Zoulim du 2015

1. Hépatite B
Fabien Zoulim
Département d’hépatologie
& INSERM U1052, Lyon

2. EPIDEMIOLOGIE DE L’HÉPATITE B

3. Ott et al, Vaccine 2012

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. 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. 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. 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. 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. 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. 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. VIROLOGIE

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. 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. 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. The viral replication cycle
Zoulim & Locarnini, Gastroenterology 2009

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. 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. 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. Cell culture models for HBV infection
Zeisel M …Zoulim F, Gut 2015

20. Animal models for HBV infection
Zeisel M …Zoulim F, Gut 2015

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

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. PHYSIOPATHOLOGIE /
IMMUNOPATHOLOGIE

26. HBV and the immune responses

27. Current model of HBV pathogenesis

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

30. Immunopathology
Fulminant hepatitis
CD8+
HBV

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

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. 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. 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. 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. 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. HISTOIRE NATURELLE ET
VIROLOGIE CLINIQUE

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. 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. 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. 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. 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. 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. 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. 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. 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. 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. Formes cliniques

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

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. 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. 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. Impact Clinique de la Variabilité
du Génome Viral

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

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

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

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

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. 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. 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. 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. DIAGNOSTICS DIFFICILES
I. Porteur inactif
II. Exacerbation

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

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

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

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. 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. Antiviraux
Persistance virale
Resistance aux antiviraux
Monitoring des traitements

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

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. 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. 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. Zoulim & Locarnini, Gastroenterology, 2009

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. 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. 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. Nucleos(t)ide analogs
The HBV life cycle
Zoulim & Locarnini, Gastroenterology 2009

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

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

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. 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. 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. • 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. • 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. • 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. 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. 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. 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

126. 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

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. 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

130. 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

131. 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

132. 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 ?

133. ?
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

134. 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

135. 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

136. Impact of rtA181 and rtN236 mutations on antiviral
drug efficacy and cross-resistance
Villet et al, J Hepatol 2008

137. 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

138. Virologic response to TDF according to ADV resistance
mutations at baseline
The Australian Experience
Patterson S J et al. Gut 2011;60:247-254

139. 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

140. 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

141. 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

142. 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 !!!

143. 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

144. Ganem and Prince, NEJM 2004

145. Perspectives of anti-HBV immune therapy

146. 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

147. 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

148. 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.

149. 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

150. 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

151. Prevention of resistance
Impact of first line therapy
• Choose an antiviral drug with
1. A potent antiviral activity
2. A high barrier to resistance

152. 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

153. 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

154. 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