Hépatite B
Fabien Zoulim
Département d’hépatologie
& INSERM U1052, Lyon
EPIDEMIOLOGIE DE L’HÉPATITE B
Ott et al, Vaccine 2012
EPIDEMIOLOGIE DE L'INFECTION A VHB
• Hépatites aigues
– VHA : 40%
– VHB : 30%
– VHC : 20%
• incidence : 300 000 infections...
MODES DE TRANSMISSION DU VIRUS DE L'HÉPATITE B EN EUROPE
sexuelle
34%
hétéro
23%
homo
11%
drogue IV
26%
inconnue
31%
hémod...
Déclaration obligatoire
de l’hépatite B en France :
résultats des 12 premiers mois de notification
Denise Antona, E Delaro...
Results
158 acute hepatitis cases
• Hospital doctor in 64% cases
• Sex ratio M/F : 2,95 (118/40)
• Median age: 37 yrs for ...
Risk exposure within 6 months preceding the acute case
Source : obligatory declaration 2003-04
• Source: obligatory declar...
Surveillance épidémiologique de
l’infection HBV
• 14 446 adultes testés
• Prévalence de l’AgHBs 0,65% (280 000 porteurs
ch...
Hépatites virales B: épidémiologie
- Vaccin mais 250 millions de porteurs
chroniques dans le monde
- 280 000 porteurs chro...
VIROLOGIE
• FAMILLE : Hepadnaviridae, seul représentant humain
•VIRUSRESISTANT:
-7 jours dans l’environnement
-pendant 5 mnà 100°C,1...
S small surface protein
M middle surface protein
L large surface protein
core capsid protein
HBeAg secreted e antigen
pol ...
The HBV genome
Tiollais, Nature 1985
déterminant a
vaccin/IgHBs
Gène pol
antiviraux
Mt pre-core
Réponse anti-HBe ?
Mt du c...
The viral replication cycle
Zoulim & Locarnini, Gastroenterology 2009
Model for sodium-dependent taurocholic cotransporting
polypeptide (NTCP) binding to preS1
Seeger C , and Mason W S Gut 201...
Model for sodium-dependent taurocholic cotransporting
polypeptide (NTCP) binding to preS1
Seeger C , and Mason W S Gut 201...
Transgenic mice
Humanized mice
Human
Chimpanzee
Gibbon
baboons
Tupaïa
Woolley monkey
Ground squirrel
American woodchuck
Pe...
Cell culture models for HBV infection
Zeisel M …Zoulim F, Gut 2015
Animal models for HBV infection
Zeisel M …Zoulim F, Gut 2015
Infection à VHB et risque de CHC
• Etude de Beasley à Taiwan
– risque relatif = 100 chez les porteurs de l'AgHBs
• Etude d...
CARCINOME HEPATOCELLULAIRE ET VIRUS
DE L'HEPATITE B
• Co-incidence de répartition géographique
VHB / CHC
• Porteurs AgHBs ...
HBV replication and its role in HCC development
Wands, NEJM 2004
Role du VHB dans l’oncogénèse hépatique
VHB
INFECTION CHRONIQUE
CARCINOGENES
CO-FACTEURS
REACTION INFLAMMATOIRE CHRONIQUE
...
PHYSIOPATHOLOGIE /
IMMUNOPATHOLOGIE
HBV and the immune responses
Current model of HBV pathogenesis
HÉPATOCYTE INFECTÉ
VHB
CTL
Fas
perforine
HÉPATOCYTE
NON INFECTÉ
IMMUNOPATHOGÉNIE
DES HÉPATITES B CHRONIQUES
AgHBc/e
HLAI
c...
IMMUNOPATHOLOGY OF HBV INFECTION
Immune tolerance
Clearance phase
Chronic hepatitis
Seroconversion
Remission
CD8+
HBV
CD8+...
Immunopathology
Fulminant hepatitis
CD8+
HBV
Hépatocyte infecté
HBV
Hépatocyte
non infecté
Phase de tolérance immunitaire
Marqueurs
AgHBe +
HBV DNA +++
ALAT = N
Foie =...
Hépatocyte infecté
HBV
CD8
Fas
perforine
Hépatocyte
non infecté
Phase de clairance immune
(hépatite chronique)
Marqueurs
A...
Hépatocyte infecté
HBs Ag
Hépatocyte
non infecté
Marqueurs
AgHBe-
anti-HBe +
HBV DNA < 2000 IU/mL
ALAT = N
Foie = rémissio...
Hépatocytes infectés
Hépatocytes
non infectés
Marqueurs
HBsAg -
anti-HBc +
Anti-HBs +/-
PCR sérum (-) / foie (+)
Clairance...
cccDNA(copies/cell)
TotalHBVDNA
(copies/cell)
cccDNA levels in the different phases of
chronic HBV infection
• HBeAg+ pati...
Inactive HBV carrier
● Not virologically inactive:
– low levels of viremia
– episomal HBV DNA in the liver
LOW-REPLICATIVE...
HISTOIRE NATURELLE ET
VIROLOGIE CLINIQUE
Histoire Naturelle de l’hépatite B
Infection aigue
Infection chronique
Tolérance immunitaire
Hépatite chronique
Portage in...
HEPATITE B AIGUE
• Incubation 1 à 6 mois
• Le plus souvent asymptomatique
– Évolution plus fréquente vers la chronicité
• ...
Laboratory Diagnosis of Acute Hepatitis B
0
100
200
300
400
500
600
700
800
900
1000
0 1 2 3 4 5 6 12 24 36 48 60
ALT
HBsA...
HEPATITE B PROLONGEE
• Définition
– Persistance réplication virale à la 8ème
semaine d’évolution :
– AgHBe + ou ADN-VHB +
...
INFECTIONS CHRONIQUES A VHB
FORMES CLINIQUES
• virus sauvage
– tolérance immunitaire
– rupture de tolérance -> lésions hép...
0
100
200
300
400
500
600
700
800
0 1 2 3 4 5 6 12 24 36 48 60
ALT
HBsAg
HBeAg
HBV DNA
Normal
Months After Exposure
Labora...
ALT
``HBsAg
HBeAg
HBV DNA
Normal
Months After Exposure
Anti-HBe
Laboratory Diagnosis of Transition of Chronic
Hepatitis B ...
0
50
100
150
200
250
300
350
400
450
0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48
ALT
HBsAg
HBV DNA
Normal ALT levels
Mo...
0,001
0,01
0,1
1
10
100
1000 ALAT
ADN-
VHB
AgHBe + anti-HBe +
UI/ml
pg/ml
AgHBs
Tolérance hép chronique p. inactif mt pré-...
Dynamic ranges of quantification
of HBV DNA assays
Amplicor HBV Monitor
v2.0 (Roche)
HBV Hybrid-Capture II
(Digene)
Ultra-...
Formes cliniques
MANIFESTATIONS
EXTRAHEPATIQUES DU VHB
• PAN
– Complexes immuns circulants HBs/anti-HBs
– Dépots artères moyens et petit ca...
TRANSMISSION VERTICALE DU VHB
• mère AgHBe +
– transmission : 90%
• mère anti-HBe +
– transmission : 10-20%
– VHB muté pré...
PRESENTATION CLINIQUE
• INFECTION PERI-NATALE
– ALT normales ou subnormales
– ADN-VHB > 1000 pg/ml
– histologie : lésions ...
Pathophysiologic Cascade of
Chronic HBV Infection
HBV Replication
(Measured by
Serum HBV DNA)
Liver
Inflammation
Worsening...
Charge virale et incidence de la cirrhose
R.E.V.E.A.L. – HBV Study
Année de suivi
Incidencecumulativedecirrhose
.2
.1
0 1 ...
