JM Pawlotsky

3. I HCV resistance to IFN-  Therapy

4. Incidence of Peg-IFN  -Ribavirin Treatment Failures 2% 0 15 30 45 60 54% 48% 58% 24% 16% 18% Genotype 1 Genotypes 2/3 PEG-IFN- α 2a+ribavirin (Fried et al) PEG-IFN- α 2a+ribavirin (Hadziyannis et al) PEG-IFN- α 2b+ribavirin (Manns et al) (Manns et al, Lancet 2001 ; Fried et al, N Engl J Med 2002 ; Hadziyannis et al, Ann Intern Med 2004)

5. Treatment Failure

6. Treatment Schedule Treatment Failure

7. Host Factors Treatment Schedule Treatment Failure

8. Disease Characteristics Host Factors Treatment Schedule Treatment Failure

9. Viral Factors Disease Characteristics Host Factors Treatment Schedule Treatment Failure

10. Viral Factors Disease Characteristics Host Factors Treatment Schedule Treatment Failure

12. Incidence of Peg-IFN  -Ribavirin Treatment Failures 2% 0 15 30 45 60 54% 48% 58% 24% 16% 18% Genotype 1 Genotypes 2/3 PEG-IFN- α 2a+ribavirin (Fried et al) PEG-IFN- α 2a+ribavirin (Hadziyannis et al) PEG-IFN- α 2b+ribavirin (Manns et al) (Manns et al, Lancet 2001 ; Fried et al, N Engl J Med 2002 ; Hadziyannis et al, Ann Intern Med 2004)

13. HCV Kinetics by Genotype EC-sponsored DITTO-Trial (Pawlotsky et al., manuscript in preparation) 0 1 2 3 4 5 6 7 HCV RNA (log IU/ml) 0 4 7 8 15 22 29 1 -7 -28 Genotype 4 Genotype 1 Genotype 3 * = significant difference, 4 and 1 vs 3 * * * * * * * Quantitative assay cutoff Qualitative assay cutoff

14. HCV Kinetics by Genotype EC-sponsored DITTO-Trial 0 1 2 3 4 5 6 7 HCV RNA (log IU/ml) 0 4 7 8 15 22 29 1 -7 -28 Genotype 4 Genotype 1 Genotype 3 * = significant difference, 4 and 1 vs 3 * * * * * * * Quantitative assay cutoff Qualitative assay cutoff (Pawlotsky et al., manuscript in preparation)

15. HCV Kinetics by Genotype EC-sponsored DITTO-Trial 0 1 2 3 4 5 6 7 HCV RNA (log IU/ml) 0 4 7 8 15 22 29 1 -7 -28 Genotype 4 Genotype 1 Genotype 3 * = significant difference, 4 and 1 vs 3 * * * * * * * Quantitative assay cutoff Qualitative assay cutoff (Pawlotsky et al., manuscript in preparation)

16. HCV Kinetics by Genotype EC-sponsored DITTO-Trial 0 1 2 3 4 5 6 7 HCV RNA (log IU/ml) 0 4 7 8 15 22 29 1 -7 -28 Genotype 4 Genotype 1 Genotype 3 * = significant difference, 4 and 1 vs 3 * * * * * * * Quantitative assay cutoff Qualitative assay cutoff (Pawlotsky et al., manuscript in preparation)

17. Viral Factors Antiviral resistance Delayed clearance Longer half-life of infected cells

19. SNP and SVR in the IDEAL Trial (Ge et al, Nature, 2009;461:399-401) IL28B

20. rs12979860 Allele and SVR (Ge et al, Nature, 2009;461:399-401)

21. rs12979860 Allele Frequency 12% 39% 49% 37% 16% 47% C/C C/T T/T Caucasian ancestry n=871 African American ancestry n=191 (Ge et al, Nature, 2009;461:399-401)

22. Geographic Distribution (Thomas et al, Nature, 2009;461:798-801)

23. Effect on HCV Kinetics (Caucasians) (Thompson et al, AASLD 2009) Δ HCV RNA (Log 10 IU/mL) C T T T - 3 . 0 - 2 . 0 - 1 . 0 0 W e e k s - 4 . 0 - 5 . 0 2 4 1 2 0 - 6 . 0 C C

24. Effect on HCV Kinetics (African Americans) Δ HCV RNA (Log 10 IU/mL) C T T T - 3 . 0 - 2 . 0 - 1 . 0 0 W e e k s - 4 . 0 - 5 . 0 2 4 1 2 0 - 6 . 0 C C (Thompson et al, AASLD 2009)

