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Fabien ZOULIM

Fabien ZOULIM. How to use virological tools for the optimal management of chronic hepatitis B. Fabien Zoulim INSERM U871 & Liver Department Lyon, France. Pathobiology and Natural History of the Disease. HBV. HBV. HBV. CD8+. CD8+. CD8+. Immunopathology of HBV Infection. Viral

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Fabien ZOULIM

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  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. HBV HBV HBV CD8+ CD8+ CD8+ Immunopathology of HBV Infection Viral replication Immune response Immune tolerance Clairance phase Chronic hepatitis 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 UI/ml pg/ml HBeAg + anti-HBe Ab + ALT 1000 9- 8- 7- 6- 5- 4- 3- 2- 1- HBV- DNA 100 10 hybridsiation 1 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) HBSAg- (7) HBeAg- (18) HBSAg- (7) HBeAg+ (63) HBeAg- (18) HBeAg+ (63) Inact. Carriers (10) Inact. Carriers (10) Werle et al, Gastroenterology 2004

  11. Serum Viral Load in Chronic Hepatitis Titre vs histology in HBeAg-negative patients Lindh et al J Viral Hepatitis 2000;7:258-67.

  12. Pre-core mutants

  13. HBeAg and Precore Mutation ATG ATG 1814 1896 1901 1762-1764 Basic Core Promoter Precore Core region region HBcAg Virion HBeAg Serum HBeAg Serum

  14. 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% ) 0 12 24 months Brunetto MR et al, J Hepatol 2002

  15. Diagnosis of inactive carrier versus HBeAg negative chronic hepatitis • Inactive Carrier • 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

  16. HBV genotypes

  17. 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

  18. Viral genotypes and IFN response (HBeAg loss) 47% 50 44% % 40 28% 30 25% 20 10 0 B n=23 C n=39 D n=103 A n=90 Jansen et al, Lancet, 2005

  19. Monitoring of Antiviral Therapy

  20. 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

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

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

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

  24. 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;44:111-122; Delmas et al Antimicrob Agents Chemother 2002; 46:425-433; Kock et al Hepatology2003; 38:1410-1418; Richman Hepatology 2000;32:866-867

  25. Telbivudine Lamivudine 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 Di Bisceglie et al., Abstract #112, AASLD 2006

  26. 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

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

  28. 180 100 80 140 ALT(U/L) (10E+6 genome eq/ml) 60 HBV DNA 100 40 60 20 20 0 0 1 100 200 300 400 595 Day 1 39 290 400 595 Codon 528 LiPA L L L/M M M Seq L L L/M M M M C T C T G G C T Codon 552 LiPA M M M/V V V Seq M M M V V Codon 555 LiPA V V V V V Seq V V V V V T A T A T G G A T Line Probe Assay Versus Sequencing for the Detection of HBV Drug Resistance 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

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

  30. 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

  31. Approaches to Management Depend on Cross-Resistance Data Resistance mutations

  32. 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

  33. 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

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

  35. Management of HBV drug resistance ~20% resistance/2 years 16% resistance/3 years Adefovir switch Lamivudine Adefovir add-on 0% resistance at 3 years 70% resistance at 5 years 38% resistance at 3 years Entecavir Switch 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

  36. 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

  37. 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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