1. T-cell HIV Vaccines Giuseppe Pantaleo, M.D. Professor of Medicine Director, Swiss Vaccine Research Institute Lausanne, Switzerland

2. Challenges In The Development Of An HIV Vaccine  Identification of immune correlates of protection  Induction of a broad neutralizing antibody response  Induction of effective T cell responses  Viral diversity  Viral escape  Super-infection  Influence of genetic background  Induction of mucosal and systemic immunity  Complexity of immunization regimens

3. Vaccine Concepts and Designs Live Vectors DNA Combination of Elements Peptide Epitopes (limited immunogenicity) Pseudovirions (pre-clinical) Live-Attenuated (Not Under Study in Humans) Whole-Killed (Not Under Study in Humans) Recombinant Viral Proteins

4. Due to the Lack of Protein-Based Env Vaccines Able To Induce Neutralizing Antibodies, Candidate HIV Vaccines Have Been Designed Primarily to Induce T-Cell Immune Responses

5. T-cell Vaccines Do Not Prevent Infection But They Will Be Eventually Associated with Control of Virus Replication and Prevention of HIV-Associated Disease T-cell HIV Vaccines Concept

6. T-Cell HIV Vaccine Concept Help CTL  Vaccine ----Virus Placebo  Virus exposure

7. Rationale for the Effectiveness of HIV T-Cell Vaccines - Certain experimental vaccines confer protection in monkeys infected with SIV - There is evidence that HIV-1-specific T-cell responses may confer protection (e.g. exposed non-infected subjects) - A small percentage (<5%) of HIV-1-infected subjects show no signs of disease progression (e.g. long-term nonprogressors) - A decrease in viral load is associated with clinical benefit

8. Low responderHigh responder Effective immunity induced by rAd5 HIV (SIV) vaccine in a SIV challenge model

9. Interpretation This cell-mediated immunity vaccine did not prevent HIV-1 infection or reduce early viral level. Mechanisms for insufficient efficacy of the vaccine and the increased HIV-1 infection rates in subgroups of vaccine recipients are being explored.

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12. Interpretation Consistent with previous trials, the MRKAd5 HIV-1 gag/pol/nefvaccine was highly immunogenic for inducing HIV-specific CD8+ T cells. Our findings suggest that future candidate vaccines have to elicit responses that either exceed in magnitude or differ in breadth or function from those recorded in this trial.

13. Messages from the Step Study The Step study marks the end of the HIV T-cell vaccine concept (at least of the Ad5 vector used alone) It draws the attention on the potential danger of pre-existing immunity It shows a potential sinergy between Ad5 pre- existing immunity and circumcision with regard to the observed increased in HIV acquisition It indicates that the magnitude and the quality (breadth) of the vaccine-induced T-cell responses (particularly CD8 T-cell responses) are not optimal

14. CONCLUSIONS This ALVAC-HIV and AIDSVAX B/E vaccine regimen may reduce the risk of HIV infection in a community-based population with largely heterosexual risk. Vaccination did not affect the viral load or CD4+ count in subjects with HIV infection. Although the results show only a modest benefit, they offer insight for future research. (ClinicalTrials.gov number, NCT00223080.)

15. Major Messages from the RV-144 Study The vaccine combination is potentially more effective (61% reduction of HIV acquisition at 1 year post vaccination) The vaccine protective effect is waning over time (31.2% at 3 years post-vaccination) The delta in the reduction of the number of infections is 18 (30 in the placebo vs. 12 in the vaccine group) at 1 year and 23 (74 in the placebo vs. 51 in the vaccine group) at 3 years Therefore, most of the protective effect is gained during the 1 st year post-vaccination

16. RV-144 Results and Immunological Issues The limited set of immunological results of the RV-144 trial do not allow to rule out or to favor any specific cellular or humoral mechanism of protection. In this regard: The same protein component of the vaccine combination was not associated to protection when used alone in the Vaxgen phase III trial. However, due to differences in the target population, i.e. high risk IVDU population in the Vaxgen trial versus low risk in the RV-144 trial, the two studies cannot be compared. The poxvirus component of the vaccine, i.e. ALVAC, was never previously tested in phase III trial.

