Alternative Serotype Adenovirus Vectors. Dan Barouch September 29, 2010

Alternative Serotype Adenovirus Vectors Dan Barouch September 29, 2010

Desired Features of a Next Generation HIV-1 Vaccine Candidate The RV144 study suggests that the development of a safe and effective HIV-1 vaccine is indeed possible Improved vaccine regimens and immunogens will likely be required Key features that would be desired in a next generation HIV-1 vaccine include: Vectors that avoid high levels of vector-specific NAbs and can be combined into a heterologous prime-boost regimen Antigens that elicit both humoral and cellular immunity and that optimize immunologic coverage of global virus diversity

Adenovirus Serotypes 26 and 35 Ad26 and Ad35 were selected for clinical evaluation following preclinical studies of a series of group B and D Ad serotypes Ad26 and Ad35 are biologically substantially different than Ad5 Baseline NAb titers (Abbink et al. J. Virol. 2007; 81:4654-4663; Mast et al. Vaccine 2010; 28:950-957) Cellular receptors (Abbink et al. J. Virol. 2007; 81:4654-4663) In vivo tropism (Waddington et al. Cell 2008; 132:397-409) Innate immune profile (Barouch et al., unpublished data) Adaptive immune phenotype (Liu et al. J. Virol. 2008; 82:4844-4852; Ahmed et al., unpublished data) Protective efficacy against SIV (Liu et al. Nature 2009; 457:87-91)

International Ad Seroepidemiology: N=2,830 Subjects in North/South America, Africa, Asia High Ad5 NAb titers have been shown to suppress the immunogenicity of Ad5 vaccine vectors Ultimate vaccine target populations Infants (N=149; South Africa) Schoolchildren (N=185; South Africa) Initial clinical trial populations Adults (N=199; Africa, low-risk community) Adults (N=263; Kenya, high-risk IAVI 01B) Adults (N=242; Uganda, low-risk RV172) Adults (N=438; Thailand, high-risk VAX003) Adults (N=222; United States, low-risk IPCAVD001) Adults (N=727; United States, high-risk VAX004) Adults (N=405; North/South America, high-risk STEP) Adults in Rwanda and South Africa pending

Baseline Ad26 and Ad35 NAb Titers Are Substantially Lower than Ad5 NAb Titers in Multiple International Human Populations (N=2,830)

Ad NAb Titers in Highest Seroprevalent Regions: High-Risk Populations in Kenya and Thailand Kenya (N=263; IAVI 01B) Thailand (N=438; VAX 003) 100000 100000 Ad NAb Titer 10000 1000 100 Ad NAb Titer 10000 1000 100 10 Ad5 Ad26 Ad35 10 Ad5 Ad26 Ad35

Ad NAb Titers in High-Risk Populations in South Africa: Interim Analysis of 240 of 1,500 Samples South Africa (N=240; MIRA) 100000 Ad NAb Titer 10000 1000 100 Titer Ad5 Ad26 Ad35 <18 9 51 89 18-200 26 43 10 200-1000 40 5 2 >1000 25 1 0 10 Ad5 Ad26 Ad35

No Suppression of 4x10 10 vp Ad26-Gag/Pol/Env/Nef in Rhesus Monkeys Pre-Immunized with Ad26-Empty (Baseline Ad26 NAb Titers 18-200) 1200 SFC / 10^6 PBMC 1000 800 600 400 200 0 Ad26 NAb Titer 253 265 380 381 383 385 326 459 474 182 218 316 87 191 116 141 34 41 <18 <18 <18 <18 <18 <18 Low Ad26 NAb (18-200) Naïve Gag Pol1 Pol2 Env1 Env2 Nef IFN-γ ELISPOT Responses at Week 2 Following Immunization

