 3,2kb
X
TATAA
U5-like
DR1
DR2
Enh1Enh2
G
R
E
0/3221
SHBs (S)
MHBs (preS2+S)
LHBs (preS2+preS2+S)

93. 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

94. VARIANTS DE L'AgHBsVARIANTS DE L'AgHBs
• Mutations ponctuelles dans le déterminant a deMutations ponctuelles dans le déterminant a de
l'AgHBs (124-147)l'AgHBs (124-147)
– aa 145 : Gly -> Argaa 145 : Gly -> Arg
– aa 126 : Ile -> Ser / Thr -> Asnaa 126 : Ile -> Ser / Thr -> Asn
• transmission mère-enfant malgré la serovaccinationtransmission mère-enfant malgré la serovaccination
(3%)(3%)
• infection du greffon hépatique malgréinfection 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 +

95. 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

96. How to Detect Occult HBV Infection
Currently there is no standardized
diagnostic assay for occult HBV infection

97. Reported Prevalence of Occult HBV Infection in HIV Positive Patients
Study Country N° of
patients
Occult
HBV
N° (%)
Methods
Hofer, 1998 Switzerland 57 51 (89%) “nested” PCR
(serial evaluation)
Torres-Baranda, 2006 Mexico 35
7 (20%) “nested” PCR
Filippini, 2006 Italy 86 17 (20%) single step PCR
Mphahlele, 2006 South Africa 140 31 (22.%) “nested” PCR
Pogany, 2005 Netherlands 93 4 (4%) single step PCR
Neau, 2005 France 160 1 (0.6%)
Santos, 2003 Brazil 101 16 (16%) single step PCR
Wagner, 2004 France 30 11 (37%) “nested” PCR
Goncales, 2003 Brazil 159 8 (5%) “nested” PCR
Nunez, 2002 Spain 85 0 Cobas Amplicor HBV
Monitor (Roche)
Piroth, 2000 France 37 13 (35%) single step PCR
Raffa, 2007 Italy “nested” PCR (liver)
Cobas Amplicor HBV
Monitor (Roche)
101 42 (41%)
Raimondo et al, J Hepaol 2007, modified

98. OBI
Cause(s) for the
failure of HBsAg detection
Suppression of
HBV replication and
gene expression
Infection by
S gene Variants
“false” OBI

99. Occult HBV infection
HBV cccDNA Integrated HBV DNA
HBV mutants Epigenetic control
HBV replication
Immune surveillance
Viral co-infections

100. OBI
SeropositiveSeropositive
SeronegativeSeronegative
HBsAg lost
during CH
HBsAg lost
during CH
HBsAg lost
after AH
HBsAg lost
after AH
Progressive antibody
disappearence
Progressive antibody
disappearence
Primary occultPrimary 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

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

102. High
prevalence
ROLE
in
HCC
Diagnostic
Tools ?
Worsen
HCV
infection ?
Co-infections ?
Therapy?
To be
improved
Specific
treatments ?
Not fully
understood ?
Occult HBV infections: unresolved issues

103. AntivirauxAntiviraux
Persistance viralePersistance virale
Resistance aux antivirauxResistance aux antiviraux
Monitoring des traitementsMonitoring des traitements

104. 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 10or 1033
copies/mLcopies/mL
Histological responseHistological response
- improvement in HAI or Metavir score- improvement in HAI or Metavir score
Combined responseCombined response
Complete 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

105. Mommeja-Marin H et al. Hepatology 2003Mommeja-Marin H et al. Hepatology 2003
Median logMedian log1010 HBV DNA level decrease from BaselineHBV DNA level decrease from Baseline
Efficacy : Correlation between HBV DNAEfficacy : Correlation between HBV DNA
response and histologic benefitresponse and histologic benefit
MedianHistologicActivityIndexMedianHistologicActivityIndex
ImprovementfromBaselineImprovementfromBaseline
11 22 33 44 55
IFNIFN

106. 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 responseEnd of treatment response
Sustained reponseSustained reponse
-> can we stop therapy ?-> can we stop therapy ?