17. RV-144 Results and Immunological Issues The results of the RV-144 trial re-underscore a number of immunological issues that have been repetitively raised in the recent past but have never been concretely addressed. These include: the need of developing an integrated vaccine-induced immune response (innate plus humoral plus adaptive) the exploration of additional mechanisms of protection beyond the conventional CD4 and cytotoxic CD8 T-cell and neutralizing antibody responses

18. What Is Next?

19. Improvement of the Current Vaccine Combination Augment (above the 61% efficacy observed at 1 year post-infection) the overall protection from infection Induce durable protection Improve both components of the vaccine, i.e. the priming component (ALVAC) and the boosting component (the Env protein)

20. 20 What Is Next? Vaccine candidates Poxvirus-based vectors - NYVAC - MVA - ALVAC Adenovirus - Ad26 - Ad35 DNA vectors Protein - gp140 monomer or trimer

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25. DNA-C + NYVAC-C Platform Harari et al. JEM, 2008; Bart et al., Vaccine, 2008; McCormack, Vaccine, 2008 Supported by the European Union, EuroVacc, ANRS and CAVD

26. TVDC Poxvirus T Cell Vaccine Discovery Consortium Clinical Trial Design  Randomized trial with a parallel group design  Open to the participants and investigators but blind to laboratory personnel  Attendance to clinics at least 14 occasions over 72 weeks 0482024284872 Weeks Group 1 (n=74) Group 2 (n=73) DNA-C (4 mg) priming at week 0, 4 and 8 for group 1 at week 0 and 4 for group 2 NYVAC-C (10 7.5 PFUs) boosting at week 24 for group 1 and at week 20 and 24 for group 2

27. TVDC Poxvirus T Cell Vaccine Discovery Consortium Proportion of Responders at Primary Endpoints (Week 26/28) ITT Analysis 3 x DNA n = 70 2 x DNA n = 70 Total n = 140 Response64 (91%)56 (80%)120 (86%) PP Analysis 3 x DNA n = 67 2 x DNA n = 68 Total n = 135 Response63 (94%)55 (81%)118 (87%) Chi 2 Test: p = 0.053; Risk difference: 11.4% (95% CI 0.0 – 22.9%) Chi 2 Test: p = 0.021; Risk difference: 13.1% (95% CI 2.2 – 24.1%)

28. TVDC Poxvirus T Cell Vaccine Discovery Consortium Proportion of Responders Over Time

29. TVDC Poxvirus T Cell Vaccine Discovery Consortium Magnitude of IFN-  ELISpot Responses at Week 26/28 Overall (SFUs/10 6 cells) Week 26Week 28 ITT Analysis 3 x DNA n=58 2 x DNA n=50 3 x DNA n=61 2 x DNA n=54 Mean (SD) Median (IQR; range) 774 (622) 545 (340-1101; 75-3454) 398 (318) 328 (178-488; 63-1514) 597 (519) 445 (170-855; 88- 2773) 357 (319) 235 (123-505; 60-1326) Mann- Whitney test p<0.001 Note:Sum of SFU/Mio cells from all peptide pools with a positive response per participant

30. TVDC Poxvirus T Cell Vaccine Discovery Consortium 30 Magnitude of IFN-  ELISpot Responses at Week 26/28 (ITT) 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 ENVOtherENVOtherENVOtherENVOther 3 x DNA2 x DNA SFUs/Mio cells Week 28Week 26Week 28Week 26 Note:Magnitude statistically different between groups for Env at both weeks but not for Gag/Pol/Nef (Other)

31. TVDC Poxvirus T Cell Vaccine Discovery Consortium Functional Profile of HIV-Specific T-Cell Responses Neg Env 1 Env 2 Subject#1042 Gr#1 010 2 3 4 5 0 10 2 3 4 5 0.033 2.99e-3 1.2e-3 100 010 2 3 4 5 0 10 2 3 4 5 0.013 2.39e-3 1.79e-3100 010 2 3 4 5 0 2 3 4 5 0.05 0.019 1.65e-399.9 010 2 3 4 5 0 2 3 4 5 0.031 0.017 3.71e-399.9 010 2 3 4 5 0 2 3 4 5 0.0690.13 7.13e-399.8 010 2 3 4 5 0 2 3 4 5 0.040.11 0.03299.8 IL-2 IFN-  TNF-α CD4 T-cell responses 010 2 3 4 5 0 2 3 4 5 0.036 0.013 0 100 010 2 3 4 5 0 2 3 4 5 8.5e-3 0 100 010 2 3 4 5 0 2 3 4 5 0.093 0.22 4.18e-399.7 010 2 3 4 5 0 2 3 4 5 0.019 0.077 0.12 99.8 010 2 3 4 5 0 2 3 4 5 0.0670.027 3.54e-399.9 010 2 3 4 5 0 2 3 4 5 0.0162.66e-3 0.017100 IL-2 TNF-α IFN-  CD8 T-cell responses

