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Hépatite B     Fabien ZoulimDépartement d’hépatologie & INSERM U871, Lyon
Natural history of hepatitis B                                                         Acute infection    Resolved infecti...
EPIDEMIOLOGIE DE L’HÉPATITE B
EPIDEMIOLOGIE DE LINFECTION A VHBAUX USA    • Hépatites aigues          – VHA : 40%          – VHB : 30%          – VHC : ...
MODES DE TRANSMISSION DU VIRUS DE LHÉPATITE B EN EUROPE                                           transfusions            ...
Déclaration obligatoire       de l’hépatite B en France : résultats des 12 premiers mois de notificationDenise Antona, E D...
Results158 acute hepatitis cases• Hospital doctor in 64% cases• Sex ratio M/F : 2,95 (118/40)• Median age: 37 yrs for male...
Risk exposure within 6 months preceding the acute case                Source : obligatory declaration 2003-04• Source: obl...
Surveillance épidémiologique de           l’infection HBV• 14 446 adultes testés• Prévalence de l’AgHBs 0,65% (280 000 por...
Hépatites virales B: épidémiologie- Vaccin mais 400 millions de porteurs chroniquesdans le monde- 280 000 porteurs chroniq...
VIROLOGIE
LE VIRUS DE L ’HEPATITE B• FAMILLE : Hepadnaviridae, seul représentanthumain•VIRUS RESISTANT :    - 7 jours dans l’environ...
HBsAgfilament                                    S       small surface protein                           sphere           ...
The HBV genome                                        déterminant a                                        vaccin/IgHBs8 g...
The viral replication cycleZoulim & Locarnini, Gastroenterology 2009
The animal models of HBV infection                   Transgenic mice                   Humanized mice                     ...
Modèles in vitro• Polymerase virale                       • Culture cellulaire   – DHBV : lysat réticulocytaire           ...
Comparative dynamics among three viruses             (Tsiang et al. Hepatology 1999)
Infection à VHB et risque de CHC• Etude de Beasley à Taiwan   – risque relatif = 100 chez les porteurs de lAgHBs• Etude de...
CARCINOME HEPATOCELLULAIRE ET VIRUS DE             LHEPATITE B• Co-incidence de répartition géographiqueVHB / CHC• Porteur...
HBV replication and its role in HCC development                                          Wands, NEJM 2004
Role du VHB dans l’oncogénèse hépatique                  REACTION INFLAMMATOIRE CHRONIQUE                       REGENERATI...
PHYSIOPATHOLOGIE /IMMUNOPATHOLOGIE
Ganem and Prince, NEJM 2004
IMMUNOPATHOGÉNIE              DES HÉPATITES B CHRONIQUES                                                RÉPONSE IMMUNITAIR...
IMMUNOPATHOLOGY OF HBV INFECTION                       CD8+                               HBVImmune tolerance             ...
Immunopathology  Fulminant hepatitis                HBV CD8+
MECHANISMS OF VIRAL CLEARANCENon cytolytic processes                           Turn-over of infected cellsTH1 cytokines wi...
Non cytolytic clearance of acute                             Wieland S et al, PNAS 2004
Hepatocyte turn-over is required for clearance of                            Summers et al, PNAS 2003 & 2004
Phase de tolérance immunitaire                                           MarqueursHépatocytenon infecté                   ...
Phase de clairance immune                 (hépatite chronique)                                           Marqueurs        ...
Phase de rémission               portage inactif de l’AgHBs                                     MarqueursHépatocytenon inf...
Clairance de l’AgHBs                                            MarqueursHépatocytes                                  HBsA...
cccDNA levels in the different phases of                                   chronic HBV infection                          ...
Inactive HBV carrier  ●                         Not virologically inactive:      –                             low levels ...
HISTOIRE NATURELLE ET VIROLOGIE CLINIQUE
Histoire Naturelle de l’hépatite B                                         Infection aigue           Seeger, Zoulim, Mason...
HEPATITE B AIGUE• Incubation 1 à 6 mois• Le plus souvent asymptomatique  – Évolution plus fréquente vers la chronicité• Pr...
Laboratory Diagnosis of Acute Hepatitis B                                                           HBsAg                 ...
HEPATITE B PROLONGEE• Définition  – Persistance réplication virale à la 8ème    semaine d’évolution :  – AgHBe + ou ADN-VH...
INFECTIONS CHRONIQUES A VHB             FORMES CLINIQUES• virus sauvage   – tolérance immunitaire   – rupture de tolérance...
Laboratory Diagnosis of Chronic Hepatitis B                                                associated with wild type virus...
Laboratory Diagnosis of Transition of Chronic                                                  Hepatitis B to The inactive...
Laboratory Diagnosis of HBeAg negative                                                              Chronic Hepatitis B   ...
UI/ml                        AgHBspg/ml             AgHBe +                             anti-HBe +  10009 log             ...
Dynamic ranges of quantification                          of HBV DNA assays                        10   102   103   104   ...
Formes cliniques
MANIFESTATIONS      EXTRAHEPATIQUES DU VHB• PAN    – Complexes immuns circulants HBs/anti-HBs    – Dépots artères moyens e...
TRANSMISSION VERTICALE DU VHB• mère AgHBe +  – transmission : 90%• mère anti-HBe +  – transmission : 10-20%  – VHB muté pr...
PRESENTATION CLINIQUE• INFECTION PERI-NATALE  – ALT normales ou subnormales  – ADN-VHB > 1000 pg/ml  – histologie : lésion...
Pathophysiologic Cascade of         HBV Replication                                                                   Live...
Charge virale et incidence de la cirrhose                                    .4                                           ...
Survie chez les patients au stade cirrhose                                      100              Patients Surviving, %    ...
Charge virale et incidence du CHC                        Chen et al; JAMA 2006
REVEAL-Incidence of HCC                                   20%   % cumulative incidence of HCC                             ...
High Baseline Serum HBV DNA Levels are                                    Associated with Increased Risk of HCC Mortality ...
Relationship Between Persistent Viremia and HCC:                              Argument For Antiviral Therapy          •   ...
Impact Clinique de la Variabilité du
VARIABILITE GENETIQUE DU VHB• Multiplication virale   » taux derreur de la transcriptase inverse• Pression de sélection   ...
8 genotypes, numerous sub-genotypes, and            recombinant forms   B6                     D1                      Wor...
Génotypes VHB chez les patients atteints d’hépatite              chronique en France                                      ...
Impact du génotype sur la                                                   séroconversion Hbe/HBs                        ...
LES MUTANTS DU GÉNOME DU VHB                                        déterminant a                                        v...
ROLE DE LA RÉGION PRÉ-C ET DE L’AgHBe • Non nécessaire à la réplication du VHB      – Culture cellulaire      – Modèles in...