Survie chez les patients au stade cirrhose
1. Weissberg et al. Ann Intern Med. 1984;101:613.
2. De Jongh et al. Gastroente...
Charge virale et incidence du CHC
Chen et al; JAMA 2006
REVEAL-Incidence of HCC
Increases with Increasing HBV DNA
Baseline Viral Level
Chen JC, et al. JAMA. 2006;295:65-73.
14.9%...
High Baseline Serum HBV DNA Levels are
Associated with Increased Risk of HCC Mortality
in HBsAg-Positive Patients
80%
84%
...
Relationship Between Persistent Viremia and HCC:
Argument For Antiviral Therapy
• Persistent replication associated with g...
Impact Clinique de la Variabilité
du Génome Viral
VARIABILITE GENETIQUE DU VHB
• Multiplication virale
» taux d'erreur de la transcriptase inverse
• Pression de sélection
»...
8 genotypes, numerous sub-genotypes, and
recombinant forms
World J Gastroenterol 2007; 13: 14-21
B6
D1
Génotypes VHB chez les patients atteints
d’hépatite chronique en FranceNumberofsubjects
F GA B C D E
0
10
20
30
40
50
60
7...
Impact du génotype sur la
séroconversion Hbe/HBs
1 Janssen, Lancet 2005; 2 Flink, Am J Gastro 2006
PEG-IFN a-2b
HBeAg Loss...
LES MUTANTS DU GÉNOME DU VHB
déterminant a
vaccin/HBIg
polymérase
antiviraux
Mt pré-core
Réponse anti-e ?
Mt core
Réponse ...
ROLE DE LA RÉGION PRÉ-C ET DE L’AgHBe
• Non nécessaire à la réplication du VHB
– Culture cellulaire
– Modèles in vivo
• Ma...
LES MUTANTS PRÉ-C (-)
• codon stop / région pré-C
TGG -> TAG en pos. 1896
– génotypes B à E (A : exceptionnel)
– arrêt tra...
HBeAg and Precore Mutation
1814 1901
Precore Core
region region
HBcAg
HBeAg
G 1896A = stop codon, TAG
ATG ATG
Virion
Serum...
HBeAg and Precore Mutation
1814 1901
Precore Core
region region
HBcAg
HBeAg
ATG ATG
Virion
Serum
Core gene
VARIANTS NÉGATIFS POUR L ’AgHBe
mRNA
Protéine
pré-C/C
PRE-C CPROMOTEUR
TAG
***
1762-1764 1896
arrêt des synthèses protéiqu...
Sélection des mutants pré-core au cours de
l’histoire naturelle de l’hépatite B chronique
0
500
1000
1500
2000
2500
temps
...
Outcome of Chronic Anti-HBe Positive Hepatitis B
0
100
200
300
400
0
100
200
300
400
0
100
200
300
400
Biochemical pattern...
Augmentation de prévalence des hépatites
chroniques avec AgHBe négatif en France
HBeAg(+)
HBeAg(-)
42%
N=119
58 %
N=164
Zo...
DIAGNOSTICS DIFFICILES
I. Porteur inactif
II. Exacerbation
Diagnosis of inactive carrier versus
HBeAg negative chronic hepatitis
• Inactive Carrier
– Persistently normal ALT levels
...
DIAGNOSTIC D'UNE EXACERBATION AIGUE
SUR HEPATITE B CHRONIQUE
• Définition : poussée cytolytique
≠ réactivation virale
• Ag...
COOH
137
149
107
99 NH2
S - S
S - S S - S
S-S
138
139
147
Tiollais P. et al., Nature 1985. Torresi J., J. Clin Virol 2002;...
Variants de l'Ag HBs
• échappement à la réponse humorale anti-HBs
– naturelle
– vaccination (transmission mère-enfant)
– i...
VARIANTS DE L'AgHBs
• Mutations ponctuelles dans le déterminant a de
l'AgHBs (124-147)
– aa 145 : Gly -> Arg
– aa 126 : Il...
Presence of HBV DNA in the liver ( serum) of
individuals testing HBsAg negative by currently
available assays
Occult HBV ...
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
Study Country N° of
patients
Occult
HBV
N° (%)
Method...
OBI
Cause(s) for the
failure of HBsAg detection
Suppression of
HBV replication and
gene expression
Infection by
S gene Var...
Occult HBV infection
HBV cccDNA Integrated HBV DNA
HBV mutants Epigenetic control
HBV replication
Immune surveillance
Vira...
OBI
Seropositive Seronegative
HBsAg lost
during CH
HBsAg lost
after AH
Progressive antibody
disappearence
Primary occult
S...
High
prevalence
ROLE
in
HCC
Diagnostic
Tools ?
Worsen
HCV
infection ?
Co-infections ?
Therapy?
To be
improved
Specific
tre...
Antiviraux
Persistance virale
Resistance aux antiviraux
Monitoring des traitements
HBeAg(+) HBeAg(-) / anti-HBe(+)
ALAT
HBV DNA
Minimal CH Moderate to severe CH Moderate to severe CHRemission
Cirrhosis
Imm...
Antivirals approved for hepatitis B
Drug Type Approved
Nucleoside analogs • Lamivudine*
• Entecavir
• Telbivudine
Nucleoti...
Endpoints of therapy
Persistence of high viral load is associated with a significant risk of progression of
the liver dise...
Treatment failure
Primary non response
Partial response
Secondary treatment failure
Antiviral drug resistance
Host factors...
Clinical definition of resistance
• Virologic Breakthrough: Rebound in serum HBV DNA levels
(e.g. 1 log10 above nadir)
• G...
Laboratory Definition of HBV Resistance to Antivirals
Laboratory Investigations
• Phenotypic Resistance: Decreased suscept...
The main differences between HIV,
HBV and HCV
H
HBV1,2
Host cell
cccDNA
Host DNA
Integrated DNA
Nucleus
H
HIV1
Host cell
H...
Si Ahmed et al. Hepatology. 2000; Yuen et al Hepatology 2001; Locarnini et al Antiviral Therapy 2004;
Villet et al Gastroe...
Lamivudine Resistance Accelerates
Progression of Liver Disease
YMDDm
WT
Placebo
5%
13%
21%
Liaw YF et al. N Engl J Med. 20...
Biochemical and Histologic
Correlates of HBV Resistance
• Rise in ALT levels
– Mild ALT elevations in most cases
– ALT fla...
ALT flares in patients with lamivudine
resistance over time
Lok et al Gastroenterology 2003; 125 : 1714-1722
6
3
LVD ADV LdT ETV TDF
0
10
20
30
40
50
60
70
80
23
Proportionofpatients(%)
46
55
71
80
0
11
18
29
5
25
0.2 0.5
1.2
0
1 2...
Drug and patient population
Resistance at year of therapy expressed as percentage of
patients
1 2 3 4 5 6
Lamivudine 23 46...
Terminal
protein
Spacer POL/RT RNaseH
1 183 349 (rt) 692 (rt 344) 845 a.a.
I(G) II(F) A B C D E
F_V_LLAQ_YMDD
*rtA181T/V a...
Zoulim & Locarnini, Gastroenterology, 2009
Multiple factors are associated with the
barrier of resistance & drug efficacy
•Adherence
•Immune status
•Prior antiviral ...
Adherence to nucleos(t)ide analogues for chronic hepatitis B in
clinical practice and correlation with virological breakth...
L(-)-SddC, 3TC
LamivudineL(-)-SddC
mitochondria
nucleus
L(-)-SddC-TP HBV DNA
Nuclear DNA
Mt DNA
L(-)-SddC-TP
L(-)-SddC-TP
...
