Hepatitis C Virus

Resistance Testing

Mark Douglas

About 170 Million People Worldwide Have Chronic HCV Infection

Blatt LM, et al. J Viral Hepat 2000;7:196-202; Yu M, et al. J Gastroenterol Hepatol 2009;24:336-45; Narahari S, et al. Infect Genet Evol 2009;9:643-5;Higuchi M, et al. Jpn J Infect Dis 2002;55:69-77; Dore GJ, et al. J Clin Virol 2003;26:171-84; Wkly Epidemiol Rec 2002;77:45-7;

Centers for Disease Control and Prevention. 2009. http://www.cdc.gov/hepatitis/statistics.htm; Di Bisceglie AM. Essentials of Hepatitis C Infection. 2009;Chen CH, et al. J Formos Med Assoc 2007;106:148-55; Negro F & Alberti A. Liver Int 2011;31(s2):1-3; Shin HR, et al. J Korean Med Sci 2005;20:985-8.M, million

SoutheastAsia

30–35 M

Western Europe

5 M

United States3–4 M

South America12–15 M

Africa30–40 M

Eastern Europe10 M

Japan2 M

China40 M

Australia0.2 M

Taiwan0.4 M

India12–13 M

Korea0.2 M

1.0–1.9%2.0–10%

Prevalence of infection>10%

<1.0%

China40 M

Korea0.2 M

Taiwan0.4 M

India12–13 M

Japan2 M

Asia-Pacific Region~90 M

Australia0.2 M

SoutheastAsia

30–35 M

• 27% of cirrhosis and 25% of HCC worldwide

• Main cause of cirrhosis, liver transplant and HCC in

Australia, USA, UK

• Causes more deaths than HIV in Australia and USA

SVR (%)

80

60

40

20

024 48 78 Peg-

IFN

IFN +

RBV

PEG +

RBVweeks

IFN

monotherapy

G2/3

6-22

18-39

35-

43

61-79

33-

36

76-82

42-46

*Range of values reported;

lower bar represents lower value

G2/3G1

ALL

ALL

Evolution of SVR rates

Adapted from Manns, Foster et

al., Nature Reviews Drug

Discovery 2007

100

G1

1989 1995 1998 2001

Potential New Drug Targets

Pockros, PJ (2010, Therap Adv Gastro, 3:191–202)

Direct Acting Antivirals (DAAs)

NS3/4A protease inhibitors

– telaprevir, boceprevir, simeprevir, paretaprevir

NS5B polymerase inhibitors

– Nucleos(t)ide analogues (sofosbuvir)

– Non-nucleoside analogues (dasabuvir)

NS5A inhib. (daclatasvir, ledipasvir, ombitasvir)

Rosen, H. R. (2011, NEJM, 364:2429–2438)

SVR (%)

80

60

40

20

024 48 78 Peg-

IFN

IFN +

RBV

PEG +

RBVweeks

IFN

monotherapy

G2/3

6-22

18-39

35-

43

61-79

33-

36

76-82

42-46

*Range of values reported;

lower bar represents lower value

G2/3G1

ALL

ALL

Evolution of SVR rates

Adapted from Manns, Foster et

al., Nature Reviews Drug

Discovery 2007

100

G1

1989 1995 1998 2001 2011

PEG +

RBV +

TPV

or BOC

63-75

G1

IFN free DAA therapy

SOF +

LDV

or DCV

(8-12w)

97-99

G1N96-99

(86)

G1R

2014

PTV/r +

OTV+

DBV+

RBV

(12w)

92-96

G1N

PEG +

RBV +

SMV

80-81

G1

PEG +

RBV +

SOF

(12w)

82-92

G1

2012

SOF +

RBV

(12w)

2013

93-97

G2

56-68

G3

79-89

G3

SOF +

RBV

(24w)

GPV +

EBV

(8-12w)

94-99

G1

SOF+

VPV

(12w)

97-99

G1

90-95

G3

But what about resistance?

