Professor Stephen Locarnini Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria 3051, AUS

1. Professor Stephen Locarnini Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria 3051, AUSTRALIA www.vidrl.org.au/publications/hep_updates.htm

2. Clinical Trial End-Points Traditional Endpoints HBeAg seroconversion Improvement in liver Histology HBV DNA reduction HBsAg seroconversion Emerging Endpoints Intrahepatic HBV cccDNA level at end-of-treatment Reduction in HBeAg and/or HBsAg Effect on Adaptive Immune Responses Effect on Innate Immune Responses

3. Treatment Endpoints in Chronic Hepatitis B

4. Options for Measuring Clinical Response Biochemical Liver enzymes Normalization of alanine aminotransferase (ALT) Normalization of aspartate aminotransferase (AST) Synthetic tests Normalization of albumin, bilirubin and INR Serological Hepatitis B e antigen (HBeAg) and s antigen (HBsAg) loss Gain of anti-HBe (HBeAg seroconversion) Gain of anti-HBs (HBsAg seroconversion) Virologic HBV DNA suppression Histological Decrease in inflammation and fibrosis Before standardizing clinical measurements of response, we must understand the measurements themselves. Virological measurement is one of the most controversial, and over the course of time has been defined in various ways. But it has evolved to the point that it should be defined as reduction in serum HBV DNA levels and loss of HBeAg in those who were initially positive, and the loss of HBsAg. Serological measurement of response has also been variously defined, but for the purposes of discussing seroconversion, it should be defined as the loss of HBeAg and gain of anti-HBe when discussing HBeAg seroconversion and the loss of HBsAg and the gain of anti-HBs when discussing HBsAg seroconversion. Less controversial are the histological and biochemical measurements defined as a decrease in inflammation, necrosis, fibrosis and the normalization of ALT levels, albumin, bilirubin and prothrombin time.Before standardizing clinical measurements of response, we must understand the measurements themselves. Virological measurement is one of the most controversial, and over the course of time has been defined in various ways. But it has evolved to the point that it should be defined as reduction in serum HBV DNA levels and loss of HBeAg in those who were initially positive, and the loss of HBsAg. Serological measurement of response has also been variously defined, but for the purposes of discussing seroconversion, it should be defined as the loss of HBeAg and gain of anti-HBe when discussing HBeAg seroconversion and the loss of HBsAg and the gain of anti-HBs when discussing HBsAg seroconversion. Less controversial are the histological and biochemical measurements defined as a decrease in inflammation, necrosis, fibrosis and the normalization of ALT levels, albumin, bilirubin and prothrombin time.

5. HBeAg Seroconversion At 1 Year

6. HBeAg Loss: at 1 Year

7. DNA Response @ 1 year in HBeAg+ Studies

8. DNA Response in HBeAg-Negative Studies: @ 1yr

9. Significant Progression ofFibrosis Can Occur in Less Than a Year*

10. HBsAg Seroconversion in HBeAg+ CHB HBsAg Loss

11. HBeAg+ CHB Serologic Response by Genotype

12. HBV DNA Tools HBV DNA: Serum Compartment HBV DNA: Liver Compartment HBV DNA: Genotype Testing (A-H) HBV DNA: HBV Mutations Testing

13. Measurement of HBV DNA load Indicator of disease activity Quantification of antiviral efficacy Dynamics on therapy may be predictor of outcome Early detection of resistant virus Occult HBV

15. FTCB-201: HBV DNA FTC + ADV HBeAg+ CHB

16. FTCB-201: Comparison Between Fast vsSlow Responders During Antiviral Treatment FTC + ADV HBeAg+ CHB

17. Hepatitis B Virus Genotype and DNA Level and Hepatocellular Carcinoma: A Prospective Study in Men 4841 male asymptomatic HBsAg carriers >30 y/o enrolled 1988.8~1992.6.30 Biochem, AFP, US/6-12 months, linking data with national death/cancer registry and chart review 70% of survivals remain in follow-up (average 12 yr) By 2002.12.31: 189 HCC, 154 had entry serum

18. Combined Risk of HCC with HBV Load and Genotype

23. Summary and Conclusion HBV viral load is a risk predictor of liver cirrhosis and HCC development, in a dose-dependent manner starting at 104 copies/ml Reduction in HBV DNA level over time was associated with an up to 6-fold reduction in HCC risk Successful antiviral therapy will be able to reduce or slow the progression of liver disease

24. HBV DNA: Liver Compartment Evolution of HBV DNA testing 1. Histology 2. Immunohistochemistry 3. Hybridization assays 4. PCR assays for intrahepatic - Total HBV DNA - HBV ccc DNA - pg & pc HBV RNA

27. Role of HBV cccDNA Viral persistence is due to maintenance of the HBV cccDNA pool in the nuclei of infected cells HBV cccDNA does not self-replicate, so is not directly affected by NA-based therapy Little is known of the significance of HBV cccDNA levels during acute infection progression to chronic infection resolution of acute or chronic infection antiviral drug therapy

