Professeur Patrice DEBREProfesseur Patrice DEBRELaboratoire d’Immunologie Cellulaire et Laboratoire d’Immunologie Cellulaire et TissulaireTissulaireINSERM U945INSERM U945

HHôpital Pitié-Salpêtrière; Parisôpital Pitié-Salpêtrière; Paris

"Vers un vaccin contre le SIDA : "Vers un vaccin contre le SIDA : rôle des cellules NK"rôle des cellules NK"

PathogenPathogen PathogenicityPathogenicity

Measles Hépatitis Polio Rabies

Diphtéria

Tétanos

Cholera

VaccinesVaccines

Vaccine

INFECTION

virus

CD4 infected cells

PATHOGENESIS

CD4 non infected cells

NK cells

PATHOGENESIS

NK cells

CD4 non infected cells

Kill

NK

CD4

+-NKp44

MHC-I NKp44L

NKp44L expression by CD4+ T cells from HIV-infected patients

% N

Kp

44

L

0

10

20

30

40

3- 3-4+ 4+8+ 8+

Control PBMC HIV+ PBMC CD4 / mm3

0 250 500 750 10000

10

20

30

40 p=0.002

% N

Kp

44

L

E/T ratio

0

10

20

30

40

100/150/1 25/112.5/1

44L+

44L-

% N

K L

ysis

Vieillard et al Proc Natl Acad Sci USA (2005)

0.1 1 10 100 1000

viral load(x 1000)

0

10

20

30

40

% N

Kp

44

L

p=0.005

NKp44L expression is induced by a specific gp41 motif

HR1 HR2

NH2

Fusion Peptide TM

517 532 558 595

643 678

3S motif3S motif

HIV-1 gp41 Env

3S : very conserved motif & spécific to HIV-1

0

10

20

30

50/1 25/1 12/1 6/1E/T ratio

+3S+3S

0

10

20

30

40

+3S 1 10 20

anti-3S (g/ml)%

NK

lysis

Vieillard et al Proc Natl Acad Sci USA (2005)Vieillard et al AIDS (2006)

NKp44L

2.7% 17.4%

3SUT

CD

4

Anti-3S Ab : a predictive value for the evolution of the disease ?

0

100

200

300

0 400 800 1200

Anti-3S (U/mL)

CD4 count/mm3

Multivariate study between 40 patients (first quartile) with a slope CD4>2.7/month and 40 patients (last quartile) with slope CD4<-6.9/month

Variable Odds-ratio IC95 p

Sexe 0.372 M 1 W 1.83 0.49-6.87Age (x10y) 1.30 0.76-2.23 0.334CD4 (x100) 0.58 0.42-0.81 0.001Log VL 1.17 0.55-2.51 0.682 Anti-3S Ab 2.89 1.6-5.17 <0.001 (x100)

Vieillard et al AIDS (2006)Unpublished data

1.3: Anti-3S Ab : a predictive value for the 1.3: Anti-3S Ab : a predictive value for the evolution of the disease ?evolution of the disease ?

Vieillard et al AIDS (2006)

363024181260

500

300

100

-100

-300

-500

Time since baseline (months)

Change in CD4 count from baseline

Anti-3S= NEGAnti-3S= NEG(7 CD4 / month)(7 CD4 / month)

Anti-3S = POSAnti-3S = POS

Anti-3SAnti-3S

HIV-1HIV-1

NKp44LNKp44L

CD4CD4 NKNKLysisLysis

Summary-1Summary-1

Conclusion-1Conclusion-1

- NKp44L is specifically expressed on CD4+ T cells from HIV-1 patients;

- NKp44L is induced par a specific peptide from the gp41 HIV-1 protein;

- Anti-3S antibodies are predictive of the evolution of the disease evolution.

