HCV resistance - theoretical basics
and practical meaning
Vladimir Chulanov, MD, PhD
The head of the Reference Centre
for Viral Hepatitis monitoring
Evolution of HCV Treatment Revolution
Drug Resistance Concepts and Terminology
■ Resistance Associated Substitution (RAS)
– amino acid change in a viral protein, which leads to a decrease in the
susceptibility of the virus to inhibitory activity of the drug;
■ Resistance Associated Varian (RAV)
– genetic variant (strain) of the virus, which has one or more resistance
associated substitutions;
■ Genetic barrier to resistance
– multiple factors which together determine the speed of selection of
viral variants resistant to the DAA;
■ Viral fitness
– the ability of the virus to survive and propagate under particular
conditions (e.g. under inhibitory activity of the drug);
Effect of HCV biology on developing resistance
High mutation rate
One error introduced per HCV virus produced
High virus production
> 1012 virus particles produced per day
8.7 x 1010 viruses with single mutations each day
4.2 x 109 viruses with double mutations each day
All possible single and double mutants are generated several times per day.
Some are unviable and are eliminated; some may confer resistance
Rong L et al. Sci Transl Med. 2010;2(30):30ra32.
3
DAA therapy can select for drug resistance
Baseline contains WT
(sensitive virus) +
resistant variants in
treatment naïve
individuals
Therapy fails to suppress
DAA-resistant
variants
Treatment
initiation
Therapy suppresses
DAA-resistant
variants
Sensitive virus
Resistant virus
Baseline RAVs ≠ treatment emergent RAVs
McHutchison JG., et al. N Engl J Med. 2009;360(18):1827-183.
Sarrazin, C. & Zeuzem, S. Gastro. 2010;138:447-62.
Hezode C., et al. N Engl J Med. 2009;360(18):1839-1850
Kwo PY., et al. Hepatology. 2008;48:1027A
Common DAA RAVs in non-SVR patients from clinical trials
Position
NS3/4 protease inhibitors
•
•
•
•
•
Boceprevir1
Telaprevir2
Simeprevir3
Asunaprevir4
Paritaprevir5
NS5B polymerase inhibitors
• Sofosbuvir (nucleoside)8
• Dasabuvir5
V36
T54
V55
Q80
S122
R155
A156
V158
D168
M175
M28
Q30
L31
H58
Y93
L159
S282
V321
A421
P495
A553
S556
D559
All DAAs are associated
with drug resistance
NS5A inhibitors
• Daclatasvir6
• Ledipasvir7
• Ombitasvir5
1. Boceprevir prescribing information. 2011. 2. Telaprevir prescribing information. 2011. 3. Simeprevir prescribing
information. 2015. 4. Asunaprevir Japan Label. October 2015. 5. Viekira Pak prescribing Information. 2015.
6. Daclatasvir prescribing information. 2015. 7. HARVONI prescribing information. 2015. 8. SOVALDI prescribing
information. 2015.
Suppression of emergent drug resistance
■ Combine ≥2 drug classes to increase resistance barrier
– Resistance barrier could vary depending on the drug
combination and GT (subtype)
■ Proof of concept studies with DCV + ASV indicated the regimen
had a low resistance barrier against GT 1a and a high resistance
barrier against GT 1b 1
■ Phase 2 study with DCV + SOF indicated the regimen had a high
resistance barrier against GT 1a and GT 1b 2
ASV, asunaprevir; DCV, daclatasvir; SOF, sofosbuvir.
1. Lok A et al. N Engl J Med. 2012;366(3):216-24; 2. Sulkowski MS et al. N Engl J Med. 2014; 370(3):211-21
6
Genetic barrier to viral resistance
■ Genetic barrier can be measured by fold change in IC50 values
IC50: Half maximal inhibitory concentration (Drug concentration
required to inhibit 50% of biochemical activity in vitro)
Minimal plasma level of drug (Cmin)
Drug level to inhibit most potent drug-resistant substitution
Fold change
resistance of
single substitution
Drug level to inhibit WT virus
7
Characteristics of HCV antiviral classes
Class
NS3 ingibitors
Target
NS3
GT activity
1 (и 4)
Resistance
barrier
DAA
Moderate
Telaprevir,Boceprevir
SImeprevir
Paritaprevir
Asunaprevir
Grazoprevir*
NS5А inhibitors
NS5A
1, 4-6
Low
Ledipasvir*
Ombitasvir
Daclatasvir
Elbasvir*
Non-NUC NS5B
inhibitors
NS5B
1
Low
Dasabuvir
NUC
NS5B inhibitors
NS5B
1-6
Very high
Sofosbuvir
8
Genetic barrier to resistance of NS3 Protease Inhibitors
(SMV-ASV)
≥ 10000
EC50 (nM)
1000
100
10
1
0,1
wt
Simeprevir
D168V
Asunaprevir
Cheng G. et al. Antimicrob Agents Chemother 2016, ePub
Fridell RA et al. Antimicrob Agents Chemother 2010; 54:3641.