LES MUTANTS PRÉ-C (-)           • codon stop / région pré-C               TGG -> TAG en pos. 1896               – génotype...
HBeAg and Precore Mutation                   G 1896A = stop codon, TAG             ATG              ATGCore gene          ...
HBeAg and Precore Mutation              ATG         ATGCore gene             1814       1901              Precore         ...
VARIANTS NÉGATIFS POUR L ’AgHBe1762-1764       1896PROMOTEUR PRE-C                             C    *       *     *       ...
Sélection des mutants pré-core au cours de  l’histoire naturelle de l’hépatite B chronique         2500                   ...
Outcome of Chronic Anti-HBe Positive Hepatitis B      400               Biochemical patterns in 164 untreated patients    ...
Augmentation de prévalence des hépatites      chroniques avec AgHBe négatif en France                            58 %     ...
Pre-core mutations     Both mutations                         No pre-core mutation     (n = 95; 33.6%)                    ...
MUTANTS PRÉ-C ET SÉVÉRITÉ HISTOLOGIQUE           LA CONTROVERSE   • Italie      – Cirrhose plus fréquente            • Bon...
HBe serotype and liver pathology                                                          HBe-positive                    ...
HÉPATITES FULMINANTES ET MUTANTS PRE-C       • Lien de causalité :           – Épidémies hépatites fulminantes           –...
DIAGNOSTICS DIFFICILES
Diagnosis of inactive carrier versus    HBeAg negative chronic hepatitis• Inactive Carrier  – Persistently normal ALT leve...
DIAGNOSTIC DUNE EXACERBATION AIGUE     SUR HEPATITE B CHRONIQUE • Définition : poussée cytolytique ≠ réactivation virale •...
PreS2                                                                                       PreS1            HBs Ag       ...
Variants de lAg HBs• échappement à la réponse humorale anti-HBs   – naturelle   – vaccination (transmission mère-enfant)  ...
VARIANTS DE LAgHBs• Mutations ponctuelles dans le déterminant a de lAgHBs (124-147)   – aa 145 : Gly -> Arg   – aa 126 : I...
Occult HBV Infection (OBI) Presence of HBV DNA in the liver (± serum) ofindividuals testing HBsAg negative by currently   ...
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                                                   Occ...
Cause(s) for the        failure of HBsAg detection      OBI                “false” OBI  Suppression of                    ...
HBV replication                      HBV cccDNA                        Integrated HBV DNA         HBV mutants             ...
Schematic representation of HBV serum marker profile in OBI and                          “false” OBI                      ...
Occult hepatitis BTorbenson M. & Thomas D.L., Lancet Inf Dis, 2002
Occult HBV infections: unresolved issues  Specific                          Diagnostic    To be  treatments ?             ...
Antiviraux   Persistance viraleResistance aux antivirauxMonitoring des traitements
HBsAg                            Immuno-active   Inactive phase                                     Occult infectionImmuno...
Antivirals approved for hepatitis BDrug Type                             Approved                Phase 3          Phase 2N...
Endpoints of therapyPersistence of high viral load is associated with a significant risk of progression of                ...
Treatment failurePrimary non response                                  Secondary treatment failurePartial response        ...
Clinical definition of resistance• Virologic Breakthrough: Rebound in serum HBV DNA levels  (e.g. 1 log10 above nadir)• Ge...
Laboratory Definition of HBV Resistance to AntiviralsLaboratory Investigations• Phenotypic Resistance: Decreased susceptib...
The main differences between HIV,                         HBV and HCV                 HIV1                                ...
Kinetics of emergence of HBV drug resistant mutantsSi Ahmed et al. Hepatology. 2000; Yuen et al Hepatology 2001; Locarnini...
Lamivudine Resistance Accelerates                                        Progression of Liver Disease                     ...
Biochemical and Histologic               Correlates of HBV Resistance  • Rise in ALT levels        – Mild ALT elevations i...
ALT flares in patients with lamivudine         resistance over time                         Lok et al Gastroenterology 200...
Incidence of drug resistance over time                                           Resistance at year of therapy expressed a...
Rates of resistance with lamivudine (LVD), adefovir (ADV), telbivudine (LdT),                 entecavir (ETV) and tenofovi...
Zoulim & Locarnini, Gastroenterology, 2009
Determinants of viral persistence & resistanceZoulim & Locarnini, Gastroenterology, 2009
Multiple factors are associated with the        barrier of resistance & drug efficacy    • Antiviral potency    • Number o...
L(-)-SddU   mitochondria                        deaminase                                                                 ...
The HBV life cycle                                            Nucleos(t)ide analogsZoulim & Locarnini, Gastroenterology 2009
Formation of the recalcitrant cccDNA: a difficult     target for antiviral therapyuncoating             CCC DNA            ...
Can we prevent cccDNA formation ?Nucleoside analogs in monotherapy or
Kinetics of Viral Loss During Antiviral Therapy with L-     FMAU (clevudine) in the woodchuck model                       ...
ADV Associated Serum HBsAg Reductions are Similar in Magnitude to cccDNA Reductions                     Serum        Total...
Immunohistochemical Staining of Patient Biopsies at         Baseline and After 48 Weeks ADV Therapy    Baseline           ...
Persistence of cccDNA after HBs seroconversion                                         Maynard et al, J Hepatol 2005
Clearance of viral infection versus selection of                 escape mutantsThe most important factors to consider:§   ...
Kinetics of spread and emergence of drug     resistant virus during antiviral therapy                                     ...
Kinetics of HBV drug resistance emergence                                                                                 ...
Partial response to adefovir dipivoxil is not due to the                        selection of DR mutants•   The top 25% pat...
Amino acid substitutions result in conformation  changes of the polymerase catalytic site  Wild-type M204/L180            ...
Definition of fitness• A parameter that quantifies the adaptation of an  organism or a virus to a given environment• For a...
Cross-resistance data for the main mutants                      and the commercially available drugs Pathway            Am...
Archiving of viral variants                                                                                   Viral quasis...
Archiving of viral variants                                                                                    Viral quasi...
Archiving of viral variants                                                                                    Viral quasi...
Phenotyping of HBV clinical isolates                                                                                      ...
Maximising the barrier to resistance                                                                                     W...
Can we detect low frequency mutants prior to or                     during therapy ?Use of pyrosequencing to detect lowfre...
Important factors involved in selection of                   MDR mutants• Use of inadequate sequential monotherapies and i...
The problem of sequential therapy with               nucleoside analogues                                                 ...
Drugs sharing cross-resistance characteristics:                                   Switching strategy  emergence of MDR mu...
A single a.a. substitution at position rt181 may be                     responsible for multidrug resistanceLVD           ...
Impact of rtA181 and rtN236 mutations on antiviral        drug efficacy and cross-resistance                              ...