107. Pyrimidine dideoxynucleoside analogues
2 ’ Fluoro-substituted
arabinosylpyrimidines
Lamivudine Emtricitabine ElvucitabineClevudine
OH
O OHN
HN
O
O
CH3
β-L-2’-deoxythymidine
telbivudine
Purine dideoxynucleoside analogues
acyclic nucleoside phosphonates carbocyclic guanosine analogue
entecaviradefovir
N
N
N
N
NH2
P O
HO
HO
O
CH3
tenofovir
PMPA

108. Anti-HBV Active Compounds
Drug type Approved Phase 3 Phase 2
Nucleoside
analogs
Lamivudine
Entecavir
Telbivudine
Emtricitabine*
Clevudine**
Elvucitabine
Valtorcitabine
Amdoxovir
Racivir
LB80380
Nucleotide
analogs
Adefovir
Tenofovir
Alamifovir
Pradefovir
Cytokines
Interferon alfa
Peg-interferon
alfa-2a
* Currently approved for HIV
** Approved in South Korea

109. L(-)-SddC, 3TCL(-)-SddC, 3TC
LamivudineLamivudineL(-)-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

110. interaction
Virion
Nucleus
Hepatocyte
translation
encapsidation
reverse transcription
pgRNADNA (-)
cccDNA
amplification
transcription
mRNA
pgRNA
AAA
AAA
AAA
AAA
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

111. Blood stream
viral load
Infected hepatocytes
Infected liver
CD8
NKT
CD4
B

112. Blood Stream
Viral Load
Infected hepatocytes
Infected Liver
Antiviral
CD8
NKT
CD4
B

113. uncoating CCC DNA
removal of protein primer
removal of RNA primer
completion of viral (+) strand DNA
ligation of DNA strands extremities
supercoiled DNA
minichromosome
viral polymerase?
DNA repair protein?
other cellular enzymes?
topoisomerase?
Acetyl transferase ?
Histones
Formation of the recalcitrant cccDNA: a difficultFormation of the recalcitrant cccDNA: a difficult
target for antiviral therapytarget for antiviral therapy
Tuttleman et al Cell 1986
Le Guerhier et al AAC 2000
Delmas et al AAC 2002
Kock et al Hepatology 2003
Antivirals ?

114. 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 un
décompresseur TIFF (non compressé)
sont requis pour visionner cette image.

115. 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

116. M0 M2 M6
L-FMAU + FTC + Ad-IFNL-FMAU + FTC + Ad-IFNγγ UntreatedUntreated
M0 M2 M6
M0 M2M1
Failure to eradicate cccDNA with a combination ofFailure 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

117. 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))
Serum
HBV
DNA
Total
Intracellular
DNA
cccDNA 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 > cccDNAserum 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

118. • 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-overSuggests 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 atImmunohistochemical Staining of Patient Biopsies at
Baseline and After 48 Weeks ADV TherapyBaseline and After 48 Weeks ADV Therapy
BaselineBaseline Week 48Week 48

119. Maynard et al, J Hepatol 2005Maynard et al, J Hepatol 2005
Persistence of cccDNA after HBs seroconversionPersistence of cccDNA after HBs seroconversion

120. Clearance of viral infection versus selection ofClearance 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 theThe rate of replication and spread of mutant virus in the
chronically infected liver (I.e. fitness of the virus: the rate ofchronically 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 onSmall changes in these factors may have profound effect on
whether treatment response is durable or subject to rapidwhether treatment response is durable or subject to rapid
reboundrebound (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