Krishnan P. et al. Antimicrob Agents Chemother 2015; 59:979-987.
Lenz O. et al. Antimicrob Agents Chemother 2010; 54:1878-1887.
McPhee F. et al. Antimicrob Agents Chemother2012; 56:3670-3681.
Effect of Q80K Mutation on SVR12 rate in patients with
HCV genotype 1a treated with Simeprevir
• Thepre-treatment prevalence of Q80K in patients with GT 1а in phase 3 trials was ~ 30%
(48% in USA and 19% in Europe)
Patient group
Prevalence
SVR12 in pts with1а
Q80K +
Q80K -
Placebo
Simeprevir
Placebo
Simeprevir
52%
58%
43%
84%
30%
47%
27%
79%
Partial response
0%
38%
17%
65%
Null response
0%
75%
0%
38%
Treatment naive
Relapce
~ 30%
10
Amino Acid Substitutions Associated with Resistance
to NS3 Protease Inhibitors
NS3 protease (180 aa)
Asunaprevir
Paritaprevir
Simeprevir
Ванипревир
Genotyp HCV: 1a – red, 1b – blue, 2 – brown, 3a – green, 4 – orange
Lontok E et al., Hepatitis C virus drug resistance-associated substitutions: State of the art summary/
Hepatology. 2015 Nov;62(5):1623-32. doi: 10.1002/hep.27934. Epub 2015 Jul 30.
Discordance in HCV Genotyping and Frequency of
Recombinant Variants of HCV
Rate of HCV genotyping discordance in clinical practice
Primary HCV
Genotyping
AmpliSens
Genotype-FL
HCV Genotype
by Sequencing
Number of
cases
1b
3a
3a
1
1a
1b
1b
2
1a+1b
1b
1b
1
2
3a
3a
1
In 4% of cases HCV
genotype can be
determined incorrectly
5/128 (3,9%)
ВСЕГО (n=128)
Rate of HCV recombinant variants in Russia
In 40 percent of cases HCV
genotype determined as 2
may be recombinant 2k/1b
4%
n=283
Reference Center for Viral Hepatitis, 2016
42%
41%
13%
2a
2c
2k
2k/1b
Prevalence of baseline NS3 polymorphisms in
HCV GT-1b (Russian population)
10
NS3
n=70
8,6
9
8
7
6
5
4,3
4
3
2
1,4
1
0
Q80K/L
Reference Center for Viral Hepatitis, 2016
S122G/T
T54S
Broad cross-resistance to NS5A inhibitors
HCV GT,
mutation
1a
1b
M28T
Q30R
L31M/V
Y93H/N
Ledipasvir
(LDV)
20x
>100x
>100x/
>100x
>1000x/
>10000x
Ombitasvir
>1000x
>100x
<3x
>100x
>10000x/
>10000x
<10x
Daclatasvir
(DCV)
>100
>1000x
>100x/
>1000x
>1000x/
>10000x
<10x
Elbasvir
20x
>100x
>10x
>1000x/
>1000x
<10x
Velpatasvir
<10x
<3x
20x/50x
>100x/
>1000x
<3x/-
ACH-3102
30x
20x
<10x
>100x/>100x
<3x/<3x
ABT-530
<3x
<3x
<3x
<10x/<10x
<3x
<3x/<3x
MK-8408
<10x
<10x
<10x
<10x
<10x
<10x
DAA
RAV per DAA class or specific DAA?
>100x
L31V
Y93H/N
>100x/20x/50x
20x/50x
>100x/-
D. Wyles. AASLD 2015
In vitro resistance profile of DCV
GT-1b1
GT-1aa,1
GT-3a3
GT-4a4
8336 7735
8000
Fold change in EC50 value
GT-2a2
6000
4000
3350
2000
1450
683
0
1850
1217
1451
749
350
23
19
141
98
320
1215
169
454
• Primary amino acids associated with DCV resistance at positions 28, 30, 31 and 93 at N-terminus of NS5A
– Predominant resistance variant(s) is GT (subtype) specific
• Fold change in EC50 value is genotype (subtype) dependent
• Resistance barrier is genotype specific: two substitutions required to substantially increase resistance in
GT-1b
– NS5A RAVs L31 and Y93H rarely observed together at baseline (< 1%)
1. Fridell et al. Antimicrob Agents Chemother. 2010;54:3641. 2. Fridell et al. J Virol. 2011;85:7312. 3. Wang et al.
Antimicrob Agents Chemother. 2013;57:611. 4. Wang et al. Antimicrob Agents Chemother. 2012;56:1588.