Impact on virus infectivity and fitnessImpact on virion release (intracellular       retention) and virologic monitoring o...
Potential risk of transmission of HBV DR mutantsClements et al, Bull WHO 2009
Management algorithm                                  Antiviral treatment                                                 ...
Management algorithm        Antiviral treatment                                        Viral load asssessment      Treatme...
Virologic Consequences of Persistent Viremia  § Infection of new hepatocytes  "  slower kinetics of clearance infected c...
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  1. 1. Hépatite B Fabien ZoulimDépartement d’hépatologie & INSERM U871, Lyon
  2. 2. Natural history of hepatitis B Acute infection Resolved infection 5% neonates Chronic infection: 400 million carriers ! 90% adults Chronic hepatitis Wild type virus HBeAg+ Pre-core mutant HBeAg- Inactive carrier Immune tolerance Reactivation Cirrhosis30-50 years Hepatocellular carcinoma Seeger, Zoulim, Mason; Fields Virology; 2007
  3. 3. EPIDEMIOLOGIE DE L’HÉPATITE B
  4. 4. EPIDEMIOLOGIE DE LINFECTION A VHBAUX USA • Hépatites aigues – VHA : 40% – VHB : 30% – VHC : 20% • incidence : 300 000 infections à VHB / an • 30 000 nouveaux porteurs chroniques / an • 3 000 décès / an
  5. 5. MODES DE TRANSMISSION DU VIRUS DE LHÉPATITE B EN EUROPE transfusions contact avec 2% porteur du VHB personnels de santé sexuelle 4% 2% homo hémodialysés 34% 11% 8% hétéro 23% inconnue 31% Asie drogue IVTransmission verticale 26%
  6. 6. Déclaration obligatoire de l’hépatite B en France : résultats des 12 premiers mois de notificationDenise Antona, E Delarocque-Astagneau, D Lévy-Bruhl département des maladies infectieuses
  7. 7. Results158 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% • Sentinel networks 91-96 – Familial 14 9,7% N=195 – Perinatal 2 1,4% –sexual 35% – Live in instiution 11 7,6% –IVDU 19% –« percutaneous » 15% – Travel in endemic 21 14,5% –No factor 35% areas 91/145 patients (63 %) had a vaccine indication (2 vaccinated ≥ 3 doses)
  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 droguesMeffre et al, J. Med Virol 2004
  10. 10. Hépatites virales B: épidémiologie- Vaccin mais 400 millions de porteurs chroniquesdans 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. LE VIRUS DE L ’HEPATITE B• FAMILLE : Hepadnaviridae, seul représentanthumain•VIRUS RESISTANT : - 7 jours dans l’environnement - pendant 5 mn à 100°C, 10 h à 60°C - à la congélation.
  13. 13. HBsAgfilament S small surface protein sphere Dane particle M middle surface protein v v L large surface protein core capsid protein HBeAg HBeAg secreted e antigen v pol polymerase HBx X protein (non-secreted)
  14. 14. The HBV genome déterminant a vaccin/IgHBs8 génotypes A to H Gène pol antiviraux Mt du core Réponse CTLMt pre-coreRéponse anti-HBe ? Tiollais, Nature 1985
  15. 15. The viral replication cycleZoulim & Locarnini, Gastroenterology 2009
  16. 16. The animal models of HBV infection Transgenic mice Humanized mice Human Chimpanzee Gibbon baboons Tupaïa Woolley monkey Ground squirrel American woodchuck Summers PNAS 1978, Mason J Virol Pekin Duck 1981, Chisari Science 1985, Grey Heron Petersen PNAS 1998, Lanford PNAS 1998
  17. 17. Modèles in vitro• Polymerase virale • Culture cellulaire – DHBV : lysat réticulocytaire – Transfection : lignées d’hépatome – HBV : baculovirus – Infection : hépatocytes primaires, HepaRG – Baculovirus ou adenovirus recombinant RC - L- U Polymerase VHB SS - DNA(-) ELONGATION CCC - Sells PNAS 1987, Wang Cell 1992, Zoulim J Virol 1994, Lanford J Virol 1995, Gripon PNAS 2002, Sprinzl J Virol 2001
  18. 18. Comparative dynamics among three viruses (Tsiang et al. Hepatology 1999)
  19. 19. Infection à VHB et risque de CHC• Etude de Beasley à Taiwan – risque relatif = 100 chez les porteurs de lAgHBs• 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)
  20. 20. CARCINOME HEPATOCELLULAIRE ET VIRUS DE LHEPATITE B• Co-incidence de répartition géographiqueVHB / CHC• Porteurs AgHBs : RR x 100 pour le CHC• CHC dans les modèles animaux de lhépatite B : – marmotte – écureuil• Présence dADN viral intégré dans les tumeurs
  21. 21. HBV replication and its role in HCC development Wands, NEJM 2004
  22. 22. Role du VHB dans l’oncogénèse hépatique REACTION INFLAMMATOIRE CHRONIQUE REGENERATION HEPATIQUE VHB CARCINOGENESINFECTION CHRONIQUE CHC CO-FACTEURS MUTAGENESE INSERTIONNELE TRANSACTIVATION DE GENES CELLULAIRES INTERACTIONS PROTEIQUES INACTIVATION DE GENES SUPPRESSEURS DE TUMEUR
  23. 23. PHYSIOPATHOLOGIE /IMMUNOPATHOLOGIE
  24. 24. Ganem and Prince, NEJM 2004
  25. 25. IMMUNOPATHOGÉNIE DES HÉPATITES B CHRONIQUES RÉPONSE IMMUNITAIRE HÉPATOCYTE CYTOKINES NON INFECTÉ CTL cytokines perforine FasANTICORPS NEUTRALISANTS AgHBc/e VHB HLAI ANTIVIRAUX HÉPATOCYTE INFECTÉ
  26. 26. IMMUNOPATHOLOGY OF HBV INFECTION CD8+ HBVImmune tolerance CD8+ HBVClearance phaseChronic hepatitis HBVSeroconversion CD8+Remission
  27. 27. Immunopathology Fulminant hepatitis HBV CD8+
  28. 28. MECHANISMS OF VIRAL CLEARANCENon cytolytic processes Turn-over of infected cellsTH1 cytokines with direct antiviral Immune mediated lysis of infected cells effect Transgenic mice Ducks Chimpanzees Woodchucks (Guidotti Science 1999, (Guo J Virol 1999 Thimme J Virol 2003) Summers PNAS 2003&2004) Antivirals Inhibition of viral DNA synthesis -> inhibition of intracellular recycling of cccDNA (Werle Gastroenterology 2004) Restoration of anti-HBV immune response (Boni Hepatology 2000)