V520-018: MRKAd5 gag ELISPOT (SFC/10 6 PBMC) vs. Baseline Ad5 Titer by Dose Level (North/South America, Caribbean, South Africa, Thailand) Study Week 8 gag ELISPOT 3 0 1 0 30 10 30 10 3 1 slope=-0.25 p-value<0. 01 1x10 9 vp/d 18 50 20 50 1 0 4608 BaselineAd5Titer slope=-0.03 p-value=0. 58 1x10 10 vp/d 18 50 20 50 1 0 4608 BaselineAd5Titer Study Week 30 gag ELISPOT 3 0 1 0 30 10 30 10 3 1 slope=-0.34 p-value<0. 01 1x10 9 vp/d 18 50 20 50 1 0 4608 BaselineAd5Titer ELISPOTpositive slope=-0.05 p-value=0.281 1x10 10 vp/d 18 50 20 50 1 0 4608 BaselineAd5Titer ELISPOTnegative Casimiro et al. unpublished 9

V520-018: MRKAd5 gag ELISPOT (SFC/10 6 PBMC) vs. Baseline Ad5 Titer by Dose Level (North/South America, Caribbean, South Africa, Thailand) Study Week 8 gag ELISPOT 3 0 1 0 30 10 30 10 3 1 slope=-0.25 p-value<0. 01 1x10 9 vp/d 18 50 20 50 1 0 4608 BaselineAd5Titer slope=-0.03 p-value=0. 58 1x10 10 vp/d 18 50 20 50 1 0 4608 BaselineAd5Titer Study Week 30 gag ELISPOT 3 0 1 0 30 10 30 10 3 1 slope=-0.34 p-value<0. 01 1x10 9 vp/d 18 50 20 50 1 0 4608 BaselineAd5Titer ELISPOTpositive slope=-0.05 p-value=0.281 1x10 10 vp/d 18 50 20 50 1 0 4608 BaselineAd5Titer ELISPOTnegative Casimiro et al. unpublished 10

International Ad Seroepidemiology: Conclusions Ultimate vaccine target populations in South Africa Ad26 and Ad35 seroprevalence in infants negligible Ad26 and Ad35 seroprevalence in adolescents low Adult clinical trial populations in US, Africa, and Asia Ad seroprevalence higher in adults than children; higher in Africa/Asia than the US; higher for Ad26 than Ad35 Ad35 seroprevalence low in all regions Ad26 seroprevalence substantial in Africa and Asia, but median Ad26 NAb titers 10-fold lower than Ad5 NAb titers Similar to recent Merck, HVTN, VRC, U. Penn. studies These relatively low NAb titers were minimally suppressive in rhesus monkeys for Ad26 and in humans for Ad5 These data support further clinical development of Ad26 and Ad35 vectors for HIV-1 in the developing world

Increased CD127, CD62L, Bcl-2 on T Lymphocytes Elicited by Ad26-Gag and Ad35-Gag Compared with Ad5-Gag in Mice Ahmed, Tan, et al. unpublished 12

Improved Functionality of T Lymphocytes Elicited by Ad26-Gag and Ad35-Gag Compared with Ad5-Gag in Mice Ad5 2% Ad26 4% Ad26/35 12% 19% 38% 60% 47% 49% 69% 5% 5% 13% 21% 57% 38% 49% 37% 66% Ad48 Single IFN-γ Ahmed, Tan, et al. unpublished Ad35 Double IFN-γ/TNF-α IFN-γ/IL-2 Ad35/26 Triple IFN-γ/TNF-α/IL-2

Improved Proliferative Potential of T Lymphocytes Elicited by Ad26-Gag and Ad35-Gag Compared with Ad5-Gag in Mice Adoptive transfer of 7,000 tetramer+ liver cells and recall with MVA-Gag Ahmed, Tan, et al. unpublished

Adaptive Immune Phenotypes Elicited by Ad Vectors: Conclusions Antigen-specific T lymphocytes elicited by Ad26 and Ad35: Re-express memory markers (CD127, CD62L, Bcl-2) at a higher frequency compared with Ad5 Demonstrate increased functionality compared with Ad5 Exhibit substantially increased proliferative potential compared with Ad5 in liver memory recall studies Quality may be more important than quantity of responses Antigen-specific T lymphocyte functionality and proliferative potential were further enhanced with Ad35-Ad26 regimens Ahmed, Tan, et al. unpublished

