Development of a Recombinant
Subunit Dengue Vaccine
Flavivirus Vaccination
Fondation Mérieux
December 8, 2010
Beth-Ann Coller
2
Recombinant Subunit Dengue Vaccine
•Hawaii Biotech originally developed subunit vaccine based on carboxy truncated envelope protein – 80E –Recently acquired by Merck.
•Drosophila S2 insect cell culture expression system is used to produce dengue subunits
•No live virus, potential to achieve balanced tetravalent immunity without interference
Crystal structure of Merck DEN2-80E protein
Steve Harrison, Harvard University
Recombinant Envelope Protein Vaccine
3
Overview of Drosophila S2 Expression System
Gene of Interest
Drosophila
Expression
Vector
Drosophila S2 Cells
Protein Secreted into
Culture Medium
Native-like
Protein Structure
pHBI-20D
Recombinant Subunit Dengue Vaccine
4
• Functional regions of Envelope are associated with
binding to cell receptors and fusion
• Envelope important in protective immune response –
key target for neutralizing Ab
• Envelope also contributes as cell mediated immune
target (CD4 and CD8)
• Balance of multivalent immunity may be manipulated
by varying antigen doses – no viral interference
• No prM protein in purified product – minimize risk of
enhancement linked to anti-prM antibodies
Rationale for Envelope Protein Based Subunit Vaccine Candidate:
Recombinant Subunit Dengue Vaccine
5
• HBI has tested dengue and WN vaccines with variety of
adjuvants in preclinical models.
• Saponin-based adjuvants and MPL/Alum in combination
with DEN antigens induce potent neutralizing responses
and protective efficacy in primates
• Access to potent adjuvants and regulatory challenges
led to focus in short term on alum-based adjuvants
• Alum based formulations immunogenic and efficacious
if properly formulated and administered.
• Focus on alum-based vaccines for initial clinical trial.
Other adjuvants may offer significant advantages in
long term (durability, dose sparing, magnitude and
broadness of immune response)
Formulation Development
Dengue Vaccine Formulation Development
6
Non-Human Primate StudiesMonovalent DEN-80E studies1 - Rhesus macaques – DEN2-80E + ISCOMATRIX
adjuvant2 - Rhesus macaques – DEN2-80E + GSK adjuvants –
Putnak et al., 2005
3 - Rhesus macaques – DEN1-80E + alum adjuvant –
stand alone and prime boost with LAV
Tetravalent DEN-80E studies
1 - Rhesus macaques – ISCOMATRIX Adjuvant –
+ DEN2 NS1 pilot study
2 - Rhesus macaques – ISCOMATRIX adjuvant –
+/- DEN2 NS1 study – large safety, immunogenicity
and efficacy study
Dengue Vaccine Preclinical Studies
7
Vaccine Formulation PRNT50
GMT
Viremia
(Cell culture)
Anamnestic
Response
5 μg 80%E + AS04-OH 175 2/3 Yes
20 μg 80%E + AS04-OH 541 0/3 Yes
5 μg 80%E + AS04-PO 114 2/3 Yes
1 μg 80%E + AS05 850 0/3 Yes
5 μg 80%E + AS05 1068 0/3 Yes
5 μg 80%E + AS08 1676 1/3 Yes
Monovalent DEN2-80E + GSK adjuvants♦ Rhesus Macaques♦ Immunogenicity and Efficacy♦ 2 doses ♦ Challenge 2 months post dose 2
Dengue Vaccine Preclinical Studies
8
Group Antigen formulation Vaccine Dosing
Days
Number of
Animals
1 3 µg each serotype 80E + 0.3 µg NS1
10 µg Adjuvant A + 10 µg Adjuvant B
0, 28, 67, 102 2
2 3 µg each serotype 80E + 0.3 µg NS1
50 µg Adjuvant A + 50 µg Adjuvant B
0, 28, 67, 102 2
3 1 µg each serotype 80E + 0.1 µg NS1
60 µg of ISCOMATRIX™ Adjuvant
0, 28, 67, 102 2
4 5 µg each serotype 80E + 0.5 µg NS1
60 µg of ISCOMATRIX™ Adjuvant
0, 28, 67, 102 2
• 4 Doses - Primary endpoint Safety – Safety Profile OK
• Immunogenicity assessed following each dose
• Challenge approximately 5 months post dose 4 with DENV2 or DENV4
Safety/Immunogenicity/Protection of Tetravalent
80E + NS1 Vaccine in Primates – Pilot Study
Dengue Vaccine Preclinical Studies
