development of a recombinant subunit dengue vaccine · recombinant subunit dengue vaccine •hawaii...

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


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:


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

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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

http://www.ewildz.com/index.html

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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



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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


50 µg Adjuvant A + 50 µg Adjuvant B

0, 28, 67, 102 2


60 µg of ISCOMATRIX™ Adjuvant

0, 28, 67, 102 2


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


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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


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,


Red: 1 µg each 80E, 0.1 µg NS1,

60 µg ISCOMATRIX™ Adjuvant

Blue: 5 µg each 80E, 0.5 µg NS1,



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Neutralizing Ab Responses in Monkeys


1

10

100

1000

0 28 67 102 130

Study Day

PR

NT

50

Tit

er

V3J

HJC

V2G

AC70

AA37

FTH

T206

AJ14


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,


Yellow: 3 µg each 80E, 0.3 µg NS1,


Red: 1 µg each 80E, 0.1 µg NS1,


Blue: 5 µg each 80E, 0.5 µg NS1,



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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


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


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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


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


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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


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)


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


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



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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


Tetravalent DEN-80E + ISCOMATRIX™ Adjuvant


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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


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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


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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

development of a recombinant subunit dengue vaccine · recombinant subunit dengue vaccine •hawaii...

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