Nucleos(t)ide analogs
The HBV life cycle
Zoulim & Locarnini, Gastroenterology 2009
uncoating CCC DNA
removal of protein primer
removal of RNA primer
completion of viral (+) strand DNA
ligation of DNA stran...
Can we prevent cccDNA formation ?
Nucleoside analogs in monotherapy or
combination therapy cannot prevent the de
novo form...
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
-6
-5
-4
-3
-2
-1
0
ChangesinHBVMarker...
• 0.8 log10 (84%) decline in cccDNA, not paralleled by a similar decline in the number of
HBcAg+ cells
• Suggests cccDNA d...
Maynard et al, J Hepatol 2005
Persistence of cccDNA after HBs seroconversion
Clearance of viral infection versus selection of
escape mutants
The most important factors to consider:
§ The rate of immu...
Kinetics of spread and emergence of drug
resistant virus during antiviral therapy
Zhou T, et al. Antimicrobial Agents and ...
Kinetics of HBV drug resistance emergence
Si Ahmed et al. Hepatology. 2000; Yuen et al Hepatology 2001; Locarnini et al An...
• cccDNA in the liver:
– Is propagated during the normal
replication cycle of HBV
– Can serve as a template for the
produc...
• cccDNA in the liver:
– Is propagated during the normal replication
cycle of HBV
– Can serve as a template for the produc...
• cccDNA in the liver:
– Is propagated during the normal replication
cycle of HBV
– Can serve as a template for the produc...
Definition of fitness
• A parameter that quantifies the adaptation of an
organism or a virus to a given environment
• For ...
Cross-resistance data for the main mutants and the commercially
available drugs
Zoulim & Locarnini Gastroenterology 2009; ...
Phenotyping of HBV clinical isolates
1. Durantel D, et al., Hepatology, 2004;40:855-64. 2. Yang H, et al., Antiv Ther, 200...
ADV rtN236T +/or rtA181V
Wild-type virus
ADV-resistant virus
LAM-resistant virus
LAM rtM204V/I ± rtL180M
ETV-resistant vir...
?
Multiple drug
resistant mutants
with complex
pattern of
mutations
+ one mutation + one mutation
Drug A Drug B
Risk of se...
103
104
105
106
107
108
109
0 20 40 60 80 100 120
Treatment (months)
HBVDNA(copies/ml)
entecavirIFN
adefovir
lamivudine
Ge...
Warner et al Hepatology 2009
Kamili et al Hepatology 2009
Villet et al Gastroenterology 2009
Impact on virus infectivity a...
Virologic Consequences of Persistent Viremia
 Infection of new hepatocytes
 slower kinetics of clearance infected cells ...
Patients heavily exposed to NUCs with low barrier to
resistance – Risk of MDR selection
• Risk of multidrug resistance by ...
?
Multiple drug
resistant mutants
with complex
pattern of
mutations
+ one mutation + one mutation
Drug A Drug B
Risk of se...
Liu et al, Antivir Ther. 2010;15(8):1185-90.
Sequential therapy with NUCs and the risk of MDR
Accumulation of
multiple mut...
A single a.a. substitution at position rt181 may be
responsible for multidrug resistance
Villet S, et al. J Hepatol. 2008;...
Impact of rtA181 and rtN236 mutations on antiviral
drug efficacy and cross-resistance
Villet et al, J Hepatol 2008
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
BL W4 W12 W24 W36 W48
N236T
A181V + N236T
A181V
A181S + N236T
A181T + N236T
A1...
Virologic response to TDF according to ADV resistance
mutations at baseline
The Australian Experience
Patterson S J et al....
Tenofovir + Emtricitabine in patients with treatment failure –
treatment intensification
0
1
2
3
4
5
6
7
Baseline M3 M6 M1...
Rescue therapy with ETV + TDF in CHB patients with advanced liver disease and complex viral resistance patterns or
showing...
Conclusions 1
• Maladie fréquente et grave
– 300 000 porteurs chroniques en france
– 1ère cause de cancer du foie dans le ...
Conclusions 2
• Différentes formes d’hépatites en fonction de
l’interaction virus / réponse immunitaire
– Portage asymptom...
Acknowledgements
Hepatology Unit INSERM U1052 Collaborations
David Durantel
Barbara Testoni
Malika Ait-Goughoulte
Souphalo...
Ganem and Prince, NEJM 2004
Perspectives of anti-HBV immune therapy
The concept of combination therapy
Entry inhibition
cccDNA
- formation
- stability / destruction
- epigenetic regulation
V...
Mason, W. S. et al. 2009 / 2010. J. Virol
Devons nous redéfinir la tolérance immunitaire et
repenser les indications théra...
Effective T-cells control virus Exhausted T-cells lose control of virus
CD8 T cells
Infected hepatocytes Infected hepatocy...
HBsAg clearance
Werle-Lapostolle B et al., Gastroenterology 2004;126: 1750-58.
Infected hepatocytes
Infected
liver
CD8
NKT...
Perspectives / Prevention of drug resistance
• First line therapy
– Use of antivirals with high antiviral potency and high...
Prevention of resistance
Impact of first line therapy
• Choose an antiviral drug with
1. A potent antiviral activity
2. A ...
6
3
LVD ADV LdT ETV TDF
0
10
20
30
40
50
60
70
80
23
Proportionofpatients(%)
46
55
71
80
0
11
18
29
5
25
0.2 0.5
1.2 0
1 2...
Zoulim & Locarnini, Gastroenterology 2009; EASL CPG J Hepatol 2009 & 2012
Mangement of antiviral drug resistance
• Impact ...
Cross-resistance data for the main mutants and the commercially
available drugs
Zoulim & Locarnini Gastroenterology 2009; ...
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Zoulim du 2015

  1. 1. Hépatite B Fabien Zoulim Département d’hépatologie & INSERM U1052, Lyon
  2. 2. EPIDEMIOLOGIE DE L’HÉPATITE B
  3. 3. Ott et al, Vaccine 2012
  4. 4. EPIDEMIOLOGIE DE L'INFECTION A VHB • 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 AUX USA
  5. 5. MODES DE TRANSMISSION DU VIRUS DE L'HÉPATITE B EN EUROPE sexuelle 34% hétéro 23% homo 11% drogue IV 26% inconnue 31% hémodialysés 8% transfusions 2% personnels de santé 2% contact avec porteur du VHB 4% Asie Transmission verticale
  6. 6. 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
  7. 7. 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)
  8. 8. 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% – Familial 14 9,7% – Perinatal 2 1,4% – Live in instiution 11 7,6% – Travel in endemic 21 14,5% areas 91/145 patients (63 %) had a vaccine indication (2 vaccinated ≥ 3 doses) • Sentinel networks 91-96 N=195 – sexual 35% – IVDU 19% – « percutaneous » 15% – No factor 35%
  9. 9. Surveillance épidémiologique de l’infection HBV • 14 446 adultes testés • Prévalence de l’AgHBs 0,65% (280 000 porteurs chroniques du VHB) • Homme 1,1% versus 0,2% femme • Naissance en zone d’endémie 4% versus 0,5% • Précarité, séjour en institution, homosexualité, usage de drogues Meffre et al, J. Med Virol 2004
  10. 10. Hépatites virales B: épidémiologie - Vaccin mais 250 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
  11. 11. VIROLOGIE
  12. 12. • FAMILLE : Hepadnaviridae, seul représentant humain •VIRUSRESISTANT: -7 jours dans l’environnement -pendant 5 mnà 100°C,10h à 60°C -à la congélation. LE VIRUS DE L ’HEPATITE B
  13. 13. S small surface protein M middle surface protein L large surface protein core capsid protein HBeAg secreted e antigen pol polymerase HBx X protein (non-secreted) v v v sphere filament Dane particle HBeAg HBsAg
  14. 14. The HBV genome Tiollais, Nature 1985 déterminant a vaccin/IgHBs Gène pol antiviraux Mt pre-core Réponse anti-HBe ? Mt du core Réponse CTL 8 génotypes A to H
  15. 15. The viral replication cycle Zoulim & Locarnini, Gastroenterology 2009
  16. 16. Model for sodium-dependent taurocholic cotransporting polypeptide (NTCP) binding to preS1 Seeger C , and Mason W S Gut 2013 in press; Yan H, et al. eLife 2012;1:e00049; Hu NJ, et al. Nature 2011;478:408–11. • Sodium-dependent transporter for taurocholic acid • Expressed at the basolateral membrane of hepatocytes • Mediates the transport of conjugated bile acids • 349 amino acid-long glycosylated transmembrane protein. • Expression controlled by hepatocyte-specific transcription factors, including HNF3 and C/EBP
  17. 17. Model for sodium-dependent taurocholic cotransporting polypeptide (NTCP) binding to preS1 Seeger C , and Mason W S Gut 2013 in press; Yan H, et al. eLife 2012;1:e00049; Hu NJ, et al. Nature 2011;478:408–11.