  29. 29. Non cytolytic clearance of acute Wieland S et al, PNAS 2004
  30. 30. Hepatocyte turn-over is required for clearance of Summers et al, PNAS 2003 & 2004
  31. 31. Phase de tolérance immunitaire MarqueursHépatocytenon infecté AgHBe + HBV DNA +++ ALAT = N Foie = N HBc/e Ag HBV Hépatocyte infecté
  32. 32. Phase de clairance immune (hépatite chronique) Marqueurs AgHBe+Hépatocytenon infecté HBV DNA > 2000 IU/mL ALAT +++ CD8 Foie: Hépatite chronique cytokines perforine Fas HBc/e Ag HBV HLAI Hépatocyte infecté
  33. 33. Phase de rémission portage inactif de l’AgHBs MarqueursHépatocytenon infecté CD8 AgHBe- CD4 anti-HBe + HBV DNA < 2000 IU/mL ALAT = N Foie = rémission HBs Ag Hépatocyte infecté Réactivation Virus sauvage Oncogénèse ou mt pre-core
  34. 34. Clairance de l’AgHBs MarqueursHépatocytes HBsAg -non infectés CD8 CD4 anti-HBc + Anti-HBs +/- B PCR sérum (-) / foie (+) Hépatocytes infectés Mutants d’échappement Oncogénèse Infections occultes
  35. 35. cccDNA levels in the different phases of chronic HBV infection Total HBV DNA cccDNA (copies/cell) (copies/cell) 3 ) 8 ) ) 7 ) ) ) 0 ) ) (6 - (1 (10 -( (6 3 (1 8 (1 -( 7 g g+ Ag r s SA g+ g- r s Ag A e rie A A rie BS Be HB ar H B Be Be r H . C H H . Ca H ct t a ac In In• HBeAg+ patients had significantly higher cccDNA (90-fold) and total HBV DNA (147- fold) levels compared to HBeAg- patients. (p<0.001, Wilcoxon tests) Werle et al, Gastroenterology 2004
  36. 36. Inactive HBV carrier ● Not virologically inactive: – low levels of viremia – episomal HBV DNA in the liver Low-replicative or latent infection Epigenetic control Pollicino et al., Gastroenterology 2006 Sirt1 PCAF CBP p300 CBP HDAC1HDAC1 Sirt1 p300 PCAF Histones HistonesLOW-REPLICATIVE STATE HIGH-REPLICATIVE STATE – spontaneously – during immunosuppression 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 Seeger, Zoulim, Mason; Guérison Fields Virology; 2007 5% nx-nés Infection chronique 90% adultes Hépatite chronique Virus sauvage (HBeAg+) Mutant pre-core (HBeAg-) Portage inactif Tolérance immunitaire Réactivation Cirrhose30-50 ans Carcinome hépatocellulaireSeeger, 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 HBsAg Anti-HBs Ab 1000 HBeAg Anti-HBe Ab IU/L and million copies/ml ALT 750 Total anti-HBcALT and HBV DNA Symptoms 500 HBV DNA IgM anti-HBc 250 0 Normal 0 1 2 3 4 5 6 12 24 36 48 60 Months After Exposure 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 lhépatopathie : controversée – association au CHC
  43. 43. Laboratory Diagnosis of Chronic Hepatitis B associated with wild type virus infection HBsAg 700 HBeAg IU/L or million copies/ml 525ALT and HBV DNA HBV DNA 350 175 ALT 0 Normal 0 1 2 3 4 5 6 12 24 36 48 60 Months After Exposure Seeger, Zoulim, Mason, Fields Virology 2007
  44. 44. Laboratory Diagnosis of Transition of Chronic Hepatitis B to The inactive Carrier State 800 HBsAg `` IU/L and million copies/ml 700 HBeAg Anti-HBe 600ALT and HBV DNA 500 400 HBV DNA 300 200 ALT 100 Normal 0 0 1 2 3 4 5 6 12 24 36 48 60 72 80 92 104 Months After Exposure Seeger, Zoulim, Mason, Fields Virology 2007
  45. 45. Laboratory Diagnosis of HBeAg negative Chronic Hepatitis B HBsAg HBeAg Anti-HBe IU/L and million copies/ml 500 ALTALT and HBV DNA 375 250 HBV DNA 125 0 Normal ALT levels 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 Months Seeger, Zoulim, Mason, Fields Virology 2007
  46. 46. UI/ml AgHBspg/ml AgHBe + anti-HBe + 10009 log ALAT ADN-8 log 100 VHB7 log 106 log5 log1 hybridation4 log 0,13 log 0,012 log PCR1 0,001 log Tolérance hép chronique p. inactif mt pré-core VHB occulte
  47. 47. Dynamic ranges of quantification of HBV DNA assays 10 102 103 104 105 106 107 108 109Amplicor HBV Monitorv2.0 (Roche)HBV Hybrid-Capture II(Digene)Ultra-sensitive HBVHybrid-Capture IIVersant HBV DNA3.0 (bDNA, Siemens)Cobas Taqman HBV(Roche)RealArt HBV LC PCR(Artus Biotech)Abbot Real-time HBV(Abbott)Versant HBV DNA 1.0(kPCR, Siemens)* *in development
  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
  52. 52. Pathophysiologic Cascade of HBV Replication Liver (Measured by Inflammation Serum HBV DNA) ALT Elevation Worsening Histology Disease Progression • Necroinflammation • Liver Failure • Fibrosis • Liver Cancer • Cirrhosis • Transplant • DeathAdapted 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.
  53. 53. Charge virale et incidence de la cirrhose .4 P <0.001 37.1% Incidence cumulative de cirrhose 1.0 x 106 n=627 1.0-9.9x105 n=344 n=3774 1.0-9.9x104 n=649 .3 300-9.9x103 n=1210 <300 n=944 23.0% .2 .1 10.0% 6.3% 5.2% 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Année de suiviR.E.V.E.A.L. – HBV Study Iloeje UH et al. Gastroenterology 2006; 130: 678-686
  54. 54. Survie chez les patients au stade cirrhose 100 Patients Surviving, % 80 60 Cirrhosis1 55% (n = 130) 40 20 Decompensated cirrhosis2 14% (n = 21) 0 0 1 2 3 4 5 Years1. Weissberg et al. Ann Intern Med. 1984;101:613.2. De Jongh et al. Gastroenterology. 1992;103:1630.
  55. 55. Charge virale et incidence du CHC Chen et al; JAMA 2006
  56. 56. REVEAL-Incidence of HCC 20% % cumulative incidence of HCC 14.9% 15% 12.2% 10% 5% 3.6% 1.3% 1.4% 0% <300 >300 - 103 > 103 - 104 >104 - 106 ≥106 Baseline HBV DNA (copies/mL)Chen JC, et al. JAMA. 2006;295:65-73.
