1. HCV resistance Understanding the mechanism and Prevention
Fabien Zoulim
Hepatology department, Hospices Civils de Lyon
INSERM U1052, Viral Hepatitis Team
Lyon University, France

2. Novel targets for antiviral intervention using the HCV life cycle
IFN + RBV ER
HCV
Endosome
Uncoating
Translation
Entry
Receptor
NS2
NS3/4A
NS4B
NS5B
NS5A + -
Replication
Progeny genomes
Golgi E1 E2
Core
Assembly
Release
Exocytosis
Nucleus
Cytoplasm
Polymerase
inhibitors
Protease inhibitors
Other viral
and cellular targets
Racanelli V, et a., Trends Immunol 2003;24:456-64;
Manns MP, et al. Nat Rev Drug Discov 2007;6:
IFN – Interferon, RBV - Ribavirin

3. Key concepts for HCV Viral persistence but a curable disease !
Viral genome heterogeneity
Resistance to polymerase & protease inhibitors
Selection of resistant mutants
Key objective: suppress resistance

4. The main differences between HIV, HBV and HCV
Host cell H
Host cell H
Host cell
HCV RNA
cccDNA
Host DNA
Host DNA
Proviral DNA
Integrated DNA
Nucleus
Nucleus
Nucleus
Life-long suppression
of viral replication
Long-term suppression
of viral replication
Definitive viral clearance
and SVR
SVR – Sustained viral response
Kieffer TL, et al. J Antimicrob Chemother 2010;65:202-12; Garrido C, et al. J Antimicrob Chemother 2010;65;320-6; Clavel F, et al. New Engl J Med 2004;350:1023-5; Zoulim F, et al. Gastroenterology 2009;137: ; Sarrazin C, et al. Gastroenterology 2010;138:

5. How does HCV resistance develop?
High replication rate with errors during replication
~1012 viruses produced per day
~1 mistake per virus produced
Continual development of viral variants
Drug-resistant variants exist before antiviral therapy
Antiviral therapy
This describes the steps involved in the development of resistance.
Suppression of wild-type
and susceptible variants
Selection and expansion
of drug-resistant mutants

6. Definitions of treatment failure
Null-Response
Breakthrough
Relapse
Partial responder
Detection limit
This illustrates the response patterns and definitions for 4 groups of patients failing therapy.
The yellow line describes patients achieving a relatively small change in HCV decline from baseline. These patients are labeled as having a Null-Response.
The red line describes patients with detectable HCV RNA at any time during treatment after previous undetectable HCV RNA with qualitative PCR-test during antiviral therapy. These patients are labeled as having a Breakthrough.
The grey line describes patients achieving a > 1 log HCV RNA decline from baseline during therapy but not achieving PCR negativity. These patients are labeled as being Partial Responders.
The blue line describes patients achieving PCR negativity by a sensitive assay and maintaining this through Week 48 of therapy. These patients are labeled as having a Relapse because upon withdrawal of therapy, HCV levels will rise above the limit of normal.
Treatment including DAA
Resistance mutants
DAA – Direct acting antiviral agents

7. The virus and its environment
The infected hepatocytes
Virus replication in hepatocytes
Hepatocyte response to infection
Antiviral drugs
Potency of antivirals
Potency shift to mutant
Barrier to resistance
The virus
Fitness of the resistant mutants
Accumulation of compensatory mutations
Cross-resistance profile

8. Emergence of drug resistant mutants
Wild-type
All single mutations likely to be generated every day
Resistant mutants
less fit than wt
HCV RNA
Hepatocytes
Viral infection
Viral replication
Resistant mutant

9. Emergence of drug resistant mutants
All single mutations likely to be generated every day
Hepatocytes
Wild-type
HCV RNA
Viral infection
Viral replication
Resistant mutants
more fit than wt
Resistant mutant
Antiviral treatment with DAA
DAA – Direct acting antiviral agents, wt – wild-type

10. Antiviral drug resistance testing
Genotypic (sequence analysis) assays
Detection of emerging mutants
Population sequencing
Clonal analysis
Ultradeep sequencing
HCV viral fitness assays
Determination of fitness, infectivity and evolution of mutants
Difficult to apply to clinical isolates
HCV phenotypic assays
Determination of drug susceptibility and cross-resistance profile / Replicon system