  18. 18. Transgenic mice Humanized mice Human Chimpanzee Gibbon baboons Tupaïa Woolley monkey Ground squirrel American woodchuck Pekin Duck Grey Heron Summers PNAS 1978, Mason J Virol 1981, Chisari Science 1985, Petersen PNAS 1998, Lanford PNAS 1998 The animal models of HBV infection
  19. 19. Cell culture models for HBV infection Zeisel M …Zoulim F, Gut 2015
  20. 20. Animal models for HBV infection Zeisel M …Zoulim F, Gut 2015
  21. 21. 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)
  22. 22. 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
  23. 23. HBV replication and its role in HCC development Wands, NEJM 2004
  24. 24. Role du VHB dans l’oncogénèse hépatique VHB INFECTION CHRONIQUE CARCINOGENES CO-FACTEURS REACTION INFLAMMATOIRE CHRONIQUE REGENERATION HEPATIQUE MUTAGENESE INSERTIONNELE TRANSACTIVATION DE GENES CELLULAIRES INTERACTIONS PROTEIQUES INACTIVATION DE GENES SUPPRESSEURS DE TUMEUR CHC
  25. 25. PHYSIOPATHOLOGIE / IMMUNOPATHOLOGIE
  26. 26. HBV and the immune responses
  27. 27. Current model of HBV pathogenesis
  28. 28. HÉPATOCYTE INFECTÉ VHB CTL Fas perforine HÉPATOCYTE NON INFECTÉ IMMUNOPATHOGÉNIE DES HÉPATITES B CHRONIQUES AgHBc/e HLAI cytokines RÉPONSE IMMUNITAIRE CYTOKINES ANTIVIRAUX ANTICORPS NEUTRALISANTS
  29. 29. IMMUNOPATHOLOGY OF HBV INFECTION Immune tolerance Clearance phase Chronic hepatitis Seroconversion Remission CD8+ HBV CD8+ HBV CD8+ HBV
  30. 30. Immunopathology Fulminant hepatitis CD8+ HBV
  31. 31. Hépatocyte infecté HBV Hépatocyte non infecté Phase de tolérance immunitaire Marqueurs AgHBe + HBV DNA +++ ALAT = N Foie = N HBc/e Ag
  32. 32. Hépatocyte infecté HBV CD8 Fas perforine Hépatocyte non infecté Phase de clairance immune (hépatite chronique) Marqueurs AgHBe+ HBV DNA > 2000 IU/mL ALAT +++ Foie: Hépatite chronique HBc/e Ag HLAI cytokines
  33. 33. Hépatocyte infecté HBs Ag Hépatocyte non infecté Marqueurs AgHBe- anti-HBe + HBV DNA < 2000 IU/mL ALAT = N Foie = rémission Phase de rémission portage inactif de l’AgHBs Réactivation Virus sauvage ou mt pre-coreOncogénèse CD8 CD4
  34. 34. Hépatocytes infectés Hépatocytes non infectés Marqueurs HBsAg - anti-HBc + Anti-HBs +/- PCR sérum (-) / foie (+) Clairance de l’AgHBs Mutants d’échappement Infections occultes Oncogénèse CD8 CD4 B
  35. 35. cccDNA(copies/cell) TotalHBVDNA (copies/cell) cccDNA levels in the different phases of chronic HBV infection • HBeAg+ patients had significantly higher cccDNA (90-fold) and total HBV DNA (147- fold) levels compared to HBeAg- patients. (p<0.001, Wilcoxon tests) 10-3 10-2 10-1 100 101 102 103 104 10-3 10-2 10-1 100 101 102 103 Werle et al, Gastroenterology 2004
  36. 36. Inactive HBV carrier ● Not virologically inactive: – low levels of viremia – episomal HBV DNA in the liver LOW-REPLICATIVE STATE HIGH-REPLICATIVE STATE – spontaneously – during immunosuppression Low-replicative or latent infection Epigenetic control Histones PCAF p300 PCAF p300 Sirt1 Sirt1 HDAC1HDAC1 Histones Pollicino et al., Gastroenterology 2006 Pollicino et al. Gastroenteroplogy 2006 Levrero et al. J Hepatol, 2009
  37. 37. HISTOIRE NATURELLE ET VIROLOGIE CLINIQUE
  38. 38. Histoire Naturelle de l’hépatite B Infection aigue Infection chronique Tolérance immunitaire Hépatite chronique Portage inactif Guérison 5% nx-nés 90% adultes Virus sauvage (HBeAg+) Mutant pre-core (HBeAg-) Cirrhose Carcinome hépatocellulaire Réactivation 30-50 ans Seeger, Zoulim, Mason; Fields Virology; 2007 Seeger, Zoulim, Mason – Fields Virology 2007
  39. 39. 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
  40. 40. Laboratory Diagnosis of Acute Hepatitis B 0 100 200 300 400 500 600 700 800 900 1000 0 1 2 3 4 5 6 12 24 36 48 60 ALT HBsAg HBeAg HBV DNA Normal Months After Exposure Symptoms Anti-HBs Ab Anti-HBe Ab IgM anti-HBc Total anti-HBc Seeger, Zoulim, Mason, Fields Virology 2007
  41. 41. 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
  42. 42. 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
  43. 43. 0 100 200 300 400 500 600 700 800 0 1 2 3 4 5 6 12 24 36 48 60 ALT HBsAg HBeAg HBV DNA Normal Months After Exposure Laboratory Diagnosis of Chronic Hepatitis B associated with wild type virus infection Seeger, Zoulim, Mason, Fields Virology 2007
  44. 44. ALT ``HBsAg HBeAg HBV DNA Normal Months After Exposure Anti-HBe Laboratory Diagnosis of Transition of Chronic Hepatitis B to The inactive Carrier State 0 100 200 300 400 500 600 700 800 0 1 2 3 4 5 6 12 24 36 48 60 72 80 92 104 Seeger, Zoulim, Mason, Fields Virology 2007
  45. 45. 0 50 100 150 200 250 300 350 400 450 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 ALT HBsAg HBV DNA Normal ALT levels Months Anti-HBeHBeAg Laboratory Diagnosis of HBeAg negative Chronic Hepatitis B Seeger, Zoulim, Mason, Fields Virology 2007
  46. 46. 0,001 0,01 0,1 1 10 100 1000 ALAT ADN- VHB AgHBe + anti-HBe + UI/ml pg/ml AgHBs Tolérance hép chronique p. inactif mt pré-core VHB occulte hybridation PCR 9 log 8 log 7 log 6 log 5 log 4 log 3 log 2 log 1 log
  47. 47. 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) Abbot Real-time HBV (Abbott) Versant HBV DNA 1.0 (kPCR, Siemens)* *in development 10 102 103 104 105 106 107 108 109 RealArt HBV LC PCR (Artus Biotech)
  48. 48. Formes cliniques
  49. 49. 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
  50. 50. 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%
  51. 51. 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
  52. 52. Pathophysiologic Cascade of Chronic HBV Infection HBV Replication (Measured by Serum HBV DNA) Liver Inflammation Worsening Histology • Necroinflammation • Fibrosis • Cirrhosis Disease Progression • Liver Failure • Liver Cancer • Transplant • 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. ALT Elevation
  53. 53. Charge virale et incidence de la cirrhose R.E.V.E.A.L. – HBV Study Année de suivi Incidencecumulativedecirrhose .2 .1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 0 .4 .3 P <0.001 n=3774 1.0 x 106 n=627 1.0-9.9x105 n=344 1.0-9.9x104 n=649 300-9.9x103 n=1210 <300 n=944 5.2% 6.3% 10.0% 23.0% 37.1% Iloeje UH et al. Gastroenterology 2006; 130: 678-686
  54. 54. Survie chez les patients au stade cirrhose 1. Weissberg et al. Ann Intern Med. 1984;101:613. 2. De Jongh et al. Gastroenterology. 1992;103:1630. 1 32 4 50 20 40 60 100 80 Cirrhosis1 (n = 130) Decompensated cirrhosis2 (n = 21) 14% 55% PatientsSurviving,% Years 0
