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

2. Guérison 30-50 ans
VHB HCA cirrhose CHC
Vaccin ANTIVIRAUX Antiviraux/IFN?
IFN Niederau N Engl J Med 1996
& Liaw N Engl J Med 2004
RESISTANCE VIRALE
Lee, N Engl J Med 1997; Lok, Hepatology 2001

3. EPIDEMIOLOGIE DE L’HÉPATITE B

5. EPIDEMIOLOGIE DE L'INFECTION A VHB
AUX USA
• Hépatites aigues
– VHA : 40%
– VHB : 30%
– VHC : 20%
• incidence : 300 000 infections à VHB / an
• 30 000 nouveaux porteurs chroniques / an
• 3 000 décès / an

6. MODES DE TRANSMISSION DU VHB
• 1108 habitants de San Francisco
• 159 (14%) anti-HBc +
• positivité des anti-HBc associée avec
– âge
– éthnie
– degré d'éducation
– toxicomanie IV
– prostitution
– nombre de partenaires sexuels
– homosexualité
– HIV / HSV 2 / syphilis

7. MODES DE TRANSMISSION DU VIRUS DE L'HÉPATITE B EN EUROPE
transfusions
contact avec 2%
porteur du VHB
4% personnels de santé
sexuelle 2%
homo hémodialysés
34% 11% 8%
hétéro
23%
inconnue
31%
Asie drogue IV
26%
Transmission verticale

8. 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

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

10. Circuit de l’information
Feuillets 2 et 3 Médecin
Biologiste à compléter prescripteur
Feuillet 1 : Relance Feuillet 2 :
parties 1-2 et parties 3-4-5
6-7 renseignées MISP de DDASS du complétées
département
d’exercice
Feuillets 1 et 2 complétés et validés
InVS
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

11. Results
158 acute hepatitis cases
• Hospital doctor in 64% cases
• Sex ratio M/F : 2,95 (118/40)
• Median age: 37 yrs for males, 36yrs for females
• Jaundice : 69%
• Hospitalisation : 46%
• Fulminant hepatitis : 3 (2 death)

12. Age distribution: comparison of the different periods
1991-94 versus 03/2003 - 02/2004
years 1991- 94
n= 151
March 03- February 04
n= 158

13. Risk exposure within 6 months preceding the acute case
Source : obligatory declaration 2003-04
• Source: obligatory declaration march 03- february 2004 N=145
– Sexual 59 40,6% No factor 43 29,6%
– IVDU 9 6,2% >1 factor 38 26,3%
– Invasive treatment 15 10,3%
– Tatoo, piercing 5 3,4%
• Sentinel networks 91-96
– Familial 14 9,7% N=195
– Perinatal 2 1,4% – sexual 35%
– Live in instiution 11 7,6% – IVDU 19%
– « percutaneous » 15%
– Travel in endemic 21 14,5%
– No factor 35%
areas
91/145 patients (63 %) had a vaccine indication (2 vaccinated ≥ 3 doses)

14. Hépatites virales B: épidémiologie
- Vaccin mais 400 millions de porteurs
chroniques dans le monde
- 280 000 porteurs chroniques en France (INVS)
- 45% ignorent leur statut
- 1 300 décès par an en France
- 60 000 avec hépatite chronique active
- Seulement 13 000 patients traités

15. VIROLOGIE

16. LE VIRUS DE L ’HEPATITE B
• FAMILLE : Hepadnaviridae, seul représentant humain
•VIRUS RESISTANT :
- 7 jours dans l’environnement
- pendant 5 mn à 100° 10 h à 60°
C, C
- à la congélation.

18. LE GÉNOME DU VIRUS DE L’HÉPATITE B
déterminant a
vaccin/IgHBs
8 génotypes
A to H Gène pol
antiviraux
Mt du core
Réponse CTL
Tiollais Nature 1985
Mt pre-core Günther Adv Virus Res 1999
Réponse anti-HBe ? Norder J Gen Virol 2003

19. Ganem & Prince, NEJM 2004

20. Réplication du génome viral. Implication pour la
persistance virale et l’intégration du génome viral
Membrane cellulaire
ARN pg
ds DNA
10% virion
ss DNA
90%
intégration
cccDNA
illégitime
RC DNA
noyau
virion cccDNA

21. Modèles Animaux Chimpanzé
VHB HUMAIN
Souris Transgéniques
Souris SCID uPa
Tupaia
MARMOTTE (WHV) CANARD (DHBV)
Summers PNAS 1978, Mason J Virol 1981, Chisari Science 1985, Petersen PNAS 1998

22. Modèles in vitro
• Polymerase virale • Culture cellulaire
– DHBV : lysat réticulocytaire – Transfection : lignées d’hépatome
– HBV : baculovirus – Infection : hépatocytes primaires, HepaRG
– Baculovirus ou adenovirus recombinant
RC -
L-
Polymerase VHB
DNA(-)
DNA(
U SS -
ELONGATION
CCC -
Sells PNAS 1987, Wang Cell 1992, Zoulim J Virol 1994,
Lanford J Virol 1995, Gripon PNAS 2002, Sprinzl J Virol 2001

23. Cycle de réplication du VHB
Zoulim & Locarnini, Gastroenterology 2009

24. Comparative dynamics among three viruses
HIV HCV HBV
(Ritonav ir) (IFN- ) (Lamivu dine)
Plasma virus
Half-life 5.8 h 2.7 - 7.2 h 24 h
Mea n viral 2.7 d 3.8 - 7.3 d 24.7 d
genera tion time
Daily t urnover 95% 94% - 99. 8% 50%
Daily produ ction 10 10 (1.1 - 10 11
(plasm a) 12.7 )*10 11
Tot al load 1.2*1 0 9 (3.8 - 5.6)*10 10 2*10 11
Infecte d ce lls
Half-life 1.6 d 2.4 - 4.9 d 10 - 10 0 d
Mea n lifespan 2.3 d 3.5 - 7.1 d 23.3 d
Daily t urnover 38% 13% - 25% 1% - 7%
(Tsiang et al. Hepatology 1999)

