"Towards a Cure”:HIV Reservoirs and Strategies to Control ThemIAS WORKSHOP, 16 & 17 JULY 2010

Purging the HIV-1 Reservoir Through the Disruption of the PD-1 Pathway

Sandrina DA FONSECA

Vaccine and Gene Therapy Institute – FloridaLaboratoire d’Immunologie - INSERM U743 - Université de Montréal

IntroductionIA

S H

IV R

ES

ER

VO

IRS

WO

RK

SH

OP,

16

& 1

7 JU

LY 2

010,

VIE

NN

A

- In viremic donors, HIV infected cells do generally survive for long enough to revert back to a resting state. However, a small fraction of these cells enter into latency and constitute a stable latent reservoir for the virus.

- Small pool of TCM (central memory) and TTM (transitional memory) latently infected CD4+T cells that persist in patients receiving HAART: main obstacle to HIV-1 eradication (N. Chomont et al, Nat. Med, 2009).

- Development of targeted strategies aimed at purging these reservoir cells needed.- Mechanisms implicated in the establishment and persistence of the HIV reservoir: not

fully understood. - Factors that interfere with CD4+T cells activation and proliferation: major impact on

the establishment and/or the maintenance of the HIV-1 reservoir ?

- HIV specific CD4+T cells are preferentially infected (DC. Douek et al, Nature, 2002) and express high levels of PD-1(CL. Day et al, Nature, 2006, L. Trautmann et al, Nat. Med, 2006). Can PD-1 play a role in the establishment of latency by repressing HIV transcription, leading to a small pool of quiescent, latently infected cells ?

Hypothesis:Role for PD-1 in the establishment and/or maintenance of a cellular reservoir for

HIV?

Part 1

A role for PD-1 in the ESTABLISHMENT of a reservoir

The establishment of a stable reservoir for HIV necessitates the establishment of viral latency = inhibition of viral production.

Does PD-1 triggering inhibit viral production in HIV infected primary CD4+T cells?

Triggering of the PD-1 pathway inhibits 95% of HIV-1 production in primary CD4+T cells

at day 3

IAS

HIV

RE

SE

RV

OIR

S W

OR

KS

HO

P, 1

6 &

17

JULY

201

0, V

IEN

NA

CD3/CD28 + IgG2CD3/CD28 + PD-L1

NS

The negative signal conferred by the PD-1/PD-L1 interaction inhibits viral production in primary CD4+T cells.

- CD4+T cells from HIV infected viremic subjects purification- Stimulated with

CD3/CD28 in the presence or absence of PD-L1.

- Viral production measured by p24 ELISA.

p24

(pg/

ml)

Time (d) Time (d)

p24

(pg/

ml)

0 3 6 91

10

100

1000

10000

100000

1000000 Donor BDonor A

0 3 6 91

10

100

1000

10000

0 3 6 90.1

1

10

100

1000

0 3 6 91

10

100

1000

10000

100000

1000000

Donor C Donor D

Ultrasensitive assay to measure viral productionIA

S H

IV R

ES

ER

VO

IRS

WO

RK

SH

OP,

16

& 1

7 JU

LY 2

010,

VIE

NN

A

1-5x106 CD4+T cells per well

PBMCs

HIV infected donor

Supernatant

Viral pellet

Viral RNA

Ultrasensitive RT-PCR

Centrifugation 17000 rpm, 30’ at 4°C

RNA extraction

DNase treatment

Blood : leukapheresis

Ficoll gradient

Stimulation CD3/CD28 +/- PD-L124 hours incubation at 37°C

Assay sensitivity : 1 RNA copy/mL

Triggering of the PD-1 pathway inhibits 60% of HIV-1 production in primary CD4+T cells

after 24h of stimulation

IAS

HIV

RE

SE

RV

OIR

S W

OR

KS

HO

P, 1

6 &

17

JULY

201

0, V

IEN

NA

Very short term effect of PD-1 triggering on HIV viral production. PD-1 triggering may act directly on the LTR.

Non stimu-lated

CD3/CD28 + IgG2

CD3/CD28 + PD-L1

0

20000

40000

60000

80000

100000

120000

Non stimu-lated

CD3/CD28 + IgG2 beads

CD3/CD28 + PD-L1 beads

0

500

1000

1500

2000

2500

3000

3500

4000

0

2

4

6

8

10

12

Non st

imula

ted

CD3/CD28

+ IgG2

CD3/CD28

+ PD-L1

0

2

4

6

8

10

12

Non st

imula

ted

CD3/CD28

+ IgG2

CD3/CD28

+ PD-L1

0

2

4

6

8

10

12

Non st

imula

ted

CD3/CD28

+ IgG2

CD3/CD28

+ PD-L1

0

2

4

6

8

10

12

Donor A Donor B Donor C

Donor D Donor E Donor F

HIV

RN

A co

pies

HIV

RN

A co

pies

- HAART naïve chronically HIV-infected subjects → Total CD4+T cells negative selection- TCR triggering (CD3/CD28) with or without co-triggering of the PD-1 pathway with an Ig-PD-L1 chimera. - Viruses pellet + Viral RNA extraction and quantification by QRT-PCR