  55. 55. Charge virale et incidence du CHC Chen et al; JAMA 2006
  56. 56. REVEAL-Incidence of HCC Increases with Increasing HBV DNA Baseline Viral Level Chen JC, et al. JAMA. 2006;295:65-73. 14.9% 12.2% 3.6% 1.4%1.3% 0% 5% 10% 15% 20% <300 >300 - 103 Baseline HBV DNA (copies/mL) %cumulativeincidenceofHCC > 103 - 104 >104 - 106 ≥106
  57. 57. High Baseline Serum HBV DNA Levels are Associated with Increased Risk of HCC Mortality in HBsAg-Positive Patients 80% 84% 88% 92% 96% 100% 0 1 2 3 4 5 6 7 8 9 10 11 12 Survival time (Years) Survivaldistributionfunction HBV DNA Negative HBV DNA Low < 105 copies/mL RR = 1.7 (0.5-5.7) HBV DNA High ≥ 105 copies/mL RR = 11.2 (3.6-35.0)p < 0.001 across viral categories http://www.fccc.edu/docs/sci_report/Evans.pdf#search=%22haimen. Accessed 1/23/07. Chen G, et al. J Hepatology 2005; 42 (suppl 2):477A. Chen G, et al. Hepatology 2005; 40 (suppl 1):594A.
  58. 58. Relationship Between Persistent Viremia and HCC: Argument For Antiviral Therapy • Persistent replication associated with greater risk of HCC • Decreased risk when viral replication declines Chen, et al. JAMA 2006 Baseline HBV DNA, (copies/mL) < 104 105 105 105 Follow-up HBVDNA, copies/mL --- < 104 104 to <105 105 Adjusted RR (95% CI) 1.0 (ref) 3.6 (1.7-7.6) 6.9 (3.4-13.8) 9.1 (5.8-14.1) P Value -- < 0.001 < 0.001 < .001 HCCIncidence RatePer100,000 0 1473 5882 8730 10,108 2.0x103 4.0x103 6.0x103 8.0x103 1.0x104 1.2x104
  59. 59. Impact Clinique de la Variabilité du Génome Viral
  60. 60. 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
  61. 61. 8 genotypes, numerous sub-genotypes, and recombinant forms World J Gastroenterol 2007; 13: 14-21 B6 D1
  62. 62. Génotypes VHB chez les patients atteints d’hépatite chronique en FranceNumberofsubjects F GA B C D E 0 10 20 30 40 50 60 70 80 90 100 30.2% 7.9% 12.5% 37.4% 11.3% 0.4 % 1.1% Zoulim et al J Viral Hepatitis 2006
  63. 63. Impact du génotype sur la séroconversion Hbe/HBs 1 Janssen, Lancet 2005; 2 Flink, Am J Gastro 2006 PEG-IFN a-2b HBeAg Loss 1 0 10 20 30 40 50 A n=90 28% 47% 44% 25% B n=23 C n=39 D n=103 Percentageofpatients(%) HBV genotype 0 3 6 9 12 15 A n=90 5% 8% 0% B n=23 C n=39 D n=103 18 15% Percentageofpatients(%) 21 HBV genotype PEG-IFN a-2b HBsAg Loss 2
  64. 64. LES MUTANTS DU GÉNOME DU VHB déterminant a vaccin/HBIg polymérase antiviraux Mt pré-core Réponse anti-e ? Mt core Réponse CTL
  65. 65. 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
  66. 66. 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
  67. 67. HBeAg and Precore Mutation 1814 1901 Precore Core region region HBcAg HBeAg G 1896A = stop codon, TAG ATG ATG Virion Serum Core gene
  68. 68. HBeAg and Precore Mutation 1814 1901 Precore Core region region HBcAg HBeAg ATG ATG Virion Serum Core gene
  69. 69. VARIANTS NÉGATIFS POUR L ’AgHBe mRNA Protéine pré-C/C PRE-C CPROMOTEUR TAG *** 1762-1764 1896 arrêt des synthèses protéiques Diminution de l’expression de l ’AgHBe
  70. 70. Sélection des mutants pré-core au cours de l’histoire naturelle de l’hépatite B chronique 0 500 1000 1500 2000 2500 temps ALAT ADN-VHB AgHBe Anti-HBe 0 20 40 60 80 100 temps sauvage Mt pré-C
  71. 71. Outcome of Chronic Anti-HBe Positive Hepatitis B 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 Biochemical patterns in 164 untreated patients after 23 months (range 12-36) monthly monitoring 0 12 24 months With flares and normalization Without flares With flares and without normalization 73 pts ( 44.5% ) 59 pts ( 36.0% ) 32 pts ( 19.5% ) Asymptomatic flare-up: 90% of cases A L T Flare-up yearly frequency: once 57.1% twice 20% < once 22.8% Brunetto MR et al, J Hepatol 2002
  72. 72. Augmentation de prévalence des hépatites chroniques avec AgHBe négatif en France HBeAg(+) HBeAg(-) 42% N=119 58 % N=164 Zoulim et al, J Viral Hepatitis 2006
  73. 73. DIAGNOSTICS DIFFICILES I. Porteur inactif II. Exacerbation
  74. 74. 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 (see EASL CPG J Hepatol 2009/2012) • HBeAg negative chronic hepatitis – Fluctuation / exacerbation of ALT – Fluctuations of HBV DNA levels usually > 2000 IU/ mL – Presence of pre-core / core promoter mutations
  75. 75. 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, biothérapie, chimiothérapie – surinfection delta / VHC
  76. 76. COOH 137 149 107 99 NH2 S - S S - S S - S S-S 138 139 147 Tiollais P. et al., Nature 1985. Torresi J., J. Clin Virol 2002; Dryden KA. et al., Mol Cell 2006 « a » determinant HBs Ag « a » determinant induces the synthesis of anti-HBs neutralizing antibodies sG145R sP120T sD144H/A/E PreS1 PreS2 SPol Pré-C C Brin(+) 2,4kb Brin(-) 3,2kb X TATAA U5-like DR1 DR2 Enh1Enh2 0/3221 SHBs (S) MHBs (preS2+S) LHBs (preS2+preS2+S)
  77. 77. 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  Risques : transmission virale + infections occultes
  78. 78. 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 +
  79. 79. Presence of HBV DNA in the liver ( serum) of individuals testing HBsAg negative by currently available assays Occult HBV Infection (OBI) Raimondo et al, J Hepatol 2008
  80. 80. How to Detect Occult HBV Infection Currently there is no standardized diagnostic assay for occult HBV infection
  81. 81. Reported Prevalence of Occult HBV Infection in HIV Positive Patients Study Country N° of patients Occult HBV N° (%) Methods Hofer, 1998 Switzerland 57 51 (89%) “nested” PCR (serial evaluation) Torres-Baranda, 2006 Mexico 35 7 (20%) “nested” PCR Filippini, 2006 Italy 86 17 (20%) single step PCR Mphahlele, 2006 South Africa 140 31 (22.%) “nested” PCR Pogany, 2005 Netherlands 93 4 (4%) single step PCR Neau, 2005 France 160 1 (0.6%) Santos, 2003 Brazil 101 16 (16%) single step PCR Wagner, 2004 France 30 11 (37%) “nested” PCR Goncales, 2003 Brazil 159 8 (5%) “nested” PCR Nunez, 2002 Spain 85 0 Cobas Amplicor HBV Monitor (Roche) Piroth, 2000 France 37 13 (35%) single step PCR Raffa, 2007 Italy “nested” PCR (liver) Cobas Amplicor HBV Monitor (Roche) 101 42 (41%) Raimondo et al, J Hepaol 2007, modified
  82. 82. OBI Cause(s) for the failure of HBsAg detection Suppression of HBV replication and gene expression Infection by S gene Variants “false” OBI
