Triple combination treatment failures are associated with poor interferon responsiveness in patients and the presence of pre-existing resistant variants, such as Q80K, which can influence treatment outcomes. Treatment failures typically result in the selection of drug-resistant viruses, though protease inhibitor-resistant viruses are progressively replaced by wild-type viruses over time after treatment ends. In contrast, NS5A inhibitor-resistant viruses appear to persist longer. The presence of pre-existing resistance variants has only a modest impact on success rates of interferon-free regimens. While resistance is observed in some treatment failures with these regimens, overall response rates remain very high even in the presence of variants.

1. Hépatite C: Résistance
aux Traitements
Prof. Jean-Michel Pawlotsky
CNR des Hépatites B, C et delta
Laboratoire de Virologie & INSERM U635
Hôpital Henri Mondor
Université Paris XII
Créteil

2. I
Mechanisms of HCV
Resistance

3. HCV RNA-Dependent
RNA Polymerase

4. • Errors during replication:
• frequent
• spontaneous
• at random positions
• Lack of “proofreading” activity
=> Accumulation of genomic mutations
=> Selection
Properties of HCV RdRp

5. Mutant Selection
• At the level of populations of infected
individuals:
• Genotypes and subtypes
• At the level of an infected individual:
• Viral quasispecies

6. Quasispecies Distribution
of Viruses

7. sensitive
resistant
Mechanisms of Resistance

8. sensitive
resistant
Drug
Mechanisms of Resistance

9. sensitive
resistant
Drug
resistant
Mechanisms of Resistance
sensitive

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

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

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

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

14. II
DAA Monotherapy

15. HCV Lifecycle

16. HCV Lifecycle
Inhibition of polyprotein
processing
(Pawlotsky JM, Antivir Ther 2012;17:1109-17)

17. 1st-wave, 1st-generation
Telaprevir (Janssen)
Boceprevir (Merck)
2nd-wave, 1st-generation
Simeprevir (Janssen)
Paritaprevir/r (Abbvie)
Asunaprevir (BMS, Japan)
Sovaprevir (Achillion)
Vedroprevir (Gilead)
Vaniprevir (Merck, Japan)
2nd-generation
Grazoprevir (Merck)
ACH-2684 (Achillion)
Narrow genotypic activity
Low barrier to resistance
All genotypes except 3
Low barrier to resistance
Pangenotypic (~)
Higher barrier to
resistance
NS3/4A Protease Inhibitors

18. NS3/4A Protease Inhibitors
(Raney et al., J Biol Chem 2010:285:22725-31)

19. Protease Inhibitor Resistance
(HCV DRAG, Forum for Collaborative HIV Research, April 2014)

20. MK-5172 Resistance Profile
MK-5172
BMS-032
simeprevir
GS-9551
boceprevir
telaprevir
0.1
1.0
10.0
100.0
1000.0
10000.0
RepliconEC90(nM)
MK-5172 BMS-032 simeprevir GS-9551 boceprevir telaprevir
(Lahser et al., AASLD 2012)

21. HCV Lifecycle
Inhibition of HCV
replication
(Pawlotsky JM, Antivir Ther 2012;17:1109-17)

22. Nucleotide analogues
Sofosbuvir (Gilead)
MK-3682 (Merck)
ACH-3422 (Achillion)
AL-335 (Janssen)
Pangenotypic
High barrier to resistance
Nucleoside/Nucleotide Analogue
Inhibitors of HCV RdRp

23. Catalytic
Site
Nucleoside/Nucleotide Analogue
Inhibitors of HCV RdRp
(Kindly provided by Dr F. Penin)

24. Sofosbuvir Acts as a
Chain Terminator
SOF
RNA chain
cannot be
elongated
3’
5’ AG C
C GA CGGG
C
5’
Template strand
Primer strand
C
U
SOF
SOF U
U
SOF

25. 2’C-Me-ATP in the catalytic site
(Migliaccio et al., J Biol Chem 2003;278:49164-70)
HCV Resistance to 2’-C-Methyl
Nucleoside Inhibitors

26. Sofosbuvir Resistance
FDA analysis of NGS data
• Post-hoc analysis of NGS data from FISSION, FUSION, POSITRON,
NEUTRINO, and pretransplant study:
• 982 subjects treated by SOF/RBV or SOF/RBV/PEG
• 224 failed treatment
Selection of 3 substitutions of interest
• L159F (n=12)
• V321A (n=5)
• S282R (n=1)
One baseline polymorphism that potentially reduced SOF
efficacy in genotype 1b (exceptional in 1a*)
• N316 (n=6, including 4 also with L159F)
(Donaldson et al., Hepatology 2014; epub ahead of print) *C316: 99.9% in 1a, 81.8% in 1b