No expression of NKp44L in CD4+ T cells infected by HIV-1 : A new escape mechanism

mediated by Nef

Fausther Bovendo et al (In press)

100 101 102 103 104

p24FITC

CD4/AD8.002

100 101 102 103 104

p24FITC

CD4/AD8.002

NKp44L

HIV-p24

HIV viruses

Relative p24+NKp44L+

NKp44L expression

100 101 102 103 1040

100

150

200

50

52.1%

UI

100 101 102 103 104

WT

71.3%

100 101 102 103 104

GAG

68.1%

100 101 102 103 1040

100

150

200

50

67.2%

POL

100 101 102 103 104

73.6%

gp120

100 101 102 103 104

gp4190.4%

104 101 102 103 104104100 101 102 103

11.7%

NEF60.2%

VIF

100100 101 102 1030

100

150

200

50

TAT

72.4%

Rec

ombi

nant

vac

cini

a vi

ruse

s

0

5

10

15

20

WT nef

*

p24+NKp44L+

(flod increased)

WT nef G2A P76/79A L168/169A

HIV Nef variants

0

5

10

15

20 * *

Effect of Nef mutants on NKp44L expression

0

10

20

30

40

50

60

70

100/1 50/1 25/1 12.5/1

E/T ratio

% NK lysis

Effect of Nef mutants on NK cytotoxicity

Conclusion-2Conclusion-2

- NKp44L is non-expressed on HIV-infected CD4+ T cells;

- Retention of NKp44L by Nef HIV protein;

However,

- The mechanism of Nef inhibition remains unknown.

3- Vaccine strategies : Macaque model3- Vaccine strategies : Macaque model« proof of concept »« proof of concept »

3.1 : Macaque model of SHIV infection3.1 : Macaque model of SHIV infection

3.2 : Preventive vaccination3.2 : Preventive vaccination

SHIV162P3-infected macaques model :Relationship between CD4 cell decreases,

NKp44L expression and anti-3S Ab.

Days post-infection

CD

4+ /

mL

1000

1500

2000

2500

2500

500

0 50 100 150 2000

5

10

15

20

25

% N

Kp

44

L

an

ti-3

S (

U /

mL)

0

50

100

150

0 50 100 150 200 2500

5

10

15

20

25

% N

Kp

44L

Vieillard et al AIDS (2008)

Proofs of concept : Immunization of macaques by the 3S peptide

KLH KLH-3S

CD

4 /

mm

3

0

500

1000

1500

2000

0 50 100 150 200

** *

Vir

al lo

ad

1

102

104

106

108

0 50 100 150 200

NS NS

Vieillard et al Proc Natl Acad Sci USA (2008)

0

10

20

30

40

50

0 50 100 150 200

% C

D4

+N

Kp

44

L+

0

500

1000

1500

-400 -200 0 200

an

ti-3

S (

U/m

L)

SHIV162P3

3S3S

3S3S

Days post-infection

Effect of 3S vaccination on CD4 apoptosis, NK activity et T cell-activation

% C

D4

+A

nn

exi n

V+

Days post-infection

0

10

20

30

40

50

UI 14 21 42 LN

*

**

*

**

% N

K lysis

Days post-infection

0

25

50

75

100

0 14 21 42 LN

**

*****

Vieillard et al Proc Natl Acad Sci USA (2008)

0 20 40 60 80

Days post-infection

% C

D4

+C

D6

9+

0

2.5

5

7.5

10

Conclusions1- Pathophysiology : role of NKp44L

- Depletion of non-infected CD4+ cells - In infected cells: Escape mechanism by Nef

2- Immunotherapic interventions

- Preventive vaccine : in macaque model, we have observed :

- high production of anti-3S Ab; - low expression of NKp44L on CD4 cells.

Stable level of the CD4 cells in absence of effect on the

viral load !

Pitié-Salpêtrière Hospital, Paris

France

Laboratory of Cellular and Tissular Immunology

Hugues Fausther Bovendo

Vincent Vieilllard

Patrice Debré

Clinical Epidemiology

Dominique Costagliola

Department of virology

Henry Agut

Daniel Candotti

Acknowlegments

CEA, Fontenay-aux-roses, France

Laboratory of Immuno-Virology

Roger Le Grand

Nathalie Bosquet

Pasteur Institute, Paris, France

Department of virology

Nathalie Sole-Foulon

Olivier Schwartz

Harvard University, Cambridge, MA

Jack Strominger