Hépatite B.pdf

Natural history of hepatitis B
Acute infection

Resolved
infection Chronic infection: 400 million carriers !
5% neonates
90% adults

Chronic hepatitis
Wild type virus HBeAg+
Pre-core mutant HBeAg- Inactive carrier
Immune tolerance

Reactivation

Cirrhosis

30-50 years Hepatocellular carcinoma
Seeger, Zoulim, Mason; Fields Virology; 2007

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

25

20
Taux /100 000

15

10

5

0
1983 1985 1987 1989 1991 1993 1995 1997
COURLY Réseau "Sentinelles"

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

Age distribution: comparison of the different periods
1991-94 versus 03/2003 - 02/2004
% de cas
40%

years 1991- 94
n= 151
30%
March 03- February 04
n= 158
20%

10%

0%
0-9 ans 10-19 ans 20-29 ans 30-39 ans 40-49 ans 50-59 ans
Classes d'âge
Réseau "Sentinelles" Déclarations obligatoires

! 60

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

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
-  Environ 15 000 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°C, 10 h à 60°C
- à la congélation.

HBsAg

filament S small surface protein
sphere
Dane particle
M middle surface protein
v v
L large surface protein

core capsid protein

HBeAg HBeAg secreted e antigen

v pol polymerase

HBx X protein (non-secreted)

The HBV genome

Tiollais, Nature 1985

The viral replication cycle

Zoulim & Locarnini, Gastroenterology 2009

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

cccDNA

noyau

virion

The animal models of HBV infection
Transgenic mice
Humanized mice

Human
Chimpanzee
Gibbon
baboons

Tupaïa

Woolley monkey

Ground squirrel

American woodchuck

Pekin Duck
Grey Heron

HIV HCV HBV
(Ritonavir ) (IFN- ) (Lamivudi n e )

Plasma virus
Half-life 5.8 h 2.7 - 7.2 h 24 h
Mean viral 2.7 d 3.8 - 7.3 d 24.7 d
generation time
Daily turnover 95% 94% - 99.8% 50%
Daily production 1010 (1.1 - 1011
(plasma) 12.7)*1011
Total load 1.2*109 (3.8 - 5.6)*1010 2*1011

Infected cells
Half-life 1.6 d 2.4 - 4.9 d 10 - 100 d
Mean lifespan 2.3 d 3.5 - 7.1 d 23.3 d
Daily turnover 38% 13% - 25% 1% - 7%

(Tsiang et al. Hepatology 1999)

HBV replication and its role in HCC development

Wands, NEJM 2004

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!

The level of viral antigen presented by hepatocytes
influences CD8 T-cell function

•  Hepatocyte antigen presentation was generally inefficient, and the quantity of
viral antigen strongly influenced CD8 T-cell antiviral function.

•  High levels of hepatitis B virus production induced robust IFN-gamma and TNF-
alpha production in virus-specific CD8 T cells,

•  while limiting amounts of viral antigen, both in hepatocyte-like cells and
naturally infected human hepatocytes, preferentially stimulated CD8 T-cell
degranulation.
•  Virus-specific CD8 T-cell function is influenced by the quantity of virus

produced within hepatocytes

Gehring AJ, et al . J Virol 2007;81:2940-9.

10 3 10 4

10 3
10 2
10 2
10 1
10 1
10 0
10 0
10 -1
-2
10 -1
10
10 -2
-3
10
10 -3

Werle et al, Gastroenterology 2004

Inactive HBV carrier
●  Not virologically inactive:
–  low levels of viremia
–  episomal HBV DNA in the liver

Low-replicative or latent infection
Epigenetic control
Pollicino et al., Gastroenterology 2006

Sirt1 PCAF CBP p300 CBP
HDAC1HDAC1 Sirt1 p300 PCAF

Histones Histones

LOW-REPLICATIVE STATE HIGH-REPLICATIVE STATE
–  spontaneously
–  during immunosuppression
Pollicino et al. Gastroenteroplogy 2006
Levrero et al. J Hepatol, 2009

HISTOIRE NATURELLE ET
VIROLOGIE CLINIQUE

Seeger, Zoulim, Mason;
Fields Virology; 2007

Laboratory Diagnosis of Acute Hepatitis B

HBsAg Anti-HBs Ab

1000
IU/L and million copies/ml

HBeAg Anti-HBe Ab

900
800 ALT
Total anti-HBc

ALT and HBV DNA

700
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

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

HBsAg
800
HBeAg
IU/L or million copies/ml

700
600
ALT and HBV DNA

500
HBV DNA
400
300
200 ALT

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

1000 ALAT
9 log
ADN-
VHB
8 log
100

7 log
10
6 log
1
5 log
4 log
0,1

3 log
0,01
2 log
0,001
1 log
Tolérance hép chronique p. inactif mt pré-core

VHB occulte

Dynamic ranges of quantification
of HBV DNA assays
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

Pathophysiologic Cascade of
Chronic HBV Infection

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.

