Prevalence of HCV NS3 pre-treatment resistance

revalence

of

HCV

NS3

pre-treatment

resistance

associated

amino

acid

variants

within

a

Scottish

cohort

Samantha

J.

Shepherd

a

,

∗

,

Tamer

Abdelrahman

b

,

Alasdair

R.

MacLean

a

,

Emma

C.

Thomson

b

,

Celia

Aitken

a

,

Rory

N.

Gunson

a

a

West

of

Scotland

Specialist

Virology

Centre,

Level

5,

New

Lister

Building,

10-16

Alexandra

Parade,

Glasgow

G31

2ER,

United

Kingdom

b

MRC–University

of

Glasgow

Centre

for

Virus

Research,

Stoker

Building,

464

Bearsden

Road,

Glasgow

G61

1QH,

United

Kingdom

a

r

t

i

c

l

e

i

n

f

o

Article

history:

Received

16

December

2014

Received

in

revised

form

4

February

2015

Accepted

6

February

2015

Keywords:

Simeprevir

HCV

Protease

inhibitor

Prevalence

RAV

a

b

s

t

r

a

c

t

Background:

Protease

inhibitors

(PI)

including

boceprevir,

telaprevir

and

simeprevir

have

revolutionised

HCV

genotype

1

treatment

since

their

introduction.

A

number

of

pre-treatment

resistance

associated

amino

acid

variants

(RAVs)

and

polymorphisms

have

been

associated

with

reduced

response

to

treatment.

Objectives:

We

measured

the

prevalence

of

RAVs/polymorphisms

in

a

PI

treatment-naïve

HCV

genotype

1

Scottish

cohort

using

Sanger

sequencing.

Study

design:

Chronically

infected,

treatment-naïve,

HCV

genotype

1

patients

(

n

=

146)

attending

NHS

Greater

Glasgow

and

Clyde

clinics

were

investigated

for

RAVs/polymorphisms

to

the

PIs

boceprevir,

telaprevir

and

simeprevir.

The

NS3/4A

region

was

amplified

by

nested

polymerase

chain

reaction.

The

1.4

kb

amplified

product

was

sequenced

using

an

ABI

3710XL

DNA

sequencer.

Sequence

analysis

was

performed

using

web-based

ReCall

(beta

2.10).

Amino

acid

positions

36,

41,

43,

54,

55,

80,

109,

122,

155,

156,

168

and

170

were

analysed

for

RAVs/polymorphisms.

Results:

Overall,

23.29%

(34/146)

of

patients

had

an

RAV

or

polymorphism

detected.

Overall,

13.69%

(20/146)

of

patients

had

HCV

virus

that

contained

the

Q8

K

polymorphism.

Other

RAVs

detected

were:

V36

M

0.70%

(1/146),

V36L

0.70%

(1/146),

T54S

6.85%

(10/146),

V55A

3.42%

(5/146)

and

V/I170A

0.68%

(1/146).

Four

patients

had

dual

combinations

of

mutations

(T54S

+

V36L;

T54S

+

V55A

and

2

patients

with

T54S

+

Q80K).

Conclusions:

Q80K

was

the

most

prevalent

baseline

polymorphism

detected

in

the

Scottish

cohort.

Simeprevir

treatment

is

not

recommended

in

patients

infected

with

the

Q80K

genotype

1a

variant.

This

highlights

the

need

for

baseline

sequencing

prior

to

administration

of

this

drug

in

this

population.

Crown

Copyright

©

2015

Published

by

Elsevier

B.V.

This

is

an

open

access

article

under

the

CC

BY-NC-ND

license

(

http://creativecommons.org/licenses/by-nc-nd/4.0/

).

1.

Background

Traditionally

genotype

1HCV

infections

have

been

the

hardest

to

treat

with

sustained

virological

response

(SVR)

rates

to

the

stan-

dard

of

care

treatment

of

ribavirin

(RBV)

co-administered

with

pegylated-interferon

alpha

(IFN)

in

the

region

of

42–50%

[1,2]

.

