T-cell responses induced by an Ad35-vectored HIV vaccine were broad, durable, polyfunctional and could inhibit HIV in a phase I trial. The vaccine induced HIV-specific T-cell responses in over 86% of subjects as measured by IFN-γ ELISPOT. Responses targeted multiple HIV proteins and were predominantly from CD8+ T-cells. T-cells exhibited multifunctionality with cytokine production and degranulation. Modest antibody responses were induced with no neutralizing antibodies detected. Further studies will evaluate prime-boost regimens to enhance the breadth and potency of immune responses.

1. T-cell responses induced by immunization withp y
Ad35-vectored HIV-1 vaccines are broad, durable,
polyfunctional and can mediate inhibition of HIV
Josephine H Cox, PhD
AIDS Vaccine 2011
Bangkok, Thailand, September 12-15th, 2011

2. Ad35-GRIN/ENV Phase I Overview
Two constructs: Ad35-GRIN & Ad35-ENV
Ad35-GRIN/ENV: Groups A-C co-formulated
together and Ad35-GRIN: Group Dtogether and Ad35 GRIN: Group D
All administered via intramuscular route at 0
and 6 months.
Subtype A
N = 56, 14/group: 10 vaccine/4 placebo
A (Low) B (Mid) C (High) D (Mid)
Ad35 GRIN/ENVAd35-GRIN/ENV
Dose escalation
2x109 vp 2x1010vp 2x1011 vp
Ad35-GRIN 1x1010 vp
Clinical Site: University of Rochester Medical Center (USA)
Vaccinations completed Sept. 2010
Follow up for 18 months post first vaccinationFollow-up for 18 months post first vaccination
Last visit of last volunteer August 2011
2

3. Safety Data
• Only Ad35 antibody negative subjects were enrolled.
• Ad35 prevalence of 4.9% low in this US populationAd35 prevalence of 4.9% low in this US population
• Ad35-GRIN/ENV is safe and generally well tolerated
- No related SAE reported
- Reactogenicity and tolerability are dose-dependent
- High dose at 2x1011 vp is responsible for severe local and/or
systemic reactions in 50% of vaccinees persisting more than 5systemic reactions in 50% of vaccinees, persisting more than 5
days in one volunteer.
- All reactions resolved spontaneously without complications or
sequellaesequellae
• All Ad35 samples (scheduled and symptomatic) tested
negative for shedding (urine and saliva).g g ( )
3

4. Immunogenicity Assessments
35
35
Weeks
0 24
Ad3
Ad3
Weeks
0, 2 and 4 post 1st 0, 1, 2, 4, 8, 14, 28, 40 and 48 post 2nd
•Immunogenicity: IFN- ELISPOT at all time points
Followed for 72 weeks total (18 months)
g y  p
•Cut-off 38 SFC/106 PBMC and 4 x background / six peptide pools
•ICS, Viral Inhibition Assay (VIA), epitope mapping,
Env Ab, p24 Ab, Ad35 neuts at selected time pointsb, p b, d35 euts at se ected t e po ts
4

5. High HIV-specific IFN- ELISPOT
Response rates maintained over time
60%
80%
100%
A Low
p
Any Antigen
0%
20%
40%
A-Low
B-Mid
C-High
D-GRIN
0%
E A
60%
80%
100%
Env Antigen
60%
80%
100%
20%
40%
60%
GRIN Antigen20%
40%
60%
0% 0%
5

6. HIV-specific IFN- ELISPOT in most individuals
maintained up to 10 months post 2nd vaccinationp p
3.5
Ad35 GRIN-ENV increasing dose Ad35 GRIN
3.0
scale)
2.5
BMC(log-s
2.0
FC/106PB
1.5
Wk2
50%
Vac.1
Wk4
50%
Wk2
86%
Vac.2
Wk4
75%
Wk40
63%
Wk2
50%
Vac.1
Wk4
56%
Wk2
100%
Vac.2
Wk4
100%
Wk40
100%
Wk2
78%
Vac.1
Wk4
70%
Wk2
89%
Vac.2
Wk4
88%
Wk40
75%
Wk2
89%
Vac.1
Wk4
90%
Wk2
86%
Vac.2
Wk4
86%
Wk40
88%
SF
38 SFC
2x109 vp 2x1010 vp 2x1011 vp 1x1010 vp
6