  57. 57. High Baseline Serum HBV DNA Levels are Associated with Increased Risk of HCC Mortality in HBsAg-Positive Patients HBV DNA Negative 100 % 98 % HBV DNA Low Survival distribution function < 105 copies/mL 96 % RR = 1.7 (0.5-5.7) 94 % 92 % 90 % 88 % HBV DNA High ≥ 105 copies/mL 86 % p < 0.001 across viral RR = 11.2 (3.6-35.0) 84 % categories 82 % 80 % 0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 10,0 11,0 12,0 Survival time (Years)http://www.fccc.edu/docs/sci_report/Evans.pdf#search=%22haimen. Accessed 1/23/07.Chen G, et al. J Hepatology 2005; 42 (suppl 2):477A.Chen G, et al. Hepatology 2005; 40 (suppl 1):594A.
  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 Rate Per 100,000HCC Incidence 1.2x104 10,108 1.0x104 8730 8.0x103 5882 6.0x103 4.0x103 1473 2.0x103 0 Baseline HBV DNA, < 104 ≥105 ≥105 ≥105 (copies/mL) Follow-up HBVDNA, --- < 104 104 to <105 ≥105 copies/mL Adjusted RR 1.0 3.6 6.9 9.1 (95% CI) (ref) (1.7-7.6) (3.4-13.8) (5.8-14.1) P Value -- < 0.001 < 0.001 < .001 Chen, et al. JAMA 2006
  59. 59. Impact Clinique de la Variabilité du
  60. 60. VARIABILITE GENETIQUE DU VHB• Multiplication virale » taux derreur de la transcriptase inverse• Pression de sélection » réponse immunitaire cellulaire / humorale » antiviraux -> possibilité de variants déchappement• Conséquences cliniques » diagnostic sérologique » traitements antiviraux
  61. 61. 8 genotypes, numerous sub-genotypes, and recombinant forms B6 D1 World J Gastroenterol 2007; 13: 14-21
  62. 62. Génotypes VHB chez les patients atteints d’hépatite chronique en France 37.4% 100 90 30.2% 80 Number of subjects 70 60 50 40 12.5% 11.3% 30 7.9% 20 10 1.1% 0.4 % 0 A B C D E F GZoulim et al J Viral Hepatitis 2006
  63. 63. Impact du génotype sur la séroconversion Hbe/HBs PEG-IFN a-2b PEG-IFN a-2b HBeAg Loss 1 HBsAg Loss 2 47% 50 21 44% Percentage of patients (%)Percentage of patients (%) 18 40 15% 15 28% 30 25% 12 20 9 8% 6 5% 10 3 0% 0 0 A B C D A B C D n=90 n=23 n=39 n=103 n=90 n=23 n=39 n=103 HBV genotype HBV genotype 1 Janssen, Lancet 2005; 2 Flink, Am J Gastro 2006
  64. 64. LES MUTANTS DU GÉNOME DU VHB déterminant a vaccin/HBIg polymérase antivirauxMt coreRéponse CTL Mt pré-core Réponse anti-e ?
  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-capsideChang 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 dAgHBe :Carman et al Lancet 1989, Okamoto et al J Virol 1990/1994, Tong et al Virology 1990
  67. 67. HBeAg and Precore Mutation G 1896A = stop codon, TAG ATG ATGCore gene 1814 1901 Precore Core region region HBcAg Virion HBeAg Serum
  68. 68. HBeAg and Precore Mutation ATG ATGCore gene 1814 1901 Precore Core region region HBcAg Virion HBeAg Serum
  69. 69. VARIANTS NÉGATIFS POUR L ’AgHBe1762-1764 1896PROMOTEUR PRE-C C * * * TAG mRNA Protéine pré-C/C 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 2500 AgHBe Anti-HBe 1875ALATADN-VHB 1250 625 100 75 50 0sauvage 25Mt pré-C 0
  71. 71. Outcome of Chronic Anti-HBe Positive Hepatitis B 400 Biochemical patterns in 164 untreated patients 300 after 23 months (range 12-36) monthly monitoring 200 With flares and normalization 73 pts 100 ( 44.5% ) 400 0 Asymptomatic flare-up: 300 90% of cases 200A Without flares 59 ptsL 100 123456789112 156 19 223 26 29 32 35 38 41 44 47 50 53 56 59 62 65 10 13 117 20 2 25 28 31 34 37 40 43 46 49 52 55 58 61 64 1 14 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60 63 66 ( 36.0% )T 0 Flare-up yearly frequency: once 57.1% 400 twice 20% 300 < once 22.8% 200 With flares and without normalization 32 pts 12345678911134 178 222 25 28 31 34 338 41 44 47 50 53 56 560 63 5 100 10 2 15 119 1 24 27 30 33 367 40 43 46 49 52 55 589 62 6 1 16 20 23 26 29 32 35 39 42 45 48 51 54 57 61 64 ( 19.5% ) 0 0 12 24 months 123456789 11111111122222222223333333333444444444455555555556666665 et al, J Hepatol 2002 10 2345678901 3456789012 4567890123 5678901234 Brunetto MR 678901234
  72. 72. Augmentation de prévalence des hépatites chroniques avec AgHBe négatif en France 58 % 42% HBeAg(+) N=119 HBeAg(-) N=164Zoulim et al, J Viral Hepatitis 2006
  73. 73. Pre-core mutations Both mutations No pre-core mutation (n = 95; 33.6%) (n = 42; 14.8%) Data unavailable (n = 12; 4.2%) Stop codon mutation (n = 55; 19.4%) Promoter mutation (n = 99; 27.9%)Lamivir cohort, Zoulim et al, J Viral Hepatitis 2006
  74. 74. MUTANTS PRÉ-C ET SÉVÉRITÉ HISTOLOGIQUE LA CONTROVERSE • Italie – Cirrhose plus fréquente • Bonino Gastroenterology 1986, Fattovich Hepatology 1988 • France – Activité idem / cirrhose plus fréquente • Zarski et al, J Hepatol 1993 • Grandjacques et al, J Hepatol 2000 • Zoulim et al, J Viral Hepatitis 2006 • Asie – Mt promoteur : activité histologique et fibrose plus importante – Mt pré-C : activité histologique moins importante • Lindh et al, J Infect Dis 1999 – Rémission histologique • Chan et al, Hepatology 1999 • Afrique – Mt promoteur : plus fréquents dans le CHC • Baptista et al, Hepatology 1999
  75. 75. HBe serotype and liver pathology HBe-positive HBe-negative 70 70Number of subjects 60 60 50 50 40 40 30 30 20 20 10 10 0 0 0-4 5-9 10-14 15-22 ≤ F2 F3 F4 Knodell score Metavir scoreLamivir cohort, Zoulim et al, J Viral Hepatitis 2006
  76. 76. HÉPATITES FULMINANTES ET MUTANTS PRE-C • Lien de causalité : – Épidémies hépatites fulminantes – Transmission souche mutée pré-C (-) – Rôle immunomodulateur de l ’AgHBe • Pas de lien de causalité – Séquençage génome complet – Pas de profil commun de mutationStuyver et al, Hepatology 1999, Sternbeck et al Hepatology 1996, Liang et al, NEJM 1991