Substantial Biological Differences Among Ad5, Ad26, and Ad35 Vaccine Vectors Ad5 Ad26 Ad35 Virus Subgroup Group C Group D Group B Seroprevalence 1 High Intermediate Low NAb Titers 1 High Low Low Cellular Receptor 2 CAR CD46 CD46 Tropism 3 Hepatic Non-hepatic Non-hepatic DC Maturation 4 Low Intermediate High Innate Profile 5 Proinflammatory Type I IFN Type I IFN Adaptive Phenotype 6 IFN-γ Polyfunctional Polyfunctional Immunologic Potency 7 High High Intermediate NHP Protective Efficacy 7 + ++ + Human Safety 8? (phase 2b) + (phase 1) + (phase 1) Human Immunogenicity 8 ++ ++ + References: 1 Thorner et al. J. Clin. Microbiol. 2006 44:3781-3783, Abbink et al. J. Virol. 2007 81:4654-4663, Mast et al. Vaccine 2010 28:950-957, Barouch et al. submitted; 2 Vogels et al. J. Virol. 2003 77:8263-8271, Abbink et al. J. Virol. 2007 81:4654-4663; 3 Waddington et al. Cell 2008 132:397-409; 4 Lore et al. J. Immunol. 2007 179:1721-1729; 5 Barouch et al. unpublished; 6 Liu et al. J. Virol. 2008; 82:4844-4852, Ahmed et al. unpublished; 7 Liu et al. Nature 2009; 457:87-91; 8 Buchbinder et al. Lancet 2008 372:1881-1893, Catanzaro et al. J. Infect. Dis. 2006 194: 1638-1649, Barouch et al. AIDS Vaccine 2009, Paris, France

HIV-1 Vaccine Clinical Development Strategy 1. Develop prototype novel Ad vectors expressing a single test antigen (VRC EnvA) for a rapid assessment of vector safety and immunogenicity in humans 2. Develop complete vaccine products involving optimal heterologous prime-boost regimens expressing multiple HIV-1 antigens (mosaic Gag/Pol/Env) for clinical development

IPCAVD 001 A phase 1 randomized, double-blind, placebo controlled dose escalation clinical trial to evaluate the safety and immunogenicity of recombinant adenovirus serotype 26 HIV-1 vaccine (Ad26.ENVA.01) in healthy, HIV-1 uninfected adults PI: Lindsey Baden, Brigham & Women s Hospital Ad26.ENVA.01 (rad26): Recombinant adenovirus serotype 26 vector vaccine is a recombinant product composed of an adenovirus serotype 26 vector ( E1/E3) that encodes a modified gp140 HIV-1 Clade A Env glycoprotein (strain 92rw020), manufactured in HER96 cells by Crucell

Protocol Schema and Study Objectives Primary Objective: To evaluate the safety and tolerability of Ad26.ENVA.01 Secondary Objective: To evaluate the immunogenicity of Ad26.ENVA.01

Ad26.ENVA.01 Safety Analysis Data remains blinded vaccine vs placebo No serious adverse events (SAEs) and no pattern of vaccineassociated adverse events (AEs) in any group Minimal to no reactogenicity in the 10 9 vp and 10 10 vp groups In the 10 11 vp group, 7 subjects exhibited moderate (N=5) or severe (N=2) reactogenicity following initial vaccination Fatigue, myalgia, chills Mild fever of 100.5 F (38.0 C) in one subject In all subjects, symptoms resolved within 24-48 hours Did not recur after second vaccination Reduced frequency and severity of reactogenicity in the 5x10 10 vp group as compared with the 10 11 vp group

Ad26.ENVA.01 Safety Analysis Reactogenicity with 10 11 vp Ad26 comparable with that reported with 10 11 vp Ad5 Merck Ad5 (Priddy et al. CID 2008; 46:1769-1781) 58% HA, 42% chills, 47% fever VRC Ad5 (Cantanzaro et al. JID 2006; 194:1638-1649) 60% moderate reactogenicity, 40% fever Ad26 vaccine safe and generally well tolerated to date at all doses studied