25. -5 -4 -3 -2 -1 0 0 4 8 12 16 20 24 Weeks of therapy HCV RNA reduction (Log 10 IU/mL) TT CT NS P=0.045 P=0.021 P=0.004 P=0.0005 VK on High-Dose Peg IFN  According to IL28B Genotype (Chevaliez et al, AASLD 2010)

26. SVR Predictors (Thompson et al, AASLD 2009) <0.0001 2.2 1.3 1.7 Fasting blood sugar < 5.6 mmol/L <0.0001 4.1 2.3 3.1 HCV RNA ≤ 600,000 IU/mL <0.0001 4.0 2.0 2.8 Caucasian vs African American 0.004 3.6 1.3 2.1 Hispanic vs African American <0.0001 6.7 4.1 5.2 rs12979860 CC vs non-CC <0.0001 4.0 1.8 2.7 METAVIR score ≤F2 p-value 95% CI Odds Ratio

28. II HCV Resistance to Ribavirin ?

30. Ribavirin’s Antiviral Effect (Pawlotsky et al., Gastroenterology 2004;126:703-14) -1.0 -0.5 0.0 0.5 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 Mean HCV RNA decrease (log IU/ml) Time (days) Controls Ribavirin monotherapy

31. Ribavirin’s Antiviral Effect (Pawlotsky et al., Gastroenterology 2004;126:703-14) -1.5 -1.0 -0.5 0.0 +0.5 0 2 4 6 8 10 12 14 Days HCV RNA changes (log IU/ml) -1.5 -1.0 -0.5 0.0 +0.5 0 2 4 6 8 10 12 14 Days HCV RNA changes (log IU/ml) -1.5 -1.0 -0.5 0.0 +0.5 0 2 4 6 8 10 12 14 Days HCV RNA changes (log IU/ml) -1.5 -1.0 -0.5 0.0 +0.5 0 2 4 6 8 10 12 14 Days HCV RNA changes (log IU/ml)

33. III HCV Resistance to Direct Acting Antivirals

35. Mechanisms of Resistance sensitive resistant

36. Mechanisms of Resistance sensitive resistant Drug

37. Mechanisms of Resistance sensitive resistant Drug resistant sensitive

38. Mechanisms of Resistance sensitive resistant Drug Stop drug resistant sensitive

39. Mechanisms of Resistance sensitive resistant sensitive resistant Drug Stop drug resistant sensitive

40. Mechanisms of Resistance sensitive resistant sensitive resistant + fit Drug Stop drug resistant sensitive

41. Mechanisms of Resistance sensitive resistant Drug Stop drug resistant sensitive resistant + very fit sensitive

42. Chronic HCV infection is curable by therapy

43. Mechanisms of Resistance sensitive resistant

44. Mechanisms of Resistance sensitive resistant Drug resistant

45. Mechanisms of Resistance sensitive resistant Drug Stop drug resistant resistant

46. HCV Life Cycle (Popescu & Dubuisson, Biol Cell 2009;102:63-74)

48. NS3/4A Protease Active Site

49. Antiviral Efficacy of NS3/4A PIs -2.8 3 days 200 mg bid Ib GS-9256 -3.9 4.5 days 600 mg bid Ib ACH-1625 -3.3 3 days 300 mg bid Ib BMS-650032 II II II II II III III Phase -4.2 7 days 400 mg bid Narlaprevir -4.7 8 days -4.0 14 days 240 mg qd BI201335 700 mg bid Vaniprevir (MK-7009) -3.8 14 days 200 mg q8h Danoprevir (RG7227) -4.1 7 days 200 mg qd TMC435 -1.6 7 days 400 mg tid Boceprevir -4.4 14 days 750 mg q8h Telaprevir Median/mean log HCV RNA reduction Duration Dose Drug

50. Asp168 Ala156 Arg155 Thr54 Val36 (Pawlotsky J-M, Ther Adv Gastroenterol 2009;2: 205-219) Amino Acid Substitutions Associated with PI Resistance

51. Resistance and Fitness (Kieffer T, et al. Hepatology 2007;46:631-9) In vivo fitness Resistance

52. Telaprevir Resistance (Reesink HW, et al. Gastroenterology 2006;131:997-1002) -5 -4 -3 -2 -1 0 1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Study Time (Days) Median HCV RNA Change from Baseline (Log 10 IU/mL) Placebo VX-950 450 mg q8h VX-950 1250 mg q12h

