Zoulim Hbv EpidéMio Marqueurs

Hépatite B

Fabien Zoulim
Département d’hépatologie
& INSERM U871, Lyon

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

EPIDEMIOLOGIE DE L’HÉPATITE B

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

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

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

Déclaration obligatoire
de l’hépatite B en France :
résultats des 12 premiers mois de notification

Denise Antona, E Delarocque-Astagneau, D Lévy-Bruhl
département des maladies infectieuses

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

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

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)

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

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)

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

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.

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

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

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

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

Cycle de réplication du VHB

Zoulim & Locarnini, Gastroenterology 2009

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)

Infection à VHB et risque de CHC

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

CARCINOME HEPATOCELLULAIRE ET VIRUS
DE L'HEPATITE B

• Co-incidence de répartition géographique
VHB / CHC
• Porteurs AgHBs : RR x 100 pour le CHC
• CHC dans les modèles animaux de l'hépatite B :
– marmotte
– écureuil
• Présence d'ADN viral intégré dans les tumeurs

HBV replication and its role in HCC development

Wands, NEJM 2004

PATHOGENIE DU CARCINOME
HEPATOCELLULAIRE
ALCOOL
VHB VHC

LESIONS HEPATIQUES CHRONIQUES DESORDRES
METABOLIQUES
ACTIVATION FACTEURS
DE CROISSANCE

REGENERATION FACTEURS
ENVIRONNEMENTAUX

ALTERATIONS GENETIQUES

CARCINOME HEPATOCELLULAIRE

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

PHYSIOPATHOLOGIE /
IMMUNOPATHOLOGIE

IMMUNOPATHOGÉNIE
DES HÉPATITES B CHRONIQUES
RÉPONSE IMMUNITAIRE
HÉPATOCYTE
CYTOKINES
NON INFECTÉ

CTL cytokines

perforine
Fas

ANTICORPS NEUTRALISANTS AgHBc/e

VHB HLAI

ANTIVIRAUX
HÉPATOCYTE INFECTÉ

IMMUNOPATHOLOGY OF HBV INFECTION

CD8+
HBV
Immune tolerance

CD8+ HBV
Clairance phase
Chronic hepatitis

HBV
Seroconversion CD8+
Remission

Immunopathology

Fulminant hepatitis

HBV

CD8+

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)

Non cytolytic clearance of acute
HBV infection in chimpanzee

Wieland S et al, PNAS 2004

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

Summers et al, PNAS 2003 & 2004

Phase de tolérance immunitaire

Marqueurs
Hépatocyte AgHBe +
non infecté
HBV DNA +++
ALAT = N
Foie = N

HBc/e Ag
HBV

Hépatocyte infecté

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

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

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

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

HISTOIRE NATURELLE ET
VIROLOGIE CLINIQUE

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

MARQUEURS SEROLOGIQUES

• Système AgHBe /anti-HBe
– distinction virus sauvage / virus muté AgHBe
négatif

• Virémie
– détection quantitative de l'ADN viral

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

Laboratory Diagnosis of Acute Hepatitis B

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

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

INFECTIONS CHRONIQUES A 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

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

Laboratory Diagnosis of Transition of Chronic
Hepatitis B to The inactive Carrier State

800
HBsAg ``

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

Laboratory Diagnosis of HBeAg negative
Chronic Hepatitis B
HBsAg
HBeAg Anti-HBe

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

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

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

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

TRANSMISSION VERTICALE DU VHB

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

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

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).

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.

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;

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.

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

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.

Charge virale et incidence du CHC

Chen et al; JAMA 2006

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.

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.

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

Impact Clinique de la Variabilité
du Génome Viral

VARIABILITE GENETIQUE DU VHB

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

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

8 genotypes, numerous sub-genotypes, and
recombinant forms

B6

D1

World J Gastroenterol 2007; 13: 14-21

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

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

LES MUTANTS DU GÉNOME DU VHB

déterminant a
vaccin/HBIg

polymérase
antiviraux

Mt core
Réponse CTL

Mt pré-core
Réponse anti-e ?