32. TVDC Poxvirus T Cell Vaccine Discovery Consortium 0 0.1 0.2 0.3 0.4 IFNg IL-2 TNFa + + + + + - + - + + - - - + + - + - - - + 0 0.1 0.2 0.3 0.4 IFNg IL-2 TNFa + + + + + - + - + + - - - + + - + - - - + Gr#1 Gr#2 Gr#1 Gr#2 Gr#1Gr#2Gr#1Gr#2 Frequency of CD4 T-cellsFrequency of CD8 T-cells CD4 T-cell responsesCD8 T-cell responses Functional Profile of HIV-Specific T-Cell Responses

33. TVDC Poxvirus T Cell Vaccine Discovery Consortium Number of Pools Recognized P=0.02 0 2 4 6 8 Number of responses per subject CD4 T cellsCD8 T cellsCD4 T cellsCD8 T cells Gr1 N=14 Gr2 N=11 Median

34. TVDC Poxvirus T Cell Vaccine Discovery Consortium Distribution of HIV Regions Targeted By CD4 T-Cell Responses Gr#1 N=44 responses Gr#2 N=28 responses GAG POL NEF ENV Number of responses ENV Gag, Pol or Nef P=0.06 Gr#1 Gr#2

35. TVDC Poxvirus T Cell Vaccine Discovery Consortium Distribution of HIV Regions Targeted By CD8 T-Cell Responses Gr#1 N=22 responses Gr#2 N=8 responses Number of responses ENVGag, Pol or Nef P=0.01 GAG POL NEF ENV Gr#1 Gr#2

36. TVDC Poxvirus T Cell Vaccine Discovery Consortium Total Magnitude of T-Cell Responses (Sum of Responding Pools) CD4 T cellsCD8 T cellsCD4 T cellsCD8 T cells 0.0 0.5 1.0 1.5 2.0 Percentage of T-cells Gr1 N=14 Gr2 N=11

37. TVDC Poxvirus T Cell Vaccine Discovery Consortium EV03/ANRS VAC20: NYVAC- and HIV-Specific CD4 and CD8 T-Cell Responses in the Gut 010 2 3 4 5 0 3 4 5 0.87 010 2 3 4 5 0 3 4 5 11.9 010 2 3 4 5 0 3 4 5 6.42 010 2 3 4 5 0 3 4 5 0.52 010 2 3 4 5 0 3 4 5 10.1 010 2 3 4 5 0 3 4 5 1.13 unstimulatedNYVACHIV Blood Gut Gated on CD3+CD4+ CFSE CD4 010 2 3 4 5 0 3 4 5 0.34 010 2 3 4 5 0 3 4 5 24 010 2 3 4 5 0 3 4 5 10.6 010 2 3 4 5 0 3 4 5 0.5 010 2 3 4 5 0 3 4 5 4.85 010 2 3 4 5 0 3 4 5 0.57 Gated on CD3+CD8+ unstimulatedNYVACHIV Blood Gut CFSE CD8

38. 38 What Is Next? Potential vaccine commbinations (with novel env protein candidates) Poxvirus-based vaccine combinations - NYVAC (2X or 4X) plus gp120/140 (2X or multiple) - DNA (3X) plus NYVAC (1X) plus gp120/140 (2X or multiple) - ALVAC (4X) plus gp120/140 (2X or multiple) Adenovirus-based vaccine combinations - Ad26 (1X) plus Ad35 (1X) plus gp120/140 (2X or multiple) - Ad26 (2X) plus MVA (1X) plus gp120/140 (2X or multiple)

39. TVDC Current and Future Poxvirus Vectors Portfolio 571 passages In CEF cells Deletion of 18 ORFs 200 passages in CEF cells Vaccinia Virus Ankara (MVA) Vaccinia Virus Copenhagen Canarypox virus MVANYVACALVAC Gene deletion Combined NYVAC Rc Plus Gene deletion NYVAC Attenuated Rc NYVAC KC Reinsertion of K1L & C7L Gene deletion mutants