53. Telaprevir Resistance (PROVE2) 0 1 2 3 4 5 6 7 0 10 20 30 40 50 60 70 80 90 HCV RNA (Log 10 IU/mL) Weeks WT A B C D E F A B C D E R155K/N V36L/M WT A40T V36L/M + A40T R155K/N + A40T V36L/M + R155K/N V36L/M + R155K/N + A40T TVR-Peg-IFN (Chevaliez S, et al., EASL 2009)

54. Telaprevir Resistance (PROVE2) HCV RNA (Log 10 IU/mL) Weeks WT A B C D E R155K/E + T42S WT A B WT C D E TVR-Peg-IFN SOC 0 1 2 3 4 5 6 7 0 8 16 24 32 40 48 56 64 72 (Chevaliez S, et al., EASL 2009)

55. Antiviral Efficacy of NUCs II II Phase -0.7 3 days 100 mg qd IDX184 -2.7 14 days 1500 mg bid R7128 Median/mean log HCV RNA reduction Duration Dose Drug

56. HCV Resistance to 2’-C-Methyl Nucleoside Inhibitors 2’C-Me-ATP in the catalytic site (Migliaccio et al., J Biol Chem 2003;278:49164-70)

57. Antiviral Efficacy of NNIs -1.5 2 days 600 mg bid II ABT-333 -3.2 3 days 750 mg bid Ib VX-222 -1.7 10 days 400 mg tid II VCH-759 Ib II II II II Phase -1.3 (1a), -3.8 (1b) 7 days 800 mg bid MK-3281 -3.1 3 days 800 mg q8h BI207127 -2.9 3 days 800 mg bid ANA598 -2.1 8 days 300 mg bid Filibuvir -1.4 8 days 40 mg bid GS-9190 Median/mean log HCV RNA reduction Duration Dose Drug

58. RdRp Resistance Mutations (courtesy of Isabel Najera, Roche) A B C D pol Fingers Thumb Palm

59. Filibuvir (Pfizer) Resistance in IFN Null-Responders (Mori et al., EASL 2010) Thumb 2 domain M423 Filibuvir

60. Antiviral Efficacy of NS5A Inhibitors -3.2 1 day 10 mg qd II BMS790052 Ib Phase -2.1 (only 1b) 5 days 233 mg q8h AZD7295 Median/mean log HCV RNA reduction Duration Dose Drug

61. BMS-790052 Resistance in vitro (Gao et al., Nature 2010;465:96-100) 1a replicon 11,000±4,000 20,000±6,000 2,100±610 7,300±1,100 8,700±1,900 4,100±360 5.9±3.7 49±13 61±15 2.6±0.3 EC50 4,000 6,000 610 1,100 1,900 360 1 19 24 1 Fold-change 117±29 L31V 11±7 Y93C 55±15 L31M 41±16 Q30R 75±31 Q30H 31±23 M28T 100 wt 20±7 Y93H 144±47 L31V 100 wt 1b replicon Replication level (% wt) Sustitution Subtype

62. Antiviral Efficacy of Cyclophylin Inhibitors -2.2 15 days 900 mg qd Ib SCY-465 II II Phase None 14 days 600 mg bid NIM 811 -3.6 14 days 1200 mg bid Alisporivir (DEBIO-025) Median/mean log HCV RNA reduction Duration Dose Drug

63. Alisporivir Resistance in vitro 3’UTR NS3 NS4 A B NS5A A NS5B 5’UTR neo HCV IRES EMCV IRES Domain I Domain II Domain III 36 213 250 342 356 447 R262Q R318W A241P D320E (Coelmont et al., EASL 2009) 3.67 R318W + D320E 1.72 A241P + R262Q + R318W 1.37 R262Q + R318W A241P + R262Q + R318W + D320E A241P + R318W A241P + R262Q 1.58 3.89 1.02 Fold-change vs wt

64. IV Triple Combination Treatment Failure

65. Prevention of DAA Resistance DAA

66. Prevention of DAA Resistance DAA Ribavirin Pegylated IFN-  +

70. Treatment Failures in SPRINT-1 Failures according to lead-in, 28wk 71 76 70 27 33 17 0 0 0 20 40 60 80 100 <0.5 Log 10 HCV RNA level at week 4 (Lead-in phase) 0.5 – <1.0 1.0 – <1.5 1.5 – <2.0 2.0 – <3.0 3.0 – <4.0 ≥ 4.0 Undetectable N=7 N=21 N=10 N=11 N=21 N=12 N=11 N=3 Treatment failures (%) (Kwo et al., AASLD 2009)