ROLE DE LA RÉGION PRÉ-C ET DE L’AgHBe

• Non nécessaire à la réplication du VHB
– Culture cellulaire
– Modèles in vivo
• Marmotte
• Canard
• Modulation de la réponse immune
– Tolérogène : souris transgéniques
– Cible de la réponse anti-capside

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

LES MUTANTS PRÉ-C (-)
• codon stop / région pré-C
TGG -> TAG en pos. 1896

– génotypes B à E (A : exceptionnel)

– arrêt traduction protéine pré-C/C

– AgHBe négatif

• mutation dans promoteur pré-C
TTAAAGG -> TTAATGA en pos. 1762 /1764

– génotypes A à E

– transcrits pré-C/C :

– synthèse d'AgHBe :

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

HBeAg and Precore Mutation
G 1896A = stop codon, TAG

ATG ATG
Core gene

1814 1901

Precore Core
region region

HBcAg Virion

HBeAg Serum

HBeAg and Precore Mutation

ATG ATG
Core gene

1814 1901

Precore Core
region region

HBcAg Virion

HBeAg Serum

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

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

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

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

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

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

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


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

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


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

DIAGNOSTICS DIFFICILES

I. Porteur inactif
II. Exacerbation

Diagnosis of inactive carrier versus
HBeAg negative chronic hepatitis
• Inactive Carrier
– Persistently normal ALT levels
– Persistently low levels of serum HBV DNA
• Threshold : 2,000 IU/ mL ?
• 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

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

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 - - - - - -

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

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

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 +

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

How to Detect Occult HBV Infection

Currently there is no standardized
diagnostic assay for occult HBV infection

Reported Prevalence of Occult HBV Infection in HIV Positive Patients
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

Cause(s) for the
failure of HBsAg detection

OBI “false” OBI

Suppression of
Infection by
HBV replication and
S gene Variants
gene expression

HBV replication

HBV cccDNA Integrated HBV DNA

HBV mutants Epigenetic control

Immune surveillance
Viral co-infections

Occult HBV infection

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

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

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 ?

Antiviraux
Persistance virale
Resistance aux antiviraux
Monitoring des traitements

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

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

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

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

Clinical definition of resistance

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

Laboratory Definition of HBV Resistance to Antivirals

Laboratory Investigations
• Phenotypic Resistance: Decreased susceptibility (in vitro testing) to
inhibition by anti-viral drugs associated with genotypic resistance.

• Cross Resistance: Mutants selected by one agent that also confer
resistance to other antiviral agents

Zoulim et al; Future Virology 2006

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.

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

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.

ALT flares in patients with lamivudine
resistance over time

Lok et al Gastroenterology 2003; 125 : 1714-1722

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

Zoulim & Locarnini, Gastroenterology, 2009

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

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

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

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

Can we prevent cccDNA formation ?
Nucleoside analogs in monotherapy or
combination therapy cannot prevent the de
novo formation of cccDNA in hepatocyte
culture and in vivo in animal experiments
(Delmas et al AAC 2000; Seigneres et al AAC 2002)
Can we clear cccDNA from a chronically
infected cell ?
The decrease of intrahepatic cccDNA during
nucleoside analog requires hepatocyte turn
over in animal experiments
(Zhu et al J Virol 2001; Litwin et al J Clin Virol 2005)

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

Zhu et al, J Virol 2001

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

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

Persistence of cccDNA after HBs seroconversion

Maynard et al, J Hepatol 2005

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

Kinetics of 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

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.

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

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.

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

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

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

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

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

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

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

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.

Cross-resistance data for the main mutants
and the commercially available drugs
Pathway Amino acid Lamivudine Telbivudine Entecavir Adefovir Tenofovir
substitutions in the
rt domain
Wild type S S S S S
L-nucleoside M204I R R I S S
L-nucleoside L180M+M204V R R I S S
Alkyl N236T S S S R I
phosphonate
Shared A181T/V I/R I/R S R I
D-Cyclopentane L180M+M204V/I R R R S S
(ETV) ±I169T±V173L±M2
50V
D-Cyclopentane L180M+M204V/I±T R R R S S
(ETV) 184G±S202I/G
MDR V173L+L180M+A1 R R S R S
81V+N36T

Zoulim & Locarnini Gastroenterology 2009

Maximising the barrier to resistance
Wild-type virus
LAM rtM204V/I ± rtL180M
LAM-resistant virus
ADV-resistant virus
ETV-resistant virus
ADV rtN236T +/or rtA181V

rtT184 or rtS202 or rtM250
ETV
rtM204V/I +/-
rtL180M

LAM + TDF – what
do we see?
LAM rtT184 or rtS202 or rtM250
then ETV rtM204V/I +/- rtL180M

TDF: what can
we expect?
TDF

Can we detect low frequency mutants prior to or
during therapy ?