25. Infection à VHB et risque de CHC
• Etude de Beasley à Taiwan
– risque relatif = 100 chez les porteurs de l'AgHBs
• Etude de Tsukuma
– risque cumumatif de CHC à 3 ans
• 12,5% chez 240 patients avec cirrhose
• 3,8% chez 677 patients avec hépatite chronique
– risque x 7 si AgHBs +
– risque X 4 si anti-HCV +
• Facteurs associés : alcool, tabac, aflatoxine
• Diminution incidence avec la vaccination de masse (Chen,
NEJM 1995)

26. CARCINOME HEPATOCELLULAIRE ET VIRUS
DE L'HEPATITE B
• Co-incidence de répartition géographique
VHB / CHC
• Porteurs AgHBs : RR x 100 pour le CHC
• CHC dans les modèles animaux de l'hépatite B :
– marmotte
– écureuil
• Présence d'ADN viral intégré dans les tumeurs

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

28. PATHOGENIE DU CARCINOME
HEPATOCELLULAIRE
ALCOOL
VHB VHC
LESIONS HEPATIQUES CHRONIQUES DESORDRES
METABOLIQUES
ACTIVATION FACTEURS
DE CROISSANCE
REGENERATION FACTEURS
ENVIRONNEMENTAUX
ALTERATIONS GENETIQUES
CARCINOME HEPATOCELLULAIRE

29. Role du VHB dans l’oncogénèse hépatique
REACTION INFLAMMATOIRE CHRONIQUE
REGENERATION HEPATIQUE
VHB CARCINOGENES
INFECTION CHRONIQUE CHC CO-FACTEURS
MUTAGENESE INSERTIONNELE
TRANSACTIVATION DE GENES CELLULAIRES
INTERACTIONS PROTEIQUES
INACTIVATION DE GENES SUPPRESSEURS DE TUMEUR

30. PHYSIOPATHOLOGIE /
IMMUNOPATHOLOGIE

31. Ganem and Prince, NEJM 2004

32. IMMUNOPATHOGÉNIE
DES HÉPATITES B CHRONIQUES
RÉPONSE IMMUNITAIRE
HÉPATOCYTE
CYTOKINES
NON INFECTÉ
CTL cytokines
perforine
Fas
ANTICORPS NEUTRALISANTS AgHBc/e
VHB HLAI
ANTIVIRAUX
HÉPATOCYTE INFECTÉ

33. IMMUNOPATHOLOGY OF HBV INFECTION
CD8+
HBV
Immune tolerance
CD8+ HBV
Clairance phase
Chronic hepatitis
HBV
Seroconversion CD8+
Remission

34. Immunopathology
Fulminant hepatitis
HBV
CD8+

35. MECHANISMS OF VIRAL CLEARANCE
Non cytolytic processes Turn-over of infected cells
TH1 cytokines with direct antiviral Immune mediated lysis of infected cells
effect
Transgenic mice Ducks
Chimpanzees Woodchucks
(Guidotti Science 1999, (Guo J Virol 1999
Thimme J Virol 2003)
Summers PNAS 2003&2004)
Antivirals
Inhibition of viral DNA synthesis
-> inhibition of intracellular recycling of cccDNA
(Werle Gastroenterology 2004)
Restoration of anti-HBV immune response
(Boni Hepatology 2000)

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

37. Hepatocyte turn-over is required for clearance of
viral infection in acute infection
Summers et al, PNAS 2003 & 2004

38. Phase de tolérance immunitaire
Marqueurs
Hépatocyte AgHBe +
non infecté
HBV DNA +++
ALAT = N
Foie = N
HBc/e Ag
HBV
Hépatocyte infecté

39. Phase de clairance immune
(hépatite chronique)
Marqueurs
Hépatocyte AgHBe+
non infecté
HBV DNA +
CTL
ALAT +++
Foie: Hépatite
cytokines chronique
perforine
Fas
HBc/e Ag
HBV
HLAI
Hépatocyte infecté

40. Phase de rémission
portage inactif de l’AgHBs
Marqueurs
Hépatocyte
non infecté
AgHBe-
anti-HBe +
HBV DNA < 104 /mL
ALAT = N
Foie = rémission
HBs Ag
Hépatocyte infecté Réactivation
Virus sauvage
Oncogénèse ou mt pre-core

41. Clairance de l’AgHBs
Marqueurs
Hépatocytes HBsAg -
non infectés
anti-HBc +
Anti-HBs +/-
HBV DNA - mais PCR +
Hépatocytes infectés Mutants d’échappement
Oncogénèse
Infections occultes

42. cccDNA levels in the different phases of
chronic HBV infection
10 3 10 4
cccDNA (copies/cell)
Total HBV DNA
10 3
(copies/cell)
10 2
1 10 2
10
0
10 1
10
-1
10 0
10
-2
10 -1
10
10 -2
-3
10
10 -3
) ) ) ) ) )
63 18 10 (7 3) 18) 10 (7
( ( ( - (6 -( ( -
g + g- rs Ag g + g rs Ag
eA eA rie BS eA ie
B r H eA B rr BS
HB H
. Ca HB H .C
a H
ct t
a ac
In In
• HBeAg+ patients had significantly higher cccDNA (90-fold) and total HBV
DNA (147- fold) levels compared to HBeAg- patients. (p<0.001, Wilcoxon
tests)
Werle et al, Gastroenterology 2004