121. LamivudineLamivudine
II IIII IIIIII IVIV
wtwt mtmt
nini
INHIBITION OF WILD TYPE VIRUS REPLICATIONINHIBITION OF WILD TYPE VIRUS REPLICATION DELAYED EMERGENCE OFDELAYED EMERGENCE OF
DRUG RESISTANT VIRUSDRUG RESISTANT VIRUS
XX
XX
XX
XX
XX
XXXX
Zhou et al AAC 1999
Kinetics of emergence of drug resistantKinetics of emergence of drug resistant
virus during antiviral therapyvirus during antiviral therapy
• Free liver space
• Mutant fitness

122. 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
Viralload
LamivudineLamivudine
Changement de la quasi-espèce virale et de la sensibilité auxChangement 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
2
1.5
1.2
1
Lamivudine
0 1,6 3,2 6,3 12,5 25 50 100 mM
3
2
1.5
1.2
Linear
ds HBV
1
IC50» 0.1µM
IC90
» 2 µM
IC50> 50µM
IC90>>100µM
Quasi-espèce
Phenotype
Quasi-espèce
Durantel, Hepatology 2004

123. Mechanisms of HBV Drug Resistance
Viral persistence
cccDNA
Long half-life
Infected cells
Long half-life
DefectiveDefective
immuneimmune
responseresponse
VirusVirus HepatocytesHepatocytes
Impairment ofImpairment of
innate responseinnate response
HostHost
Selective pressure
Antivirals or others
Viral polymerase
spontaneous error rate
VirusVirus
Selection of escapeSelection of escape
mutantsmutants
Treatment failureTreatment failure
Replication fitness
Replication space
Viral quasi-species
Immune response
Drug PK
Zoulim Antivir Res 2004;64:1–15

124. 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 EEDD
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 N236T
I233V ?
LdT M204I
TDF A194T ?
M204I/V

125. Mechanisms of resistance

126. Treatment failure
Primary non response
Partial response
Secondary treatment failure
Antiviral drug resistance
Host factors
Drug metabolism
Patient’s compliance
Drug factors
Antiviral potency
Drug factors
Barrier to resistance
Viral factors
Resistant mutants

127. Partial response to adefovir dipivoxil is not due to the
selection of DR mutants
• The top 25% patients (quartile 1): > 4.91 log10 reduction in serum HBV DNA at week 48.
• In Q2: 3.52 to 4.90 log10 reduction of viral load.
• In Q3: 2.22 to 3.51 log10 reduction in viral load.
• The bottom 25% of patients (Q4):< 2.22 log10 reduction in HBV DNA levels at week
48.
• Phenotypic analysis of viral strains: Q4 as sensitive to ADV as Q1 strains
• Documented Drug Compliance (% of days without taking ADV)
• Wilcoxon rank sum test, P=0.01 Durantel et al, Antiviral Therapy, 2008
Virological Response
Q1 (best response)
(n=38)
Virological Response
Q2
(n=38)
Virological Response
Q3
(n=38)
Virological Response
Q4 (worse response)
(n=38)
Median 99% 99% 99% 97%a
range 86-100% 41*-100% 91-100% 70-100%

128. 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

129. 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

130. 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

131. 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

132. Villet, Billioud et al, Gastroenterology 2008
0
50
100
150
200
250
300
350
400
wt #1 #2 #3 #4 Mutant
Mutantreplicationcapacity/wt(%)
Viral replication capacity in the presence of both
antivirals (LAM + ADV)

133. Mutant
wt #1 #2 #3 #4
1,7 kb
A
B
wt #1 #2 #3 #4 Mutant
Mutantinfectivity/wt(%)
0
20
40
60
80
100
120
Villet, Billioud et al, Gastroenterology 2008
Infectivity of the mutants in HepaRG cells
Impact of mutations in the overlapping S gene
HDV hybrids with HBV mutant envelopes
HDV replication in HepaRG cells as a reporter of infection

134. 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
HBVreplication
Primary resistance
mutations
Secondary resistance mutations
/ compensatory resistance mutations

135. 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 181
ADV
204 ± 180
Wild-type
204 + 180
LAM
Wild-type
Wild-type
ETV
184 / 202 /250
ADV-R
ETV-R
ETV-R
LAM-R
TDF ?