Ad26.ENVA.01 Immunogenicity Analysis Data remains blinded vaccine vs placebo 36 subjects (10 vaccinees, 2 placebos per group): Group 1: 10 9 vp Group 2: 10 10 vp Group 3: 10 11 vp Samples (laboratory coded to maintain blinding): Immunogenicity assays at weeks 0, 2, 4, 6, 8, 24, 26, 28, 52 Vaccines administered at weeks 0, 4, 24 Immunologic Assays: EnvA ELISA assay EnvA IFN-γ ELISPOT assay (validated)

EnvA ELISA Responses Group 1 (10 9 vp) Consistent Env Ab titers following week 0 and week 4 prime Increased following week 24 boost and durable to week 52 Log EnvA ELISA Titer 5.0 4.0 3.0 2.0 1.0 1 2 3 4 5 6 7 8 9 10 11 12 week 0 2 4 6 8 24 26 28 52 Log EnvA ELISA Titer 5.0 4.0 3.0 2.0 1.0 0 2 4 6 8 24 26 28 52 Week No Response No Response No Response: samples #1 and #12 EnvA ELISA Titer Mean Median Week 6: 1,380 1,778 Week 24: 217 300 Week 26: 6,138 5,623 Week 52: 1,278 1,000

EnvA ELISA Responses Group 2 (10 10 vp) Higher Env Ab titers in Group 2 compared with Group 1 Increased following week 24 boost and durable to week 52 Log EnvA ELISA Titer 5.0 4.0 3.0 2.0 1.0 13 14 15 16 17 18 19 20 21 22 23 24 * week 0 2 4 6 8 24 26 28 52 Log EnvA ELISA Titer 5.0 4.0 3.0 2.0 1.0 0 2 4 6 8 24 26 28 52 Week No Response No Response * samples not available No Response: samples #14 and #23 EnvA ELISA Titer Mean Median Week 6: 3,436 3,000 Week 24: 509 300 Week 26: 12,654 10,000 Week 52: 2,726 3,000

EnvA ELISA Responses Group 3 (10 11 vp) Comparable Env Ab titers in Group 3 compared with Group 2 Increased following week 24 boost and durable to week 52 Log EnvA ELISA Titer 5.0 4.0 3.0 2.0 1.0 25 26 27 28 29 30 31 32 33 34 35 36 * week 0 2 4 6 8 24 26 28 52 Log EnvA ELISA Titer 5.0 4.0 3.0 2.0 1.0 0 2 4 6 8 24 26 28 52 Week No Response No Response * samples not available No Response: samples #29 and #35 EnvA ELISA Titer Mean Median Week 6: 4,406 10,000 Week 24: 759 1,000 Week 26: 12,525 30,000 Week 52: 2,411 3,000

EnvA ELISPOT Responses Group 1 (10 9 vp) Consistent Env T cell responses following week 0 and week 4 prime Increased slightly following week 24 boost and durable to week 52 Log SFC / 10^6 PBMC 4.0 3.0 2.0 1.0 1 2 3 4 5 6 7 8 9 10 11 12 week 0 2 4 6 8 24 26 28 52 Log SFC / 10^6 PBMC 4.0 3.0 2.0 1.0 0 2 4 6 8 24 26 28 52 Week No Response No Response No Response: samples #1 and #12 EnvA SFC/10 6 PBMC Mean Median Week 6: 296 359 Week 24: 172 272 Week 26: 302 327 Week 52: 207 214

EnvA ELISPOT Responses Group 2 (10 10 vp) Comparable Env T cell responses in Group 2 compared with Group 1 Increased slightly following week 24 boost and durable to week 52 Log SFC / 10^6 PBMC 4.0 3.0 2.0 1.0 13 14 15 16 17 18 19 20 21 22 23 24 * week 0 2 4 6 8 24 26 28 52 Log SFC / 10^6 PBMC 4.0 3.0 2.0 1.0 0 2 4 6 8 24 26 28 52 Week No Response No Response * samples not available No Response: samples #14 and #23 EnvA SFC/10 6 PBMC Mean Median Week 6: 90 97 Week 24: 91 151 Week 26: 147 112 Week 52: 104 145