43. HISTOIRE NATURELLE ET
VIROLOGIE CLINIQUE

44. Histoire Naturelle de l’hépatite B
Infection aigue Seeger, Zoulim, Mason;
Guérison Fields Virology; 2007
5% nx-nés Infection chronique
90% adultes
Hépatite chronique
Virus sauvage (HBeAg+)
Mutant pre-core (HBeAg-) Portage inactif
Tolérance immunitaire
Réactivation
Cirrhose
30-50 ans Carcinome hépatocellulaire

45. MARQUEURS SEROLOGIQUES
• Système AgHBe /anti-HBe
– distinction virus sauvage / virus muté AgHBe
négatif
• Virémie
– détection quantitative de l'ADN viral

46. HEPATITE B AIGUE
• Incubation 1 à 6 mois
• Le plus souvent asymptomatique
– Évolution plus fréquente vers la chronicité
• Prodromes:
– Maladie sérique : arthralgies, urticaire,
acrodermatite etc. ..
• Formes ictériques : + graves que VHA et VHC
– Durée de l’ictère : jusqu’à 4 mois
• Evolution : chronicité 5 à 10%
• Hépatites fulminantes

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

48. HEPATITE B PROLONGEE
• Définition
– Persistance réplication virale à la 8ème
semaine d’évolution :
– AgHBe + ou ADN-VHB +
• Evolution
– Chronicité : 8 cas / 10
• Traitement : IFN
– Guérison : 7 à 8 cas / 10

49. INFECTIONS CHRONIQUES A VHB
FORMES CLINIQUES
• virus sauvage
– tolérance immunitaire
– rupture de tolérance -> lésions hépatocytaires : HCA
– séroconversion anti-HBe spontanée (portage inactif) :
5-10% /an
– > diminution significative réplication virale
– > amélioration signes histologiques
• virus muté pré-C (-)
– sélection au moment de la séroconversion anti-HBe
– dépend du génotype viral
– immunopathologie ?
– sévérité de l'hépatopathie : controversée
– association au CHC

50. Laboratory Diagnosis of Chronic Hepatitis B
associated with wild type virus infection
HBsAg
800
HBeAg
IU/L or million copies/ml
700
ALT and HBV DNA
600
500
HBV DNA
400
300
ALT
200
100
Normal
0
0 1 2 3 4 5 6 12 24 36 48 60
Months After Exposure
Seeger, Zoulim, Mason, Fields Virology 2007

51. Laboratory Diagnosis of Transition of Chronic
Hepatitis B to The inactive Carrier State
800
HBsAg ``
IU/L and million copies/ml
700
HBeAg Anti-HBe
600
ALT and HBV DNA
500
400 HBV DNA
300
200 ALT
100
Normal
0
0 1 2 3 4 5 6 12 24 36 48 60 72 80 92 104
Months After Exposure
Seeger, Zoulim, Mason, Fields Virology 2007

52. Laboratory Diagnosis of HBeAg negative
Chronic Hepatitis B
HBsAg
HBeAg Anti-HBe
IU/L and million copies/ml
450
400 ALT
ALT and HBV DNA
350
300
250
200
HBV DNA
150
100
50
Normal ALT levels
0
0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 Months
Seeger, Zoulim, Mason, Fields Virology 2007

53. AgHBs
UI/ml
UI/
pg/ml
pg/
AgHBe + anti-HBe +
1000 ALAT
9 log
ADN-
8 log
100 VHB
7 log
10
6 log
1 hybridation
5 log
4 log
0,1
3 log
0,01
2 log
PCR
0,001
1 log
Tolérance hép chronique p. inactif mt pré-core VHB occulte

54. Dynamic ranges of quantification
of HBV DNA assays
10 102 103 104 105 106 107 108 109
Amplicor HBV Monitor
v2.0 (Roche)
HBV Hybrid-Capture II
(Digene)
Ultra-sensitive HBV
Hybrid-Capture II
Versant HBV DNA
3.0 (bDNA, Siemens)
Cobas Taqman HBV
(Roche)
RealArt HBV LC PCR
(Artus Biotech)
Abbot Real-time HBV
(Abbott)
Versant HBV DNA 1.0
(kPCR, Siemens)*
*in development

55. Formes cliniques

56. MANIFESTATIONS
EXTRAHEPATIQUES DU VHB
• PAN
– Complexes immuns circulants HBs/anti-HBs
– Dépots artères moyens et petit calibre
– Traitement : plasmaphéreses, corticoides, antiviraux
(vidarabine / IFN / famciclovir / lamivudine)
• Glomérulonéphrites
• Cryoglobulinémies
• Guillain-Barré
• Myocardite

57. TRANSMISSION VERTICALE DU VHB
• mère AgHBe +
– transmission : 90%
• mère anti-HBe +
– transmission : 10-20%
– VHB muté pré-C (-) : hépatites fulminantes
• chronicité chez l’enfant : 90%

58. PRESENTATION CLINIQUE
• INFECTION PERI-NATALE
– ALT normales ou subnormales
– ADN-VHB > 1000 pg/ml
– histologie : lésions minimes
• INFECTION POST-NATALE
– ALT élevées
– ADN-VHB < 1000 pg/ml
– histologie : hépatite modérée à sévère
• CARCINOME HEPATOCELLULAIRE : 30 ANS

59. Histoire naturelle de l’infection chronique
par le virus de l’hépatite B
en Alaska
• McMahon BJ, Ann Intern Med 2001;135(9):759-68
• 1536 natifs d’Alaska : 641 AgHBe+, 83 anti-HBe+
• Probabilité d’éliminer l’Ag HBe à 10 ans : 72,5 %.
• Elimination de l’Ag HBs chez 106 porteurs
chroniques du VHB (7 %)
• Incidence des événements cliniques: 2,3/1000
porteurs/année
• Incidence du CHC: 1,9/1000 porteurs/année (2,3 chez
l’homme; 1,2 chez la femme).