136. Archiving of resistant mutants

137. • cccDNA in the liver:
– Is propagated during the normal
replication cycle of HBV
– Can serve as a template for the
production of new virus
Archiving of viral variants
Viral quasispecies
cccDNA variants
Liver
Majority population
Minority variants
Resistant variants
Blood circulation
Zhou et al, AAC 1999; Zoulim F. Antivir Res. 2004. Zoulim F & Perillo R. J Hepatol. 2008

138. • 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
Liver
Majority population
Minority variants
Resistant variants
Zhou et al, AAC 1999; Zoulim F. Antivir Res. 2004. Zoulim F & Perillo R. J Hepatol. 2008

139. • 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
Liver
Majority population
Minority variants
Resistant variants
Blood circulation
Zhou et al, AAC 1999; Zoulim F. Antivir Res. 2004. Zoulim F & Perillo R. J Hepatol. 2008

140. Multidrug resistance

141. 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

142. The Problem of Sequential Therapy
with Nucleoside Analogs
+ one mutation + one mutation
?
Multiple drug resistant
mutants with complex
pattern of mutations
Drug A
Drug B
Risk of selection of MDR mutants by sequential therapy,
especially when using drugs sharing cross-resistance characteristics

143. 103
104
105
106
107
108
109
0 20 40 60 80 100 120
Treatment (months)
HBVDNA(copies/ml)
entecavir
IFN
adefovir
lamivudine
Genotype H
lamivudine
Drugs sharing cross-resistance characteristics:
Switching strategy emergence of MDR mutant
L180M+S202G+M204V
L180M+M204V
Villet et al, J Hepatol 2007

144. Treatment(months)
lamivudineentecavir
0 20 40 60 80 100
1
L180M+M204V
M204V
wt
V173L+L180M+M204V
L180M+M204V
V173L+L180M+M204V
L180M+S202G+M204V
I169L+L180M+S202G+M204V
V173L+P177S+L180M+S202G+M204V
V173L+P177S+L180M+S202G+M204V
L180M+A181G+S202G+M204V
L180M+S202G+M204V
L180M+A181G+S202G+M204V
wt
% clones in the quasi-species
27/0
0
11
34
36
- Lamivudine therapy: Selection of a main population harboring the V173L+L180M+M204V
mutations = primary resistance mutations
- Entecavir therapy: Selection of three populations, all harboring the L180M+S202G+M204V
mutations = secondary resistance mutations
Genotypic analysis of the viral quasi-species
during lamivudine and entecavir therapy
Lamivudine
rebound
Entecavir
rebound
Villet et al, J Hepatol 2007

145. 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
HBVDNA(Meq/ml)
lamivudine
adefovir
HBIg tenofovir
V173L+L180M+A181V+N236TL180M+M204V
Liver transplantation

146. 0 10 20 30 40 50 60 70 80 90 100
1
Lamivudine+adefovirtreatment(months)
1
8
24
34
38
40
42 to 50
Viral rebound
0
16
26
30
32
34 to 42
Timepost-transplantation(months)
% of variants in the
viral quasi-species
Accumulation of mutations and selection of a complex mutant
YMDDYMDD
Terminal
Protein spacer Pol/RT RNaseH
V173L L180M A181V N236T
Pre-S/S gene
P120S
dominant HBV
mutant
L180M+M204I
wt
V173L+L180M+A181V+M204V
M204I
V173L+L180M+A181V
V173L+L180M+A181V+M204V+N236T
V173L+L180M+A181V+N236T
V173L+L180M+A181V+N236T
V173L+L180M+A181V+N236T
V173L+L180M+A181V+M204V+N236T
V173L+L180M+A181V+M204V
V173L+L180M+A181V+M204V
V173L+L180M+A181V+M204V
V173L+L180M+A181V+M204V
L180M+M204I
V173L+L180M+A181V+M204I
I169V+L180M+T184I+M204V
V173L+L180M+A181V+N236T