Use of pyrosequencing to detect
low frequency mutants

•May detect mutants representing
as low as 0.1% of the viral
population

•The clinical significance for
treatment choice or adaptation
needs to be determined by
prospective studies

Important factors involved in selection of
MDR mutants
• Use of inadequate sequential monotherapies and inadequate treatment
adaptation
• Incomplete viral suppression
– > Persistent replication in the presence of antiviral pressure

• Use of drugs sharing cross-resistance characteristics
– One mutation may confer resistance to several drugs
– > Persistent replication

• Accumulation of mutations
• Wide replication space (liver transplantation)

The problem of sequential therapy with
nucleoside analogues

?
Drug A Drug B
Multiple drug
resistant mutants
+ one mutation + one mutation with complex
pattern of
Risk of selection of MDR mutants by sequential therapy mutations
- drugs sharing cross-resistance characteristics
- incomplete viral suppression
- liver transplantation
Yim et al, Hepatologyal. J Hepatol. 2008;48:S2-19.Gastroenterology 2006 & 2009
Zoulim F, et
2006; Villet et al

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

adefovir
IFN
Genotype H entecavir
lamivudine lamivudine
109

108
HBV DNA (copies/ml)

107

106

L180M+M204V
105

104
L180M+S202G+M204V

103

0 20 40 60 80 100 120

Treatment (months)

Villet et al, J Hepatol 2007

Genotypic analysis of the viral quasi-species
during lamivudine and entecavir therapy

L180M+A181G+S202G+M204V
36 V173L+P177S+L180M+S202G+M204V
L180M+S202G+M204V
Treatment (months)
entecavir Entecavir
L180M+A181G+S202G+M204V rebound
V173L+P177S+L180M+S202G+M204V
34 I169L+L180M+S202G+M204V
L180M+S202G+M204V
1
V173L+L180M+M204V
11
L180M+M204V

V173L+L180M+M204V
Lamivudine
lamivudine

L180M+M204V
27/0 M204V rebound
wt

0 wt

0 20 40 60 80 100
% clones in the quasi-species

- 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
Villet et al, J Hepatol 2007

Role of cross-resistance, inefficacy of viral load suppression,
and replication space, in MDR mutant selection
HBIg tenofovir
adefovir
Genotype E lamivudine

108 Liver transplantation

107
HBV DNA (Meq/ml)

106

105

104
L180M+M204V V173L+L180M+A181V+N236T
103

102
0 500 1000 1500 2000 2500 3000 3500

days of treatment

Villet et al Gastroenterology 2006

Lamivudine+adefovir treatment (months) Accumulation of mutations and selection of a complex mutant

Time post-transplantation (months)
42 to 50 V173L+L180M+A181V+N236T 34 to 42
V173L+L180M+A181V+N236T
40 V173L+L180M+A181V 32
V173L+L180M+A181V+M204V+N236T
V173L+L180M+A181V+M204V
V173L+L180M+A181V+M204V+N236T
38
1 V173L+L180M+A181V+N236T 30 Viral rebound
V173L+L180M+A181V+M204V
1 V173L+L180M+A181V+M204V
34 L180M+M204I 26
M204I

V173L+L180M+A181V+M204V
24 I169V+L180M+T184I+M204V 16
V173L+L180M+A181V+N236T
wt

V173L+L180M+A181V+M204V
8 V173L+L180M+A181V+M204I 0
L180M+M204I

0 10 20 30 40 50 60 70 80 90 100 % of variants in the
viral quasi-species

Terminal spacer Pol/RT RNaseH
YMDD
Protein

V173L L180M A181V N236T dominant HBV
mutant
Pre-S/S gene

P120S

A single a.a. substitution at position rt181 may be
responsible for multidrug resistance

LVD ADV LVD+ADV
Patient #1 Patient #3 Patient #4 Patient #8
(67 months) (37 months) (31 months) (47 months)

Patient #7 Patient #5 Patient #6 Patient #9
(30 months) (44 months) (36 months) (19 months)

LVD+TDF Patient #2 LVD+ADV+TDF Patient #10
(23 months) (7 months) wt N236T + N238T
A181V M204V
A181T M204I
A181V + N236T L80V
A181T + N236T L80V + M204I
N236T
Villet S, et al. J Hepatol. 2008;48:747-55.