27. Sofosbuvir Resistance
FDA analysis of NGS data
(Donaldson et al., Hepatology 2014; epub ahead of print)

28. Sofosbuvir Resistance
FDA analysis of NGS data
(Donaldson et al., Hepatology 2014; epub ahead of print)
Interaction with the catalytic triad
C316 N316

29. Thumb-1 inhibitors
Beclabuvir (BMS)
Thumb-2 inhibitors
GS-9669 (Gilead)
Palm-1 inhibitors
Dasabuvir (Abbvie)
Genotype 1 ~only
Low barrier to resistance
Genotype 1 ~only
Low barrier to resistance
Non-Nucleoside Inhibitors (NNI)
Genotype 1 ~only
Low barrier to resistance

30. Non-Nucleoside Inhibitors (NNI)
Thumb I
BMS-791325
Thumb II
GS-9669
Palm I
Dasabuvir
Palm II
A
B
C
D
(Adapted from Dr F. Penin)

31. HCV NNI Resistance Mutations
(courtesy of Isabel Najera, Roche)
499
495
496
316
365
201
482
423
419
95
176
451
414
411
448
142 96
282
499
495
496
316
365
201
482
423
419
95
176
451
414
411
448
142 96
282
499
495
496
316
365
201
482
423
419
95
176
451
414
411
448
142 96
282
FingersThumb
Palm
A
B
C
D

32. HCV Lifecycle
Inhibition of HCV
assembly and release
Inhibition of HCV
replication
(Pawlotsky JM, Antivir Ther 2012;17:1109-17)

33. 1st-generation
Daclatasvir (BMS)
Ledipasvir (Gilead)
Ombitasvir (Abbvie)
2nd-generation
Elbasvir (Merck)
ACH-3102 (Achillion)
GS-5816 (Gilead)
Genotypes 1 and 4, other
genotypes variable
Low barrier to resistance
Pangenotypic
Slightly higher barrier
to resistance
NS5A Inhibitors

34. NS5A Protein
Domain III
Domain II
Domain I
 Required for HCV RNA
replication
 Required for HCV viral
particle assembly
 May be involved in the
release of HCV particles
NS5A Dimer
ER membrane
Cytosol
ER lumen

35. NS5A Inhibitor Resistance
(HCV DRAG, Forum for Collaborative HIV Research, April 2014)

36. MK-8742 Resistance Profile
(Merck, unpublished data)
MK-8742
Daclatasver
GS-5885
0.001
0.01
0.1
1
10
100
1000
RepliconEC90(nM)
MK-8742 Daclatasver GS-5885

37. GS-5816, a 2nd-Generation
NS5A Inhibitor
(Gilead, data on file)

38. Barrier to Resistance
Low-barrier drug High-barrier drug
Protease inhibitors
NS5A inhibitors
Non-nucleoside RdRp inhibitors
Nucleoside/nucleotide analogue
RdRp inhibitors

39. III
Triple Combination of PegIFNa,
Ribavirin and a DAA

40. Triple Combination Treatment Failures
Depend on the Patient’s IFN Responsiveness
29%
%ofpatientswithSVR
0
10
20
30
40
50
60
70
80
90
100
BOC/RGT
82%
IFN nonresponsive
(<1 log HCV RNA
decrease during lead-in)
IFN-responsive
(≥1 log HCV RNA
decrease during lead-in)
(Poordad et al., N Engl J Med 2011;364:1195-206)

41. Pre-existing Resistant Variants May Influence the
Outcome of Triple Combination Treatment
(Simeprevir + PegIFNa + Ribavirin)
(Jacobson et al., AASLD 2013; Choe et al., AASLD 2013; FDA AVDAC, Oct 24, 2013)
*Q80K prevalence in the US: GT 1a, 32.5%; GT 1b, 0.1%
58%
84%
SVR24rate(%)
0
10
20
30
40
50
60
70
80
90
100
N=84 N=165
85%
N=267
1a Q80K 1a no Q80K 1b

