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Fabien ZOULIM.

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Presentation on theme: "Fabien ZOULIM."— Presentation transcript:

1 Fabien ZOULIM

2 Fabien Zoulim INSERM U871 & Liver Department Lyon, France
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
Viral replication Immune response CD8+ HBV Immune tolerance CD8+ HBV Clairance phase Chronic hepatitis CD8+ HBV Seroconversion Remission Guidotti, Science 1999; Guo, J. Virol 2000; Kakimi J Exp Med 2000; Zhu J Virol 2001

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

6 Natural history of hepatitis B
Acute infection Lee, N Engl J Med 1997 Lok, Hepatology 2001 Ganem, NEJM 2004 Recovery Chronic infection Chronic hepatitis Wild type virus (HBeAg+) Pre-core mutant (HBeAg-) Inactive carrier Immune tolerance Reactivation Cirrhosis 30-50 years Hepatocellular carcinoma

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

8 Monitoring of Viral Load

9 HBsAg 9- 8- 7- 6- 5- 4- 3- 2- 1- 1000 100 10 1 0,1 0,01 0,001 HBeAg +
UI/ml pg/ml HBeAg + anti-HBe Ab + ALT 1000 9- 8- 7- 6- 5- 4- 3- 2- 1- HBV- DNA 100 10 1 hybridsiation 0,1 0,01 PCR 0,001 Tolerance chronic hepatitis inactive carrier pre-core mt occult HBV

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

11 Serum titre Histology (inflammation) < 104 31/37 had HAI < 3
Serum Viral Load in Chronic Hepatitis Titre vs histology in HBeAg-negative patients Serum titre Histology (inflammation) < 104 31/37 had HAI < 3 > 2  105 15/22 had HAI > 4 > 107 5/6 had HAI > 7 Lindh et al J Viral Hepatitis 2000;7:

12 Pre-core mutants

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


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

16 Diagnosis of inactive carrier versus HBeAg negative chronic hepatitis
Persistently normal ALT levels Persistently low levels of serum HBV DNA Threshold : 103 or 104 copies / mL ? Wild type genome; sometimes pre-core mutations The key : careful monitoring ! HBeAg negative chronic hepatitis Fluctuation / exacerbation of ALT Fluctuations of HBV DNA levels usually below 106 copies / mL Presence of pre-core / core promoter mutations

17 HBV genotypes

18 HBV genotypes Influence on the type of pre-core or BCP mutation
Impact on the outcome of infection and severity of liver disease (HCC) Impact on IFN response No 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 IFN response (HBeAg loss)
Viral genotypes and IFN response (HBeAg loss) 47% 50 44% % 40 28% 30 25% 20 10 A n=90 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 < 104 copies/mL: decreased liver damage - HBV DNA < 103 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 NKT CD4 CD8 B Infected liver Blood circulation Viral load
Infected hepatocytes Infected liver Blood circulation Viral load NKT CD8 CD4 B cccDNA

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

24 (Log10 copies/mL Log10copies/cell)
Reductions in Serum HBV DNA, Total Intrahepatic HBV DNA and cccDNA During ADV Therapy Werle et al, Gastroenterology 2004 (Log10 copies/mL Log10copies/cell) Median 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


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) HBeAg Positive, n=921 HBeAg Negative, n=446 Telbivudine Lamivudine Di Bisceglie et al., Abstract #112, AASLD 2006

28 Clinical Definition of HBV Resistance to Antivirals
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
Spacer Pol/RT RNaseH Terminal protein 1 183 349 692 845 a.a. (rt1) (rt 344) GVGLSPFLLA YMDD I(G) II(F) A B C D E V173L L180M M204I/V LAM / FTC ADV A181V N236T I233V ETV I169T T184G S202G/I M250V LdT M204I TDF A194T ? * All ETV resistance requires background YMDD 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 ; Schildgen et al NEJM 2006; Villet et al J Hepatol 2007

30 Line Probe Assay Versus Sequencing for the Detection of HBV Drug Resistance
20 40 60 80 100 HBV DNA (10E+6 genome eq/ml) ALT(U/L) 140 180 1 595 200 300 400 Codon 528 LiPA Seq Codon 552 Codon 555 L M V 39 290 L/M M/V Day T A G C Sequencing of PCR products Can detect any new mutation Line probe assay Very sensitive (minor species and low viremia) Nafa et al Hepatology 2000; Lok et al. J Clin Microbiol. 2002

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


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 Requirement for the most sensitive / quantitative assays
Management of chronic HBV infection Low levels of replication : inactive carriers / occult infection Early detection / prediction of reactivation Treatment eligibility Monitoring 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


37 Incidence of Resistance in Nucleoside Naive Patients
resistance mutations % of patients with 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
resistance mutations % of patients with Lampertico et al AASLD 2006; Colonno et al AASLD 2006

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

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

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

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