Triggering of the PD-1 pathway inhibits 45% of HIV-1 production in primary CD4+T cells

at day 3 in the presence of ARV

IAS

HIV

RE

SE

RV

OIR

S W

OR

KS

HO

P, 1

6 &

17

JULY

201

0, V

IEN

NA

- The inhibition mediated by PD-L1 is still observed in the presence of ARV

- PD-1 directly impacts on viral production

- CD4+T cells from HIV infected viremic subjects purification- Stimulated with CD3/CD28 in the presence or

absence of PD-L1, and ARV- Viral production measured by p24 ELISA.

CD3/CD28 + IgG2CD3/CD28 + PD-L1

NS

Donor A

Donor B0 3 6 9

0

20

40

60

80

100

120

140

160

180

200

0 3 6 90

20

40

60

80

100

120

140

160

180

p24

(pg/

ml)

p24

(pg/

ml)

Time (d)

Inhibition restricted to PD-1high cellsIA

S H

IV R

ES

ER

VO

IRS

WO

RK

SH

OP,

16

& 1

7 JU

LY 2

010,

VIE

NN

A

Inhibition of viral production was exclusively observed in PD-1 high cells indicating the specificity of this pathway

p24

(pg/

mL)

p24

(pg/

mL)

0,0

100,0

200,0

300,0

400,0

0,0

200,0

400,0

600,0

800,0

1000,0

1200,0

PD-1 High CD4+T cells PD-1 Low CD4+T cells

CD3/CD28+PD-L1

CD3/CD28+IgG2

NS CD3/CD28+PD-L1

CD3/CD28+IgG2

NS

- Total CD4+T cells form viremic HIV infected patients isolation- PD-1high and PD-1low subsets highly purified by cell sorting- Stimulation with different combination of triggering antibodies (CD3/CD28 with PD-L1 or the

corresponding IgG isotype).

Part 2

A role for PD-1 in the MAINTENANCE of a reservoir

Sorted PD-1high cells preferentially harbor HIV-1 integrated DNA when compared to their

PD-1low counterparts

IAS

HIV

RE

SE

RV

OIR

S W

OR

KS

HO

P, 1

6 &

17

JULY

201

0, V

IEN

NA

PD-1 expression correlates with the reservoir size. Impact of PD-1 signaling on the HIV reservoir?

Inte

grat

ed H

IV D

NA

copi

es

per 1

06 CD

4 T

cells

% PD-1+ CD4 T cells 0 10 20 30

1

10

100

1000

10000

= 0.45p = 0.01

0

100

200

300

400

500

Cm Tm Em

PD-1Hi CD4 T cellsPD-1Lo CD4 T cells

HIV

DN

A co

pies

per

106 c

ellsSorted PD-1 high cells are enriched in total and integrated HIV DNA compared to sorted PD-1 low cells : PD-1 high cells constitute a preferential reservoir for the virus.

The disruption of the PD-1/PD-L1 interaction enhances the spontaneous release of HIV-1

virions by CD4+T cells

IAS

HIV

RE

SE

RV

OIR

S W

OR

KS

HO

P, 1

6 &

17

JULY

201

0, V

IEN

NA

HIV production is induced after blocking the PD-1 pathway suggesting a role for PD-1 in the maintenance of HIV latency.

Donor A Donor B Donor C

Mock

a-PD-1 blocking Ab

Isotype control200

400

600

0

p24

(pg/

mL)

If PD-1 triggering inhibits viral production, blocking the PD-1/PD-L1 interaction should increase viral production.

- CD4+T cells isolated from HIV infected subjects - CD4+T cells incubated with an anti-PD-1 antibody that prevents the interaction between PD-1

and its ligands

- PD-1+ Central and Transitional memory CD4+T cells are enriched for HIV integrated DNA

- PD-1 receptor may be used as a specific marker to target HIV-1 reservoir cells

- PD-1 receptor triggering inhibits viral production. Role in the establishment of a reservoir?

- Blocking PD-1/PD-L1 interaction induces viral production. Role in the maintenance of a reservoir?

Can we purge the HIV reservoir by disrupting the PD-1 negative pathway in HAART individuals?

ConclusionIA

S H

IV R

ES

ER

VO

IRS

WO

RK

SH

OP,

16

& 1

7 JU

LY 2

010,

VIE

NN

A

"Towards a Cure”:HIV Reservoirs and Strategies to Control ThemIAS WORKSHOP, 16 & 17 JULY 2010

Acknowledgments

Vaccine and Gene Therapy Institute, FloridaUniversité de Montréal, CHUM, INSERM U743Nicolas ChomontRafick-Pierre Sékaly

Université de Montréal, CHUM, INSERM U743Mohamed El Far

Royal Victoria Hospital, Mc Gill UniversityMohamed-Rachid BoulasselJean-Pierre Routy