60. Pathophysiologic Cascade of
Chronic HBV Infection
HBV Replication
HBV Replication Liver
Liver
(Measured by
(Measured by Inflammation
Inflammation
Serum HBV DNA)
Serum HBV DNA)
ALT
ALT
Elevation
Elevation
Worsening Histology
Worsening Histology Disease Progression
Disease Progression
• Necroinflammation
• Necroinflammation • Liver Failure
• Liver Failure
• Fibrosis
• Fibrosis • Liver Cancer
• Liver Cancer
• Cirrhosis
• Cirrhosis • Transplant
• Transplant
• Death
• Death
Adapted from: Lavanchy D. Journal of Viral Hepatitis, 2004, 11, 97–107. Chen JC, et al. JAMA. 2006;295:65-73. Iloeje U. H, et al.
Gastroenterology. 2006;130:678-86.

61. Normal Aminotransferase Levels and
Risk of Mortality from Liver Diseases
59.0
>100
30.0
50-99
19.2
Elevated
40-49 9.5
ALT
30-39
2.9
20-29 Normal
1.0
<20
0 10 20 30 40 50 60 70 80 90
• Korea Medical Insurance Corporation Risk ratio (95% CI)
– 94,533 men; 47,522 women
– 35-59 yrs old
– Relative risk for liver mortality compared with AST and ALT <20 IU/l
Kim HC et al. BMJ 2004; 328:983
al. 2004;

62. AgHBeAg et risque de CHC
• 11,893 Taiwanese men; 92,359 person-years
follow-up
12
Cumulative incidence (%)
HBsAg+
10 HBeAg+
8
6
4
HBsAg+, HBeAg -
2
0 HBsAg -, HBeAg -
0 2 4 6 8 10
Year
Yang et al. N Engl J Med. 2002;347:168-174.

63. Charge virale et incidence de la cirrhose
.4
P <0.001
Incidence cumulative de cirrhose
37.1%
1.0 x 106 n=627
1.0-9.9x105 n=344
n=3774
1.0-9.9x104 n=649
.3 300-9.9x103 n=1210
<300 n=944
23.0%
.2
.1 10.0%
6.3%
5.2%
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Année de suivi
R.E.V.E.A.L. – HBV Study Iloeje UH et al. Gastroenterology 2006; 130: 678-686

64. Survie chez les patients au stade cirrhose
100
Patients Surviving, %
80
60 Cirrhosis1 55%
(n = 130)
40
20 Decompensated cirrhosis2 14%
(n = 21)
0
0 1 2 3 4 5
Years
1. Weissberg et al. Ann Intern Med. 1984;101:613.
2. De Jongh et al. Gastroenterology. 1992;103:1630.

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

66. REVEAL-Incidence of HCC
Increases with Increasing HBV DNA
20% Baseline Viral Level
% cumulative incidence of HCC
14.9%
15%
12.2%
10%
5% 3.6%
1.3% 1.4%
0%
<300 >300 - 103 > 103 - 104 >104 - 106 ≥106
Baseline HBV DNA (copies/mL)
Chen JC, et al. JAMA. 2006;295:65-73.

67. High Baseline Serum HBV DNA Levels are
Associated with Increased Risk of HCC Mortality
in HBsAg-Positive Patients
HBV DNA
Negative
100%
HBV DNA Low
96% < 105 copies/mL
copies/
RR = 1.7 (0.5-5.7)
(0.5-5.7)
92%
88%
HBV DNA High
≥ 105 copies/mL
copies/
p < 0.001 across viral RR = 11.2 (3.6-35.0)
(3.6-35.0)
84% categories
80%
0 1 2 3 4 5 6 7 8 9 10 11 12
Survival time (Years)
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.

68. Relationship Between Persistent Viremia and HCC:
Argument For Antiviral Therapy
• Persistent replication associated with greater risk of HCC
• Decreased risk when viral replication declines
Rate Per 100,000
HCC Incidence
1.2x104 10,108
1.0x104 8730
8.0x103 5882
6.0x103
4.0x103 1473
2.0x103
0
Baseline HBV DNA,
(copies/mL)
< 104 ≥105 ≥105 ≥105
Follow-up HBVDNA,
copies/mL
--- < 104 104 to <105 ≥105
Adjusted RR 1.0 3.6 6.9 9.1
(95% CI) (ref) (1.7-7.6) (3.4-13.8) (5.8-14.1)
P Value -- < 0.001 < 0.001 < .001
Chen, et al. JAMA 2006

69. Impact Clinique de la Variabilité
du Génome Viral

70. VARIABILITE GENETIQUE DU VHB
• Multiplication virale
» taux d'erreur de la transcriptase inverse
• Pression de sélection
» réponse immunitaire cellulaire / humorale
» antiviraux
-> possibilité de variants d'échappement
• Conséquences cliniques
» diagnostic sérologique
» traitements antiviraux

71. VARIABILITE GENETIQUE DU VHB
• SOUS-TYPES : acides aminés et déterminants HBs
– boucle 139-147 -> det a
– 122 -> det d ou y
– 127 -> det w1-4
– 160 -> det w ou r
• GENOTYPES : variabilité de séquence génomique
– du génome complet : 8%
– du gène S : 4%
– 8 génotypes A à H
• MUTANTS DU VHB
– mutations ponctuelles / délétions / insertions