The

development

of

non-structural

protein

3

(NS3)

protease

inhibitors

(PIs)

including

telaprevir,

boceprevir

and

simeprevir,

has

substan-

tially

improved

outcome

in

these

patients

with

SVR

rates

now

approaching

80%

in

both

treatment-naive

patients

and

relapsers

[3–6]

.

Newer

PI

based

IFN-free

regimens

show

even

greater

poten-

tial

and

lower

toxicity.

For

example,

the

combination

of

simeprevir

and

the

NS5B

polymerase

inhibitor

sofosbuvir

increased

the

SVR

∗

Corresponding

author.

Tel.:

+44

141

2018722.

E-mail

address:

Samantha.Shepherd@ggc.scot.nhs.uk

(S.J.

Shepherd).

to

over

90%

in

genotype

1

patients

[7]

.

NS5A

inhibitors

daclatasvir

or

ledipasvir

when

used

with

sofosbuvir

have

also

generated

SVR

rates

>90%

[8,9]

.

Further

breakthroughs

are

expected

as

other

com-

binations

of

antivirals

become

available

which

promise

to

offer

improvements

in

SVR,

shortened

duration

of

treatment

and

lower

pill

burden.

A

number

of

pre-treatment

resistance

associated

amino

acid

variants

(RAVs)

within

NS3

are

associated

with

reduced

response

to

PI–IFN

regimens.

For

example,

RAVs

at

position

156

(A156T/V)

and

R155K

have

been

shown

to

reduce

the

effectiveness

of

all

cur-

rent

PIs

[10–13]

.

Substitutions

at

the

D168

locus

(D168T/Y/H/A/V/I)

result

in

high-level

resistance

to

simeprevir

(>300

fold)

and

the

other

2nd

generation

PIs

only

[13–15]

.

Resistance

polymorphisms

Q80K

or

R

have

been

shown

to

negate

the

benefit

of

adding

simeprevir

to

pegylated

IFN

and

RBV

[16]

and,

for

this

reason,

it

is

recommended

in

the

license

that

patients

infected

with

geno-

type

1a

HCV

who

have

evidence

of

Q80K/R

mutations

are

not

http://dx.doi.org/10.1016/j.jcv.2015.02.005

1386-6532/Crown

Copyright

©

2015

Published

by

Elsevier

B.V.

This

is

an

open

access

article

under

the

CC

BY-NC-ND

license

(

http://creativecommons.org/licenses/by-nc-nd/4.0/

).

S.J.

Shepherd

et

al.

/

Journal

of

Clinical

Virology

65

(2015)

50–53

51

considered

for

treatment

with

simeprevir.

RAVs

at

amino

acid

posi-

tions

36,

41,

43,

54,

55,

109,

122

and

170

have

also

been

reported

[11,13,14,17,18]

.

However,

their

significance

is

currently

uncertain

with

most

reports

suggesting

that

they

only

have

a

minor

effect

on

overall

SVR

rates.

Only

a

few

studies

have

examined

the

prevalence

of

the

aforementioned

RAVs

at

baseline

[19–23]

.

Knowing

their

fre-

quency,

can

be

used

to

plan

treatment

policies

and

will

determine

the

usefulness

of

baseline

testing

prior

to

treatment.

2.

Objectives

We

measured

the

prevalence

of

natural

resistance

polymor-

phisms

in

a

protease

inhibitor

treatment-naïve

HCV

genotype

1

Scottish

cohort

using

Sanger

sequencing.

3.

Study

design

3.1.

Patients

Stored

plasma

samples,

taken

between

August

2013

and

March

2014

for

146

chronically

infected

HCV

genotype

1

patients

attend-

ing

clinics

within

NHS

Greater

Glasgow

&

Clyde,

were

used

in

this

study.