Prevalence of baseline NS5A polymorphisms in
HCV GT-1b
20
18,4
Japan
Proportion with NS5A polymorphism (%)
18
Korea or Taiwan
Non-Asian countries
16
15,0
14
12,8
12,0 12,0
11,5
12
11,7
10,4
9,6
10
8
7,2
7,2
6,1
6
6,1
4,8
4,9
4,3
4
2,4
2
0
1,4
27 6 7
374 125 489
43 12 30
374 125 489
45 15 30
374 125 489
16 3 24
374 125 489
56 13 35
374 125 489
69 16 57
374 125 489
L28M
R30Q
L28M or R30Q
(without
L31F/I/M/V or
Y93H)
L31F/I/M/V
Y93H
L31F/I/M/V or
Y93H
HCV, hepatitis C virus; GT-1b, genotype 1 subtype b; NS5A, non-structural protein 5A;
RAV, resistance-associated variant.
Adapted from McPhee et al. Adv Ther. 2015;32:637.
Prevalence of baseline NS5A polymorphisms in
HCV GT-1b (Russia population)
NS5A
20
n=88
18
%
16
14
12
10,2
10
8,0
8
6
4
8,0
4,5
2,3
1,1
2
0
L28M
R30Q
Reference Center for Viral Hepatitis, 2016
L31M
P58S
Y93H
L31M+Y93H
Daclatasvir+Asunaprevir SVR rates by previous
treatment and baseline RAV status
Non-Asian countries (N = 485)
100
97,0
93,5
92,4
91,4
90
80
70
60
53,8
50
40
39,1
38,6
28,6
SVR12
(%)
30
20
10
22
57
400
428
7
13
128
132
6
21
134
145
9
23
138
151
0
All patients
Treatment-naïve
Prior
non-responders
IFN
ineligible/intolerant
Клиническое значение мутаций рез-ти зависит от исходных характеристик
пациента (предыдущий опыт ПВТ, степень фиброза печени и т.п.)
McPhee et al. 2015 APASL Conference Poster
Efficacy of DCV + ASV in elderly patients with GT-1 (Japan)
N = 159 with SVR4 determinable:
• Mean age 71 years
• Prior SMV, 2.5%
• NS5A L31 RAV, 8.8%
• NS5A Y93 RAV, 17.6%
N = 180 patients with GT-1 received DCV + ASV*
in Fukuoka, Japan (Sept 2014–Sept 2015)
SVR4 in GT-1 patients
SVR4 (%)
100
88,0
97,0
89,7
P <0.0001
80
88,3
85,7
94,9
P <0.0001
P = 0.0054
60,7
60
46,4
40
25,0
20
0
Overall
Yes
No
Completed
DCV + ASV
No
Yes
Prior SMV
No
Yes
NS5A L31
No
Yes
NS5A Y93
*DCV 60 mg QD + ASV 100 mg BID for 24 weeks
ASV, asunaprevir; BID, twice daily; DCV, daclatasvir; GT-1, genotype 1; NS5A, non-structural protein 5A; QD, once daily;
RAV, resistance-associated variant; SMV, simeprevir; SVR4, sustained virologic response at post-treatment follow-up Week 4
Adapted from Motomura et al. AASLD 2015; Poster 1157
No impact of NS5A RAVs on outcomes with LDV/SOF
in patients with GT-1
Patients with SVR12 (%)
100
99
99
187
189
504
509
GT-1 patients: 12 weeks of LDV/SOF
With NS5A RAVs
No NS5A RAVs
99
90
96
96
80
60
40
20
79
88
298
300
26
27
65
68
0
LDV/SOF
No cirrhosis
Tx naive
LDV/SOF
No cirrhosis
Tx experienced
LDV/SOF
Cirrhosis
• Baseline NS5A RAVs did not have a clinically meaningful impact on treatment outcomes with
LDV/SOF as when analysed according to cirrhosis status and prior treatment status
GT-1, genotype-1; LDV, ledipasvir; NS5A, non-structural protein 5A; RAV, resistance-associated variant; SOF,
sofosbuvir; SVR12, sustained virologic response at post-treatment follow-up Week 12; Tx, treatment
Adapted from Zeuzem et al. AASLD 2015; Oral presentation 91
Baseline NS5A RAVs did not have significant effect
on response to DCV+SOF treatment
ALLY-1
N = 113
ALLY-2
N = 203
ALLY-3
N = 152
ALLY-3+
N = 152
• Patients with cirrhosis or post-liver
transplant
• GT 1 to 6
• DCV + SOF + RBV, 12 weeks
22 patients with BL RAVs
18 achieved SVR1
• Patients with HIV coinfection
• GT 1 to 6
• DCV + SOF, 8 or 12 weeks
Y93 – 7 of 8 SVR
L31 – 7 of 8 SVR
R30 – 11 of 13 SVR2
• Patients with GT 3 infection
• Treatment-naive or treatment-experienced
• DCV + SOF, 12 weeks
•
•
•
BL RAVs did not
measurably affect on
SVR3
Patients with GT 3 infection
Pts with advanced fibrosis or liver cirrhosis
DCV + SOF+ RBV, 12 or 16 weeks