71. Treatment Failures in SPRINT-1 Failures according to lead-in, 48wk 0 20 40 60 80 100 <0.5 Log 10 HCV RNA level at week 4 (Lead-in phase) 0.5 – <1.0 1.0 – <1.5 1.5 – <2.0 2.0 – <3.0 3.0 – <4.0 ≥ 4.0 Undetectable N=9 N=13 N=17 N=10 N=14 N=17 N=12 N=9 (Kwo et al., AASLD 2009) 56 38 35 20 21 18 8 0 Treatment failures (%)

72. SVR According to Lead-in (SPRINT-2, non-black) 29% 39% 82% % of patients with SVR 0 10 20 30 40 50 60 70 80 90 100 BOC/RGT BOC/PR48 (Poordad et al., AASLD 2010) 82% <1 log HCV RNA decrease ≥ 1 log HCV RNA decrease

73. SVR According to Lead-in (RESPOND-2, non-black) 33% 34% 79% % of patients with SVR 0 10 20 30 40 50 60 70 80 90 100 BOC/RGT BOC/PR48 (Bacon et al., AASLD 2010) 73% <1 log HCV RNA decrease ≥ 1 log HCV RNA decrease

74. Telaprevir Rollover Study 107 0 20 40 60 80 100 37% 55% 75% 97% 59% Patients With Undetectable HCV RNA (%) TOTAL N=117 Prior null-response N=51 Prior partial response N=29 Prior breakthrough N=8 Prior relapse N=29 (Berg et al., EASL 2010)

75. REALIZE Trial-Telaprevir Arms 0 20 40 60 80 100 31% 57% 86% 65% Patients With Undetectable HCV RNA (%) TOTAL Prior null-response Prior partial response Prior relapse (Vertex press release, Sept 7, 2010)

77. Incidence of HCV resistance (Hézode et al., N Engl J Med 2009;360:1839-50) T12P12 T12PR12 T12PR24 Arm 2 16 - 4 22 Relapse 8 9 - 2 19 Breakthrough 2 13 1 3 19 Relapse - - 1 - 1 Breakthrough 3 4 1 - 8 Relapse 4 - - - 4 Breakthrough High-level resistance Low-level resistance Wild-type VL <LOD N Event

78. DAA Resistance -5 -4 -3 -2 -1 0 1 Study Time Median HCV RNA Change from Baseline (Log 10 IU/mL)

79. DAA Resistance -5 -4 -3 -2 -1 0 1 Wild-type, sensitive HCV Study Time Median HCV RNA Change from Baseline (Log 10 IU/mL)

80. DAA Resistance -5 -4 -3 -2 -1 0 1 Wild-type, sensitive HCV Resistant HCV Study Time Median HCV RNA Change from Baseline (Log 10 IU/mL)

81. Triple Combo Failure -5 -4 -3 -2 -1 0 1 Study Time Median HCV RNA Change from Baseline (Log 10 IU/mL)

82. Triple Combo Failure -5 -4 -3 -2 -1 0 1 Study Time Median HCV RNA Change from Baseline (Log 10 IU/mL) Potent IFN  -ribavirin effect

83. Triple Combo Failure -5 -4 -3 -2 -1 0 1 Wild-type, sensitive HCV Study Time Median HCV RNA Change from Baseline (Log 10 IU/mL) Potent IFN  -ribavirin effect

84. Triple Combo Failure -5 -4 -3 -2 -1 0 1 Wild-type, sensitive HCV Resistant HCV Study Time Median HCV RNA Change from Baseline (Log 10 IU/mL) Potent IFN  -ribavirin effect

85. Triple Combo Failure -5 -4 -3 -2 -1 0 1 Wild-type, sensitive HCV Resistant HCV Study Time Median HCV RNA Change from Baseline (Log 10 IU/mL) CURED Potent IFN  -ribavirin effect

86. Triple Combo Failure -5 -4 -3 -2 -1 0 1 Study Time Median HCV RNA Change from Baseline (Log 10 IU/mL) Moderate IFN  -ribavirin effect

87. Triple Combo Failure -5 -4 -3 -2 -1 0 1 Study Time Median HCV RNA Change from Baseline (Log 10 IU/mL) Moderate IFN  -ribavirin effect Wild-type, sensitive HCV

88. Triple Combo Failure -5 -4 -3 -2 -1 0 1 Study Time Median HCV RNA Change from Baseline (Log 10 IU/mL) Moderate IFN  -ribavirin effect Wild-type, sensitive HCV Resistant HCV