  83. 83. Occult HBV infection HBV cccDNA Integrated HBV DNA HBV mutants Epigenetic control HBV replication Immune surveillance Viral co-infections
  84. 84. OBI Seropositive Seronegative HBsAg lost during CH HBsAg lost after AH Progressive antibody disappearence Primary occult Schematic representation of HBV serum marker profile in OBI and “false” OBI „false“ OBI S gene escape mutants HBV DNA levels comparable to overt infection HBV DNA levels < 200 UI/ml
  85. 85. High prevalence ROLE in HCC Diagnostic Tools ? Worsen HCV infection ? Co-infections ? Therapy? To be improved Specific treatments ? Not fully understood ? Occult HBV infections: unresolved issues
  86. 86. Antiviraux Persistance virale Resistance aux antiviraux Monitoring des traitements
  87. 87. HBeAg(+) HBeAg(-) / anti-HBe(+) ALAT HBV DNA Minimal CH Moderate to severe CH Moderate to severe CHRemission Cirrhosis Immunotolerant phase Immuno-active phase Inactive phase Low replication Reactivation phase Cirrhosis 109-1012 IU/mL >2000-<109 IU/mL <2000 IU/mL >2000 IU/mL Inactive cirrhosis Adapted from Fattovich G. Sem Liver Dis. 2003 Treatment indicated Treatment indicated HBsAg Occult infection
  88. 88. Antivirals approved for hepatitis B Drug Type Approved Nucleoside analogs • Lamivudine* • Entecavir • Telbivudine Nucleotide analogs • Adefovir dipivoxil • Tenofovir* Cytokines • Interferon alfa • Pegylated Interferon alfa-2a
  89. 89. 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 No replication = No resistance Viral suppression Histological and clinical 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
  90. 90. Treatment failure Primary non response Partial response Secondary treatment failure Antiviral drug resistance Host factors Drug metabolism Patient’s compliance Drug factors Antiviral potency Drug factors Barrier to resistance Viral factors Resistant mutants Zoulim et al Hepatol 2008; EASL CPG J Hepatol 2009; Lancet Infect Dis 2012
  91. 91. 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
  92. 92. 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
  93. 93. The main differences between HIV, HBV and HCV H HBV1,2 Host cell cccDNA Host DNA Integrated DNA Nucleus H HIV1 Host cell Host DNA Proviral DNA Nucleus H HCV1,3 Host cell Host DNA Nucleus HCV RNA Life-long suppression of viral replication Definitive viral clearance and SVR Long-term suppression of viral replication 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.
  94. 94. 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. Kinetics of emergence of HBV drug resistant mutants
  95. 95. Lamivudine Resistance Accelerates Progression of Liver Disease YMDDm WT Placebo 5% 13% 21% Liaw YF et al. N Engl J Med. 2004;351:1521-1531
  96. 96. 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.
  97. 97. ALT flares in patients with lamivudine resistance over time Lok et al Gastroenterology 2003; 125 : 1714-1722
  98. 98. 6 3 LVD ADV LdT ETV TDF 0 10 20 30 40 50 60 70 80 23 Proportionofpatients(%) 46 55 71 80 0 11 18 29 5 25 0.2 0.5 1.2 0 1 2 3 4 5 1 2 3 4 5 1 2 1 2 3 4 5 1 2 3 0 0 Option to add emtricitabine at week 72* *Patients confirmed to be viraemic at Week 72 or beyond could add emtricitabine to TDF at the discretion of the investigator. Clinical data on the safety and efficacy of emtricitabine and TDF in CHB are pending Rates of resistance with lamivudine (LVD), adefovir (ADV), telbivudine (LdT), entecavir (ETV) and tenofovir (TDF) among NA-naïve patients 4 0 High barrier to resistance Adapted from Gish, Jia, Locarnini & Zoulim, Lancet Infect Dis 2012
  99. 99. Drug and patient population Resistance at year of therapy expressed as percentage of patients 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 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 Incidence of drug resistance over time 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
  100. 100. Terminal protein Spacer POL/RT RNaseH 1 183 349 (rt) 692 (rt 344) 845 a.a. I(G) II(F) A B C D E F_V_LLAQ_YMDD *rtA181T/V and/or rtN236T cause reduced sensitivity *rtA194T association with rtL180M+rtM204V (to be confirmed) LMV resistance/ rtL80I rtL180M rtM204V/I LdT resistance rtA181T/V ADV resistance rtA181T/V rtN236T TDF resistance* ? ETV resistance rtL180M rtM204I/V rtT184*** rtS202**** rtM250I/V rtl169T ***S/A/I/L/G/C/M ****C/G/I Zoulim F & Locarnini Gastroenterology 2009;137:1593-1608. rtV173L * Role of complex mutants: rtA181T+rtN236T ?
  101. 101. Zoulim & Locarnini, Gastroenterology, 2009
  102. 102. Multiple factors are associated with the barrier of resistance & drug efficacy •Adherence •Immune status •Prior antiviral exposure •Metabolism •Body mass Patient Antiviral Drug •Antiviral potency •Number of mutations needed to overcome drug suppression •Level of exposure to drug •Chemical structure Virus Locarnini S, et al. Antivir Ther. 2004;9:679–93. Locarnini S, et al. Antivir Ther. 2007;12:H15-H23. 3. Ghany M & Liang TJ. Gastroenterology 2007;132:1574-85. Zoulim F, et al. Antiviral Res. 2004;64:1-15. Locarnini S, et al. J Hepatol. 2003;39:S124-S132.; Zoulim & Locarnini Gastroenterology 2009 •Replication fitness and space •Persistence of archived mutations as cccDNA •Pre-existing mutations
  103. 103. Adherence to nucleos(t)ide analogues for chronic hepatitis B in clinical practice and correlation with virological breakthroughs W. Chotiyaputta et al, Journal of Viral Hepatitis, Volume 19, Issue 3, pages 205-212, 14 JUL 2011
  104. 104. L(-)-SddC, 3TC LamivudineL(-)-SddC mitochondria nucleus L(-)-SddC-TP HBV DNA Nuclear DNA Mt DNA L(-)-SddC-TP L(-)-SddC-TP cytoplasm kinase L(-)-SddU deaminase Bridges; Progress in Liver Disease 1995
  105. 105. Nucleos(t)ide analogs The HBV life cycle Zoulim & Locarnini, Gastroenterology 2009
  106. 106. uncoating CCC DNA removal of protein primer removal of RNA primer completion of viral (+) strand DNA ligation of DNA strands extremities supercoiled DNA minichromosome viral polymerase? DNA repair protein? other cellular enzymes? Topoisomerase (TDP2) ? Acetyl transferase ? Histones Formation of the recalcitrant cccDNA: a difficult target for antiviral therapy Tuttleman et al Cell 1986 Le Guerhier et al AAC 2000 Delmas et al AAC 2002 Kock et al Hepatology 2003 Cortes Ledesma et al Nature 2009 Antivirals ?