11. Evolution of NS3 protease variants in patients treated with telaprevir monotherapy
Sarrazin C, et al. Gastroenterology 2007;132:
NS3 – Non structural protein 3

12. Towards the suppression of resistance
Factors affecting the selection of resistant variants during antiviral treatment
Drug selective pressure:
Antiviral potency
Drug concentration at the site of replication
Genetic barrier to resistance:
Number of mutations required for loss of activity
Viral fitness: Relative rate of replication of resistant strains
IFN and ribavirin backbone to maintain antiviral pressure on resistant mutants
IFN – Interferon

13. Peg-IFN & Ribavirin inhibit PI resistant mutant replication
Kieffer TL, et al. AASLD 2006
Peg-IFN – Pegylated interferon, PI – Protease inhibitor

14. Failure of triple therapy is the result of the failure of IFN – Ribavirin to clear PI resistant mutants !
IFN – Interferon, PI – Protease inhibitor

15. Virologic monitoring of triple therapy

16. Example of virologic breakthrough
DAA stopped + sequencing 10
106
105
104
103
102
101
HCV - RNA (UI/ml) N 2 N 4 Y 6 Y 8 Y 10 Y 12 Y
vBT
Confirmed v BT
DAA – Direct acting antiviral agents,
v BT – Virologic breakthrough

17. Example of virologic breakthrough
Importance of the timing of virologic monitoring 10
106
105
104
103
102
101
HCV - RNA (UI/ml) N 2 N 4 Y 6 Y 8 Y 10 Y 12 Y
vBT
Confirmed v BT
v BT – Virologic breakthrough

18. Example of virologic breakthrough
Importance of sequencing and mutation detection 10
106
105
104
103
102
101
HCV - RNA (UI/ml)
V36M
R155K N 2 N 4 Y 6 Y 8 Y 10 Y 12 Y
vBT
Confirmed v BT
v BT – Virologic breakthrough

19. Loss of telaprevir resistant variants after treatment cessation
1.0
0.9
Médiane de retour des variants “sauvages” en séquençage direct = 7 mois (IC95% : 5-8)
0.8
0.7
0.6
médiane
Probabilité
0.5
0.4
0.3
0.2
0.1
0.0 2 4 6 8 10 12 14 16 18
Suivi après l’échec thérapeutique (mois)
Sullivan J. et al. EASL 2011.

20. Loss of telaprevir resistant variants after treatment cessation
1.0
0.9 1a
0.8
0.7
0.6
médiane
Probabilité
0.5
0.4 1b
0.3
0.2
0.1
0.0 2 4 6 8 10 12 14 16 18
Suivi après l’échec thérapeutique (mois)
Sullivan J. et al. EASL 2011.

21. Optimizing anti-HCV regimens to maximize SVR and minimize resistance
The drugs
Antiviral potency
Pharmacokinetics (intracellular concentration of the inhibitor)
The virus
Genetic barrier to resistance
Fitness of resistant variants
Cross-resistance
The host
Treatment adherence
IFN response:
Genetics / IL28B gene polymorphism & others
ISGs expression
The treatment regimen
Treatment duration / Dosage of the drug /Dosing interval
Side effects
IFN – Interferon, IL28 – Interleukin 28B, ISGs – Interferon stimulated genes,
SVR – Sustained virologic response

22. All-oral HCV treatment GT-1

23. Site of action of BI compounds
NS3/4A Protease inhibitor BI
NS5B pocket I Polymerase inhibitor BI

24. Serine protease domain
Cross-resistance between polymerase and protease inhibitors is unlikely
NS2–NS3 protease
NS3 protease C E1 E2 p7
NS2
NS3
NS4A
NS4B
NS5A
NS5B
Core
Envelope
Serine protease domain
Telaprevir; SCH
T54
R155
A156
D168
V36
R1479* (R1626)
S96
N142
S282
C316
M414
M419
P495
T423
Telaprevir; BI
NM283*
Telaprevir; BI ; SCH
HCV-796† BI
Telaprevir
*nucleoside; †non-nucleoside
BI207127†
Sarrazin C, et al. Gastroenterol. 2007;132:1767–77; Tong X, et al. Antiviral Res. 2006;70:28–38; De Francesco R, et al. Nat. 2005;436:953–60; Le Pogam S, et al. Virol. 2006;351:349–59; Villano S, et al. 57th AASLD 2006, Boston, MA, October 27–31, 2006