The

patients

consisted

of

141

treatment

naïve

patients

and

5

treatment

relapsers

who

had

previously

been

treated

with

pegy-

lated

IFN

and

RBV.

The

majority

of

the

patients

(

n

=

140)

were

subtype

1a

and

six

subtype

1b.

All

patients

had

a

detectable

HCV

RNA

tested

by

Abbott

RealTime

HCV

(detection

limit

12

IU/ml).

3.2.

RNA

extraction

RNA

was

extracted

using

the

NucliSens

easyMag

(BioMerieux).

Using

the

on-board

lysis

protocol,

1000

l

of

sample

was

eluted

to

60

l.

3.3.

PCR

amplification

and

sequencing

procedure

The

NS3/4A

region

was

amplified

by

nested

polymerase

chain

reaction

(PCR)

using

a

method

and

primers

supplied

by

Dr

Richard

Harrigan

(British

Columbia

Centre

for

Excellence

in

HIV/AIDS).

The

1st

round

primer

sequences

were:

5

TTCAGCCTGGACC-

CTACCTTTACCAT

3

(position

4731–4756),

5

ATGGAGATCAAG-

GTCATCACGTGGGG

3

(position

3276–3301)

and

5

GTGGCCG-

TAGAGCCTGTCGTCTTC

3

(position

3246–3269).

The

2nd

round

primer

sequences

were:

5

GACTTCGACTCTGTGATAGACTGCAAC

3

(position

4680–4706),

5

TCAAGGTCATCACGTGGGGGGCGGA

3

(position

3283–3307)

and

5

TACCGGCGACTTCGACTCGGT-

GAT

3

(position

4673–4696).

The

1st

round

PCR

amplification

was

carried

out

using

a

Qiagen

OneStep

RT-PCR

kit

and

the

2nd

round

with

the

Expand

High

Fidelity

PCR

system

(Roche

Diagnostics

GmbH).

Sanger

sequencing

was

performed

on

the

ABI

3710XL

DNA

sequencer

with

Big

Dye

v3.1.

The

1.4

kb

sequence

was

analysed

using

web-based

ReCall

beta

v2.10

(

http://pssm.cfenet.ubc.ca/home/index

).

Using

the

amino

acid

fea-

ture

on

ReCall,

the

following

amino

acid

positions

were

analysed:

36,

41,

43,

54,

55,

80,

109,

122,

155,

156,

168

and

170.

4.

Results

There

was

no

evidence

of

RAVs

at

position

155,

156

and

168

in

any

of

the

sequences

analysed

(

Table

1

).

The

polymorphism

Q80K

was

found

in

13.69%

(20/146)

of

patients

sequenced.

Other

RAVs

were

found

at

the

following

frequencies:

0.70%

(1/146)

V36M,

0.70%

(1/146)

V36L,

6.85%

(10/146)

T54S

3.42%

(5/146)

V55A

and

0.68%

(1/146)

V/I170A.

Four

patients

were

identified

as

having

dual

combinations

of

mutations

(T54S

+

V36L;

T54S

+

V55A

and

2

patients

with

T54S

+

Q80K).

5.

Discussion

This

study

analysed

sequences

from

the

NS3/4A

serine

pro-

tease

region

of

146

genotype

1

patients

(140

were

genotype

1a

and

6

were

genotype

1b).

The

low

level

of

subtype

1b

patients

in

this

study

is

a

reflection

of

the

Scottish

population,

where

subtype

1a

predominates.

This

is

also

a

reflection

of

the

UK

pop-

ulation

as

a

whole

[24]

.

Overall

23.29%

of

patients

tested

had

NS3

RAVs/polymorphisms

without

prior

exposure

to

PIs.

No

high-level

resistant

RAVs

were

detected

at

positions

155,

156

or

168.

Other

prevalence

studies

in

treatment-naive

patients

have

shown

that

these

three

key

resistance

mutations

either

occur

at

a

very

low

level

(<0.9%)

or

not

at

all

[21.25,26]

.