72. 8 genotypes, numerous sub-genotypes, and
recombinant forms
B6
D1
World J Gastroenterol 2007; 13: 14-21

73. Génotypes VHB chez les patients atteints
d’hépatite chronique en France
37.4%
100
90
30.2%
80
Number of subjects
70
60
50
40 12.5%
11.3%
30
7.9%
20
10 1.1%
0.4 %
0
A B C D E F G
Zoulim et al J Viral Hepatitis 2006

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

75. LES MUTANTS DU GÉNOME DU VHB
déterminant a
vaccin/HBIg
polymérase
antiviraux
Mt core
Réponse CTL
Mt pré-core
Réponse anti-e ?

76. ROLE DE LA RÉGION PRÉ-C ET DE L’AgHBe
• Non nécessaire à la réplication du VHB
– Culture cellulaire
– Modèles in vivo
• Marmotte
• Canard
• Modulation de la réponse immune
– Tolérogène : souris transgéniques
– Cible de la réponse anti-capside
Chang et al, J. Virol 1987; Schlicht et al J. Virol 1987; Chen J. Virol 1992; Millich et al PNAS

77. LES MUTANTS PRÉ-C (-)
• codon stop / région pré-C
TGG -> TAG en pos. 1896
– génotypes B à E (A : exceptionnel)
– arrêt traduction protéine pré-C/C
– AgHBe négatif
• mutation dans promoteur pré-C
TTAAAGG -> TTAATGA en pos. 1762 /1764
– génotypes A à E
– transcrits pré-C/C :
– synthèse d'AgHBe :
Carman et al Lancet 1989, Okamoto et al J Virol 1990/1994, Tong et al Virology 1990

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

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

80. VARIANTS NÉGATIFS POUR L ’AgHBe
1762-1764 1896
PROMOTEUR PRE-C C
* * *
TAG
mRNA
Protéine
pré-C/C
arrêt des synthèses protéiques
Diminution de l’expression de l ’AgHBe

81. Main pre-c/core promoter mutations observed in vivo
Basic core promoter
LEF
Pre-C mRNA
AGGTCA HNF4
1762 64 66 68
GGGGGAGGAGATTAGGTTAAAGGTCTTTGTATTAGGAGGCTGTAGGCATAAATT
TGA TTA
GGTTAATNATTA HNF1 TTG
HNF3 WTRTTKRY
Deletion 63-70
Insertion (RGTTAATYATTA) at 74/75 Insertion (TTG) at 66/67
Mutation AGG to TCA and insertion TA at 65/66

82. Sélection des mutants pré-core au cours de
l’histoire naturelle de l’hépatite B chronique
AgHBe Anti-HBe
ALAT
2500
ADN-VHB 2000
1500
1000
500
0 temps
100
sauvage
80
60
Mt pré-C 40
20
0 temps

83. Outcome of Chronic Anti-HBe Positive Hepatitis B
Biochemical patterns in 164 untreated patients
after 23 months (range 12-36) monthly monitoring
400
With flares and normalization 73 pts
300
( 44.5% )
200
100
Asymptomatic
flare-up:
0 90% of cases
400
A Without flares 59 pts
300
L ( 36.0% )
200 Flare-up yearly
T
100 frequency:
once 57.1%
0 twice 20%
< once 22.8%
With flares and without normalization
400
32 pts
300
( 19.5% )
200
100
0
0 12 24
months
Brunetto MR et al, J Hepatol 2002

84. Augmentation de prévalence des hépatites
chroniques avec AgHBe négatif en France
62% 48% HBeAg(+)
N=119 HBeAg(-)
N=164
Zoulim et al, J Viral Hepatitis 2006

85. Pre-core mutations
Both mutations No pre-core mutation
(n = 95; 33.6%) (n = 42; 14.8%)
Data unavailable
(n = 12; 4.2%)
Stop codon mutation
(n = 55; 19.4%)
Promoter mutation
(n = 99; 27.9%)
Lamivir cohort, Zoulim et al, J Viral Hepatitis 2006

86. HBe serotype and pre-core mutations
90
80 HBe-positive
Number of subjects
70 HBe-negative
60
50
40
30
20
10
0
No pre-core Stop codon Promoter Both
mutation mutation mutation mutations
Lamivir cohort, Zoulim et al, J Viral Hepatitis 2006

87. MUTANTS PRÉ-C ET SÉVÉRITÉ HISTOLOGIQUE
LA CONTROVERSE
• Italie
– Cirrhose plus fréquente
• Bonino Gastroenterology 1986, Fattovich Hepatology 1988
• France
– Activité idem / cirrhose plus fréquente
• Zarski et al, J Hepatol 1993
• Grandjacques et al, J Hepatol 2000
• Zoulim et al, J Viral Hepatitis 2006
• Asie
– Mt promoteur : activité histologique et fibrose plus importante
– Mt pré-C : activité histologique moins importante
• Lindh et al, J Infect Dis 1999
– Rémission histologique
• Chan et al, Hepatology 1999
• Afrique
– Mt promoteur : plus fréquents dans le CHC
• Baptista et al, Hepatology 1999

88. HBe serotype and liver pathology
HBe-positive
HBe-negative
70 70
Number of subjects
60 60
50 50
40 40
30 30
20 20
10 10
0 0
0-4 5-9 10-14 15-22 ≤ F2 F3 F4
Knodell score Metavir score
Lamivir cohort, Zoulim et al, J Viral Hepatitis 2006

