Professeur Patrice DEBRE Laboratoire d’Immunologie Cellulaire et Tissulaire INSERM U945

1. "Vers un vaccin contre le SIDA : rôle des cellules NK" Professeur Patrice DEBRE Laboratoire d’Immunologie Cellulaire et Tissulaire INSERM U945 Hôpital Pitié-Salpêtrière; Paris

2. Pathogenicity Pathogen Measles Hépatitis Polio Rabies Diphtéria Tétanos Cholera Vaccines

3. INFECTION virus CD4 infected cells PATHOGENESIS CD4 non infected cells NK cells Vaccine  PATHOGENESIS  CD4 non infected cells  NK cells

4. Kill 40 30 20 CD4 10 0 NKp44L MHC-I 40 NKp44 44L+ - + 30 % NK Lysis 20 44L- NK 10 0 100/1 50/1 25/1 12.5/1 E/T ratio NKp44L expression by CD4+ T cells from HIV-infected patients 40 p=0.002 30 % NKp44L % NKp44L 20 10 0 3- 4+ 8+ 4+ 3- 8+ 0 250 500 1000 750 Control PBMC HIV+ PBMC CD4 / mm3 40 p=0.005 30 % NKp44L 20 10 0 0.1 10 100 1 1000 viral load(x 1000) Vieillard et al Proc Natl Acad Sci USA (2005)

5. 30 20 10 0 NKp44L expression is induced by a specific gp41 motif +3S UT 3S CD4 2.7% 17.4% % NK lysis 50/1 25/1 12/1 6/1 E/T ratio 40 NKp44L 30 20 HIV-1 gp41 Env 10 Fusion Peptide 0 HR1 HR2 TM +3S 1 10 20 NH2 517 532 643 678 558 595 anti-3S (mg/ml) 3S motif 3S : very conserved motif & spécific to HIV-1 Vieillard et al Proc Natl Acad Sci USA (2005) Vieillard et al AIDS (2006)

6. Anti-3S Ab : a predictive value for the evolution of the disease ? 300 200 Anti-3S (U/mL) 100 0 0 400 800 1200 CD4 count/mm3 Multivariate studybetween 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 0.49-6.87 Age (x10y)1.30 0.76-2.23 0.334 CD4 (x100)0.58 0.42-0.81 0.001 Log VL1.17 0.55-2.51 0.682 Anti-3S Ab2.891.6-5.17 <0.001 (x100) Vieillard et al AIDS (2006) Unpublished data

7. 500 300 100 Change in CD4 count from baseline Anti-3S = POS -100 Anti-3S= NEG (7 CD4 / month) -300 -500 0 6 12 18 24 30 36 Time since baseline (months) Vieillard et al AIDS (2006) 1.3: Anti-3S Ab : a predictive value for the evolution of the disease ?

8. Anti-3S NK Lysis HIV-1 CD4 NKp44L Summary-1

9. Conclusion-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.

10. No expression of NKp44L in CD4+ T cells infected by HIV-1 : A new escape mechanism mediated by Nef 200 UI WT GAG 150 68.1% 52.1% 71.3% 100 50 0 104 103 102 100 101 104 101 100 103 100 102 101 102 104 103 200 POL gp120 gp41 150 90.4% 73.6% 67.2% 100 50 * 20 0 101 102 100 103 104 100 103 104 100 101 101 103 104 102 102 15 10 5 0 WT Dnef NKp44L HIV-p24 Recombinant vaccinia viruses VIF NEF 200 TAT 60.2% 11.7% 150 72.4% Relative p24+NKp44L+ 100 50 0 100 104 104 101 103 100 102 104 100 101 101 103 102 102 103 NKp44L expression Fausther Bovendo et al (In press) HIVviruses

11. * * 20 15 10 p24+NKp44L+ (flod increased) 5 0 P76/79A WT Dnef G2A L168/169A HIV Nef variants Effect of Nef mutants on NKp44L expression

12. Effect of Nef mutants on NK cytotoxicity 70 60 50 40 % NK lysis 30 20 10 0 100/1 50/1 25/1 12.5/1 E/T ratio

13. Conclusion-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.

14. 3- Vaccine strategies : Macaque model « proof of concept » 3.1 : Macaque model of SHIV infection 3.2 : Preventive vaccination

15. 2500 25 150 25 2000 20 20 100 1500 15 15 CD4+ /mL anti-3S (U /mL) % NKp44L 1000 10 % NKp44L 10 50 500 5 5 0 0 0 0 0 50 100 150 200 250 0 50 100 150 200 250 SHIV162P3-infected macaques model : Relationship between CD4 cell decreases, NKp44L expression and anti-3S Ab. Days post-infection Vieillard et alAIDS (2008)

16. SHIV162P3 3S 1500 NS NS 3S 108 3S 106 1000 3S anti-3S (U/mL) 104 500 102 0 1 -400 200 -200 0 0 50 100 150 200 50 2000 ** * 40 1500 30 % CD4+NKp44L+ 1000 20 500 10 0 0 0 50 100 150 200 0 50 100 150 200 Proofs of concept : Immunization of macaques by the 3S peptide Viral load CD4 / mm3 Days post-infection KLH KLH-3S Vieillard et al Proc Natl Acad Sci USA (2008)

17. Effect of 3S vaccination on CD4 apoptosis, NK activity et T cell-activation 10 ** 50 7.5 40 ** 5 30 * * 20 2.5 10 0 0 % CD4+CD69+ UI 14 21 42 LN Days post-infection 100 ** ** ** * 75 0 20 40 60 80 Days post-infection 50 25 0 0 14 21 42 LN % CD4+AnnexinV+ % NK lysis Days post-infection Vieillard et al Proc Natl Acad Sci USA (2008)

18. Conclusions 1- 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 !

19. Acknowlegments 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 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