147. 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

148. Perspectives
Prevention of drug resistance
• First line therapy
– Use of antivirals with high antiviral potency and high barrier to
resistance
– Combination therapy with complementary drugs
• 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

149. Antiviraux
Monitoring et resistance
Aspects cliniques

150. Clinical Definition of HBV Resistance to Antivirals
Clinical
• 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

151. 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 ofLine 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
HBVDNA
(10E+6genomeeq/ml)
ALT(U/L)
0
20
60
100
140
180
1 595100 200 300 400
Codon 528 LiPA
Seq
Codon 552 LiPA
Seq
Codon 555 LiPA
Seq
1
L
L
M
M
V
V
39
L
L
M
M
V
V
290
L/M
L/M
M/V
M
V
V
400
M
M
V
V
V
V
595
M
M
V
V
V
V
Day
T A T A T G
C T C M T G
G A T
G C T

152. 600
500
200
150
100
50
0
8
7
6
5
4
3
0 6 12 18 24 30 36
Lamivudine
Months42
ALT(U/L)
HBVDNAlogcopies/mL
M
M
V
M
M/V
V
L
M
V
L/M
M
V
M
M/V
V
L
M
V
M
V
V
Codon 180
Codon 204
Codon 207
M
V
V
Genotypic resistance
Dynamics of Resistance Emergence
Genotypic Resistance
Si Ahmed et al. Hepatology 2000;32:1078–88

153. 600
500
200
150
100
50
0
8
7
6
5
4
3
0 6 12 18 24 30 36
Lamivudine
Months42
PCR assay
ALT(U/L)
M
M
V
M
M/V
V
L
M
V
L/M
M
V
M
M/V
V
L
M
V
M
V
V
Codon 180
Codon 204
Codon 207
M
V
V
HBVDNAlogcopies/mL
Rebound of serum
HBV DNA*
> 1 log10
copies/mL
Dynamics of Resistance Emergence
Virologic Breakthrough
Si Ahmed et al. Hepatology 2000;32:1078–88

154. 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
M
M
V
M
M/V
V
L
M
V
L/M
M
V
M
M/V
V
L
M
V
M
V
V
Codon 180
Codon 204
Codon 207
M
V
V
ALT(U/L)
HBVDNAlogcopies/mL
Worsening of liver disease
Dynamics of Resistance Emergence
Clinical Breakthrough
Si Ahmed et al. Hepatology 2000;32:1078–88

155. Sequence of Events in
Resistance to antiviral Therapy
Time
Antiviral drug
HBVDNA(log10IU/mL)
ALT(IU/L)
4
6
5
3
2 ULN
1
0
Detection of
Genotypic Resistance
Nadir
1 log10
Virologic
Breakthrough
Biochemical
Breakthrough
Genotypic
resistance
Virological
Breakthrough
Biochemical
Breakthrough

156. 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

157. Incidence of Resistance in
Nucleoside Naive Patients
%ofpatientswith
resistancemutations
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 1
year 2
year 3
year 4
year 5

158. Incidence of Resistance in
Lamivudine Refractory Patients
%ofpatientswith
resistancemutations
0
10
20
30
40
Adefovir
switch
Adefovir
add-on
Entecavir
switch
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

159. 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.

160. ALT flares in patients with lamivudine
resistance over time
QuickTime™ et un
décompresseur TIFF (LZW)
sont requis pour visionner cette image.
Lok et al Gastroenterology 2003; 125 : 1714-1722

161. 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

162. 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 mutations
VR: virologic breakthrough
ALT: biochemical breakthrough*
M M + VR M + VR + ALT

163. 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: ?

