2. 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
3.
4.
5.
6.
7. Déclaration obligatoire
de l’hépatite B en France :
résultats des 12 premiers mois de notification
Denise Antona, E Delarocque-Astagneau, D Lévy-Bruhl
département des maladies infectieuses
8. Incidence of acute hepatitis B in France
Sentinel networks 1991-1996 et Lyon (COURLY) 1983-1997
25
20
Taux /100 000
15
10
5
0
1983 1985 1987 1989 1991 1993 1995 1997
COURLY Réseau "Sentinelles"
9. 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. 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
12. Risk exposure within 6 months preceding the acute case
Source : obligatory declaration 2003-04
13. 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
14.
15. 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.
16. 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)
19. 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
20. 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
21.
22. 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)
34. 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.
42. 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
47. 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
50. 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
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
72. 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
73.
74.
75.
76. HBeAg and Precore Mutation
G 1896A = stop codon, TAG
ATG ATG
Core gene
79. 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
82. 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
92. 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
93.
94.
95. 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
96. How to Detect Occult HBV Infection
Currently there is no standardized
diagnostic assay for occult HBV infection
97. Reported Prevalence of Occult HBV Infection in HIV Positive Patients
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
98. Cause(s) for the
failure of HBsAg detection
OBI “false” OBI
Suppression of
Infection by
HBV replication and
S gene Variants
gene expression
100. HBV replication
HBV cccDNA
Integrated HBV DNA
HBV mutants
Epigenetic control
Immune surveillance
Viral co-infections
Occult HBV infection
101. 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
103. 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 ?
104.
105. 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
106. 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
107. 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
108. 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
109. 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
110. 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
111. 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.
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)
% 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
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 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
120. The HBV life cycle
Nucleos(t)ide analogs
Zoulim & Locarnini, Gastroenterology 2009
121. 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
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)
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
Werle et al, Gastroenterology 2004
125.
126.
127.
128. 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.
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. 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
133. 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
134. 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
135. 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
136. 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
137. 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.
138.
139.
140.
141.
142.
143. 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
144. 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
145. 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)
146. 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
148. 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
149. 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
150. 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
153. 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
154. Potential risk of transmission of HBV DR mutants
Clements et al, Bull WHO 2009
155. 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 ?
156. 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
157. 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
158. 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
159. 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
160. 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).
161. 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
162. 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
163. Rescue therapy in patients at the time of virologic breakthrough
Drug A
Drug B
Serum HBV DNA (Log10 copies/mL)
8
6
and ALT (x ULN)
4
2
0
M0
ALT
M6
Month of therapy
M12
M18
M24
M30
M36
ALT
HBV DNA
164. Early add-on therapy to prevent drug resistance
Drug A
Serum HBV DNA (Log10 copies/mL)
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
165. 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
Serum HBV DNA (Log10 copies/mL)
7
6
5
4 MDR ?
3
2
M0 M3 M6 M9 M12 M15 M18 M21 M24
Month of therapy
outgrowth of drug resistant mutant ?
166. 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
167. De novo combination therapy to prevent drug resistance
Drug A
Serum HBV DNA (Log10 copies/mL)
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
168. 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
169. 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
170. Study 106 – Treatment-Experienced Patients ‡
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.
171. Study 106 – Treatment-Experienced Patients ‡
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.
172. Study 106 – Treatment-Experienced Patients ‡
Mean HBV DNA
by Baseline LAM-R and Treatment
Berg T, et al., AASLD 2010; Oral# 136.
173. Study 106 – Treatment-Experienced Patients ‡
Mean HBV DNA
by Baseline ADV-R and Treatment
Berg T, et al., AASLD 2010; Oral# 136.
176. 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
177. 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)