  77. 77. DIAGNOSTICS DIFFICILES
  78. 78. 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)• HBeAg negative chronic hepatitis – Fluctuation / exacerbation of ALT – Fluctuations of HBV DNA levels usually > 2000 IU/ mL – Presence of pre-core / core promoter mutations
  79. 79. DIAGNOSTIC DUNE 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
  80. 80. PreS2 PreS1 HBs Ag Pol S 0/3221 GR E Brin(-) 3,2kb Brin(+) 2,4kbSHBs (S) TATAA « a » determinant U5-likeMHBs (preS2+S) DR1 Enh2 Enh1 C DR2LHBs (preS2+preS2+S) S-S sP120T Pré-C X 137 S- S 138 149 107 S-S S-S 147 139 sG145R sD144H/A/E 99 NH2 S-S COOH « a » determinant induces the synthesis of anti-HBs neutralizing antibodies Tiollais P. et al., Nature 1985. Torresi J., J. Clin Virol 2002; Dryden KA. et al., Mol Cell 2006
  81. 81. Variants de lAg 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
  82. 82. VARIANTS DE LAgHBs• Mutations ponctuelles dans le déterminant a de lAgHBs (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 +
  83. 83. Occult HBV Infection (OBI) Presence of HBV DNA in the liver (± serum) ofindividuals testing HBsAg negative by currently available assays Raimondo et al, J Hepatol 2008
  84. 84. How to Detect Occult HBV Infection Currently there is no standardized diagnostic assay for occult HBV infection
  85. 85. Reported Prevalence of Occult HBV Infection in HIV Positive Patients Occult HBV Study Country N° of N° (%) Methods patients Hofer, 1998 Switzerland 57 51 (89%) “nested” PCR (serial evaluation) Torres-Baranda, 2006 Mexico 35 7 (20%) “nested” PCR Filippini, 2006 Italy 86 17 (20%) single step PCR Mphahlele, 2006 South Africa 140 31 (22.%) “nested” PCR Pogany, 2005 Netherlands 93 4 (4%) single step PCR Neau, 2005 France 160 1 (0.6%) Cobas Amplicor HBV Monitor (Roche) Santos, 2003 Brazil 101 16 (16%) single step PCR Wagner, 2004 France 30 11 (37%) “nested” PCR Goncales, 2003 Brazil 159 8 (5%) “nested” PCR Nunez, 2002 Spain 85 0 Cobas Amplicor HBV Monitor (Roche) Piroth, 2000 France 37 13 (35%) single step PCR Raffa, 2007 Italy 101 42 (41%) “nested” PCR (liver) Raimondo et al, J Hepaol 2007, modified
  86. 86. Cause(s) for the failure of HBsAg detection OBI “false” OBI Suppression of Infection byHBV replication and S gene Variants gene expression
  87. 87. HBV replication HBV cccDNA Integrated HBV DNA HBV mutants Epigenetic controlImmune surveillanceViral co-infections Occult HBV infection
  88. 88. Schematic representation of HBV serum marker profile in OBI and “false” OBI OBI „false“ OBI HBV DNA levels < 200 UI/ml Seropositive S gene Seronegative escape mutants Primary occult HBV DNA levels HBsAg lost comparable to after AH overt infectionHBsAg lost Progressive antibodyduring CH disappearence
  89. 89. Occult hepatitis BTorbenson M. & Thomas D.L., Lancet Inf Dis, 2002
  90. 90. Occult HBV infections: unresolved issues Specific Diagnostic To be treatments ? improved High prevalence Tools ?Co-infections ?Therapy? Worsen ROLE HCV in infection ? HCC Not fully understood ?
  91. 91. Antiviraux Persistance viraleResistance aux antivirauxMonitoring des traitements
  92. 92. HBsAg Immuno-active Inactive phase Occult infectionImmunotolerant Reactivation phase phase Low replication phase HBeAg(+) HBeAg(-) / anti-HBe(+) HBV DNA109-1012 IU/mL >2000-<109 IU/mL <2000 IU/mL >2000 IU/mL ALAT Minimal CH Moderate to severe CH Remission Moderate to severe CH Cirrhosis Inactive cirrhosis Cirrhosis Treatment indicated Treatment indicated Adapted from Fattovich G. Sem Liver Dis. 2003
  93. 93. Antivirals approved for hepatitis BDrug Type Approved Phase 3 Phase 2Nucleoside analogs • Lamivudine* • Emtricitabine* • Entecavir • Clevudine** • TelbivudineNucleotide analogs • Adefovir dipivoxilCytokines • Tenofovir* alfa • Interferon • IL7 • Pegylated Interferon •IFN Lambda alfa-2a •Vaccine therapy *Currently approved for HIV **development on hold
  94. 94. Endpoints of therapyPersistence of high viral load is associated with a significant risk of progression of the liver disease and of HCC Aim of antiviral therapy: HBV DNA < 10-15 IU/mL by real-time PCR assays Viral suppression No replication =Histological and clinical No resistance improvement Chen CJ, et al. JAMA 2006. Iloeje UH, et al. Gastroenterology 2006. Chen C, et al. Am J Gastroenterol 2006. Zoulim & Perrillo J Hepatol 2008. Zoulim & Locarnini Gastroenterology 2009
  95. 95. Treatment failurePrimary non response Secondary treatment failurePartial response Antiviral drug resistanceHost factors Drug factorsDrug metabolism Barrier to resistancePatient’s compliance Viral factorsDrug factors Resistant mutantsAntiviral potency Zoulim et al Hepatol 2008; EASL CPG J Hepatol 2009; Lancet Infect Dis 2012
  96. 96. 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
  97. 97. Laboratory Definition of HBV Resistance to AntiviralsLaboratory 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
  98. 98. The main differences between HIV, HBV and HCV HIV1 HBV1,2 HCV1,3 Host cell Host cell Host cell HCV RNA cccDNA Host DNAHost DNA H Host DNA H H Proviral DNA Integrated DNA Nucleus Nucleus Nucleus Life-long suppression Long-term suppression Definitive viral clearance of viral replication of viral replication and SVR Adapted from 1. Sorriano V, et al. J Antimicrob Chemother 2008;62:1-4. 2. Locarnini S and Zoulim F. Antiviral Therapy 2010;15 (suppl 3):3-14. 3. Sarrazin C and Zeuzem S. Gastroenterology 2010;138:447-462.
  99. 99. Kinetics of emergence of HBV drug resistant mutantsSi 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.
