ngs clinical implications in...
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Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
NGS clinical implications in virology
Laboratory of molecular virology and immunology-Physiopathology and therapeutics of chronic viral hepatitis
French National Center of viral hepatitis B,C and delta
C.Rodriguez, S.Chevaliez, JM.Pawlotsky
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
NGS for an exhaustive overview of host/virus interaction/adaptation/response
- GWAS - Exome - ChIP Seq - …
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
Viral extraction
from patient serum
RT. Amplification. emPCR.
pyrosequencing
Analysis
Data collection
Softwares are protected under IDDN
Ultra-Deep Pyrosequencing (UDPS) process
% of each mutations
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
Viral adaptation capacity evaluation
Static studies at baseline
- Primary resistance : HBV HCV HIV
- HIV genotropism
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
Adapted from Simen et al., JID 2009
HIV Resistant Minority variants and virological failure
2 NRTI+NNRTI 2 NRTI+IP NRTI+NNRTI+PI
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
Adapted from Simen et al., JID 2009
HIV Resistant Minority variants and virological failure
2 NRTI+NNRTI 2 NRTI+IP NRTI+NNRTI+PI
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
HBV Resistant Minority Variants and virological failure
HBV DNA level
V173L L180M A181V/T T184 S/A/I/L R202G M204V/I N236T (log
IU/mL)
Pt 1 6.6 <0.1% <0.1% 0,47% <0.1% 0,12% 0,31% 0,25%
Pt 2 7.8 <0.1% <0.1% 0,18% <0.1% <0.1% <0,1% 0,12%
Pt 3 6.8 <0.1% <0.1% 0,22% <0.1% <0.1% 0,32% <0.1%
Pt 4 5.5 <0.1% <0.1% <0.1% 0,17% <0.1% 0,16% 0,12%
Pt 5 7.6 <0.1% <0.1% 0,33% <0.1% <0.1% <0.1% <0.1%
Pt 6 6.2* <0.1%* <0.1%* <0.1%* <0.1%* <0.1%* <0.1%* <0.1%*
Pt 7 6.1 <0.1% <0.1% 0,33% <0.1% <0.1% 0,20% <0.1% Primary ADV resistance Primary LAM, FTC, LdT, ETV resistance Secondary ’fitness’
*Low number of sequences at baseline for this patient
Rodriguez et al., submitted
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
Primary ADV resistance Primary LAM, FTC, LdT, ETV resistance Secondary ’fitness’
HBV DNA level
V173L L180M A181V/T T184S/A/I/L R202G M204V/I N236T
(log IU/mL)
A 5.2 <0.1% <0.1% 0,47% <0.1% <0.1% <0.1% <0.1%
B 6.5 <0.1% <0.1% <0.1% 0,34% <0.1% <0.1% <0.1%
C 7.1 <0.1% 6,32% <0.1% <0.1% <0.1% 9,57% <0.1%
D 6.4 <0.1% <0.1% <0.1% <0.1% 0,31% <0.1% <0.1%
E 5.7 <0.1% <0.1% 0,39% <0.1% <0.1% 0,37% <0.1%
Rodriguez et al., submitted
HBV Resistant Minority Variants and success of treatment
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
Patie
nt
IFN
RB
V TL
V
stat
ut
geno
nb se
q
V36A
/M
T54A
/S
V55A
Q80
R/K
R155
K/T
/Q
A156
S/T
/V
D168
A/V/
T/H
I170
A/T
KHB NR 1a 8405 90.0% 0.4% 0.5%
SF NR 1a 4101 1.1% 0.2% LP Rel 1b 609 0.5% 0.5% 0.2% DT Rel 1b 877 29.4% 1.3% SM Rel 1a 3762 2.9% SG Rel 1b 3825 4.2% PB SVR 1a 4643 11.1% 0.7% 0.3% 0.3% IM SVR 1a 4190 0.5% NT SVR 1a 1338 0.6% 1.8% HM SVR 1a 1103 0.6% AZ Rel 1a 8324 100.0% 6.0% 3.2% 0.3% VS SVR 1b 2685 0.3% ES SVR 1b 1572 0.2% 0.2% 0.8% SC NR 1b 2259 0.2%
NJ SVR 1b 3252 0.4% 0.2%
AP SVR 1a 17981 1.3% 0.5% 7.8% 0.2%
ML SVR 1a 6338 47.4% 0.4%
JK SVR 1b 11009 20.0% 0.4%
Preexisting Resistant HCV Variants to Protease Inhibitors at Baseline
NR : non responder Rel : Reponder relapser SVR : Sustained virological response
Chevaliez S, Rodriguez C et al, in preparation