  107. 107. 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)
  108. 108. Kinetics of Viral Loss During Antiviral Therapy with L- FMAU (clevudine) in the woodchuck model Zhu et al, J Virol 2001
  109. 109. ADV Associated Serum HBsAg Reductions are Similar in Magnitude to cccDNA Reductions -6 -5 -4 -3 -2 -1 0 ChangesinHBVMarkers fromBaseline (log10copies/cell(ml)) Serum HBV DNA Total Intracellular DNA cccDNA Serum HBsAg  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
  110. 110. • 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 Immunohistochemical Staining of Patient Biopsies at Baseline and After 48 Weeks ADV Therapy Baseline Week 48
  111. 111. Maynard et al, J Hepatol 2005 Persistence of cccDNA after HBs seroconversion
  112. 112. 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
  113. 113. Kinetics of spread and emergence of drug resistant virus during antiviral therapy Zhou T, et al. Antimicrobial Agents and Chemotherapy 1999; 43: 1947-1954. antiviral wt ni Free liver space Mutant fitness      I II III IV INHIBITION OF WILD TYPE VIRUS REPLICATIONS DELAYED EMERGENCE OF DRUG RESISTANT VIRUS ni = non-infected wt = wild type mt = mutant type mt
  114. 114. Kinetics of HBV drug resistance emergence Si Ahmed et al. Hepatology. 2000; Yuen et al Hepatology 2001; Locarnini et al Antiviral Therapy 2004; Villet et al Gastroenterology 2006 J Hepatol 2007 & 2008; Pallier et al J Virol 2007; Yim et al Hepatology 2006. Treatment begins Drug-resistant variant Drug-susceptible virus Naturally—occurring viral variants Time HBVreplication Primary resistance mutations Secondary resistance mutations / compensatory resistance mutations
  115. 115. • cccDNA in the liver: – Is propagated during the normal replication cycle of HBV – Can serve as a template for the production of new virus Archiving of viral variants Viral quasispecies cccDNA variants Liver Majority population Minority variants Resistant variants Blood circulation Zhou et al, AAC 1999; Zoulim F. Antivir Res. 2004. Zoulim F & Perillo R. J Hepatol. 2008
  116. 116. • cccDNA in the liver: – Is propagated during the normal replication cycle of HBV – Can serve as a template for the production of new virus • It is believed that viral variants with antiviral resistance may be archived in this way Archiving of viral variants Viral quasispecies cccDNA variants Blood circulation Liver Majority population Minority variants Resistant variants Zhou et al, AAC 1999; Zoulim F. Antivir Res. 2004. Zoulim F & Perillo R. J Hepatol. 2008
  117. 117. • cccDNA in the liver: – Is propagated during the normal replication cycle of HBV – Can serve as a template for the production of new virus • It is believed that viral variants with antiviral resistance may be archived in this way Archiving of viral variants Viral quasispecies cccDNA variants Liver Majority population Minority variants Resistant variants Blood circulation Zhou et al, AAC 1999; Zoulim F. Antivir Res. 2004. Zoulim F & Perillo R. J Hepatol. 2008
  118. 118. 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
  119. 119. Cross-resistance data for the main mutants and the commercially available drugs Zoulim & Locarnini Gastroenterology 2009; Liver Int 2013 Pathway Amino Acid Substitutions in the rt Domain LMV LdT ETV ADV TFV Wild-type S S S S S L-Nucleoside (LMV/LdT) M204I/V R R I S S Acyclic phosphonate (ADV) N236T S S S R I Shared (LMV, LdT, ADV) A181T/V R R S R I Double (ADV, TFV) A181T/V + N236T R R S R R D-Cyclopentane (ETV) L180M+M204V/I ± I169 ± T184 ± S202 ± M250 R R R S S Multi-Drug Resistance A181T+N236T+ M250V R R R R R
  120. 120. Phenotyping of HBV clinical isolates 1. Durantel D, et al., Hepatology, 2004;40:855-64. 2. Yang H, et al., Antiv Ther, 2005;10:625-33. Southern blot analysis Patient serum PCR cloning Whole genome HBV clones Transfection HepG2 Huh7 IC50 reference strain IC50 mutant Fold resistance = Wild-type virus Increasing antiviral concentration Cell culture plate Patient’s virus SS - RC - lamivudine adefovir
  121. 121. ADV rtN236T +/or rtA181V Wild-type virus ADV-resistant virus LAM-resistant virus LAM rtM204V/I ± rtL180M ETV-resistant virus rtT184 or rtS202 or rtM250 ETV rtM204V/I rtL180M+/- TDF TDF: what can we expect? rtM204V/I +/- rtL180M LAM then ETV rtT184 or rtS202 or rtM250 LAM + TDF – what do we see? Maximising the barrier to resistance
  122. 122. ? Multiple drug resistant mutants with complex pattern of mutations + one mutation + one mutation Drug A Drug B Risk of selection of MDR mutants by sequential therapy - drugs sharing cross-resistance characteristics - incomplete viral suppression - liver transplantation The problem of sequential therapy with nucleoside analogues Zoulim F, et al. J Hepatol. 2008;48:S2-19. Yim et al, Hepatology 2006; Villet et al Gastroenterology 2006 & 2009
  123. 123. 103 104 105 106 107 108 109 0 20 40 60 80 100 120 Treatment (months) HBVDNA(copies/ml) entecavirIFN adefovir lamivudine Genotype H lamivudine Drugs sharing cross-resistance characteristics: Switching strategy  emergence of MDR mutant L180M+S202G+M204V L180M+M204V Villet et al, J Hepatol 2007
  124. 124. Warner et al Hepatology 2009 Kamili et al Hepatology 2009 Villet et al Gastroenterology 2009 Impact on virus infectivity and fitness Impact on virion release (intracellular retention) and virologic monitoring of breakthrough Impact on vaccine prophylaxis efficacy
  125. 125. 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
  126. 126. Patients heavily exposed to NUCs with low barrier to resistance – Risk of MDR selection • Risk of multidrug resistance by sequential accumulation of resistance mutations • Risk of partial response, even with the newest NUCs -> long-term impact ?