  100. 100. Lamivudine Resistance Accelerates Progression of Liver Disease Placebo (N=215) YMDDm (N=209) (49%) Wild Type (N=221) Placebo 21%% With disease progression 25 YMDDm 13% 19 13 WT 5% 6 0 0 9 18 27 36 Time after randomization (Months) Liaw YF et al. N Engl J Med. 2004;351:1521-1531
  101. 101. 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 developmentLai 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 Hepatitis2006;13:278-288 ; Fung et al J Hepatol 2005;43:937-943; Liaw et al NEJM 2004;351:1521-1531.
  102. 102. ALT flares in patients with lamivudine resistance over time Lok et al Gastroenterology 2003; 125 : 1714-1722
  103. 103. Incidence of drug resistance over time Resistance at year of therapy expressed as percentage of patients Drug and patient population 1 2 3 4 5 6Lamivudine 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 -Entecavir (naïve) 0.2 0.5 1.2 1.2 1.2 1.2Entecavir (LAM resistant) 6 15 36 46 51 57 CL Lai Clin Infect Dis 2003; CL Lai NEJM 2007; Hadzyiannis Gastroenterology 2006; Marcellin NEJM 2008; CL Lai & Chang NEJM 2006; Zoulim & Locarnini Gastroenterology 2009
  104. 104. Rates of resistance with lamivudine (LVD), adefovir (ADV), telbivudine (LdT), entecavir (ETV) and tenofovir (TDF) among NA-naïve patients 80 80 71 High barrier to resistance 70 Proportion of patients (%) 60 55 50 46 40 30 29 25 Option to add 23 emtricitabine at 20 18 week 72* 11 10 5 1.2 3 0 0 0 0 0 0.2 0.5 0 1 2 3 4 5 1 2 3 4 5 1 2 1 2 3 4 5 6 1 2 3 4 LVD ADV LdT ETV TDF *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 pendingAdapted from Gish, Jia, Locarnini & Zoulim, Lancet Infect Dis 2012
  105. 105. Zoulim & Locarnini, Gastroenterology, 2009
  106. 106. Determinants of viral persistence & resistanceZoulim & Locarnini, Gastroenterology, 2009
  107. 107. Multiple factors are associated with the barrier of resistance & drug efficacy • Antiviral potency • Number of mutations needed to overcome drug suppression • Level of exposure to drug • Chemical structure Antiviral Virus Drug • Adherence Patient • Replication fitness and space • Immune status • Persistence of archived mutations as cccDNA • Prior antiviral exposure • Metabolism • Pre-existing mutations • Body massLocarnini 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
  108. 108. L(-)-SddU mitochondria deaminase L(-)-SddC, 3TCMt DNA L(-)-SddC-TP L(-)-SddC Lamivudine kinase L(-)-SddC-TP HBV DNA nucleus L(-)-SddC-TP cytoplasm Nuclear DNA Bridges; Progress in Liver Disease 1995
  109. 109. The HBV life cycle Nucleos(t)ide analogsZoulim & Locarnini, Gastroenterology 2009
  110. 110. Formation of the recalcitrant cccDNA: a difficult target for antiviral therapyuncoating CCC DNA supercoiled DNA minichromosome Topoisomerase (TDP2) ? removal of protein primer Acetyl transferase ? Histones removal of RNA primer completion of viral (+) strand DNA ligation of DNA strands extremitiesAntivirals ? Tuttleman et al Cell 1986 viral polymerase? Le Guerhier et al AAC 2000 DNA repair protein? Delmas et al AAC 2002 other cellular enzymes? Kock et al Hepatology 2003 Cortes Ledesma et al Nature 2009 Boeck et al Plos Pathogen 2010
  111. 111. Can we prevent cccDNA formation ?Nucleoside analogs in monotherapy or
  112. 112. Kinetics of Viral Loss During Antiviral Therapy with L- FMAU (clevudine) in the woodchuck model Zhu et al, J Virol 2001
  113. 113. ADV Associated Serum HBsAg Reductions are Similar in Magnitude to cccDNA Reductions Serum Total HBV Intracellular cccDNA Serum DNA DNA 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
  114. 114. Immunohistochemical Staining of Patient Biopsies at Baseline and After 48 Weeks ADV Therapy Baseline Week 48• 0.8 log10 (84%) decline in cccDNA, not paralleled by a similar decline in the number of HBcAg+ cells• Suggests cccDNA depleted primarily by non-cytopathic mechanisms or that cell turn-over occurred but was associated with infection of new cells during therapy
  115. 115. Persistence of cccDNA after HBs seroconversion Maynard et al, J Hepatol 2005
  116. 116. Clearance of viral infection versus selection of escape mutantsThe 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
  117. 117. Kinetics of spread and emergence of drug resistant virus during antiviral therapy antiviral wt mt Ò Ò Ò Ò Free liver space Mutant fitness Ò ni = non-infected wt = wild type Ò mt = mutant type Ò ni I II III IV INHIBITION OF WILD TYPE VIRUS REPLICATIONS DELAYED EMERGENCE OF DRUG RESISTANT VIRUSZhou T, et al. Antimicrobial Agents and Chemotherapy 1999; 43: 1947-1954.
  118. 118. Kinetics of HBV drug resistance emergence Drug-susceptible virus Treatment begins Naturally—occurring viral variants Drug-resistant variant Secondary resistance mutations / compensatory resistance mutations HBV replication Primary resistance mutations TimeSi Ahmed et al. Hepatology. 2000; Yuen et al Hepatology 2001; Locarnini et al Antiviral Therapy 2004; Villet et al Gastroenterology 2006 JHepatol 2007 & 2008; Pallier et al J Virol 2007; Yim et al Hepatology 2006.
  119. 119. Partial response to adefovir dipivoxil is not due to the selection of DR mutants• The top 25% patients (quartile 1): > 4.91 log10 reduction in serum HBV DNA at week 48.• In Q2: 3.52 to 4.90 log10 reduction of viral load.• In Q3: 2.22 to 3.51 log10 reduction in viral load.• The bottom 25% of patients (Q4):< 2.22 log10 reduction in HBV DNA levels at week 48.• Phenotypic analysis of viral strains: Q4 as sensitive to ADV as Q1 strains• Documented Drug Compliance (% of days without taking ADV) Virological Response Virological Response Virological Response Virological Response Q1 (best response) Q2 Q3 Q4 (worse response)Median 99% (n=38) 99% (n=38) 99% (n=38) 97% a (n=38)range 86-100% 41*-100% 91-100% 70-100%• Wilcoxon rank sum test, P=0.01 Durantel et al, Antiviral Therapy, 2008
  120. 120. Amino acid substitutions result in conformation changes of the polymerase catalytic site Wild-type M204/L180 LVDr M204V/L180M L180 L180M M204 M204V LVD-TP LVD-TP LVDr M204V/L180M M204V reduces pocket size L180M Steric clash between lamivudine and V204 M204V Minimal steric clash between entecavir and ETV-TP V204 Langley DR, et al. J Virol. 2007;81:3992-4001.