EnvA ELISPOT Responses Group 3 (10 11 vp) Comparable Env T cell responses in Group 3 compared with Group 1 Increased slightly following week 24 boost and durable to week 52 Log SFC / 10^6 PBMC 4.0 3.0 2.0 1.0 25 26 27 28 29 30 31 32 33 34 35 36 * week 0 2 4 6 8 24 26 28 52 Log SFC / 10^6 PBMC 4.0 3.0 2.0 1.0 0 2 4 6 8 24 26 28 52 Week No Response No Response * samples not available No Response: samples #29 and #35 EnvA SFC/10 6 PBMC Mean Median Week 6: 236 245 Week 24: 307 309 Week 26: 441 417 Week 52: 312 282

Ongoing Studies Group 4: 5x10 10 vp dose, week 0, 24 regimen Group 5: 1x10 10 vp dose, week 0, 24 regimen Aims: To fine tune Ad26 vector dose for future studies To compare 1 (week 0) vs 2 (week 0, 4) priming immunizations To assess EnvA-specific T lymphocyte functionality by ICS

Preliminary Comparison of EnvA ELISPOT Responses in Group 3 (10 11 vp; wk 0/4/24) vs Group 4 (5x10 10 vp; wk 0/24) Comparable EnvA ELISPOT responses following 1 vs 2 Ad26 primes Group 3 Group 4 Log SFC / 10^6 PBMC 4.0 3.0 2.0 1.0 0 2 4 6 8 24 26 28 52 Week Log SFC / 10^6 PBMC 4.0 3.0 2.0 1.0 TBD 0 2 4 6 8 24 26 28 52 Week EnvA SFC/10 6 PBMC Mean Median Week 6: 236 245 Week 24: 307 309 Week 26: 441 417 Week 52: 312 282 EnvA SFC/10 6 PBMC Mean Median Week 6: 314 437

EnvA IFN-γ ICS Responses Group 4 Total, Central, Effector Memory CD8+ and CD4+ T Cells Multiple T lymphocyte subpopulations elicited by Ad26.ENVA.01 10 Unfractionated 10 Central Memory 10 Effector Memory % CD8 T Cells 1 0.1 0.01 % CD8 CM T Cells 1 0.1 0.01 % CD8 EM T Cells 1 0.1 0.01 0.001 0 2 4 6 8 0.001 0 2 4 6 8 0.001 0 2 4 6 8 1 1 1 % CD4 T Cells 0.1 0.01 % CD4 CM T Cells 0.1 0.01 % CD4 EM T Cells 0.1 0.01 0.001 0 2 4 6 8 0.001 0 2 4 6 8 0.001 0 2 4 6 8 Week

First-in-Human Evaluation of a Prototype Ad26.ENVA.01 Vaccine Vector: Conclusions Ad26.ENVA.01 is safe and immunogenic in humans at doses of 10 9, 10 10, and 10 11 vp Reactogenicity reduced with 5x10 10 vp dose as compared with 10 11 vp dose; supports future studies with 4x10 10 vp dose Immunogenicity of 10 9 vp in humans unexpected based on lack of immunogenicity of 10 9 vp in NHP studies, suggesting that Ad vectors are optimal in their natural host species Durability of humoral and cellular immune responses >52 weeks Responses (humoral>cellular) increased by homologous vector boost immunizations Ad26 is a promising HIV-1 vaccine vector for further clinical development

Immunogenicity of Heterologous Ad35/Ad26 Regimens in Rhesus Monkeys 6000 Prime at week 0; Boost at week 12 SFC / 10^6 PBMC 4000 2000 Env Pol Gag 0 week 0 2 4 12141824 0 2 4 12141824 0 2 4 12141824 0 2 4 12141824 Ad5 Ad5 Ad5HVR48 Ad26 Ad35 Ad26 Ad48 Ad26 High-dose, homologous, IV SIVmac251 challenge studies currently in progress Low-dose, heterologous, IR SIVmac251 challenge studies currently in progress