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
Prediction of HIV-1 coreceptor usage
Rodriguez C et al, submitted
Sensitivity 0.5 log decrease
Sensitivity 1 log decrease
Sensitivity 2 log decrease
Specificity 0.5 log decrease
Specificity 1 log decrease
Specificity 2 log decrease
PPV 0.5 log decrease
PPV 1 log decrease
PPV 2 log decrease
NPV 0.5 log decrease
NPV 1 log decrease
NPV 2 log decrease
Consensus g2p
UDPS g2p
UDPS pyrotrop
20%
40%
60%
80%
100%
0%
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
Dynamic viral adaptation evaluation
Dynamic studies overtime - HBV resistance to NRTIs
- HCV resistance to IP - HIV resistance to HAART - HIV resistance to Vaccine
Static studies at baseline - HBV primary resistance - HCV primary resistance
- HIV genotropism
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
0
50
100
J0 T1 T2 T3 T4 T5 T6
0
50
100
J0 T1 T2 T3 T4 T5 T6
0
50
100
J0 T1 T2 T3 T4 T5 T6
High polymoprphism
Low polymorphism
Resistance
Modeling resistance
% o
f mut
atio
n in
the
w
hole
qua
sispe
cies
%
of m
utat
ion
in th
e
who
le q
uasis
peci
es
% o
f mut
atio
n in
the
w
hole
qua
sispe
cies
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
0
50
100
J0 T1 T2 T3 T4 T5 T6
0
50
100
J0 T1 T2 T3 T4 T5 T6
0
50
100
J0 T1 T2 T3 T4 T5 T6
High polymoprphism
Low polymorphism
Resistance
Modeling resistance
% o
f mut
atio
n in
the
w
hole
qua
sispe
cies
%
of m
utat
ion
in th
e
who
le q
uasis
peci
es
% o
f mut
atio
n in
the
w
hole
qua
sispe
cies
Slope > 0
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
Analysis of Resistant Variant Dynamics with Pyrodyn©
Time point 1
A145T: 1%
Time point 2
A145T: 5%
Time point 3
A145T: 12%
Time point 4
A145T: 25%
Time point 5
A145T: 40%
G194H: 1% G194H: 8% G194H: 7% G194H: 4% G194H: 6%
- Mathematical modeling of each substitutions
- Relevance testing of the model - Selection - Graphic creation
Pyrodyn©
- Substitutions linkage in variants Pyrolink©
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
UDPS based on known resistance
to ADV
UDPS+Pyrodyn© (resistance
modelisation)
Dynamics of HBV Resistant Populations on ADV Therapy
Rodriguez et al., submitted
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
UDPS + Pyrodyn©
+1 log of viral load Population sequencing UDPS Cloning and
sequencing
% o
f var
iant
s in
the
who
le
quas
ispec
ies
months
2
4
6
8
Viral load (Log IU/mL)
0
HBV Prediction of resistance emergence
Rodriguez et al., submitted
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
Dynamics of HCV Resistant Populations on TVR Therapy
H28Q
V36M
Q41R
Y52C
S54T
H57L
P96HR155K
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0
29
57
85Mutations of NS3 4A
% o
f mut
atio
ns in
the
who
le q
uasi
spec
ies
Days of treatment
Viral load log IU/mL
1
2
3
4
5
6
7
8
Chevaliez S, Rodriguez C et al, in preparation
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona Chevaliez S, Rodriguez C et al, in preparation
Dynamics of HCV Resistant Populations on TVR Therapy
Presented at the 10th Eu Meeting on HIV & Hepatitis, 28-30 March 2012, Barcelona
Conclusions
• UDPS at baseline : – Presence of HIV minority resistant variants at baseline is
associated with an increase risk of failure – Individual risk to predict resistance remains difficult – Best prediction of response with MVC treatment
• UDPS in dynamics
– Detection of new viral mutations – Prediction of resistance emergence
• in HBV • in HIV ?