The

majority

of

patients

had

the

nat-

urally

occurring

polymorphism

Q80K

(13.69%).

The

prevalence

of

Q80K

in

the

Scottish

cohort

is

similar

to

that

found

in

other

Euro-

pean

studies;

France

10.5%;

Italy

10.1%;

London

16%

and

Sweden

5.7%

[20,22,23,27]

.

Q80K

prevalence

in

the

USA

has

been

reported

at

higher

prevalence

levels

of

37%

and

47%

[19,21]

.

Mutational

dif-

ferences

between

genotype

1

subtypes

and

clades

within

subtype

1

may

reflect

differences

seen

between

American

and

European

patients

[19,28,29]

.

Studies

have

also

highlighted

that

Q80K

is

more

likely

to

occur

in

patients

with

subtype

1a

HCV

than

subtype

1b

[20,21]

.

The

V36L/M,

T54S,

V55A

and

V/I170A

mutations

detected

in

this

study

are

low

level

resistance

RAVs

that

have

little

effect

on

SVR

rates

in

patients

treated

with

triple

therapy

[21,30]

.

These

inde-

terminate

or

low

level

RAVs

have

been

reported

at

a

prevalence

of

between

0.2%

and

11%

[20,21,25–27]

.

The

mutations

V36M,

V36L

and

V/I170A

do

not

appear

to

be

detrimental

to

viral

fitness

com-

pared

with

high

level

resistance

mutations

and

may

explain

the

presence

of

these

mutations

within

untreated

populations

[10,31]

.

In

this

study,

T54S

was

found

at

a

prevalence

of

7.53%

within

the

Scottish

cohort.

This

mutation

confers

low

level

resistance

to

both

boceprevir

and

telprevir

but

not

simeprevir

[13,32,33]

.

T54S

has

been

identified

in

7.5%

treatment-naive

patients

in

Sweden

and

2.8%

in

Italy

[20,22]

.

In

this

study

four

(2.74%)

subtype

1a

patients

were

found

to

have

RAV

combinations,

which

all

contained

T54S

with

another

mutation

(T54S

+

V36L;

T54S

+

V55A

and

T54S

+

Q80K).

Bae

et

al.

[19]

found

that

the

combination

of

T54S

and

Q80K

did

not

increase

drug

resistance

to

simeprevir

but

did

reduce

resistance

to

bocepre-

vir

and

teleprevir

when

compared

to

the

single

mutation

T54S

(<3

fold–5

fold).

The

combination

of

V36L

+

T54S

has

been

reported

pre-

viously

[20]

.

It

is

unclear

if

this

combination

substantially

increases

resistance

to

PIs

but

the

mutational

combination

of

V36M

+

T54S

increases

viral

fitness

compared

to

a

virus

with

T54S

only

[18]

.

Combination

RAV

at

positions

54

and

55

have

been

shown

to

reduce

response

to

triple

therapy

containing

boceprevir

[34]

.

This

study

examined

the

frequency

of

NS3

variants

detected

by

Sanger

sequencing.

Sanger

sequencing

will

only

detect

these

vari-

ants

at

a

frequency

of

>20%.

Next

generation

sequencing

(NGS)

can

detect

lower

frequency

variants

by

measuring

population

variants

that

occur

at

<1%

[35]

.

As

a

result

studies

using

NGS

will

likely

detect

RAVs

at

an

increased

frequency.

NGS

studies

of

the

NS3

region

in

treatment-naive

patients,

have

again

identified

Q80K

as

the

most

prevalent

baseline

mutation

with

∼

42%

harbouring

this

polymor-

phism

[36,37]

.

Currently,

European

guidelines

have

six

suggested

treatment

options

for

genotype

1

patients

with

simeprevir

recommended

within

two

of

these

treatment

protocols

[38]

.

This

study

confirms

that

high-level

resistance

RAVs

155,

156

and

168

are

rare

within

the

treatment-naïve

population

in

the

West

of

Scotland.

