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 ?