Charge virale et incidence de la cirrhose
.4
P <0.001
37.1%
Incidence cumulative de cirrhose

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.

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 du CHC

Chen et al; JAMA 2006

REVEAL-Incidence of HCC
Increases with Increasing HBV DNA
Baseline Viral Level
20%
% 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%
Survival distribution function

96%

92%

88%

p < 0.001 across viral
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

B6

D1

World J Gastroenterol 2007; 13: 14-21

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 (%)
44%
Percentage of patients (%)

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

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

ATG ATG
Core gene

HBeAg and Precore Mutation

ATG ATG
Core gene

Main pre-c/core promoter mutations observed in vivo

Basic core promoter

LEF

AGGTCA

HNF4

GGGGGAGGAGATTAGGTTAAAGGTCTTTGTATTAGGAGGCTGTAGGCATAAATT

GGTTAATNATTA

HNF1

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

2500
2000
1500
1000
500
0 temps
100
80
60
40
20
0 temps

Outcome of Chronic Anti-HBe Positive Hepatitis B

400

300

200

100

0
400

300

200

100

0

400

300

200

100

0

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

Lamivir cohort, Zoulim et al, J Viral Hepatitis 2006

Pichoud et al, J hepatol 2000

25 10000000000

1000000000

20 100000000

10000000

15 1000000

100000

10 10000 ALT

pre-S1
1000

5 100 bDNA

10 PCR

0 1
months
0 1 2 5 9 12 13 16

PreS2
PreS1

HBs Ag
Pol S

0/3221

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

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

Occult HBV Infection Is Associated to Hypermethylated and Deacetylated
HBV cccDNA-bound histones

cccDNA-ChIP
Input 1 2 3 4 5 6 IgG

Occult HBV

Overt HBV

1: HP1 4: HDAC1
2: SUV39 5: Ac.H3
3: MECP2 6: Ac.H4
Sirt1 Sirt1
MeCP2
HDAC1 HDAC1 HP1 Suv39
AcH3/AcH4 Core
TF TF TF
TF TF TF
Histones Methylated
H3/H4 H3/H4
and DNA

Pollicino et al., unpublished

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
Seronegative escape mutants

Primary occult HBV DNA levels
HBsAg lost
comparable to
after AH
overt infection

HBsAg lost Progressive antibody
during CH 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
prevalence
Tools ?

Co-infections ?
Therapy? ROLE
Worsen
HCV in
infection ? HCC Not fully
understood ?

HBsAg

Immuno-active Inactive phase Occult infection
Immunotolerant Reactivation phase
phase Low replication
phase

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 •  IL7
•  Pegylated Interferon • IFN Lambda
alfa-2a • Vaccine therapy

*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

The main differences between HIV,
HBV and HCV
HIV1 HBV1,2 HCV1,3
Host cell Host cell Host cell HCV RNA

cccDNA
Host DNA
Host DNA H Host DNA H H
Proviral DNA Integrated DNA
Nucleus Nucleus Nucleus

Life-long suppression Long-term suppression Definitive viral clearance
of viral replication of viral replication and SVR

Adapted from 1. Sorriano V, et al. J Antimicrob Chemother 2008;62:1-4. 2. Locarnini S and Zoulim F. Antiviral Therapy 2010;15 (suppl 3):3-14. 3.
Sarrazin C and Zeuzem S. Gastroenterology 2010;138:447-462.

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)
% With disease progression

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 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

Determinants of viral persistence

Zoulim & Locarnini, Gastroenterology, 2009

Bridges; Progress in Liver Disease 1995

The HBV life cycle

Nucleos(t)ide analogs

Zoulim & Locarnini, Gastroenterology 2009

uncoating! CCC DNA! supercoiled DNA!
minichromosome!

Topoisomerase (TDP2) ?!
Acetyl transferase ?!
removal of protein primer!
Histones!
removal of RNA primer!
completion of viral (+) strand DNA!
ligation of DNA strands extremities!

Antivirals ? Tuttleman et al Cell 1986
viral polymerase?! Le Guerhier et al AAC 2000
DNA repair protein?! Delmas et al AAC 2002
other cellular enzymes?! Kock et al Hepatology 2003
Cortes Ledesma et al Nature 2009
Boeck et al Plos Pathogen 2010

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)

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

Werle et al, Gastroenterology 2004

Kinetics of spread and emergence of drug
resistant virus during antiviral therapy
antiviral

wt

mt   
Free liver space

Mutant fitness
 ni = non-infected
wt = wild type
 mt = mutant type


ni

I II III IV
INHIBITION OF WILD TYPE VIRUS REPLICATIONS DELAYED EMERGENCE OF
DRUG RESISTANT VIRUS
Zhou T, et al. Antimicrobial Agents and Chemotherapy 1999; 43: 1947-1954.