30. cccDNA and Natural History of HBV Natural history study (patients=98) - Chronic active hepatitis (HBeAg+ve, n=63) - Chronic active hepatitis (HBeAg-ve precore mutant, n=18) - Inactive carriers (HBeAg -ve, n=10) - HBsAg clearance (HBsAg -ve, anti-HBc+ve, anti-HBs+ve, n=7)

33. quantitative real-time PCR assay used to measure cccDNA in biopsies

34. Reduction in serum HBsAg during ADV therapy is significant compared to PBO (p<0.001)

35. ADV Associated Serum HBsAg Reductions are Similar in Magnitude to cccDNA Reductions

36. Changes in Serum HBsAg are Correlated with Changes in cccDNA Titer

37. Changes in Serum HBsAg are Correlated with Changes in Total Intracellular and Serum HBV DNA

38. Conclusions 48 Weeks of adefovir dipivoxil therapy results in a significant 73% decrease in serum HBsAg titer reductions similar in magnitude to that of cccDNA (84%) ADV-associated changes in HBsAg are significantly and positively correlated with changes in cccDNA, total intracellular HBV DNA, and serum HBV DNA HBsAg reduction confirms that adefovir dipivoxil therapy reduces the reservoir of transcriptionally active viral cccDNA

39. HBV cccDNA Levels as a Predictor of Sustained Virological Response IFN & LMV combination vs LMV monotherapy (patients = 47) Evaluated serum HBV load, intrahepatic HBV DNA and cccDNA as predictors of response

41. cccDNA Levels as a Predictor of Reactivation After Chemotherapy HBsAg positive lymphoma (patients = 22) Treated with intense chemotherapy Evaluated pre-chemotherapy serum HBV load, intrahepatic HBV DNA and cccDNA as predictors of reactivation

43. A real-time PCR method for quantifying cccDNA from liver tissue has been established HBeAg +ve patients have significantly more cccDNA than HBeAg -ve patients 48 weeks of ADV therapy resulted in a decrease in cccDNA levels cccDNA levels can give a greater predictive value of response than alternative measures for a range of clinical therapies Larger scale trials to determine if quantification of cccDNA may provide an indicator of the efficacy of antiviral therapy an independent predictor of outcome

44. Clinical Trial End-Points Traditional Endpoints HBeAg seroconversion Improvement in liver Histology HBV DNA reduction HBsAg seroconversion Emerging Endpoints Intrahepatic HBV cccDNA level at end-of-treatment Reduction in HBeAg and/or HBsAg Effect on Adaptive Immune Responses Effect on Innate Immune Responses

49. Randomized, Double-Blind Study Comparing Adefovir Dipivoxil (ADV) Plus Emtricitabine (FTC) Combination Therapy Versus ADV Alone in HBeAg (+) Chronic Hepatitis B: Efficacy and Mechanisms of Treatment Response GKK Lau1, H Cooksley2, R Ribeiro3, K Powers3, S Bowden4, H Mommeja-Marin5, J Sorbel5, E Mondou5, F Rousseau5, S Lewin6, A Perelson3, S Locarnini4, and N Naoumov2 1Department of Medicine, Queen Mary Hospital, Hong Kong SAR, China; 2Institute of Hepatology, University College London, London, UK; 3Los Alamos National Laboratory, Los Alamos, NM, USA; 4Victorian Infectious Diseases Reference Laboratory, Victoria, Australia; 5Gilead Sciences Inc, Durham, NC, USA; 6Infectious Diseases Unit, Alfred Hospitaland Department of Medicine, Monash University, Melbourne, Australia

50. Introduction A short-term viral dynamic study of LAM+famciclovir suggested an advantage for combination therapy1 Longer term studies have not consistently shown greater virologic response with combination treatment in patients with chronic HBV LdT+LAM versus LdT2 ADV+LAM versus LAM3 Additive anti-HBV activity has been demonstrated in vitro for adefovir plus emtricitabine (FTC)4 FTCB-201 is an investigator-initiated pilot phase 2 study of ADV+FTC versus ADV monotherapy for 96 weeks FTC is currently approved only for HIV infection Adefovir dipivoxil (ADV) is an approved nucleotide only for chronic HBV

51. AIMS To compare the efficacy of a new combination therapy of ADV plus FTC versus ADV alone To examine early HBV kinetics and virus-specific T-cell reactivity to gain understanding of the mechanisms of successful HBV control

52. Methods ? FTCB-201 Study Design

54. FTCB-201 Key Entry Criteria Nucleos(t)ide treatment-naïve HBeAg+ chronic hepatitis B Serum HBV DNA > 0.7 MegaEq/mL* ALT > 1.3 times to 10 times upper limit Compensated liver disease No co-infection with HCV, HDV, or HIV, No treatment with IFN within 6 months