89. HÉPATITES FULMINANTES ET MUTANTS PRE-C
• Lien de causalité :
– Épidémies hépatites fulminantes
– Transmission souche mutée pré-C (-)
– Rôle immunomodulateur de l ’AgHBe
• Pas de lien de causalité
– Séquençage génome complet
– Pas de profil commun de mutation
• Sélection des mutants par la réponse immunitaire cytotoxique
dirigée contre la souche à l ’origine de l ’HF
Stuyver et al, Hepatology 1999, Sternbeck et al Hepatology 1996, Liang et al, NEJM 1991

90. DIAGNOSTICS DIFFICILES
I. Porteur inactif
II. Exacerbation

91. Diagnosis of inactive carrier versus
HBeAg negative chronic hepatitis
• Inactive Carrier
– Persistently normal ALT levels
– Persistently low levels of serum HBV DNA
• Threshold : 2,000 IU/ mL ?
• HBeAg negative chronic hepatitis
– Fluctuation / exacerbation of ALT
– Fluctuations of HBV DNA levels usually below 6
log IU/ mL
– Presence of pre-core / core promoter mutations

92. DIAGNOSTIC D'UNE EXACERBATION AIGUE
SUR HEPATITE B CHRONIQUE
• Définition : poussée cytolytique
≠ réactivation virale
• Ag HBe + initialement
– rupture de tolérance immunitaire
– séroconversion anti-HBe
– très fréquent chez patients asiatiques
• Anti-HBe + initialement
– réactivation virus sauvage : -> AgHBe +
– réactivation virus muté pré-C (-)
– corticothérapie
– surinfection delta / VHC

93. Pichoud et al, J hepatol 2000
interferon
HBeAg + + - - - - + -
Anti-HBe Ab - + + + + + - +
25 10000000000
1000000000
20 100000000
10000000
15 1000000
case#6 10
100000
10000 ALT
Genotype A 1000
pre-S1
5 100 bDNA
10 PCR
0 1
months
0 1 2 5 9 12 13 16
pre-C promoter
WT - - + + - +
MT + + + - + -
pre-C region
WT + + + + + +
M2 - - - - - -
M4 - - - - - -
M2+M4 - - - - - -

94. PreS2
PreS1
Pol S
HBs Ag 0/3221
GR
E
Brin(-) 3,2kb
Brin(+) 2,4kb
SHBs (S) TATAA
MHBs (preS2+S) « a » determinant U5-like
DR1
Enh2 Enh1
C DR2
LHBs (preS2+preS2+S) S-S
sP120T Pré-C
X
137 S- S
138 149
107 S-S S-S 147
139 sG145R
sD144H/A/E
99 NH2 S-S
COOH
« a » determinant induces the synthesis of
anti-HBs neutralizing antibodies
Tiollais P. et al., Nature 1985. Torresi J., J. Clin Virol 2002; Dryden KA. et al., Mol Cell 2006

95. Variants de l'Ag HBs
• échappement à la réponse humorale anti-HBs
– naturelle
– vaccination (transmission mère-enfant)
– immunoprophylaxie (transplantation hépatique)
• infection active malgré Ac anti-HBs
• sérologie AgHBs faussement négative
á Risques : transmission virale + infections occultes

96. VARIANTS DE L'AgHBs
• Mutations ponctuelles dans le déterminant a de
l'AgHBs (124-147)
– aa 145 : Gly -> Arg
– aa 126 : Ile -> Ser / Thr -> Asn
• transmission mère-enfant malgré la serovaccination
(3%)
• infection du greffon hépatique malgré
Immunoglobulines anti-HBs
• hépatites chroniques avec anti-HBc et anti-HBs +

97. Occult HBV Infection (OBI)
Presence of HBV DNA in the liver (± serum) of
individuals testing HBsAg negative by currently
available assays
Raimondo et al, J Hepatol 2008

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

99. Reported Prevalence of Occult HBV Infection in HIV Positive Patients
Occult
Study Country N° of HBV Methods
patients N° (%)
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%) Cobas Amplicor HBV
Monitor (Roche)
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 101 42 (41%) “nested” PCR (liver)
Raimondo et al, J Hepaol 2007, modified

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

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

102. Schematic representation of HBV serum marker profile in OBI and
“false” OBI
OBI „false“ OBI
HBV DNA levels
< 200 UI/ml
Seropositive S gene
S gene
Seropositive Seronegative
Seronegative escape mutants
escape mutants
Primary occult HBV DNA levels
HBsAg lost
HBsAg lost Primary occult
comparable to
after AH
after AH overt infection
HBsAg lost
HBsAg lost Progressive antibody
Progressive antibody
during CH
during CH disappearence
disappearence

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

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

105. Antiviraux
Persistance virale
Resistance aux antiviraux
Monitoring des traitements

106. HBsAg
Immunotolerant Immuno-active Inactive phase Occult infection
Reactivation phase
phase phase Low replication
HBeAg(+) HBeAg(-) / anti-HBe(+)
HBV DNA
109-1012 IU/mL >2000-<109 IU/mL <2000 IU/mL >2000 IU/mL
ALAT
Minimal CH Moderate to severe CH Remission Moderate to severe CH
Cirrhosis Inactive cirrhosis Cirrhosis
Treatment indicated Treatment indicated
Adapted from Fattovich G. Sem Liver Dis. 2003