Potential risk of transmission of HBV DR mutants

Clements et al, Bull WHO 2009

Management algorithm
Antiviral treatment
Viral load asssessment

Treatment failure
Check compliance
Viral genome sequence analysis

Wild type virus HBV drug resistant mutant

Check compliance Primary non response
Add-on therapy
based on cross-resistance data
Switch to more potent drug
Zoulim and Perrillo, J Hepatol, 2008; EASL CPG J Hepatol 2009

Management algorithm

Antiviral treatment
Viral load asssessment

Treatment response

Check for HBe/HBs seroconversion on a
regular basis (6 monthly)

Zoulim and Perrillo, J Hepatol, 2008; EASL CPG J Hepatol 2009

Virologic Consequences of Persistent Viremia

Infection of new hepatocytes
á slower kinetics of clearance infected cells and cccDNA

Increases the risk of occurrence and subsequent selection
of HBV mutations responsible for drug resistance

On-treatment prediction of HBV drug resistance

Le Guerhier et al Antimicrob Agents Chemoter 2000;44:111-122; Delmas et al Antimicrob Agents
Chemother 2002; 46:425-433; Kock et al Hepatology2003; 38:1410-1418; Richman Hepatology
2000;32:866-867

Viral Load at Week 24 is a Predictor of Resistance at Week
104 of Therapy (Telbivudine vs. Lamivudine trial)
HBeAg Positive, n=921 HBeAg Negative, n=446
100% 100%

80% 80%
% of patients with resistance

% of patients with resistance
60%
60% 60% 56%
50%
45%
41%
40% 40%
30%
29%
25% 24%
20%
20% 20%
12%
9%
4% 5% 6%
2%
0% 0%
< QL, QL - 3, 3 to 4, > 4, < QL, QL - 3, 3 to 4, > 4,
n=203,146 n=57,63 n=83,79 n=115,175 n=178,157 n=18,20 n=16,24 n=10,23

Telbivudine Lamivudine

Lai et al , NEJM, 2007

HBeAg Seroconversion at 2 Years
vs. Antiviral Effect at Week 24
60%

46%
Percent 39%
HBeAg 40%
Seroconversion
19%
20%

6%

0%
Below QL QL to 3 log 3 to 4 log > 4 log

Serum HBV DNA Level at Week 24

HBeAg Positive Patients, Combined Treatment Groups
Lai et al , NEJM, 2007

Comment adapter le traitement ?
Wild type

LAM ADV

LAM-R

ADV-R

LAM
+
ADV

Zoulim Antivir Res 2004; 64: 1-15. Villeneuve et al J Hepatol 2003. Lampertico et al
Gastroenterology 2007

Patients with lamivudine resistance:
adefovir add-on strategy
3-yr cumulative probability
Patients with virological breakthrough

100 Virologic breakthrough* 100 Virologic breakthrough* and
ADV resistance**

Patients with ADV-R
80 80
ADV mono ADV mono
60 ADV+LAM 60 ADV+LAM

40 40
P<0.001 30% P<0.001
20 20
16%
6%
0 0 0%
0 3 6 9 12 15 18 21 24 27 30 33 36 Months 0 3 6 9 12 15 18 21 24 27 30 33 36
273 268 256 225 201 158 61 Patients 229 225 217 194 179 146 57
255 238 223 213 200 177 103 still at risk 242 227 214 205 200 174 92

* > 1 log rebound of HBV DNA compared to on-treatment nadir
** N236T or A181T-V in patients with a virological breakthrough
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).

The problem of sequential therapy
and switching strategy
LAM LAM
10
ADV 300
9
250
8
(Log10 copies/mL)
Serum HBV DNA

L180M+ 200
7 N236T
M204V

ALT (IU/L)
6
Reverted to wild 150
type
5
100
4

50
3

2
janv-98 janv-99 janv-00 janv-01 janv-02 janv-03 janv-04 janv-05

HBV DNA ∆ ALT

Villeneuve et al, J Hepatol 2003

Rescue therapy in patients with clinical breakthrough

Drug A
Serum HBV DNA (Log10 copies/mL)

Drug B
8
and ALT (x ULN)

6

4
2
0
M0

M6

ALT of therapy
M12

Month
M18

M24

M30

M36
ALT HBV DNA

Rescue therapy in patients at the time of virologic breakthrough

Drug A
Drug B
8

6
and ALT (x ULN)

