Rome, 7-9 June, 2017

Virological failure to Protease inhibitors

in Monotherapy is linked to the

presence of signature mutations in Gag

without changes in HIV-1 replication

Oscar Blanch-Lombarte

European Meeting on HIV & Hepatitis

Gag

p17 p24 p2 p7 p1 p6 Protease

Mutations Protease

Cleavage sites

Structural proteins

PI

Monotherapy Why do PIs fail?

CypA

binding

loop

Contribution of Gag mutations to PI susceptibility

Contribution of Gag mutations to PI susceptibility

AIM: to identify HIV-1 mutational patterns involved in

virological failure through the characterization of Gag

and protease genes

Gag

p17 p24 p2 p7 p1 p6 Protease

PI

Monotherapy

Why do patients with good adherence and treated with boosted PIs develop

virological failure to the treatment without mutations in the protease?

CSM

Structural proteins

Mutations Protease

Patients selection criteria

Selection criteria:

1) Initiation of PI monotherapy as ART-

simplification strategy with DRV/r or LPV/r

2) No previous PI-exposure known

3) Sustained virological suppression

(<50 copies/ml) for at least 6 months from

treatment initiation

Criteria of virological failure: Two viral

loads >50 copies/ml

520 patients who received LPV/r

or DRV/r monotherapy

511 Discarded

9 patients with VF on LPV/r

or DRV/r monotherapy

7 patients with one cross-sectional

sample in VF

2 patients with two

longitudinal samples in VF

7 plasma samples to amplify

full-length Gag-PR

4 plasma samples to amplify

full-length Gag-PR

5 Gag-PR

successfully amplified

2 Gag-PR

amplification failed

Study Subjects

4 Gag-PR

successfully amplified

Clinical follow-up and Sampling

Pt6 DRV/r

Pt1 LPV/r Pt2 LPV/r Pt4 DRV/r

Pt5 LPV/r Pt8 LPV/r

Pt9 LPV/r

Pt3 LPV/r

Pt7 LPV/r

CD

4+ T

-lym

phocyte

s/m

l V

iral lo

ad (R

NA

copie

s/m

l)