107. Endpoints of therapy
Persistence of high viral load is associated with a significant risk of progression of
the liver disease and of HCC
Aim of antiviral therapy:
HBV DNA < 10-15 IU/mL by real-time PCR assays
Viral suppression No replication
=
Histological and clinical No resistance
improvement
Chen CJ, et al. JAMA 2006. Iloeje UH, et al. Gastroenterology 2006. Chen C, et al.
Am J Gastroenterol 2006. Zoulim & Perrillo J Hepatol 2008. Zoulim & Locarnini Gastroenterology 2009

108. Antivirals approved for hepatitis B
Drug Type Approved Phase 3 Phase 2
Nucleoside analogs • Lamivudine* • Emtricitabine* • Elvucitabine
• Entecavir • Clevudine** • Valtorcitabine
• Telbivudine • Amdoxovir
• Racivir
• LB80380
Nucleotide analogs • Adefovir dipivoxil • Alamifovir
• Tenofovir* • Pradefovir
Cytokines • Interferon alfa
• Pegylated Interferon
alfa-2a
*Currently approved for HIV
**development on hold

109. Treatment failure
Primary non response Secondary treatment failure
Partial response Antiviral drug resistance
Host factors Drug factors
Drug metabolism Barrier to resistance
Patient’s compliance
Viral factors
Drug factors Resistant mutants
Antiviral potency
Zoulim & Perrillo J Hepatol 2008; EASL CPG J Hepatol 2009

110. Clinical definition of resistance
• Virologic Breakthrough: Rebound in serum HBV DNA levels
(e.g. 1 log10 above nadir)
• Genotypic Resistance: Detection of mutations known to
confer resistance while on therapy
• Virologic Breakthrough with Genotypic Resistance: Viral
rebound associated with a mutation(s) known to cause
resistance.
• Primary non response: <1log10 decrease of viral load after 3
months
• Partial response: detectable HBV DNA levels during therapy
Zoulim & Perrillo, J Hepatol 2008; EASL CPG, J Hepatol 2009

111. Laboratory Definition of HBV Resistance to Antivirals
Laboratory Investigations
• Phenotypic Resistance: Decreased susceptibility (in vitro testing) to
inhibition by anti-viral drugs associated with genotypic resistance.
• Cross Resistance: Mutants selected by one agent that also confer
resistance to other antiviral agents
Zoulim et al; Future Virology 2006

112. Kinetics of emergence of HBV drug resistant mutants
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.

113. Lamivudine Resistance Accelerates
Progression of Liver Disease
25 Placebo (N=215)
YMDDm (N=209) (49%) Placebo 21%
20 Wild Type (N=221)
15
YMDDm 13%
10
WT 5%
5
0
0 6 12 18 24 30 36
Time after randomization (Months)
Liaw YF et al. N Engl J Med. 2004;351:1521-1531

114. Biochemical and Histologic
Correlates of HBV Resistance
• Rise in ALT levels
– Mild ALT elevations in most cases
– ALT flares with acute exacerbations and liver failure:
especially patients with liver cirrhosis and/or pre-core
mutant infection
• Progression of liver disease
– Progressive worsening of liver histology
– Clinical deterioration, liver decompensation, HCC
development
Lai et al Clin Infect Dis 2003; 36: 687-696; Dienstag et al Gastroenterology 2003;124:105-117 ; Lok et al Gastroenterology
2003; 125 : 1714-1722; Hadziyannis et al Hepatology 2000;32:847-851; Si Ahmed et al Hepatology 2000; Zoulim et al J Viral
Hepatitis 2006;13:278-288 ; Fung et al J Hepatol 2005;43:937-943; Liaw et al NEJM 2004;351:1521-1531.

115. ALT flares in patients with lamivudine
resistance over time
Lok et al Gastroenterology 2003; 125 : 1714-1722

116. Incidence of drug resistance over time
Resistance at year of therapy expressed as percentage of
patients
Drug and patient population 1 2 3 4 5 6
Lamivudine 23 46 55 71 80 -
Telbivudine HBeAg-Pos 4.4 21 - - - -
Telbivudine HBeAg-Neg 2.7 8.6 - - - -
Adefovir HBeAg-Neg 0 3 6 18 29 -
Adefovir (LAM-resistant) Up to 20% - - - - -
Tenofovir 0 0 0 - - -
Entecavir (naïve) 0.2 0.5 1.2 1.2 1.2 1.2
Entecavir (LAM resistant) 6 15 36 46 51 57
CL Lai Clin Infect Dis 2003; CL Lai NEJM 2007; Hadzyiannis Gastroenterology 2006;
Marcellin NEJM 2008; CL Lai & Chang NEJM 2006; Zoulim & Locarnini Gastroenterology 2009

117. Zoulim & Locarnini, Gastroenterology, 2009

118. HBV
HBV HBV
HBV hepatocytes
hepatocytes
viral polymerase spontaneous cccDNA long half-life infected cells long
mutant archiving
error rate half-life
mutant wild type
defective immune
response
viral quasi- viral
species persistence
host
host
selective pressure selection of
selection of
antivirals or others escape mutants
escape mutants
replication fitness
replication space
immune response
drug pharmacodynamics
Virus spread in the
treatment failure liver
+
Zoulim et al., Gastroenterology 2009
Disease progression / HCC development

119. L(-)-SddU
mitochondria deaminase
L(-)-SddC, 3TC
Mt DNA L(-)-SddC-TP L(-)-SddC Lamivudine
kinase
L(-)-SddC-TP HBV DNA
nucleus
L(-)-SddC-TP
cytoplasm
Nuclear DNA
Bridges; Progress in Liver Disease 1995