  127. 127. ? Multiple drug resistant mutants with complex pattern of mutations + one mutation + one mutation Drug A Drug B Risk of selection of MDR mutants by sequential therapy - drugs sharing cross-resistance characteristics - incomplete viral suppression - liver transplantation The problem of sequential therapy with nucleoside analogues Zoulim F, et al. J Hepatol. 2008;48:S2-19.Yim et al, Hepatology 2006; Villet et al Gastroenterology 2006 & 2009
  128. 128. Liu et al, Antivir Ther. 2010;15(8):1185-90. Sequential therapy with NUCs and the risk of MDR Accumulation of multiple mutations on the same viral genome Complete change of the viral quasi-species
  129. 129. A single a.a. substitution at position rt181 may be responsible for multidrug resistance Villet S, et al. J Hepatol. 2008;48:747-55. wt A181V A181T A181V + N236T A181T + N236T N236T N236T + N238T M204V M204I L80V L80V + M204I LVD LVD+TDF LVD+ADV+TDF Patient #1 (67 months) Patient #7 (30 months) Patient #2 (23 months) Patient #3 (37 months) Patient #10 (7 months) Patient #5 (44 months) Patient #4 (31 months) Patient #6 (36 months) Patient #9 (19 months) Patient #8 (47 months) LVD+ADVADV
  130. 130. Impact of rtA181 and rtN236 mutations on antiviral drug efficacy and cross-resistance Villet et al, J Hepatol 2008
  131. 131. 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% BL W4 W12 W24 W36 W48 N236T A181V + N236T A181V A181S + N236T A181T + N236T A181T wt #1051 (L180L/M,A181A/V/T,A194A/T,S202S/I,N236N/T) Week 0 4 8 12 16 20 24 28 32 36 40 44 48 HBVDNA(log10cp/mL) 0 1 2 3 4 5 6 7 8 9 10 Viral load BL viral load = 8.75log Treatment: TDF Adherence : 95.2% Patient 1051 data: LLOD Evolution of viral genome during Tenofovir therapy in patients who previously failed ADV Impact of persisting low viremia levels on treatment outcome ? Impact of persisting resistant mutants ? Lavocat et al, AASLD 2010 & Ms submitted
  132. 132. Virologic response to TDF according to ADV resistance mutations at baseline The Australian Experience Patterson S J et al. Gut 2011;60:247-254
  133. 133. Tenofovir + Emtricitabine in patients with treatment failure – treatment intensification 0 1 2 3 4 5 6 7 Baseline M3 M6 M12 Time after TDF+FTC initiation (months) HBVDNA(log10 IU/mL) HBV DNA kinetics after TDF+FTC initiation in 59 patients with treatment intensification Si-Ahmed et al, Antiviral Research 2011 Time to undetectable DNA (<50 IU/mL) 14121086420 Probability 1,0 ,8 ,6 ,4 ,2 0,0 ≤ 4 logs > 4 logs Viral load initiation
  134. 134. Rescue therapy with ETV + TDF in CHB patients with advanced liver disease and complex viral resistance patterns or showing partial antiviral responses to preceeding therapies (Virgil network) ETV + TDF combination in patients with treatment failure Petersen J, et al. J Hepatol 2012 HBV DNA Viremia 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 Baseline 3 6 9 12 15 18 21 24 10 6 Δ 3 log10 c/mL reduction P=0.0001 LLoD HBVDNA[IU/ml] Months
  135. 135. 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 – 15 000 sont traitées • Persistance virale – Pas d’éradication du génome viral – Surveillance prolongée, possibilité de réactivations
  136. 136. 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 !!!
  137. 137. Acknowledgements Hepatology Unit INSERM U1052 Collaborations David Durantel Barbara Testoni Malika Ait-Goughoulte Souphalone Luangsay Marion Gruffaz Nathalie Isorce Fanny Lebossé Maelenn Fournier Julie Lucifora Maud Michelet Judith Fresquet LabEx C. Caux, Lyon CRCL U. Hasan, Lyon CIRI T. Henry, Lyon CIRI FL. Cosset, Lyon CIRI M. Levrero, Rome M. Tommasino, IARC IHU
  138. 138. Ganem and Prince, NEJM 2004
  139. 139. Perspectives of anti-HBV immune therapy
  140. 140. The concept of combination therapy Entry inhibition cccDNA - formation - stability / destruction - epigenetic regulation Viral core functions Other viral targets Stimulating innate responses Specific ligands Stimulating adpative responses Co-inhibitory signals Co-stimulatory signals Therapeutic vaccination Functional cure / control Real cure ? Viral targets Immune modulation
  141. 141. Mason, W. S. et al. 2009 / 2010. J. Virol Devons nous redéfinir la tolérance immunitaire et repenser les indications thérapeutiques ? Observation d’une expansion clonale des hépatocytes - Cellules qui n’expriment pas les antigènes viraux - Diminution de la charge virale malgré l’absence de lésion hépatique mesurable - L’une des premières étapes du CHC Tolérance Immunitaire - Presque tous les hepatocytes sont infectés - Viremies > 10E9 copies/mL - Devrions nous réaliser une biopsie lorsque la charge virale diminue sans élévation des ALAT ? Et penser à un traitement antiviral ? Zoulim & Mason, W. S. Gut 2012
  142. 142. Effective T-cells control virus Exhausted T-cells lose control of virus CD8 T cells Infected hepatocytes Infected hepatocytes INF-g TNF-a IL-2 Granzyme Perforin Specific immunomodulation of existing T-cells e.g. PD-1 blockade1,2 Patients who have resolved HBV Patients with chronic HBV Restoration of defective T-cell immune control 1. Fisicaro P, et al. Gastroenterology 2010;138:682–93. 2. Fisicaro P, et al. Gastroenterology 2012;143:1576–85 Figure adapted from Nebbia G, et al. Q J Med 2012;105:109–13 and Freeman G, et.al. J Exp Med 2006;203(10):2223–7.
  143. 143. HBsAg clearance Werle-Lapostolle B et al., Gastroenterology 2004;126: 1750-58. Infected hepatocytes Infected liver CD8 NKT CD4 B cccDNA Antivirals Clearance of HBsAg? Blood circulation viral load
  144. 144. 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
  145. 145. Prevention of resistance Impact of first line therapy • Choose an antiviral drug with 1. A potent antiviral activity 2. A high barrier to resistance
  146. 146. 6 3 LVD ADV LdT ETV TDF 0 10 20 30 40 50 60 70 80 23 Proportionofpatients(%) 46 55 71 80 0 11 18 29 5 25 0.2 0.5 1.2 0 1 2 3 4 5 1 2 3 4 5 1 2 1 2 3 4 5 1 2 3 0 0 Option to add emtricitabine at week 72* *Patients confirmed to be viraemic at Week 72 or beyond could add emtricitabine to TDF at the discretion of the investigator. Clinical data on the safety and efficacy of emtricitabine and TDF in CHB are pending Rates of resistance with lamivudine (LVD), adefovir (ADV), telbivudine (LdT), entecavir (ETV) and tenofovir (TDF) among NA-naïve patients 4 0 High barrier to resistance 5 0 Gish, Jia, Locarnini, Zoulim, Lancet Infect Dis 2012
  147. 147. Zoulim & Locarnini, Gastroenterology 2009; EASL CPG J Hepatol 2009 & 2012 Mangement of antiviral drug resistance • Impact of second line therapy – Early treatment adaptation to prevent accumulation of mutations – Choice always based on cross-resistance data – Add-on strategy versus switch ? • Good results with TDF switch • Some cases of suboptimal responses • Combination to increase the barrier to resistance
  148. 148. Cross-resistance data for the main mutants and the commercially available drugs Zoulim & Locarnini Gastroenterology 2009; Liver Int 2013 Pathway Amino Acid Substitutions in the rt Domain LMV LdT ETV ADV TFV Wild-type S S S S S L-Nucleoside (LMV/LdT) M204I/V R R I S S Acyclic phosphonate (ADV) N236T S S S R I Shared (LMV, LdT, ADV) A181T/V R R S R I Double (ADV, TFV) A181T/V + N236T R R S R R D-Cyclopentane (ETV) L180M+M204V/I ± I169 ± T184 ± S202 ± M250 R R R S S Multi-Drug Resistance A181T+N236T+ M250V R R R R R

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