42. Triple Combination Failures are Associated
with the Selection of DAA-Resistant Variants
• Protease inhibitor-containing treatment failures
in Phase III trials:
• Boceprevir: 53% have dominant resistant virus
• Telaprevir: 77% have dominant resistant virus
• Simeprevir: >90% have dominant resistant virus
(Sullivan et al., Clin Infect Dis 2013;57:221-9; Barnard et al., Virology 2013;444:329-36; Jacobson et al., Lancet
2014;384:403-13; Manns et al., Lancet 2014;384:414-26)

43. Median time to wild-type by population
sequencing =7 months (95% CI: 5-8)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Probability
0 2 4 6 8 10 12 14 16 18
Time after treatment failure (months)
median
(Sullivan et al., Clin Infect Dis 2013;57:221-9)
DAA-Resistant Viruses are Progressively
Replaced by Wild-type Sensitive Viruses
after Treatment Failures (Telaprevir)

44. (Lenz et al., EASL 2014)
DAA-Resistant Viruses are Progressively
Replaced by Wild-type Sensitive Viruses
after Treatment Failures (Simeprevir)

45. Summary
• Triple combination treatment failures are the result of
poor IFN responsiveness, which is patient-dependent
• Pre-existing resistant variants may influence the
outcome of triple combination treatment (Q80K with
simeprevir).
• Triple combination failures (telaprevir, boceprevir,
simeprevir,…) are associated with the selection of
DAA-resistant viruses
• Protease inhibitor-resistant viruses are progressively
replaced by wild-type viruses after treatment failure

46. • In contrast with viruses resistant to protease inhibitors,
NS5A inhibitor-resistant viruses appear to persist for
years, maybe forever, after treatment failure (no data in
combination with PegIFNa and ribavirin)
• No resistant viruses are selected by the triple
combination of PegIFNa, ribavirin and sofosbuvir due
to the high barrier to resistance of sofosbuvir
Summary

47. IV
IFN-Free DAA Regimens

48. Questions
• Does the presence of pre-existing resistance
substitutions at treatment initiation impact SVR with
IFN-free regimens?
• Is resistance going to be observed in patients receiving
IFN-free regimens who fail on treatment (breakthrough)
or after treatment (relapse)?
• How long will resistant viruses persist after treatment,
and will this impair retreatment?

49. Questions
• Does the presence of pre-existing resistance
substitutions at treatment initiation impact SVR with
IFN-free regimens?
• Is resistance going to be observed in patients receiving
IFN-free regimens who fail on treatment (breakthrough)
or after treatment (relapse)?
• How long will resistant viruses persist after treatment,
and will this impair retreatment?

50. SVR According to the Presence of
NS5A RAVs at Baseline (ION-1, -2, -3)
(Dvory-Sobol et al., International Workshop on Antiviral Drug Resistance, June 2014)
Cutoff for NS5A RAVs detection: 1%

51. Sofosbuvir + Simeprevir ± RBV
COSMOS Cohort 2- Gen 1, Naive and NR, F3-F4
(Lawitz et al., Lancet 2014; epub ahead of print)
93%
SVR12rate(%)
0
10
20
30
40
50
60
70
80
90
100
SOF+SIM+RBVAll patients SOF+SIM+RBVSOF+SIM
N=80 N=27 N=14 N=24
12 weeks 24 weeks
93%93%
SOF+SIM
N=15
100%
94%

52. Sofosbuvir + Simeprevir ± RBV
COSMOS Pooled cohorts 1 & 2- Subtype 1a, Q80K
(Lawitz et al., Lancet 2014; epub ahead of print)
88%
SVR12rate(%)
0
10
20
30
40
50
60
70
80
90
100
SOF+SIM+RBVAll patients SOF+SIM+RBVSOF+SIM
N=80 N=27 N=14 N=24
12 weeks 24 weeks
83%
89%
SOF+SIM
N=15
100%
88%

53. Summary
• The detection of pre-existing resistance-associated
variants at baseline may be associated with a very
modest (if any) reduction in SVR rates with some
IFN-free regimens
• The SVR rates remain very high in patients
harboring such variants, the presence of which
does not contra-indicate therapy with the given
regimen

54. Questions
• Does the presence of pre-existing resistance
substitutions at treatment initiation impact SVR with
IFN-free regimens?
• Is resistance going to be observed in patients receiving
IFN-free regimens who fail on treatment (breakthrough)
or after treatment (relapse)?
• How long will resistant viruses persist after treatment,
and will this impair retreatment?