4
2
0
M0

M6

ALT
M12

Month of therapy
M18

M24

M30

M36
ALT
HBV DNA

Early add-on therapy to prevent drug resistance

Drug A

Drug B
8
and ALT (x ULN)

6
4
2
0
M0

M6

ALT of therapy
Month of therapy
M12

Month
M18

M24

M30

M36
ALT
HBV DNA

Very Early Add-on Therapy to Keep Viral
Load as Low as Possible
1. Start with a drug having a high genetic barrier for resistance
2. Add a drug with a different cross-resistance profile
8

Drug A
7
Drug A
+
6
Drug B
5

4 MDR ?
3

2
M0 M3 M6 M9 M12 M15 M18 M21 M24
Month of therapy
outgrowth of drug resistant mutant ?

Rationale for de novo Combination Therapy

á Combination of drugs without cross-resistance

Drug B Drug A
resistant
mutant

wt
Wild type

Low risk of
Drug A Drug B selection of MDR
resistant
mutant

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

De novo combination therapy to prevent drug resistance

Drug A
Drug B

8
and ALT (x ULN)

6
4
2
0
M0

M6

ALT of therapy
Month of therapy
M12

Month
M18

M24

M30

M36
ALT
HBV DNA

Preventing L-Nucleosides Resistance
with de novo Combination Therapy
100
Incidence of resistance* (%)

Marcellin 1 Lau 2 Lai 3 Sung 4 Lau 5
80

60

40 34%

21% 20%
18%
20 11% 12%
5% 2%
1% 0% 0%
0
LAM LAM LAM LAM LAM LAM LdT LAM LAM FTC FTC
+Peg +Peg +LdT +ADV +ADV
* After 1- year therapy

1 Marcellin et al. N Engl J Med 2004; 351: 1206-17 3 Lai et al. Hepatology 2003;38:262A
2 Lau et al. Hepatology 2004;40:171A 4 Sung et al. J Hepatol 2003 ;38 (suppl 2):25-26
5 Lau et al. Hepatology 2004:40:666A

Perspectives / Prevention of drug resistance
• First line therapy
– Use of antivirals with high antiviral potency and high barrier to
resistance
– Combination therapy with complementary drugs to increase the
barrier to resistance
• Second line treatment
– Add-on strategies with complementary drugs preferred to
sequential monotherapies
– Early treatment adaptation to prevent accumulation of
mutations
– Choice always based on cross-resistance data

Perspectives beyond the guidelines

• Can we clear cccDNA and/or HBsAg ?
new treatment strategies
new treatment targets

• Early treatment intervention to prevent disease progression ?
screening program
non invasive evaluation of liver disease / biomarkers

• Can we prevent prevent HCC development ?
decreased risk of HCC if HBsg clearance <50 yrs (Yuen et al, Gastroenterology 2008)

HBsAg clearance
Blood circulation
viral load
Antivirals

Infected NKT

liver
CD4
CD8 Clearance of
B
HBsAg?

cccDNA

Infected hepatocytes

Werle-Lapostolle B et al., Gastroenterology 2004;126: 1750-58.

Clinical example of HbsAg clearance
* * HBs Ag
Lamivudine 100 mg/day IU/ml

Viral load
Detection treshold
HBs Ag
Positivity cut off: 0.05

Viral load
Log Copies/ml

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

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

Conclusions 1
• Maladie fréquente et grave
– 300 000 porteurs chroniques en france
– 1ère cause de cancer du foie dans le monde
– 1300 décès par an en France
• Maladie méconnue
– Souvent asymptomatique, ou symptomes non spécifiques
– Seulement 60 000 personnes connaissent leur maladie
– 13 000 sont traitées
• Persistance virale
– Pas d’éradication du génome viral
– Surveillance prolongée, possibilité de réactivations

Conclusions 2

• Différentes formes d’hépatites en fonction de
l’interaction virus / réponse immunitaire
– Portage asymptomatique / hépatite chronique / cirrhose /
cancer du foie
• Impact de la variabilité du génome viral
- Role dans la persistance virale et la résistance aux antiviraux
- Echappement diagnostique
• Nécessité d’un dépistage et traitement précoce des
formes chroniques
• Prévention par la vaccination !!!

Zoulim Hbv EpidéMio Marqueurs

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