However,

52

S.J.

Shepherd

et

al.

/

Journal

of

Clinical

Virology

65

(2015)

50–53

Table

1

NS3/4A

mutations

detected

in

a

group

of

PI

treatment

naive

patients.

Mutation

list

was

adapted

from

Lenz

et

al.

[13]

;

Leggewie

et

al.

[27]

;

Forns

et

al.

[6]

;

Povada

et

al.

[29]

;

Schneider

and

Sarrazin

[41]

.

Amino

acid

position Mutation

Prevalence

in

Scottish

cohort

(

n

=

146)

Drug

V36

M

L

G

1/146

(0.68%)

1/146

(0.68%)

0.00%

Boceprevir,

telaprevir,

simeprevir

Q41

R

0.00%

F43

S

I

V

0.00%

0.00%

0.00%

Simeprevir

T54

S

A

10/146

(6.85%)

0.00%

Boceprevir,

telaprevir,

simeprevir

V55

A

5/146

(3.42%)

a

Boceprevir,

telaprevir

Q80

K

R

20/146

(13.69%)

0.00%

Simeprevir

R109

K

0.00%

Boceprevir

S122

R

0.00%

Simeprevir

R155

K

T

0.00%

0.00%

Boceprevir,

telaprevir,

simeprevir

A156

S

T

V

0.00%

0.00%

0.00%

Boceprevir,

telaprevir,

simeprevir

D168

A

V

I

T

H

E

0.00%

0.00%

0.00%

0.00%

0.00%

0.00%

Simeprevir

V/I170

A

1/146

(0.68%)

Boceprevir,

telaprevir

a

1/5

of

the

sequences

was

a

wild

type/resistant

mixture

(V55A/V).

Q80K

is

common

(13.69%)

and

baseline

sequencing

prior

to

therapy

should

be

considered

when

considering

simeprevir/IFN

treatment

in

genotype

1a

patients.

It

is

possible

that

such

testing

will

only

be

a

temporary

measure

since

newer

dual

therapies

may

largely

overcome

the

negative

effect

of

the

Q80K

mutation

[39,40]

.

Funding

Janssen

Pharmaceutical

funded

the

implementation

of

Q80K

testing

in

the

West

of

Scotland

Specialist

Virology

Centre.

TA

is

funded

by

the

MRC

(G0801822)

and

ECT

by

the

Welcome

Trust

(WT102789).

Competing

interests

None

declared.

Ethical

approval

Not

required.

Acknowledgements

The

authors

would

like

to

thank

Prof

Richard

Harrigan

for

sup-

plying

the

method

used

in

this

study.

We

would

like

to

thank

Catherine

Frew

Gillespie

for

data

analysis

during

the

set

up

of

this

project.

The

study

idea

was

conceived

by

RNG

and

CA.

Work

was

performed

by

SJS,

TA,

and

ARM.

The

manuscript

was

written

by

SJS,

TA,

ECT

and

ARM.

All

authors

have

read

the

final

manuscript.

References

[1]

M.P.

Manns,

J.G.

McHutchison,

S.C.

Gordon,

V.K.

Rustgi,

M.

Shiffman,

R.

Reibdollar,

et

al.,

Peginterferon

alfa-2b

plus

ribavirin

compared

with

interferon

alfa-2b

plus

ribavirin

for

initial

treatment

of

chronic

hepatitis

C:

a

randomised

trial,

Lancet

358

(2001)

958–965.

[2]

M.N.

Fried,

M.L.

Shiffman,

K.R.

Reddy,

C.

Smith,

G.

Marinos,

F.L.

Gonc

̧

ales

Jr.,

et

al.,

Peginterferon

alfa-2a

plus

ribavirin

for

chronic

hepatitis

C

virus

infection,

N.

Engl.

J.

Med.

347

(2002)

975–982.

[3]

I.M.

Jacobson,

J.G.