55. The Henri Mondor Experience
161 patients treated
in IFN-free regimens
in clinical trials
Protease inhibitors
received
Paritaprevir/r
Simeprevir
Faldaprevir
Asunaprevir
Danoprevir/r
NS5A inhibitors
received
Daclatasvir
Ombitasvir
Ledipasvir
Nucleotide analogue
received
Sofosbuvir

56. The Henri Mondor Experience
161 patients treated
in IFN-free regimens
in clinical trials
13 did not achieve
an SVR
8 relapses 3 breakthroughs 2 intolerant

57. The Henri Mondor Experience
Region Observed substitutions
NS3 protease
V36M, D103N, R155K
I71V
R155K
Q80K, R155K
A147V, D168V
A4M, V36M, R155K
P146S, Q80L
NS5A
Y93C
L31V, Y93H
NS5B RNA polymerase
L433F
Q65R, S189N/S
S47E

58. • Of the 23 patients who had a relapse, 15
had NS5A RAVs at the time of relapse
• Of the 15 with NS5A RAVs, 9 had the
variants detectable at baseline, whereas 6
did not.
• No patient selected the S282T substitution
Sofosbuvir/Ledipasvir FDC ± RBV
ION-3-Phase III, Gen 1, Rx-naïve
(Kowdley et al., N Engl J Med 2014;370:1879-88)

59. Treatment Failures in
SAPPHIRE-I
Patient GT
Type of
Virologic Failure
NS3 NS5A NS5B
1 1a
On-treatment failure at
Week 12
R155K,
D168V
Q30R S556G, 559N
2 1a Relapse at PT Week 2 D168V M28T S556G
3 1a Relapse at PT Week 2 V36A, D168V M28T none
4 1a Relapse at PT Week 8 none M28V*, H58P* none
5 1a Relapse at PT Week 8 D168V Q30R Y561H
6 1a Relapse at PT Week 8 D168V Q30R none
7 1a Relapse at PT Week 12 D168V Y93N* S556G
8 1b Relapse at PT Week 2 Y56H, D168V L31M*, Y93H* S556G*
(Feld et al., N Engl J Med 2014;370:1594-603)

60. Summary
• The majority of patients receiving an IFN-free
regimen will achieve an SVR
• However, 5 to 10% of patients in clinical trials,
possibly more in real-life, fail to achieve an SVR
• At the time of breakthrough or relapse, these
patients harbor viruses that are resistant to one or
more of the DAA administered (protease inhibitor,
NS5A inhibitor, non-nucleoside inhibitor of RdRp)

61. Questions
• Does the presence of pre-existing resistance
substitutions at treatment initiation impact SVR with
IFN-free regimens?
• Is resistance going to be observed in patients receiving
IFN-free regimens who fail on treatment (breakthrough)
or after treatment (relapse)?
• How long will resistant viruses persist after treatment,
and will this impair retreatment?

62. (Lenz et al., EASL 2014)
Replacement of PI-Resistant
Viruses by Wild-Type Viruses

63. 0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Probability
0 2 4 6 8 10 12 14 16 18
Time after treatment failure (months)
(McPhee et al., Hepatology 2013;58:902-911; Wang et al. Antimicrob Agents Chemother 2013;57:2054-65)
Persistence of NS5A Inhibitor-
Resistant Viruses
?

64. Summary
• NS3 protease inhibitor-resistant viruses disappear
by population sequencing within 18 months on
average in patients who failed to achieve an SVR
• The time needed to clear variants that are
resistant to NS5A inhibitors or non-nucleoside
inhibitors of the HCV polymerase is unknown
• Whether persistence of such variants as dominant
species after treatment impacts retreatment
strategies is unknown

65. V
Clinical Utility of HCV
Resistance Testing

66. Resistance Testing Methods
• No commercially available assays yet
• In-house population sequencing of NS3
protease, NS5A and polymerase regions
• Potential interest of ultra-deep
pyrosequecing

67. HCV Resistance Testing-1
• Given the very high SVR rates with IFN-
free regimens, systematic pretreatment
resistance testing in treatment-naïve
patients or in patients who failed
PegIFNa-ribavirin therapy should not be
performed

68. HCV Resistance Testing-2
• Resistance testing at treatment failure is
not recommended, because most of these
patients harbor viruses that are resistant
to one or more of the administered DAAs

69. HCV Resistance Testing-3
• Resistance testing could be considered
at retreatment of patients who failed
DAA-containing regimens, provided that
data is generated to inform treatment
decisions based on the results of
resistance testing

70. Follow me on Twitter
@JMPawlotsky