40. 40 Conclusions T-cell vaccines remain an important component of the overall HIV vaccine strategy They serve as the priming component in combination regimens with env proteins It is conceivable that improved T-cell vaccines may substantially impact the magnitude, quality and durability of the antibody response induced by env protein vaccines The Step and RV-144 efficacy trials have indicated that the current NHPs challenge model is not suitable for the prediction of the clinical efficacy of vaccine candidates in humans The evaluation of improved vaccine combinations in efficacy clinical trials is the only strategy for the correct evaluation of the vaccine effectiveness

41. Acknowledgements Vaccine Immunotherapy Centre Div. of Immunology and Allergy CHUV – Lausanne, Switzerland Pierre-Alexandre Bart Erika Castro David Bonnet Kim Ellefsen-Lavoie Alexandre Harari St. Mary Hospital, Imperial College London, UK Jonathan Weber Rebecca Chandler Lucy Garvey Ken Legg Ngaire Latch University of Regensburg, Germany Bernd Salzberger Ralf Wagner Hans Wolf Birgit Fritsch Falitsa Mandraka Gabriele Birkenfeld Caspar Franzen Josef Köstler ANRS Jean-Francois Delfraissy Yves Levy Anne de Saunière Véronique Rieux EuroVacc Foundation Song Ding Sanofi Pasteur, France Jim Tartaglia Claude Meric Collaboration for AIDS Vaccine Discovery (CAVD) Nina Russell Jose Esparza European Commission EuroVacc Foundation ANRS CAVD & All Study Volunteers Cochin, Paris, France Odile Launay Pierre Loulergue Yvette Henin Henri Mondor, Paris, France Yves Lévy Jean-Daniel Lelièvre Christine Lacabaratz Tenon, France Gilles Pialoux Marseille, France Isabelle Poizot-Martin Catherine Farnarier Toulouse, France Lise Cuzin Florence Nicot INSERM CTU U897, France Genevieve Chene Philippe Reboud Inga Tschöpe Carine Grondin Valérie Boilet MRC CTU, London, UK Sheena McCormack Abdel Babiker Wolfgang Stöhr Liz Brodnicki Patrick Kelleher Mary Rauchenberger Shabana Khan

42. TVDC Poxvirus T Cell Vaccine Discovery Consortium Proportion of Responders at Week 26/28 per Peptide Group ITT Analysis 3 x DNA n = 70 2 x DNA n = 70 Total n = 140 Env63/70 (90%)54/69 (78%)117/139 (84%) Gag/Pol/Nef27/70 (39%)17/70 (24%)44/140 (31%) PP Analysis 3 x DNA n = 67 2 x DNA n = 68 Total n = 135 Env62/67 (93%)53/67 (79%)115/134 (86%) Gag/Pol/Nef26/67 (39%)17/68 (25%)43/135 (32%)

43. TVDC Poxvirus T Cell Vaccine Discovery Consortium Primary Immunogenicity Endpoints ITT Analysis 3 x DNA n = 70 2 x DNA n = 69 Total n = 139 Response 26 (37%)15 (22%)41 (30%) PP Analysis 3 x DNA n = 67 2 x DNA n = 67 Total n = 134 Response25 (37%)15 (22%)40 (30%) Chi 2 Test: p = 0.047; Risk difference:15.4%(95% CI 0.5 – 30.3%) Risk atio: 1.7(95% CI 1.0 – 2.9) Chi 2 Test: p = 0.059; Risk difference:14.9%(95% CI -0.3 – 30.2%) Risk ratio: 1.7(95% CI 1.0 – 2.9)

44. TVDC Poxvirus T Cell Vaccine Discovery Consortium Magnitude of IFN-  ELISpot Responses at Week 26/28 by Peptide Group (median (IQR) SFUs/10 6 cells) Week 26Week 28 ITT analysis 3 x DNA n=21 2 x DNA n=13 3 x DNA n=21 2 x DNA n=13 Gag/Pol/Nef 180 (120-331)109 (85-174)145 (83-229)113 (83-218) p=0.12P=0.82 Week 26Week 28 ITT analysis 3 x DNA n=58 2 x DNA n=48 3 x DNA n=59 2 x DNA n=54 Env 539 (315-1013)294 (182-496)442 (170-833)217 (123-488) p<0.001p=0.003

45. Challenges in HIV Vaccine Development 30 commercially available effective vaccines 16 vaccine derived from live replcation competent attenuated pathogens 12 vaccine derived from pathogen modifications Only 2 vaccines, HPV and HBV derived from synthetic products