McHutchison,

G.

Dusheiko,

A.

Di

Bisceglie,

K.R.

Reddy,

N.H.

Bzowej,

Telparevir

for

previously

untreated

chronic

hepatitis

C

virus

infection,

N.

Engl.

J.

Med.

364

(2011)

2405–2416.

[4]

F.

Poordad,

J.

McCrone,

B.R.

Bacon,

S.

Bruno,

M.P.

Manns,

M.S.

Sulkowski,

et

al.,

Boceprevir

for

untreated

chronic

HCV

genotype

1

infection,

N.

Engl.

J.

Med.

364

(2011)

1195–1206.

[5]

I.M.

Jacobson,

G.J.

Dore,

G.R.

Foster,

M.W.

Fried,

M.

Radu,

V.V.

Rafalsky,

et

al.,

Simeprevir

with

pegylated

interferon

alfa

2a

plus

ribavirin

in

treatment-naive

patients

with

chronic

hepatitis

C

virus

genotype

1

infection

(QUEST-1):

a

phase

3,

randomised,

double-blind,

placebo-controlled

trial,

Lancet

384

(2014)

403–413.

[6]

X.

Forns,

E.

Lawitz,

S.

Zeuem,

E.

Gane,

J.P.

Bronowicki,

P.

Andreone,

et

al.,

Simeprevir

with

peginterferon

and

ribavirin

leads

to

high

rates

of

SVR

in

patients

with

HCV

genotype

1

who

relapsed

after

previous

therapy:

a

phase

3

trial,

Gastroenterology

146

(2014)

1669–1679.

[7]

E.

Lawitz,

M.S.

Sulkowski,

R.

Ghalib,

M.

Rodriguez-Torres,

Z.M.

Youossi,

A.

Corregidor,

et

al.,

Simeprevir

plus

sofosbuvir,

with

or

without

ribavirin,

to

treat

chronic

infection

with

hepatitis

C

virus

genotype

1

in

non-responders

to

pegylated

interferon

and

ribavirin

and

treatment-naive

patients:

the

COSMOS

randomised

study,

Lancet

384

(2014)

1756–1765.

[8]

M.S.

Sulkowski,

D.F.

Gardiner,

M.

Rodriguez-Torres,

K.R.

Reddy,

T.

Hassanein,

I.

Jacobson,

et

al.,

Daclatasvir

plus

sofosbuvir

for

previously

treated

or

untreated

chronic

HCV

infection,

NEJM

370

(2014)

211–221.

[9]

N.

Afdhal,

S.

Zeuzem,

P.

Kwo,

M.

Chojkier,

N.

Gitlin,

M.

Puoti,

et

al.,

Ledipasvir

and

sofosbuvir

for

untreated

HCV

genotype

1

infection,

NEJM

370

(2014)

1889–1898.

[10]

X.

Tong,

R.

Chase,

A.

Skelton,

T.

Chen,

J.

Wright-Minogue,

B.A.

Malcolm,

Identification

and

analysis

of

fitness

of

resistance

mutations

against

the

HCV

protease

inhibitor

SCH,

Antiviral

Res.

70

(2006)

28–38.

[11]

T.L.

Kieffer,

C.

Sarrazin,

J.S.

Miller,

M.W.

Welker,

N.

Forestier,

H.

Reesnik,

et

al.,

Telaprevir

and

pegylated

interferon-alpha-2a

inhibit

wild-type

and

resistant

genotype

1

hepatitis

C

virus

replication

in

patients,

Hepatology

46

(2007)

631–639.

[12]

C.

Sarrazin,

T.L.

Kieffer,

D.

Bartels,

B.

Hanzelka,

U.

Müh,

M.

Welker,

et

al.,

Dynamic

hepatitis

virus

genotype

and

phenotypic

changes

in

patients

treated

with

the

protease

inhibitor

telaprevir,

Gastroenterology

132

(2007)

1767–1777.