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.

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

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
±M250V
D-Cyclopentane L180M+M204V/I R R R S S
(ETV) ±T184G±S202I/G
MDR V173L+L180M R R S R S
+A181V+N36T

Zoulim & Locarnini Gastroenterology 2009

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

Southern blot
analysis
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.

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, Hepatology al. J Hepatol. 2008;48:S2-19.
Zoulim F, et 2006; Villet et al Gastroenterology 2006 & 2009

Drugs sharing cross-resistance characteristics:
Switching strategy  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
Entecavir
Treatment (months)

rebound
entecavir
L180M+A181G+S202G+M204V
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 Pol/RT
spacer RNaseH
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.

Impact on virus infectivity and fitness
Impact on virion release (intracellular
retention) and virologic monitoring of
breakthrough
Impact on vaccine prophylaxis efficacy

Warner et al Hepatology 2009
Kamili et al Hepatology 2009
Villet et al Gastroenterology 2009

Potential risk of transmission of HBV DR mutants

Clements et al, Bull WHO 2009

Conclusions – Main issues regarding
viral resistance and persistence
1) Persistence of cccDNA
A major driver of viral persistence
Can we quantify HBsAg as a surrogate / non invasive marker ?

2) Viral quasi-species
Role of more sophisticated methodologies to detect HBV variants, i.e.
Ultradeep sequencing ?

3) Viral fitness
A major determinant in the barrier to resistance
Is monotherapy sufficient on the long-term to maintain a high
barrier to resistance ?

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
 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

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 Δ 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
Month
M12
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

ALT
M6

Month of therapy
M12

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

ALT
M6

Month of therapy
M12

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

7

6

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

Drug B Drug A
resistant
mutant

wt

Wild type

Drug A Drug B
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

ALT
M6

Month of therapy
M12

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

Study 106 – Treatment-Experienced Patients ‡

Study 106: TDF Versus FTC/TDF for Treatment of CHB
in Patients with Persistent Viral Replication Receiving ADV

Double Blind
RANDOMIZATION 1:1

Tenofovir DF 300 mg Blinded TDF Blinded TDF Blinded TDF
(TDF) or or or End of Study
OL FTC/TDF OL FTC/TDF OL FTC/TDF

FTC 200 mg / Tenofovir Blinded FTC/TDF Blinded FTC/TDF Blinded FTC/TDF
DF 300 mg or or or
(FTC/TDF) OL FTC/TDF OL FTC/TDF OL FTC/TDF

Final Study Results
Week 24* Week 48 Week 96 Week 1682010)
(AASLD

*FromWeek 24 on, patients patients with confirmed4HBV DNA ≥ 400 HBV DNA ≥ 69IU/mL) had the option to add FTC (as fixed dose
* From Week 24 on,
with confirmed (within weeks) plasma copies/mL (69 IU/mL could switch to
Berg T, et al., AASLD 2010; Oral# 136. FTC/TDF) or discontinueFTC/TDF trial and initiatefrom the trial and initiate therapy
open label (OL) from the or discontinue commercially available commercially available therapy

Primary Efficacy Analysis:
Comparison of the Two Treatment Strategies
% of Patients with HBV DNA < 400 copies/mL (69 IU/mL)

82% FTC/TDF
82% TDF
Percentage (%)

ITT: NC=F*

Two patients on study at Week 168 had HBV DNA ≥400 copies/mL
*NC=F, Non-completer counted as failure in this ITT analysis, including patients who switched to open-label FTC/TDF fixed-dose combination
Berg T, et al., AASLD 2010; Oral# 136.


Mean HBV DNA (log10c/mL) by Study Visit
Mean (95% CI) HBV DNA (log10 copies/mL)

2.26 TDF
2.24 FTC/TDF

* Includes patients who switched to open-label FTC/TDF fixed-dose combination
Bert T, et al., AASLD 2010; Oral# 136.

Mean HBV DNA
by Baseline LAM-R and Treatment


Mean HBV DNA
by Baseline ADV-R and Treatment


Viral load
100%
90%
80%
70%
60%
A181T
50%
A181T + N236T
40%
wt
30%
LLOD 20%
10%
0%
BL W4 W12 W24 W48

Patient 1046 data:
BL viral load = 6.85log
Treatment: TDF
Adherence : 68%

Lavocat & Zoulim, AASLD 2010.

Viral load
100%
90%
80%
N236T
70%
A181V + N236T
60%
A181V
50%
A181S + N236T
40%
A181T + N236T
LLOD
30%
A181T
20%
wt
10%
0%
BL W4 W12 W24 W36 W48

Patient 1051 data:
BL viral load = 8.75log
Treatment: TDF
Adherence : 95.2%
Impact of persisting low viremia levels on treatment outcome ?
Lavocat & Zoulim, AASLD 2010.

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)

Hépatite B.pdf

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