164. Age: ≥ 50 years old
Alcohol consumption
Place of birth: Asia
HBV DNA: > 5 x 106
IU/ml
Metavir score: ≥ F3
LiPA genotype: C
0.1110
Odds ratio
Zoulim et al, J Viral Hepatitis, 2006
Multivariate logistic analysis: VIRAL LOAD was the only parameter associatedMultivariate 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)

165. 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

166. 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

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

168. 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
Monitor
at 12-24 weeks
Early non reponse
Monitor
every 12 weeks
Switch to more
potent agent*
Zoulim & Perrillo, J Hepatol in press

169. 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

170. 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

171. Months
ADV mono
Patientswithvirologicalbreakthrough
273 268 256 225 201 158 61
30%
6%
P<0.001
ADV+LAM
255 238 223 213 200 177 103 PatientswithADV-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

172.  HBV DNA ∆ ALT
The problem of sequential therapy
and switching strategy
Villeneuve et al, J Hepatol 2003
N236T
SerumHBVDNA
(Log10copies/mL)
ALT(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

173. Resistance to Lamivudine / Telbivudine
Add:
• ADV
• TDF
Switch 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

174. 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

175. 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

176. M0 M6
M12M18M24M30 M36
ALT
0
2
4
6
8
ALT HBV DNA
Month of therapy
Rescue therapy in patients with clinical breakthrough
Drug A
Drug B
SerumHBVDNA(Log10copies/mL)
andALT(xULN)

177. M0 M6
M12 M18 M24 M30 M36
ALT
0
2
4
6
8
ALT
HBV DNA
Month of therapy
Rescue therapy in patients at the time of virologic breakthrough
Drug A
Drug B
SerumHBVDNA(Log10copies/mL)
andALT(xULN)

178. M0 M6
M12 M18 M24 M30 M36
ALT
0
2
4
6
8
ALT
HBV DNA
Month of therapyMonth of therapy
Early add-on therapy to prevent drug resistance
Drug A
Drug B
SerumHBVDNA(Log10copies/mL)
andALT(xULN)

179. 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 A
Drug ADrug A
++
Drug BDrug B
Month of therapy
1. Start with a drug having a high genetic barrier for resistance
2. Add a drug with a different cross-resistance profile
outgrowth of drug resistant mutant ?
MDR ?

180. 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 ofLow risk of
selection of MDRselection of MDR
Clavel et al NEJM 2004;350:1023-35 ; Zoulim Antiviral Res 2004;64: 1-15

181. M0 M6
M12 M18 M24 M30 M36
ALT
0
2
4
6
8
ALT
HBV DNA
Month of therapyMonth of therapy
De novo combination therapy to prevent drug resistance
Drug A
Drug B
SerumHBVDNA(Log10copies/mL)
andALT(xULN)

182. Preventing L-Nucleosides Resistance
with de novo Combination Therapy
1 Marcellin et al. N Engl J Med 2004; 351: 1206-17
2 Lau et al. Hepatology 2004;40:171A
3 Lai et al. Hepatology 2003;38:262A
4 Sung et al. J Hepatol 2003 ;38 (suppl 2):25-26
5 Lau et al. Hepatology 2004:40:666A
* After 1- year therapy
20%18%
34%
21%
2%1%
11% 12%
5%
0
20
40
60
80
100
Sung 4
Marcellin 1
Lau 2
Lai 3
LAMLAM LAM LAM LAM
+ADV
LAM
+Peg
LAM
+Peg
LAM
+LdT
Incidenceofresistance*(%)
LdT FTC FTC
+ADV
0% 0%
Lau 5

183. 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

184. 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

185. Conclusions 2
• Différentes formes d’hépatites en fonction deDifférentes 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 desNécessité d’un dépistage et traitement précoce des
formes chroniquesformes chroniques
• Prévention par la vaccination !!!Prévention par la vaccination !!!