89. Triple Combo Failure -5 -4 -3 -2 -1 0 1 Study Time Median HCV RNA Change from Baseline (Log 10 IU/mL) Moderate IFN  -ribavirin effect Wild-type, sensitive HCV Resistant HCV CURED or RELAPSE with RESISTANT VIRUS

90. Triple Combo Failure -5 -4 -3 -2 -1 0 1 Study Time Median HCV RNA Change from Baseline (Log 10 IU/mL) Modest or null IFN  -ribavirin effect

91. Triple Combo Failure -5 -4 -3 -2 -1 0 1 Study Time Median HCV RNA Change from Baseline (Log 10 IU/mL) Modest or null IFN  -ribavirin effect Wild-type, sensitive HCV

92. Triple Combo Failure -5 -4 -3 -2 -1 0 1 Study Time Median HCV RNA Change from Baseline (Log 10 IU/mL) Modest or null IFN  -ribavirin effect Wild-type, sensitive HCV Resistant HCV

93. Triple Combo Failure -5 -4 -3 -2 -1 0 1 Study Time Median HCV RNA Change from Baseline (Log 10 IU/mL) Modest or null IFN  -ribavirin effect Wild-type, sensitive HCV Resistant HCV RELAPSE or BREAKTHROUGH with RESISTANT VIRUS

94. V Prevention of Treatment Failures

96. SPRINT-2 Trial Boceprevir, Na ïve, Genotype 1 Weeks on therapy 36 0 24 12 48 60 72 *RVR = undetectable HCV RNA at week 4 of boceprevir treatment (i.e. at week 8) Boceprevir 800 mg tid + Peg-IFN  2b + RBV Follow-up Peg-IFN  2b + RBV Peg-IFN  2b + RBV Follow-up 4 Peg-IFN  2b + RBV Follow-up Peg-IFN  2b + RBV Follow-up no RVR Boceprevir 800 mg tid + Peg-IFN  2b + RBV Peg-IFN  2b + RBV RVR 28 N > 1000

97. REALIZE Trial Telaprevir, Nonresponders , Genotype 1 Weeks on therapy 36 0 24 12 48 60 72 *eRVR = undetectable HCV RNA at week 4 and week 12 Telaprevir 750 mg q8h + Peg-IFN  2a + RBV Follow-up Peg-IFN  2a + RBV Peg-IFN  2a + RBV Peg-IFN  2a + RBV Follow-up 28 4 Peg-IFN  2a + RBV Follow-up Telaprevir 750 mg q8h + Peg-IFN  2a + RBV N = 260 N = 260 N = 130

99. 55% 26% 34% 39% 21% 11% 6% 53% 55% 0% 10% 20% 30% 40% 50% 60% 70% W4 W12 W24 < 1 log 10 HCV-RNA decrease 1 to 2 log 10 HCV-RNA decrease ≥ 2 log 10 HCV-RNA decrease Undetectable HCV-RNA ( <15 IU/mL) 1% 8% 22% (Hézode et al., AASLD 2010) High-Dose Pegylated IFN-  and Ribavirin in Non-Responders

100. GS-9256 (PI) + Tegobuvir (NNI) 0 7 1 4 2 1 2 8 0 1 2 3 4 5 6 7 8 HCV RNA IU/mL (Log) Days (Zeuzem et al., AASLD 2010) GS-9256 + tegobuvir ( < 2 5 I U / m L )

101. GS-9256 (PI) + Tegobuvir (NNI) 0 7 1 4 2 1 2 8 0 1 2 3 4 5 6 7 8 Days (Zeuzem et al., AASLD 2010) HCV RNA IU/mL (Log) GS-9256 + tegobuvir GS-9256 + tegobuvir + ribavirin ( < 2 5 I U / m L )

102. BMS-650032 (PI) + BMS-790052 (NS5A) (Lok et al., AASLD 2010) 0 1 2 3 4 6 8 10 12 1 2 3 4 5 6 7 0 1 2 3 4 6 8 10 12 1 2 3 4 5 6 7 Weeks HCV RNA (log 10 ) HCV RNA (log 10 ) No Peg/ribavirin +Peg/ribavirin Weeks

103. Danoprevir + RG7128 Combo INFORM-1 Trial (Gane et al., Lancet 2010; published online) Days Days Days Danoprevir, 900 mg bid + RG7128 Danoprevir, 900 mg bid + pegIFN  and ribavirin Increasing doses of danoprevir and RG7128

104. Conclusions