Resistance associated variants

(RAVs)

DAA Resistance - sequencing

Drug-susceptible quasispecies

Drug-resistant quasispecies

Treatment begins

Vir

al L

oad

Selection of resistantquasispecies

Incomplete suppressionInadequate potencyInadequate drug levelsInadequate adherencePreexisting resistance

Zeuzem S. http://www.clinicaloptions.com/Hepatitis/TreatmentUpdates/Future Generations.aspx Accessed 6/2011.

Market Introduction and Emergence of

Resistance (Selected Drugs)

The Race Against Drug Resistance: A Report of the Center for Global Development’s

Drug Resistance Working Group. Nugent R, Back E, Beith A. 2010

NS3

Boceprevir, telaprevir - genotype 1

Simeprevir - genotype 1, 4

Paritaprevir - genotype 1

Grazoprevir - pan genotypic

RAV prevalence 0.1 - 3.1% (likely due to fitness cost)

except Q80K (5-48%)

RAV persistence wk48 post treatment 9%

NS5A

Daclatasvir (BMS) - pan genotypic

Ombitasvir (AbbVie) - pan genotypic

Ledipasvir (Gilead) - Genotype 1

Elbasvir (MSD) - genotypes 1, 3, 4, 6

Velpatasvir (Gilead) - pan genotypic

RAV prevalence 0.3-3.5%

RAV persistence high– up to 85% 48 wks post treatment)

NS5B

Nucleos(t)ide analogue

– Sofosbuvir (Pan genotypic)

– RAV prevalence 0.0%

– RAV persistence 0.0% by Wk 4 post treatment

Non nucleoside analogue

– Dasabuvir (genotype 1)

– RAV prevalence 0.2-3.1%

– RAV persistence at wk48 post treatment 57%

HCV Resistance:

Does it matter?

0

10

20

30

40

50

60

70

80

90

100

PEG/RBV (n=44) PEG/RBV/SMV (n=84) PEG/RBV (n=83) PEG/RBV/SMV (n=165)

SVR12

%

QUEST-1/2: PEG-IFN/RBV/Simeprevir (24-48 weeks)

Genotype 1 treatment naïve

Jacobson I et al. AASLD 2013

GT1a + Q80K GT1a – Q80K

Q80K Prevalence

Q80K in Western Sydney?

Samples tested 378 (Gt 1a)

Age: mean 31 yrs

Gender: Male: 270 (71%)

Correctional centre (annonymous):

119 (31%)

Of 378 samples:

Mutation Number %

Q80K 21 5.6

V55A 16 4.2

T54S 10 2.6

Q80L 5 1.3

Q80R 3 0.8

R155K 2 0.5

Q80H 1 0.3

IFN-free therapy?

Combination DAA high cure rates

Failure more likely if baseline RAVs,

particularly multiple NS5A RAVs

Patients failing DAA therapy have

high levels of RAVs

Methods of detecting resistance

Population (Sanger) sequencing

High throughput (Next generation)

sequencing

– Better sensitivity with NGS - down to

<1% (Sanger ~20%)

» clinical relevance not yet determined

– Bioinformatics (big data) with NGS

Raw data - self processedThe more complicated stucture for variation detection

Sequence DNA

Roche 454 Titanium

Life Technologies SOLiD4 or 5500XL

Illumina Genome Analyzer II or

HiSeq

Pacific Biosciences

IonTorrent

Quality control

BWA Trimmer

Trimmomatic

Quake

Map to reference

SMALT

BWA

SSAHA2

Bowtie

Bfast

Bioscope

SHRiMP

NovoAlign

Stampy

SOAP2

CLCBio

Call variations

CASAVA1.8

Samtools

GATK

SoapSNP

SSAHA-SNP

FreeBayes

CLCBio traditional

CLCBio probabilistic

With thanks to Grant Hill-Cawthorne

Bioinformatics

Known Resistance Variants

Summary

HCV is curable

High cure rates with DAAs

Resistance can emerge and may

affect (re)-treatment response

Reliable, high-throughput assays

required to detect RAVs

Next Generation Sequencing most

sensitive, becoming feasible