Expanded Breadth by Mosaic Compared with Consensus or Natural Gag/Pol/Env Antigens in Rhesus Monkeys The Ad26 mosaic vaccine yielded many more Gag, Pol, and Env (A) epitopespecific T lymphocyte responses as well as (B) numbers of epitope response regions to PTE peptides than did the Ad26 M consensus, clade B + clade C, or optimal natural clade C vaccines P = 1 x 10-11, Poisson regression P = 2 x 10-7, Poisson regression Barouch et al. Nat. Med. 2010; 16:319-323

Mosaic Env Antigens Elicit Noninferior ELISA and NAb Responses Compared with Consensus or Natural Sequence Env Antigens The mosaic vaccine elicited comparable ELISA and Tier 1 C (MW965.26) NAb titers (P = NS) and increased Tier 1 B (SF162.LS) NAb titers compared with the M consensus and optimal natural C clade vaccine (P = 0.02) These data suggest the utility of Ad prime, Env protein boost regimens Barouch et al. Nat. Med. 2010; 16:319-323

Proposed Next-Generation HIV-1 Vaccine Candidate: Heterologous Adenovirus Mosaic Vaccine NIH IPCAVD U19 AI078526 Program We aim to develop a global HIV-1 vaccine candidate for clinical evaluation Vectors that avoid high levels of vector-specific NAbs and can be combined into a heterologous prime-boost regimen Heterologous Ad35/Ad26 prime-boost regimen Antigens that elicit both humoral and cellular immunity and that optimize immunologic coverage of global virus diversity 2-valent mosaic Gag/Pol/Env antigens Ad35 and Ad26 vectors expressing mosaic HIV-1 Gag/Pol/Env are currently being manufactured for clinical studies by Crucell

Clinical Development Timeline 2010 : Evaluation of prototype Ad35/Ad26 prime-boost vector regimens in US, East Africa, South Africa (IPCAVD004 / IAVIB003) Collaborators: IAVI, HVTN, NIAID, Ragon Institute 2011 : Evaluation of Ad35/Ad26 mosaic Gag/Pol/Env vaccine Collaborators: IAVI, HVTN, NIAID, Ragon Institute 2011 : Evaluation of Ad26/MVA mosaic Gag/Pol/Env vaccine Collaborators: MHRP (NIAID, US Army) 2012 : Evaluation of Ad prime / Env protein boost regimens

Acknowledgements Beth Israel Deaconess, Harvard Medical School Peter Abbink Kara Brandariz Annalena La Porte Jinyan Liu Lauren Peter Faye Stephens Raphael Dolin Michael Seaman New England Primate Research Center Angela Carville Keith Mansfield LANL Bette Korber Emory University Wendy Tan Rafi Ahmed Brigham & Women s, Harvard Medical School Lindsey Baden Brian Engelson Patrick Falahee Jen Johnson Jane Kleinjan Kathleen Krause Caesar Lopez Jamie Minchelo Lizanne Noble Alka Patel Robert Tucker Teresa Votto Stephen Walsh Marissa Wilck Daniel Worrall Safety Monitoring Committee Paul Goepfert Michael Keefer Peter Wright Crucell Holland BV MHRP Jaap Goudsmit Jerome Kim Sandra Kik Mary Marovich Maria Grazia Pau Nelson Michael Jerry Sadoff Merlin Robb Hanneke Schuitemaker Mo Weijtens Gerrit Jan Weverling Jort Vellinga IAVI Jim Ackland Jo Cox Pat Fast Jill Gilmour Tom Hassell Wendy Komaroff Wayne Koff Dagna Laufer Angela Lombardo Eddy Sayeed Devika Zachariah HVTN Larry Corey Jim Kublin Cecilia Morgan Ragon Institute of MGH, MIT, and Harvard Zaza Ndhlovu Bruce Walker CAVD, Bill & Melinda Gates Foundation DAIDS, NIAID, NIH Elizabeth Adams Chris Butler Massimo Cardinali Woody Dubois Alan Fix Dale Lawrence Mary Ann Luzar Rebecca Sheets Edith Swann Michael Pensiero