55. Baseline Characteristics

56. FTCB-201: HBV DNA FTC + ADV HBeAg+ CHB

57. Early HBV kinetics identified two subsetsClearance of virus (<4700 copies/ml) by week 12

59. Comparison between Fast Vs Slow responders during antiviral treatment

60. Conclusions The combination of ADV + FTC demonstrated significantly greater antiviral activity compared to ADV monotherapy Different patterns of virus-specific T-cell reactivity have been identified during antiviral treatment Assessment of early virological response and T-cell reactivity may provide a basis for individualized approach and optimization of mono or combination therapies in CHB

61. IMPAIRED TOLL-LIKE RECEPTOR EXPRESSION IN CHRONIC HEPATITIS B: IMPLICATIONS FOR PATHOGENESIS AND THERAPY Visvanathan K1, Skinner N1, Riordan S2, Sozzi V3 , Edwards R3, Chang J4, Lewis S4 and Locarnini S3.

62. Immune Based Approach:The Innate Immune System Evolutionarily ancient Universal - all multicellular organisms Constitutive - germ-line Immediate pathogen response (hours) Components: Pattern Recognition Central Pattern recognition receptors: pathogen-assoc. molecular patterns Cell-surface eg Toll-like receptors Secreted Intracellular Phagocytes eg. dendritic cells Antimicrobial peptides eg. cathelicidins, defensins Alternate complement pathway

63. TLR’s: What Are They? Type I integral membrane glycoproteins Members of larger superfamily that includes IL-1 receptors (considerable homology in cytoplasmic regions) Highly conserved

64. TLR Signaling

65. TOLL 2: Precore Interaction

66. TOLL 2: Precore Interaction

67. Quantitative HBeAg levels and patterns of TLR-2 and TLR-4 expression on CD14+ monocytes during potent antiviral therapy for chronic hepatitis B (CH-B) Alexander JV Thompson, Stephen A Locarnini, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria, Australia; George KK Lau, Queen Mary Hospital, University of Hong Kong, Hong Kong, Hong Kong Special Administrative Region of China; Nikolai V Naoumov, University College London, London, United Kingdom; Paul V Desmond, St. Vincent’s Hospital, Fitzroy, Victoria, Australia; Herve Mommeja-Marin, Gilead Sciences, Inc., Durham, NC; Hui Chee-Kin, Queen Mary Hospital, University of Hong Kong, Hong Kong, Hong Kong Special Administrative Region of China; Narelle Skinner, Kumar Visvanathan, Murdoch Children’s Research Institute, Parkville, Victoria, Australia

68. Background FTCB-201 Prospective, randomized, double-blinded, phase II Adefovir (ADV) monotherapy vs ADV / emtricitabine (FTC) HBeAg-positive CH-B, compensated liver disease Mono-infected NA naïve - no IFN for at least 6/12 96 week follow-up Primary endpoints: Early viral kinetics Virus-specific T cell immunity

69. Background Queen Mary Hospital, Hong Kong N = 30 Mono = 16, Combo = 14 Genotype B = 9, C = 21 Results: Fast responders: N = 11 Undetectable VL at 12 weeks (<300/ml), median log drop 5 Vs 3 Lower baseline VL, cccDNA Combination therapy Enhanced T-cell reactivity (HBsAg, HBcAg) - ELISPOT No patient seroconverted at 96 weeks

70. Samples N=15 Adefovir monotherapy – n = 9 Adefovir / emtricitabine therapy – n = 6 Fast responders – n = 7 Slow responders – n = 8 Genotype B = 4 (2 FR, 2 SR) Genotype C = 11 (6 SR, 5FR)

71. Samples N=15 Adefovir monotherapy – n = 9 Adefovir / emtricitabine therapy – n = 6 Fast responders – n = 7 Slow responders – n = 8 Genotype B = 4 (2 FR, 2 SR) Genotype C = 11 (6 SR, 5FR) Methods Flow cytometry CD14+ PerCP (monocytes) TLR-2 FITC,TLR-4 PE Qantitiative HBeAg assay Murex Microtitre Plate Paul Erhlich Institute Standard Generate a standard curve and perform multiple dilutions on patient's serum

72. HBeAg Vs VL Vs TLR2

73. Summary Data:TLR2 – Fast Vs Slow Responder

74. Conclusions HBV down-regulates expression of TLR2 on PBMC’s, even when viral load is only modest. Down-regulation of TLR2 expression improves with treatment that improves viral load HBeAg+ve strains showed marked TLR2 suppression as compared to the HBeAg-ve strains This virus-related depression of TLR may contribute to the development of persistent HBV infection. Link between HBeAg titre and TRL-2 suppression.

75. Precore/core protein production

76. Precore/core protein production

77. TOLL 2: Precore Interaction

78. TOLL 2: Precore Interaction

80. Clinical Trial End-Points Traditional Endpoints HBeAg seroconversion Improvement in liver Histology HBV DNA reduction HBsAg seroconversion Emerging Endpoints Intrahepatic HBV cccDNA level at end-of-treatment Reduction in HBeAg and/or HBsAg Effect on Adaptive Immune Responses Effect on Innate Immune Responses