25. BI 207127 + BI 201335 Combination: suppresses emergence of resistance
BI X EC50
Long term selection of resistance in vitro is effectively suppressed by BI and
BI combination
BI X EC50
EC50 – Median estimated concentration

26. 400 mg TID BI 207127 + 120 mg BI 201335 + RBV (n=15)
BI BI : SOUND-C1
400 mg TID BI mg BI RBV (n=15)
100000
10000
1000
100 10 1
HCV RNA (IU/mL)
GT-1a
GT-1b
GT-6e
Treatment day
BI mg/ BI /RBV
BI / PegIFN/RBV
LLOQ
LLOD
600 mg TID BI mg BI RBV (n=17)
100000
10000
1000
100 10 1
GT-1a
GT-1b
HCV RNA (IU/mL)
LLOQ
LLOD
Treatment day
BI mg/ BI /RBV
BI / PegIFN/RBV
Zeuzem S, et al. Gastroenterology 2011;141:
LLOQ, lower limit of quantification; LLOD, lower limit of detection,
GT - genotype

27. SOUND-C1 trial Multi-centre, open-label, randomised phase Ib study in
treatment-naïve, HCV genotype-1 (GT-1) patients
400 mg BI  TID mg BI QD + RBV
120 mg BI QD + PegIFN alfa 2a/RBV
PegIFN alfa 2a/ RBVa
Follow-up
n = 15
n = 17
600 mg BI  TID mg BI QD + RBV
120 mg BI QD + PegIFN alfa 2a/RBV
PegIFN alfa 2a/ RBVa
Follow-up
Day 1
Week 4
Week 24
Week 48
Week 72
SOUND-C1 study G1 naive exclude cirrosisis
High sustained virologic response following interferon-free treatment of chronic HCV GT1 infection for four weeks with HCV protease inhibitor BI , polymerase inhibitor BI and ribavirin, followed by BI and PegIFN/Ribavirin –
Initial virological response decreased with lower dose 400 MG
Note initial to week 4 IFN free then IFN; IFN sparing thus
Futility rules:
Virological breakthrough (increase in HCV RNA by > 1 log, or HCV RNA ≥ 100 IU/mL after < 25 IU/mL)
Lack of EVR (> 2 log decrease at Week 12)
Lack of undetectable HCV RNA at Week 24
aPatients without eRVR (HCV RNA ≤ 25 IU/mL Week 4, undetectable Weeks 5 to 18) continued PegIFN/RBV up to Week 48
TID, three times daily; QD, once daily
Zeuzem S, et al. AASLD Abstract LB-7
EVR – Early virologic response, eRVR – extended Rapid virologic response,
PegIFN/RBV – Pegylated interferon/ribavirin

28. SOUND-C1 Virologic response
400 mg TID 600 mg TID
100 80 60 40 20
Proportion of patients (%)
RVR eRVR EOT and SVR 12 high; higher with 600 mg
11/15
17/17
4/15
12/17
14a/15
17/17
11/15
16b/17
RVR eRVR EoT SVR12
aIncludes patient #61102 switched to Peg/RBV;
bPatient #41203 outstanding
RVR, rapid virologic response (after 4 weeks of treatment); eRVR, HCV RNA < 25 IU/mL at Week 4 and undetected at Week 5 to 18; EoT, undetectable HCV RNA at the end of all treatment
Zeuzem S, et al. AASLD Abstract 249
EOT – End of treatment, SVR – Sustained virologic response

29. SOUND-C2 trial Multi-centre, open-label, randomised phase IIb study in
treatment-naïve, HCV genotype-1 (GT-1) patients;
Including ~15% cirrhotics
A (n=81)
B (n=81)
C (n=81)
D (n=81)
E (n=81)
TID + RBV
Follow-up
TID + RBV
Follow-up
TID + RBV
BID + RBV
Follow-up
TID no RBV
Follow-up
SOUND-C2is a 5-arm,open-label,randomized,phase Iibstudy evaluating efficacy
and safety of several interferon-free all-oral combination regimens of these compounds for up to
Weeks of treatment
Methods:362 TN HCV GT-1 5 arms
arms:
Prespecified interim analysis is all patients completed 12 weeks.
Randomization was stratified by HCV subtype (1a vs 1b) and IL28B genotype
Day 1 Week 12 Week 16 Week 26 Week 40
Interim analysis
Zeuzem S, et al. AASLD Abstract LB-15
GT – Genotype, RBV - Ribavirin