Weeks after treatment initiation

T1

T1 T1 T2

T2 T1

T1 T1 T1 T1

T1

Virological failure to DRV/r or LPV/r occurs in

absence of protease drug resistance mutations Gag

p17 p24 p2 p7 p1 p6 Protease

Protease

PatientsTime ARV

Resistance

associated

mutations

Other

mutations

Pt1 T1 LPV/r - K20T

Pt2 T1 LPV/r - -

T1 LPV/r - -

T2 LPV/r - -

T1 DRV/r - -

T2 DRV/r - A71V

Pt5 T1 LPV/r - L10V

Pt6 T1 DRV/r - -

Pt7 T1 LPV/r - -

Pt3

Pt4

Sequencing and direct comparison with HXB2

Patients Time ARV p17 p17/p24 p24 p24/p2 p2 p2/p7 p7 p7/p1 p1/p6 p6

Pt1 T1 LPV/r

E12D, R15K, K30Q, S67A,

R76K, L78V, R91K, K95R,

K103R, N126R

-A146P, S148T, V215M, T242N,

N252S, R286K, T303V, E312D- - -

Q386P,

K411R- -

S465F, Q476P,

K481Q, L483K,

Y484S, S498*

Pt2 T1 LPV/r

V7L, E12K, R15Q, K18R,

K28Q, K30R, I34L, L61I,

G62Q, R76K, T81A, T84V,

E93D, S111I

- E312D - N372G

S373P,

T375L,

M377L

I389T,

R406K- -

S465F, T471I,

E477D, P478S,

R490K

T1 R15K, K30R, E93D, K113Q -

V159I, S165N, Q199E, E203D,

I223V, G248A, N252S, E260D,

T280V, R286K, S310T, G357S

- V370A

A374S,

T375V,

I376M

I389T,

K411R- - T456S, S465L

T2 R15K, K30R, E93D, K113Q -

V159I, S165N, V168I, Q199E,

E203D, I223V, G248A, N252S,

E260D, T280V, R286K, S310T,

G357S

- V370A

A374S,

T375V,

I376M

I389T,

K411R- - T456S, S465L

T1

K28Q, E55G, R76K, E93D,

K95R, K113Q, T122P,

H124K

- L268M, R286K, A326S - -R380K,

N382H

R387K,

K388R- -

T456I, S465F,

E477G, I479T

T2

K28Q, E55G, G62E, R76K,

R91K, E93D, K95R, K113Q,

T122P, H124K

- L268M, R286K, A326S - -T375A

R380K,

N382H

R387K,

K388R- S451N

T456I, S465F,

E477G, I479T

Pt5 T1 LPV/r

E12K, R15K, K18R, K26N,

K30R, S54A, E55D, R58K,

G62E, Q69K, V82I, A83S,

Q90K, R91N, E93D, K95T,

E99A

-

S173T, E203D, V215M, M228I,

G248T, N252S, P255A, T280V,

E312D, A340G, M347S

- V370M I376P

I389T,

V390I,

T401L,

K411R,

R429K

R429K,

K436R-

T456S, E460A,

R464K, S465F,

T470A, I479P,

D480E

Pt6 T1 DRV/rE12K, R76K, R91G, K95R,

K103R, K110C, Q117L-

I147L, V159I, S173T, I223V,

G248A, N252S, T280V, R286K,

E312D, G357S

- V370AS373P,

M378V

I389T,

K418R,

D425E

- -

S465F, E477G,

P478T, L483I,

L486W

Pt7 T1 LPV/r D121N N131H A179T, A209T, T318K - - R380KR384G,

E398QI437V - S465F, R490K

Pt3 LPV/r

Pt4 DRV/r

p17 p24 p2 p7 p6

Gag

p17 p24 p2 p7 p1 p6 Protease

Presence of previously described Gag mutations

• We identified previously described mutations associated to resistance or

exposure of PIs in CSM and in Gag structural regions

p17 p24 p2 p7 p1 p6

K436Ra,b,c

(1%)

S451Na

(1%)

R76Ka,c

(55%) I389Ta

(44%)

V370Aa

(33%)

a Mutations associated with exposure to PI in vivo b Mutations associated with exposure to PI in vitro c Mutations associated with PI resistance

I437Va,b,c

(1%) E12Kb

(33%)

T81Aa,c

(1%)

CSM

Structural proteins

Mutations

Novel Gag mutations identified by VESPA

p17 p24 p2 p7 p1 p6

*

*** *

*

p<0.01 (*)

p<0.0001 (***)

Are those mutations generated by PI treatment or are mutations naturally as polymorphism?

• Mutations at positions K95R, E203D, V215M and R286K were statistically more

prevalent in PIs treatment patients than naïve sequences

2000 HIV-1 subtype B treatment

naïve sequences of Gag

(Los Alamos sequence database)

Methods

• Cloning of recombinant virus

cDNA Gag-PR

from our patients Recombinant

virus

3’ HIV

genome

P83.10-GFP

Electroporation

GFP

gene

Harvest virus

• Generation of Gag-protease viral stocks

• Replication kinetics

Pt4

Viral replication

(Slope)

8 10

6

4

364

1

7

2

6

5

9

3

7

2

5

1

8

66

10

100

6

7

100

74

1911

179

10

2

70

100

HXB2

Gag-Pol Consensus B4

1

2

3

7

9

10

8

5

6

100

7474

0.005

Mutations in HIV-1 Gag do not affect viral replication

*** ***

**

***

**

***

*** **

I54V and V82A in PR

(mutations that affect replication

as previously described)

p<0.001 (**)

p<0.0001 (***)

Drug susceptibility to LPV and DRV

T1 T1 T1 T1 T2 T2

Pt1 Pt3 Pt4 Pt6

LPV (nM)

T1 T1 T1 T1 T2 T2

Pt1 Pt3 Pt4 Pt6

DRV (nM)

Patients ARV

Pt3 LPV/r

Pt1 LPV/r

Pt4 DRV/r

Pt6 DRV/r

Take Home Messages

We identified previously described mutations in Gag associated to resistance

or exposure to PIs in patients with VF in monotherapy.

• Gag CSM K436R (1%), I437V (1%) and S451N (1%)

• Structural regions of Gag R76K (55%), I389T (44%), E12K (33%),

V370A (33%) and T81A (1%)

Although we have limited number of patients, VESPA provided a novel

signature pattern of mutations in Gag including residues K95R, E203D,

V215M and R286K.

Most of the virus in patients with VF in PI monotherapy preserved their

replication.

We identified recombinant virus with reduced susceptibility to LPV and DRV

treatment during VF.

Gag mutations were directly involved in PIs monotherapy in the absence of

protease resistance mutations. However, additional studies are needed to

identify the specific sites involved in PI resistance.

VIRus Immune Escape and VACcine Group

Oscar Blanch-Lombarte

Alba Ruiz

Esther Jiménez-Moyano

Ruth Peña

Marta Colomer

Clara Francés

Àuria Eritja

Julia G Prado P31

José Ramón Santos

Roger Paredes

Bonaventura Clotet

Duncan Njenda

Ujjwal Neogi