9
Anti-DEN-1 Neutralizing Ab Response
1
10
100
1000
10000
0 28 67 102 130
Study Day
PR
NT
50 T
iter
V3J
HJC
V2G
AC70
AA37
FTH
T206
AJ14
Anti-DEN-2 Neutralizing Ab Response
1
10
100
1000
10000
0 28 67 102 130
Study Day
PR
NT
50
Tit
er
V3J
HJC
V2G
AC70
AA37
FTH
T206
AJ14
Neutralizing Ab Responses in Monkeys
Dark blue: 3 µg each 80E, 0.3 µg NS1,
10 µg each Adjuvant A & Adjuvant B
Yellow: 3 µg each 80E, 0.3 µg NS1,
50 µg each Adjuvant A & Adjuvant B
Red: 1 µg each 80E, 0.1 µg NS1,
60 µg ISCOMATRIX™ Adjuvant
Blue: 5 µg each 80E, 0.5 µg NS1,
60 µg ISCOMATRIX™ Adjuvant
Dengue Vaccine Preclinical Studies
10
Neutralizing Ab Responses in Monkeys
Anti-DEN-3 Neutralizing Ab Response
1
10
100
1000
0 28 67 102 130
Study Day
PR
NT
50
Tit
er
V3J
HJC
V2G
AC70
AA37
FTH
T206
AJ14
Anti-DEN-4 Neutralizing Ab Response
1
10
100
1000
0 28 67 102 130
Study Day
PR
NT
50 T
iter
V3J
HJC
V2G
AC70
AA37
FTH
T206
AJ14
Dark blue: 3 µg each 80E, 0.3 µg NS1,
10 µg each Adjuvant A & Adjuvant B
Yellow: 3 µg each 80E, 0.3 µg NS1,
50 µg each Adjuvant A & Adjuvant B
Red: 1 µg each 80E, 0.1 µg NS1,
60 µg ISCOMATRIX™ Adjuvant
Blue: 5 µg each 80E, 0.5 µg NS1,
60 µg ISCOMATRIX™ Adjuvant
Dengue Vaccine Preclinical Studies
11
DENV-2 Challenge
Protection
X
Anti-DEN-2 Virus Neutralizing Response
1
10
100
1000
10000
100000
0 28 67 102 130 278 322
Study Day
PR
NT
50
Tit
er
V3J
AC70
FTH
AJ14
Ch
alle
nge
Dengue Vaccine Preclinical Studies
12
DENV-4 Challenge
Anti-DEN-4 Virus Neutralizing Response
1
10
100
1000
10000
0 28 67 102 130 278 322
Study Day
PR
NT
50
Tit
er
HJC
V2G
AA37
T206
Challe
nge
Protection
X
X
Dengue Vaccine Preclinical Studies
13
Group Dose 1 Dose 2 Dose 3
1 LAV DEN1-80E DEN1-80E
2 DEN1-80E DEN1-80E LAV
3 DEN1-80E DEN1-80E DEN1-80E
4 Placebo Placebo Placebo
Dengue Vaccine Preclinical Studies
Monovalent DEN1-80E + Alhydrogel• Rhesus Macaques • Recombinant alone or as heterologous prime-
boost with LAV• 3 doses administered @ 0, 1, 2 months • Challenge 2 months post dose 3 with wild type
DEN1 (WP74)• Immunogenicity and Efficacy
14
1
10
100
1000
10000
Day 0 Day 28 Day 56 Day 84 Day112 Day140
80E/LAV
LAV/80E
80E/80E
Placebo
PR
NT
50
GM
T
Dengue Vaccine Preclinical Studies
Monovalent DEN1-80E + Alhydrogel• Safety – No adverse observations• Immunogenicity – Virus neutralizing antibody
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GroupViremia –
# Animals (mean # days)
1 - LAV/DEN1/DEN1 0/3
2 - DEN1/DEN1/LAV 0/3
3 - DEN1/DEN1/DEN1 0/3
4 - Alhydrogel Placebo3/3
(4.33)
Dengue Vaccine Preclinical Studies
Monovalent DEN1-80E + Alhydrogel
Efficacy - Post Challenge Viremia Results
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Formulation DEN 80%E DEN-2 NS1 ISCOMATRIX™
A 1 µg each 80%E - 60 µg
B 1 µg each 80%E 0.1 µg 60 µg
Placebo - - 60 µg
Dengue Vaccine Preclinical Studies
Tetravalent DEN-80E + ISCOMATRIX™ Adjuvant• Rhesus Macaques• +/- DEN2-NS1 • 3 doses @ 0, 2, 4 months • 3 groups of 12 monkeys each• challenge 5 months post dose 3• 3 monkeys challenged with each serotype• Immunogenicity and Efficacy
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• Safety - no adverse local reactions or impact on clinical chemistry/hematology parameters
• Post dose 3 PRNT50 titers
- Representative results
PRNT
Virus Serotype
DEN-1 DEN-2 DEN-3 DEN-4
GMTs 672 947 574 280
Tetravalent DEN-80E + ISCOMATRIX™ Adjuvant
Dengue Vaccine Preclinical Studies
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Group Challenge Virus Viremia –
# Animals (mean # days)
1 μg each
Tetravalent 80E
+ ISCOMATRIX™ Adjuvant
DEN1 0/3 (0)
DEN2* 0/3 (0)
DEN3 0/3 (0)
DEN4* 0/3 (0)
1 μg each
Tetravalent 80E
+ 0.1 μg NS1
+ ISCOMATRIX™ Adjuvant
DEN1 1/3 (0.33)
DEN2* 0/3 (0)
DEN3 0/3 (0)