30. Results: Antiviral activity; antiviral response assessment up to week 12 (5)
patients with HCV RNA <LLOD at Week 12 by IL28 GT and viral subtype
(per protocol analysis)
100 80 60 40 20
Proportion of patients with HCV RNA <LLOD at Week 12 (%)
Interim analysis by two variables
60% group A SVR 12)
69% 1b 49% 1a effect of host immune response
C/C Non-C/C C/C Non-C/C
GT-1a GT-1b
TID + RBV 22/25 39/61 24/26 79/92
BID + RBV 6/7 10/22 10/11 32/36
TID no RBV 3/3 2/9 7/7 13/20
LLOD – Lower limit of detection, IL28b Interleukin 28b, GT - genotype
Zeuzem S, et al. AASLD Abstract LB-15

31. All-oral HCV treatment GT-1
BMS BMS

32. BMS BMS
Phase IIa study of BMS plus BMS for 24 weeks
HCV GT-1b Japanese null responders
BMS mg QD + BMS mg BID
Follow-up
n=10 (GT-1b)
Week 24 48 72
Daclastavir and Asunaprevir to be mentioned
Japanese non response (non serotic)
Chayama K, et al. Hepatology. 2011;54(Suppl. S1): Abstract LB-4
GT – Genotype

33. BMS-650032/BMS-790052: virologic response (in GT1b only)
One patient discontinued at Week 2; HCV RNA was undetectable after 24 weeks’ follow-up
Proportion of patients with undetectable HCV RNA (%)
9 patients completed 24 weeks of treatment; 1 patient discontinued after 2 weeks, however, this patient achieved SVR24
In all 9 patients who completed treatment, HCV RNA was undetectable from treatment Week 8 through post-treatment Week 24 (SVR24)
In the patient who discontinued, HCV RNA remained undetectable for >24 weeks after discontinuation
No viral breakthrough was observed
2 serious AEs: grade 3 pyrexia and grade 4 hyperbilirubinemia
4/10
9/10
9/10
9/10
Week 4
Week 12
EOT
SVR24
EOT – End of treatment, SVR – Sustained virologic response
Chayama K, et al. Hepatology. 2011;54(Suppl. S1): Abstract LB-4

34. All-oral HCV treatment GT2/3
PSI-7977

35. PSI-7977 + RBV (GT-2/3) (n=10 per arm)
phase II, randomized, open-label trial of PSI-7977 plus RBV, with or without PegIFN, in treatment-naïve HCV GT-2 and GT-3 patients; cirrhotics excluded
PSI + R 12 weeks n=10
PSI  mg + RBV
PSI + PR 4 weeks n=10
PSI  mg + PegIFN/RBV
PSI  mg + RBV
PSI + PR 8 weeks n=10
PSI  mg + PegIFN/RBV
PSI  mg + RBV
PSI + PR 12 weeks n=10
PSI  mg + PegIFN/RBV
Week 4 8 12
Gane EJ, et al. AASLD Abstract 34

36. PSI-7977 + RBV (GT-2/3) (n=10 per arm)
No viral breakthrough observed
PSI-7977 very well tolerated, with no attributable safety signal, no treatment discontinuations and no treatment emergent laboratory abnormalities
100
100
100
100
100
100
100
100
Gane EJ, et al. AASLD Abstract 34

37. PSI-7977 + RBV (GT-2/3) (n=10 per arm)
100
100
100
100 60
Gane EJ, et al. AASLD Abstract 34
RBV - Ribavirin

38. Conclusions: Current and future HCV treatment regimens
PegIFN
Ribavirin
HCV
Protease
Inhibitor*
Polymerase
Inhibitor**
Inhibitor‘
Inhibitor
**1
Inhibitor 2***
SOC
Standard of Care
NSOC
New Standard of Care
Dual-oral / QUAD
2 direct acting antivirals
quadruple
Triple-oral
3 direct acting antivirals +/ -
HCV
NS5A inhibitor
HCV
NS5A inhibitor OR
HCV
NS5A inhibitor