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Fabien ZOULIM. How to use virological tools for the optimal management of chronic hepatitis B Fabien Zoulim INSERM U871 & Liver Department Lyon, France.

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Presentation on theme: "Fabien ZOULIM. How to use virological tools for the optimal management of chronic hepatitis B Fabien Zoulim INSERM U871 & Liver Department Lyon, France."— Presentation transcript:

1 Fabien ZOULIM

2 How to use virological tools for the optimal management of chronic hepatitis B Fabien Zoulim INSERM U871 & Liver Department Lyon, France

3 Pathobiology and Natural History of the Disease

4 Immunopathology of HBV Infection Immune tolerance Clairance phase Chronic hepatitis SeroconversionRemission CD8+ HBV CD8+ HBV CD8+ HBV Immuneresponse Viralreplication Guidotti, Science 1999; Guo, J. Virol 2000; Kakimi J Exp Med 2000; Zhu J Virol 2001

5 Phases of the disease Immunotolerance phaseImmunotolerance phase - High viral load and normal ALT levels Immunoactive phase / chronic hepatitisImmunoactive phase / chronic hepatitis - Viral replication and elevation of ALT levels Inactive carrier stateInactive carrier state - Low viral load and normal ALT levels ReactivationReactivation - Wild type virus or pre-core mutant Resolved InfectionResolved Infection - Clearance of HBsAg Fattovich, J Hepatol 2003

6 Natural history of hepatitis B Acute infection Chronic infection Immune tolerance Chronic hepatitis Inactive carrier Recovery Wild type virus (HBeAg+) Pre-core mutant (HBeAg-) Cirrhosis Hepatocellular carcinoma Reactivation years Lee, N Engl J Med 1997 Lok, Hepatology 2001 Ganem, NEJM 2004

7 Virological monitoring Viral load Viral genome heterogeneity Liver damage Reactivation Drug resistance Viral persistence Treatment response Drug resistance

8 Monitoring of Viral Load

9 0,001 0,01 0, ALT HBV- DNA HBeAg + anti-HBe Ab + UI/mlpg/ml HBsAg Tolerance chronic hepatitis inactive carrier pre-core mt occult HBV hybridsiation PCR

10 cccDNA (copies/cell) Total HBV DNA (copies/cell) Evolution of Intrahepatic cccDNA During the Natural History HBeAg+ (63) Inact. Carriers (10) HBSAg- (7) HBeAg- (18) HBeAg+ (63) Inact. Carriers (10) HBSAg- (7) HBeAg- (18) Median Werle et al, Gastroenterology 2004

11 Serum Viral Load in Chronic Hepatitis Titre vs histology in HBeAg-negative patients Serum titre Histology(inflammation) < /37 had HAI < 3 > 2  /22 had HAI > 4 > /6 had HAI > 7 Lindh et al J Viral Hepatitis 2000;7:

12 Pre-core mutants

13 HBeAg and Precore Mutation Precore Coreregion HBcAg HBeAg ATG Virion Serum Basic Core Promoter HBeAg Serum


15 Outcome of Chronic HBeAg Negative Hepatitis B Biochemical patterns in 164 untreated patients after 23 months (range 12-36) monthly monitoring months With flares and normalization Without flares With flares but without normalization 73 pts ( 44.5% ) 59 pts ( 36.0% ) 32 pts ( 19.5% ) Asymptomaticflare-up: 90% of cases ALT Flare-up yearly frequency: once 57.1% twice 20% < once 22.8% Brunetto MR et al, J Hepatol 2002

16 Diagnosis of inactive carrier versus HBeAg negative chronic hepatitis Inactive CarrierInactive Carrier –Persistently normal ALT levels –Persistently low levels of serum HBV DNA Threshold : 10 3 or 10 4 copies / mL ?Threshold : 10 3 or 10 4 copies / mL ? –Wild type genome; sometimes pre-core mutations –The key : careful monitoring ! HBeAg negative chronic hepatitisHBeAg negative chronic hepatitis –Fluctuation / exacerbation of ALT –Fluctuations of HBV DNA levels usually below 10 6 copies / mL –Presence of pre-core / core promoter mutations

17 HBV genotypes

18 Influence on the type of pre-core or BCP mutationInfluence on the type of pre-core or BCP mutation Impact on the outcome of infection and severity of liver disease (HCC)Impact on the outcome of infection and severity of liver disease (HCC) Impact on IFN responseImpact on IFN response No clear impact on response to nucleoside analogsNo clear impact on response to nucleoside analogs Zhang J Med Virol 1996, Orito Hepatology 2001, Mayerat J Viral Hepat 1999; Wai Hepatology 2002, Jansen Lancet 2005 Pichoud et al, Hepatology 1999; Grandjacques J Hepatol 2000; Si Ahmed et al, Hepatology 2000; Yang et al, Gastroenterology 2004

19 A n=90 % 28% 47% 44% 25% Viral genotypes and IFN response (HBeAg loss) B n=23 C n=39 D n=103 Jansen et al, Lancet, 2005

20 Monitoring of Antiviral Therapy

21 Goals and types of response Virological response -HBV DNA < 10 4 copies/mL: decreased liver damage - HBV DNA < 10 3 copies/mL: decreased risk of resistance Biochemical response - normalization of ALT levels Histological response - improvement in HAI or Metavir score Combined response / Complete response Timing during therapy Initial response / Maintained response End of treatment response / Sustained reponse Hoofnagle, J Hepatol 2003