7. IFN- ELISPOT responses of modest magnitude
across all vaccine-expressed HIV proteins
35
Gag RT Pol/Int Nef Env
3.0
3.5
og-scale)
2.5
PBMC(lo
2.0
SFC/106
1.5
GrpA
43%
GrpB
88%
GrpC
33%
GrpD
86%
GrpA
57%
GrpB
88%
GrpC
56%
GrpD
71%
GrpA
71%
GrpB
38%
GrpC
33%
GrpD
71%
GrpA
29%
GrpB
38%
GrpC
11%
GrpD
57%
GrpA
86%
GrpB
75%
GrpC
67%
Median
SFC/m
158 79 100 158 126 251 158 200 126 79 251 316 158 50 79 100 63 126 79
7
2 weeks post second vaccination

8. Multiple HIV proteins targeted by each volunteer
Number of visits
(maximum 10) with
Breadth of the response as
defined by number of positive
responses to any of the six
positive responses to
any peptide pool
responses to any of the six
(four for Group D) peptide
pools at any time point
Gro p MedianGroup Median
A - Low 7.5 3.5
B - Mid 9 5
C - High 9 5C - High 9 5
D - GRIN 10 4
Data up to 40 weeks post second vaccination
8

9. Median of 4 epitopes recognized per
volunteer (n = 25 mapped)volunteer (n = 25 mapped)
Pool ID
#
Volunteers
Unique
*regions
Median #
*regions RangePool ID Volunteers regions
mapped
regions
recognized
Range
Gag 12 >8 1 1-3
RT 14 17 2 1 3RT 14 >17 2 1-3
Int 12 >8 1 1-4
Nef 6 >2 1 1-2
Env 12 >14 2 1-4
Median # epitopes recognized / volunteer 4
Range of epitopes recognized / volunteer 1-8
9*Unique region = either 2 peptides sharing a sequence or single peptide

10. Flow Cytometry Panel: IFN-/IL-2/CD107/TNF-,
CD4/CD8 memory and homing markersCD4/CD8, memory and homing markers
CD4+: 0.028% IFNg+
CD8+: 2.18% IFNg+ NEF ELISPOT: 1060 SFC/m
Example shown is Nef stimulation from sample atExample shown is Nef stimulation from sample at
2 weeks post 2nd vaccination (Group D, GRIN)
10

11. Predominantly CD8+ T-cell Responses
to Gag, RT and Int
>0.5
Any Env1 Env2 Gag INT NEF RT
CD8 ANY Env 1 Env 2 Gag Int Nef RT
/TNF
0.2
0.3
0.4
-2/CD107/
Baseline
& Placebo
0.13
0.0
0.1
Bl P B C D B C D B C D B C D B C D B C D
Any Env1 Env2 Gag INT NEF RT
eIFN-/IL-
B - Mid
C - HighCD4 ANY Env 1 Env 2 Gag Int Nef RT
0.3
0.4
>0.5
Any Env1 Env2 Gag INT NEF RT
ePositive
C High
D - GRIN
0.06
0.1
0.2
0.3
%Cytokine
11
0.06
0.0
Bl P B C D B C D B C D B C D B C D B C D
%
Responses at 2 weeks post 2nd vaccine

12. Potent and broad CD8-mediated VIA activity
Placebo Low dose Mid dose High dose
CRFAE-01 ELI (AD) IIIB(B) NL4-3(B) U455(A)
inhibitionLog10
247Fv2(C) 97ZA012(C) CBL4(D) CH077(B) CH106(B)
CD8+ T cell HIV-1 inhibition to several isolates in most vaccinated individuals 12

13. ENV-A (UG037)- and Gag-p24 (IIIB)-
specific ELISA antibodiesspecific ELISA antibodies
104
Post 1st
Vaccination Post 2nd
Vaccination
rs
104
Post 1st
Vaccination Post 2nd
Vaccination
rs
103
A - Low
B - Mid
C - High
anEnvtiter
103
A - Low
B - Mid
C - High
D - GRIN
anGagtiter
102
GeomMea
102
GeomMea
0 10 20 30 40
10
Weeks post Vaccination
0 10 20 30 40
10
Weeks post Vaccination
4 w Post 1st 2 w Post 2nd
A - Low 8/10 (80) 9/9 (100)
B - Mid 9/10 (90) 8/8 (100)
4 w Post 1st 2 w Post 2nd
A - Low 0/10 0/9 (0)
B - Mid 0/10 3/8 (38)
C High 0/10 8/9 (89)
13
C - High 10/10 (100) 9/9 (100) C - High 0/10 8/9 (89)
D - GRIN 0/10 5/7 (71)