  121. 121. 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
  122. 122. Cross-resistance data for the main mutants and the commercially available drugs Pathway Amino acid Lamivudine Telbivudine Entecavir Adefovir Tenofovir substitutions in the rt domain Wild type S S S S S L-nucleoside M204I R R I S S L-nucleoside L180M+M204V R R I S S Alkyl N236T S S S R I phosphonate Shared A181T/V I/R I/R S R I D-Cyclopentane L180M+M204V/I R R R S S (ETV) ±I169T±V173L ±M250V D-Cyclopentane L180M+M204V/I R R R S S (ETV) ±T184G±S202I/G MDR V173L+L180M R R S R S +A181V+N36TZoulim & Locarnini Gastroenterology 2009
  123. 123. Archiving of viral variants Viral quasispecies Liver Majority population Minority variants Resistant variants cccDNA variants • cccDNA in the liver: – Is propagated during the normal replication cycle of HBV – Can serve as a template for the production of new virus Blood circulationZhou et al, AAC 1999; Zoulim F. Antivir Res. 2004. Zoulim F & Perillo R. J Hepatol. 2008
  124. 124. Archiving of viral variants Viral quasispecies Liver Majority population Minority variants Resistant variants cccDNA variants • cccDNA in the liver: – Is propagated during the normal replication cycle of HBV – Can serve as a template for the production of new virus • It is believed that viral variants with antiviral resistance may be archived in this way Blood circulationZhou et al, AAC 1999; Zoulim F. Antivir Res. 2004. Zoulim F & Perillo R. J Hepatol. 2008
  125. 125. Archiving of viral variants Viral quasispecies Liver Majority population Minority variants Resistant variants cccDNA variants • cccDNA in the liver: – Is propagated during the normal replication cycle of HBV – Can serve as a template for the production of new virus • It is believed that viral variants with antiviral resistance may be archived in this way Blood circulationZhou et al, AAC 1999; Zoulim F. Antivir Res. 2004. Zoulim F & Perillo R. J Hepatol. 2008
  126. 126. Phenotyping of HBV clinical isolates Cl ne A in Southern blot o ra Cl e D Cl e C eE Cl A Cl b St analysis e on on on on Whole genome La HBV clones PCR Transfection cloning Patient HepG2 serum Huh7 lamivudine adefovir Cell culture plate RC - Wild-type virus SS - Patient’s virus Fold resistance = IC50 mutant IC50 reference strain Increasing antiviral concentration1. Durantel D, et al., Hepatology, 2004;40:855-64. 2. Yang H, et al., Antiv Ther, 2005;10:625-33.
  127. 127. Maximising the barrier to resistance Wild-type virus LAM rtM204V/I ± rtL180M LAM-resistant virus ADV-resistant virus ETV-resistant virus ADV rtN236T +/or rtA181V rtT184 or rtS202 or rtM250 ETV rtM204V/I +/- rtL180M LAM + TDF – what do we see? LAM rtT184 or rtS202 or rtM250then ETV rtM204V/I +/- rtL180M TDF: what can we expect? TDF
  128. 128. Can we detect low frequency mutants prior to or during therapy ?Use of pyrosequencing to detect lowfrequency mutants•May detect mutants representing aslow as 0.1% of the viral population•The clinical significance fortreatment choice or adaptation needsto be determined by prospectivestudies
  129. 129. Important factors involved in selection of MDR mutants• Use of inadequate sequential monotherapies and inadequate treatment adaptation• Incomplete viral suppression – > Persistent replication in the presence of antiviral pressure• Use of drugs sharing cross-resistance characteristics – One mutation may confer resistance to several drugs – > Persistent replication• Accumulation of mutations• Wide replication space (liver transplantation)
  130. 130. The problem of sequential therapy with nucleoside analogues ? Drug A Drug B Multiple drug resistant mutants+ one mutation + one mutation with complex pattern ofRisk of selection of MDR mutants by sequential therapy mutations - drugs sharing cross-resistance characteristics - incomplete viral suppression - liver transplantation Yim et al, Hepatology al. J Hepatol. 2008;48:S2-19. Zoulim F, et 2006; Villet et al Gastroenterology 2006 & 2009
  131. 131. Drugs sharing cross-resistance characteristics: Switching strategy  emergence of MDR mutant adefovir IFNGenotype H entecavir lamivudine lamivudine 109 108 HBV DNA (copies/ml) 107 106 L180M+M204V 105 104 L180M+S202G+M204V 103 0 20 40 60 80 100 120 Treatment (months) Villet et al, J Hepatol 2007
  132. 132. A single a.a. substitution at position rt181 may be responsible for multidrug resistanceLVD ADV LVD+ADV Patient #1 Patient #3 Patient #4 Patient #8 (67 months) (37 months) (31 months) (47 months) Patient #7 Patient #5 Patient #6 Patient #9 (30 months) (44 months) (36 months) (19 months)LVD+TDF Patient #2 LVD+ADV+TDF Patient #10 (23 months) (7 months) wt N236T + N238T A181V M204V A181T M204I A181V + N236T L80V A181T + N236T L80V + M204I N236T Villet S, et al. J Hepatol. 2008;48:747-55.
  133. 133. Impact of rtA181 and rtN236 mutations on antiviral drug efficacy and cross-resistance Villet et al, J Hepatol 2008
  134. 134. Impact on virus infectivity and fitnessImpact on virion release (intracellular retention) and virologic monitoring of breakthroughImpact on vaccine prophylaxis efficacyWarner et al Hepatology 2009Kamili et al Hepatology 2009Villet et al Gastroenterology 2009
  135. 135. Potential risk of transmission of HBV DR mutantsClements et al, Bull WHO 2009
  136. 136. Management algorithm Antiviral treatment Viral load asssessment Treatment failure Check compliance Viral genome sequence analysis Wild type virus HBV drug resistant mutantCheck compliance Primary non response Add-on therapy based on cross-resistance data Switch to more potent drug Zoulim and Perrillo, J Hepatol, 2008; EASL CPG J Hepatol 2009
  137. 137. Management algorithm Antiviral treatment Viral load asssessment Treatment responseCheck for HBe/HBs seroconversion on a regular basis (6 monthly) Zoulim and Perrillo, J Hepatol, 2008; EASL CPG J Hepatol 2009
  138. 138. 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 resistanceLe Guerhier et al Antimicrob Agents Chemoter 2000;44:111-122; Delmas et al Antimicrob AgentsChemother 2002; 46:425-433; Kock et al Hepatology2003; 38:1410-1418; Richman Hepatology2000;32:866-867
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