120. The HBV life cycle
receptor ? Hepatocyte
Virion
polymerase
interaction entry Nucleus
cccDNA formation transcription
pgRNA
AAA
AAA
AAA
AAA
RC DNA cccDNA mRNA
cccDNA
amplification
translation
DNA (+) DNA (-) pgRNA
ER
(+) strand encapsidation
synthesis reverse transcription ER
virion secretion
viral proteins
secretion
HBsAg
Nucleoside
analogs HBeAg
Zoulim et al Future Virology 2006

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

122. 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)

123. Kinetics of Viral Loss During Antiviral Therapy with L-
FMAU (clevudine) in the woodchuck model
Zhu et al, J Virol 2001

124. ADV Associated Serum HBsAg Reductions are
Similar in Magnitude to cccDNA Reductions
Serum Total
0 HBV Intracellular cccDNA Serum
Changes in HBV Markers
(log 10 copies/cell(ml))
DNA DNA HBsAg
-1
from Baseline
-2
-3
-4
-5
-6
48 weeks of ADV resulted in significant reductions in :
serum HBV DNA > total intrahepatic HBV DNA > cccDNA
Changes in HBsAg levels correlated with cccDNA changes
-> 14 years of therapy to clear completely viral cccDNA
Werle et al, Gastroenterology 2004

125. Immunohistochemical Staining of Patient Biopsies at
Baseline and After 48 Weeks ADV Therapy
Baseline Week 48
• 0.8 log10 (84%) decline in cccDNA, not paralleled by a similar decline in the number of
HBcAg+ cells
• Suggests cccDNA depleted primarily by non-cytopathic mechanisms or that cell turn-over
occurred but was associated with infection of new cells during therapy

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

127. Clearance of viral infection versus selection of
escape mutants
The most important factors to consider:
§ The rate of immune killing of infected hepatocytes
§ The rate of replication and spread of mutant virus in the
chronically infected liver (I.e. fitness of the virus: the rate of
spread to uninfected hepatocytes)
§ Small changes in these factors may have profound effect on
whether treatment response is durable or subject to rapid
rebound (Litwin et al J Clin Virol 2005)
§ These factors may be subject to therapeutic intervention

128. Kinetics of emergence of drug resistant virus
during antiviral therapy
Lamivudine
wt mt
X X X
X
X • Free liver space
X • Mutant fitness
X
ni
I II III IV
INHIBITION OF WILD TYPE VIRUS REPLICATION DELAYED EMERGENCE OF
DRUG RESISTANT VIRUS
Zhou et al AAC 1999

129. Kinetics of HBV drug resistance emergence
Drug-susceptible virus
Treatment begins
Naturally—occurring viral variants
Drug-resistant variant
Secondary resistance mutations
HBV replication
/ compensatory resistance mutations
Primary resistance
mutations
Time
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.

130. 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)
Virological Response Virological Response Virological Response Virological Response
Q1 (best response) Q2 Q3 Q4 (worse response)
(n=38) (n=38) (n=38) (n=38)
Median 99% 99% 99% 97% a
range 86-100% 41*-100% 91-100% 70-100%
• Wilcoxon rank sum test, P=0.01
Durantel et al, Antiviral Therapy, 2008

131. Amino acid substitutions result in conformation
changes of the polymerase catalytic site
Wild-type M204/L180 LVDr M204V/L180M
L180 L180M
M204 M204V
LVD-TP LVD-TP
LVDr M204V/L180M
M204V reduces pocket size
L180M Steric clash between lamivudine and V204
M204V
Minimal steric clash between entecavir and
ETV-TP V204
Langley DR, et al. J Virol. 2007;81:3992-4001.

132. Polymerase gene mutations may result in decreased
inhibitory activity of antivirals
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
Jacquard et al, Antimicrob Agents Chemother 2006

133. 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

134. HBIg
adefovir
lamivudine wt
Mutant #1
Mutant #2
Mutant #3
Mutant #4
Polymerase clonal genetic analysis
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
Villet et al, Gastroenterology 2006

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

136. 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
Mutant
A
wt #1 #2 #3 #4
1,7 kb
B
Mutant infectivity / wt (%)
120
100
80
60
40
20
0
wt #1 #2 #3 #4 Mutant
Villet, Billioud et al, Gastroenterology 2008

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

138. Archiving of viral variants
Viral quasispecies
Liver
Majority population
Minority variants
Resistant variants
cccDNA variants
• cccDNA in the liver:
– Is propagated during the normal replication
cycle of HBV
– Can serve as a template for the production of
new virus
• It is believed that viral variants with antiviral
resistance may be archived in this way
Blood circulation
Zhou et al, AAC 1999; Zoulim F. Antivir Res. 2004. Zoulim F & Perillo R. J Hepatol. 2008

139. Archiving of viral variants
Viral quasispecies
Liver
Majority population
Minority variants
Resistant variants
cccDNA variants
• cccDNA in the liver:
– Is propagated during the normal replication
cycle of HBV
– Can serve as a template for the production of
new virus
• It is believed that viral variants with antiviral
resistance may be archived in this way
Blood circulation
Zhou et al, AAC 1999; Zoulim F. Antivir Res. 2004. Zoulim F & Perillo R. J Hepatol. 2008

140. Phenotyping of HBV clinical isolates
Cl ne A in
o ra
Southern blot
Cl e D
Cl e C
eE
Cl A
Cl b St
e
on
on
on
on
analysis
La
Whole genome
HBV clones
PCR Transfection
cloning
Patient HepG2
serum Huh7
lamivudine adefovir
Cell culture plate
RC
-
Wild-type
virus SS -
Patient’s
Fold resistance
virus IC50 mutant
=
IC50 reference strain
Increasing antiviral concentration
1. Durantel D, et al., Hepatology, 2004;40:855-64. 2. Yang H, et al., Antiv Ther, 2005;10:625-33.