1. Fred Poordad; HEPATOLOGY, VOL. 63, NO. 5, 2016; 2. D.L. Wyles; N Engl J Med 2015;373:714-25;
3. David R. Nelson; HEPATOLOGY 2015;61:1127-1135; 4. Vincent Leroy; HEPATOLOGY, VOL. 00, NO. 00, 2016
Y93 – 1 of 2 SVR
A30 – 6 of 6 SVR4
* Combination is not yet approved in Russia
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Amino Acid Substitutions Associated with Resistance
to NS5B Inhibitors
NS5B polymerase (591 a.о.) – NUC inhibitors
Sofosbuvir
591 aa
NS5B polymerase (591 a.о.) – non-NUC inhibitors
Dasabuvir
591 aa
Genotyp HCV: 1a – red, 1b – blue, 2 – brown, 3a – green, 4 – orange
Lontok E et al., Hepatitis C virus drug resistance-associated substitutions: State of the art summary/
Hepatology. 2015 Nov;62(5):1623-32. doi: 10.1002/hep.27934. Epub 2015 Jul 30.
Prevalence of baseline NS5B polymorphisms in
HCV GT-1b (Russian population)
NS5B
40
35
34
30
23
25
20
15
10
5
n=65
n=60
L159F*
S556G
0
* Chulanov V.P. et al. AASLD 2014
Reference Center for Viral Hepatitis, 2016
SVR12 in GT 1b Patients With or Without RAVs at Baseline
Treated with SOF+RBV 16 or 24 weeks
90
80
80
65
70
% of SVR
74
60
50
40
25
30
20
10
3/12
13/20
8/10
17/23
0
16 weeks
24 weeks
L158F
Chulanov V.P. et al. AASLD 2014
WT
24
Methods for Detection of RAS
Method
Sensitivity (percentage of
mutant in viral population
which can be detected)
Characteristics
Direct (population)
sequencing
10-20%
Relatively simple, reliable, wildly
used in lab. diagnostics
5-10%
Time-consuming, labor
intensive, can be used in
molecular research labs only
<1%
High-throughput, expensive
equipment and reagents,
qualified staff required
Cloning-sequencing
Next Generation Sequencing
(NGS)
or Deep Sequencing
Impact of GT1b NS5A-Y93H on SVR24
Comparison of Direct Sequencing vs. NGS
Direct Sequencing
Next-Generation Sequencing
100
100
93 100
86
80
48
44
% SVR
SVR24 (%)
% SVR
83
76
80
60
47
40
20
0
93
86
181
211
Total BL
NS5A
Sequences
16
33
10-100
7
16
>60-100
7
15
>20-60
3
3
>10-20
59
60
41
33
40
20
165
178
0
Not
Detected
(<10)
185
215
Total BL
NS5A
Sequences
27
46
1-100
7
17
>60-100
2
6
>20-60
5
6
>10-20
13
17
1-10
158
169
Not
Detected
(<1)
% Y93H in Patient Viral Population
% Y93H in Patient Viral Population
■ Optimal SVR rates with the DCV/ASV regimen can be obtained by
screening out patients with NS5A-Y93H using direct sequencing
– Exploratory NGS not required as comparable SVR rates obtained using both
sequencing methods
APASL 2015; poster 1725
26
To Test or Not To Test?
■ The breadth of the use of resistance mutations data depends on the
availability of reliable and standardized method of detection
■ Clinical utility of RASs is determined by multiple factors: IC50 fold change,
RASs combination, patient population, treatment regiment etc.
■ RAS testing is most likely not required in populations and regimens with
SVR >99%;
■ RAS testing is most likely clinically useful and cost-effective in populations
and regimens with suboptimal SVR rates (<90-95%?) (treatment
experienced patients, cirrhosis, combination of DAA with low genetic
barrier, when the shortest possible treatment is considered etc.)
■ RAS testing is mandatory in patients who failed to respond to DAA for
tailoring retreatment option (testing of two samples, baseline and after
treatment failure, is most useful).
General Considerations for Treatment Decisions in
Patients with RAVs
■ Switch DAA class;
■ Add SOF in treatment regimen;
■ Add RBV in treatment regimen;
■ Treat longer (up to 24 weeks);
■ Use triple combination of DAA from different classes;
■ Use combination of high genetic barrier DAA with PEG+RBV
■ Wait for new second generation DAAs with good resistance
profile.
28