22 Blood circulation Viral load Infected hepatocytes Infected liver CD8 NKT CD4 B cccDNA

23 Blood circulation Viral load Infected hepatocytes Infected liver Antivirals CD8 NKT CD4 B Werle et al, Gastroenterology 2004 cccDNA

24 Median (Log 10 copies/mL Log 10 copies/cell) Reductions in Serum HBV DNA, Total Intrahepatic HBV DNA and cccDNA During ADV Therapy  48 weeks of ADV resulted in significant reductions in : serum HBV DNA > total intrahepatic HBV DNA > cccDNA -> 14 years of therapy to clear completely viral cccDNA Werle et al, Gastroenterology 2004


26 Virologic Consequences of Persistent Viremia 1) Infection of new hepatocytes  slower kinetics of clearance of infected cells and cccDNA 2) Increases the risk of occurrence and selection of HBV mutations responsible for drug resistance 3) On-treatment prediction of HBV drug resistance Le Guerhier et al Antimicrob Agents Chemoter 2000;44: ; Delmas et al Antimicrob Agents Chemother 2002; 46: ; Kock et al Hepatology2003; 38: ; Richman Hepatology 2000;32:

27 Viral Load at Week 24 is a Predictor of Resistance at Week 104 of Therapy (Telbivudine vs. Lamivudine trial) TelbivudineLamivudine HBeAg Positive, n=921 HBeAg Negative, n=446 Di Bisceglie et al., Abstract #112, AASLD 2006

28 Clinical Definition of HBV Resistance to Antivirals Clinical Genotypic Resistance: Detection of mutations in the HBV genome, known to confer resistance, which develop during anti-viral therapy Virologic Breakthrough: Rebound in serum HBV DNA levels following the development of genotypic resistance Clinical Breakthrough: Virologic breakthrough with increased ALT levels or worsening histology 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

29 HBV drug resistance mutations Allen et al. Hepatology 1998;27:1670–7; Gish et al. J Hepatol 2005;43:60–6; Qi et al. J Hepatol 2004;40(Suppl 1):20–1; Tenney et al. AAC 2004;48:3498–507; Lai et al. Gastroenterology 2005;129:528–36; Sheldon et al. Antivir Ther 2005;10:727–34; Delaney et al. AAC 2006 ; Schildgen et al NEJM 2006; Villet et al J Hepatol 2007 RNaseH 845 a.a. Terminal protein Spacer Pol/RT ABCED YMDD V173L L180MM204I/V GVGLSPFLLA I(G)II(F) (rt1)(rt 344) LAM / FTC ETV I169T T184G S202G/I M250V ADV A181VN236T I233V LdT M204I * All ETV resistance requires background YMDD mutations TDF A194T ?

30 Nafa et al Hepatology 2000; Lok et al. J Clin Microbiol Line Probe Assay Versus Sequencing for the Detection of HBV Drug Resistance Can detect any new mutation Very sensitive (minor species and low viremia) Line probe assay Sequencing of PCR products

31 log copies/ml Graph adapted from J. Hepatol., 39, S3-S25, 2003 HBV DNA Quantification Dynamic Range of HBV DNA Detection


33 Strategies for Monitoring Treatment Response and Detecting HBV Drug Resistance Viraemia levels and ALT every 3 months - Antiviral response and potency - Persisting viraemia - Early detection of drug resistance Serologic assays - HBeAg/Anti-HBeAb: every 6 months in HBeAg+ patients - HBsAg/Anti-HBsAb: when HBV DNA < limit of detection Genotypic assays - In multidrug experienced patients - At the time of virologic breakthrough - When viral load is not suppressed for long period of time

34 Approaches to Management Depend on Cross-Resistance Data Resistance mutations

35 Conclusions Management of chronic HBV infectionManagement of chronic HBV infection –Low levels of replication : inactive carriers / occult infection –Early detection / prediction of reactivation –Treatment eligibility Monitoring of antiviral therapyMonitoring of antiviral therapy –Early virological response –Viral breakthrough / drug resistance –Genotypic assays –Individualized treatment adaptation for 2nd or 3rd line treatment to avoid multidrug resistance Requirement for the most sensitive / quantitative assays


37 Incidence of Resistance in Nucleoside Naive Patients % of patients with resistance mutations Lai et al CID 2003; Hadzyiannis et al Gastroenterology 2006; Colonno et al AASLD 2006; Di Bisceglie et al AASLD 2006

38 Incidence of Resistance in Lamivudine Refractory Patients % of patients with resistance mutations Lampertico et al AASLD 2006; Colonno et al AASLD 2006

39 Management of HBV drug resistance Lamivudine Adefovir switch Adefovir add-on Entecavir Switch Adefovir Entecavir ~20% resistance/2 years 16% resistance/3 years 0% resistance at 3 years 38% resistance at 3 years Lamivudine add-on Entecavir add-on Telbivudine add-on ? Adefovir add-on Tenofovir add-on* ? 70% resistance at 5 years 30% resistance at 5 years resistance at 5 years ? Telbivudine Adefovir add-on Tenofovir add-on* ? resistance at 5 years ? * Not yet approved for HBV therapy

40 Mechanisms of HBV Drug Resistance Viral persistence cccDNA Long half-life Infected cells Long half-life Defective immune response VirusHepatocytes Impairment of innate response Host Selective pressure Antivirals or others Viral polymerase spontaneous error rate Virus Selection of escape mutants Treatment failure Replication fitness Replication space Viral quasi-species Immune response Drug PK Zoulim Antivir Res 2004;64:1–15

41 The Hepadnavirus Genome and its Variability « a » determinant vaccine/HBIg RT domain antivirals pre-core mt anti-e response ? core mt CTL response 8 genotypes A to H

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