DEN4* 0/3 (0)
ISCOMATRIX™
Adjuvant Control
DEN1 3/3 (5)
DEN2* 3/3 (2.3)
DEN3 3/3 (3.7)
DEN4* 2/3 (1.7)
• Efficacy - Post Challenge Viremia Data
* Amplification on C6/36 prior to plaquing on Vero
Dengue Vaccine Preclinical Studies
Tetravalent DEN-80E + ISCOMATRIX™ Adjuvant
19
Preclinical Toxicity Testing
Group Formulation
1 Vehicle Control - PBS
2 Alhydrogel ‘85’ Adjuvant Control
3 Antigen Control – Tetravalent 80E + DEN2 NS1
4 DEN1-80E adsorbed on Alhydrogel ‘85’
5 Tetravalent 80E + DEN2 NS1 adsorbed on Alhydrogel ‘85’
• GLP Rat repeat dose safety study to assess local and systemic toxicity of vaccine antigens alone or formulated with ISCOMATRIX™Adjuvant
• No significant toxicity/reactogenicity found with vaccine antigens or formulated vaccine
• GLP Rabbit repeat dose safety study to assess local and systemic toxicity of antigens alone or in alum based formulation
• No toxicity/reactogenicity with any formulation
Dengue Vaccine Preclinical Safety Studies
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Group Formulation DEN1 ELISA
EC50 GMT
1 Vehicle Control – PBS <250
2 Alhydrogel ‘85’ Adjuvant Control <250
3 Antigen Control – Tetravalent 80E + DEN2 NS1 5229
4 DEN1-80E adsorbed on Alhydrogel ‘85’ 9816*
5 Tetravalent 80E + DEN2 NS1 adsorbed on Alhydrogel ‘85’9429
* PRNT GMT – 236
• GLP Rabbit repeat dose safety study to assess local and systemic toxicity of antigens alone or in alum based formulation
• Immunogenicity results post dose 3
Preclinical Toxicity Testing
Dengue Vaccine Preclinical Safety Studies
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Phase 1 Monovalent Clinical Study
• Placebo-controlled, double-blind study – 6 active, 2 placebo per cohort
• Dose escalation – low and high doses
• 3 doses administered at 0, 1, 2 months
• Primary endpoint safety
• Immunogenicity evaluation – virus neutralizing antibody
• Last subject, last visit in June 2010. Study still blinded.
COHORT TREATMENT
1
N=8
Low dose DEN1-80E – 10 µg
+ Alhydrogel – 1.25 mg Al
2
N=8
High dose DEN1-80E – 50 µg
+ Alhydrogel – 1.25 mg Al
Recombinant Subunit Clinical Study
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Virus Neutralizing Antibody Preliminary Results
1
10
100
Week
0
Week
2
Week
4
Week
6
Week
8
Week
10
GM
T P
RN
T50 T
iters
Cohort 1 10µg +Alum
Cohort 2 50µg +Alum
Placebo
Dose 1 Dose 2 Dose 3
Recombinant Subunit Clinical Study
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Conclusions:
• Based on preliminary, unaudited, blinded data -monovalent DEN1-80E vaccine adjuvanted with aluminum hydroxide is immunogenic in healthy, adult, flavivirus-naïve volunteers – consistent with preclinical data.
• Study included a dose escalation with monitoring by a Safety Committee - no safety signals
• Demonstrates potential of the recombinant proteins.
• Plans for testing of tetravalent product under development
Recombinant Subunit Clinical Study
24
Acknowledgements
Walter Reed Army Institute for Research
Ken Eckels Rick Millward Stephen Thomas
David Bradley Amy Dean Robert Putnak
Support from:
U.S. Department of Defense, National Institutes of Allergy and Infectious Disease, Pediatric
Dengue Vaccine Initiative, State of Hawaii, Merck, and Hawaii Biotech Investors
Saint Louis University Center for Vaccine Development
Sarah George Nicole Purcell Carolyn Stefanski Linda Eggemeyer
All the Study Volunteers
Johns Hopkins University University of Texas Medical Branch
Anna Durbin Robert Tesh
Janece Lovchik
CSL Clinical and Statistical Support
Debbie Drane Martin Pearse Elenie Bartzokis
Steve Honey Gary Stevens
Hawaii Biotech, Inc.
Jon Ruckle Maile O’Connell Tim Martyak Mike Thorne
Vidya Pai Robbin Henley David Clements Michele Yelmene