14. T-cell response summary for B001 Ad35 trial
High response rate by ELISPOT
>86% across all groups post 2nd
Magnitude of response is modest but maintained over
time
Greater magnitude to GRIN proteins in the absence ofGreater magnitude to GRIN proteins in the absence of
ENV but no enhancement of VIA activity
Good breadth; all proteins targeted
CD8+ T-cell HIV-1 inhibition in most vaccinated individuals
to several isolates
Fl t tFlow cytometry
Predominantly CD8+ response
Multifunctional (cytokines + degranulation)Multifunctional (cytokines + degranulation)
Induction of T cell memory responses (data not
shown)
14

15. Antibody response summary for B001 Ad35 trial
Anti-HIV ENV: 100% responders post second
administration with modest titers
No HIV Nabs to multi-clade panel (Monogram BioSciencesNo HIV Nabs to multi clade panel (Monogram BioSciences,
data not shown)
Anti-HIV Gag p24: no antibody post 1st, modest titer
post 2ndpost 2nd
Anti-Ad35 neutralization response rates and titers are
modest even after 2 vaccinations and lower than seen
with Ad5 (data not shown)
Low-prevalence serotype Ad vectors may exhibit
i d f t d i i it dimproved safety and immunogenicity as compared
with Ad5 vectors.
15

16. Next steps
 Ad35-GRIN/ENV is immunogenic at the dose with an acceptable safety
profile (2x1010 vp)
 Evaluate the safety and immunogenicity of Ad35-based HIV vaccine in
heterologous prime-boost regimens, to assess potential enhancement of the
breadth and potency of cellular and humoral immune responses as follows:
Ad26 vector (Dan Barouch / Crucell, ongoing)
Ad35 ENV d Ad26 ENV P t tAd35-ENV and Ad26-ENV Prototype
Mosaic Immunogens to optimize cellular immunologic coverage of
global HIV-1 sequence diversity
Adjuvanted Protein (GSK ongoing)Adjuvanted Protein (GSK, ongoing)
Ad35-GRIN and Adjuvanted GSK investigational HIV vaccine
formulation 1 and 2
Electroporated (EP) DNA and IL-12 (Profectus start late 2011)Electroporated (EP) DNA and IL 12 (Profectus, start late 2011)
Ad35-GRIN-Env and multi-antigenic DNA (HIV-MAG), IL-12 and EP
Mucosal delivery – Sendai virus (SeV)
Ad35-GRIN and SeV-Gag prototype in pre-clinical developmentAd35 GRIN and SeV Gag prototype in pre clinical development
16

17. Acknowledgements
University of RochesterUniversity of Rochester
Medical Center
Michael Keefer
Lottie Hachaambwa
Catherine Bunce
IAVI
Eddy Sayeed, Angela Lombardo
Wendy Komaroff, Sabrina Welsh
Catherine Bunce
Mhorag Hay
EMMES
Kelley Loughran
Burc Barin
James Ackland, Devika Zachariah
Kamaal Anas, Jean-Louis Excler
Patricia Fast
Burc Barin
Carol Smith
IAVI Human Immunology
Laboratory, Imperial College
Lorna Clark
Justyna Czyzewska
Laura Seamons
Aggeliki SpentzouLaboratory, Imperial College
London
Hong Wan
Ambreen Ashraf
Phil Bergin
Justyna Czyzewska
Christopher Farrance
Natalia Fernandez
Dilbinder Gill
P t H
Aggeliki Spentzou
Tony Tarragona
Paramesh Chetty
Gwynn Stevens
Jill GilPhil Bergin
Emmanuel Cormier
Jana Carga
Michelle Cashin-Cox
Hannah Cheeseman
Peter Hayes
Vanaja Kakarla
Jakub Kopycinski
Francesco Lala
Jill Gilmour
Hannah Cheeseman Andrew Raxworthy-Cooper
18

19. Back- up slide

20. Range of CD8+ T-cell memory and other
phenotypes observedphenotypes observed
No trend towards a particular memory phenotype. Majority of
responses lack
homing markers
d B7
Data is across study arms and visits
in samples with >0.2% IFN- production
and are B7-

21. Ad35-specific neutralization antibody responses
100 500
60
80
responders
300
400
GMT
0
20
40
%Ad35
0
100
200
0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2
0
D - GRINA - Low B - Mid C - High
0 = baseline
1 4 k t fi t i1 = 4 weeks post first vaccine
2 = 2 weeks post second vaccine