Download Dengue Vaccines Based on Alphavirus Expression Vectors and

Dengue Vaccines Based on Alphavirus Expression Vectors and Adjuvant Systems
Laura White1, Melissa Mattocks1, Wahala Wahala2, John Gibbs1, Daniel Tonkin1, Jill Whitley1, Vorraphun Yingsiwaphat4, Idia Rodriguez3, Melween Martinez3, Carlos Sariol3, Aravinda de Silva2 and Robert Johnston1.
Global Vaccines Inc., Research Triangle Park, NC1; University of North Carolina, Chapel Hill, NC2; Caribbean Primate Research Center, University of Puerto Rico-Medical Sciences Campus, San Juan, PR3, Chulalongkorn University, Bangkok, Thailand4
VRP AS EXPRESSION VECTOR: STUDIES IN MACAQUES
<10
281
0
0
-
48
17,186
4
4
3-7
1.52 – 2.09
<10
163
Vaccine
4
0
0
-
162
10,937
C
αDV3
III
Immune sera from VRP immunized or virus infected macaques was
depleted of DENV3 EDIII binding antibodies by incubation with EDIIIMBP or control MBP protein bound to amylose beads.
**
1024
8
512
64
32
BP
MB
P
BP
n tr
o l-
I- M
Co
B
16
Post-prime
Post-boost
P
P
+V
A
P
PIV
PIV
-V
A
+V
A
P
-V
A
PIV
PIV
ug
8
nV
RP
+
ug
nV
RP
+
iD
V4
8
V4
iD
4C
4B
4A
3C
# IFN-γ+ ce
ells per 106
16
8
4
8
D II
3E
A
64
ELISA endpoint titer log10
FNeut₅₀
32
Macaques were immunized at 0 and 6
weeks with 10 ug of purified formalininactivated DENV4 alone, or with nVRP
adjuvant at doses of 106, 107 or 108 IU,
or the nVRP3A adjuvant at 10^7 IU. N= 6
animals/group. There is a significant
increase in the Neut50 titers for all the
adjuvanted groups compared to antigen
only, with average fold increases of 5 X
after the prime and 27X after the boost.
Protection results are pending.
16
10. Anti-vector immunity after prime does
not interfere with the boost
256
4
3 weeks post boost
1024
512
256
128
64
32
16
8
4
nVRP adjuvant significantly enhances the neutralizing antibody responses to iDV4 after a single
immunization. No significant differences between VRP doses of 106, 107 and 108 IU.
256
128
TV-VRP Immune sera was depleted of DENV3 EDIII binding antibodies
by incubation with EDIII-MBP or control MBP protein bound to amylose
beads, followed by neutralization assay against each serotype (αDV1-4).
1024
ug
4
iD
V4
3 weeks post prime
64
4
DV
I- M
D II
32
16
4
4
3E
64
I- M
8
iD
V4
+n
ug
4
P IV
4. nVRP adjuvant + iDV4 in macaques
1024
256
128
D II
32
16
512
3E
64
DV
256
128
Anti DENV4 Neut5 0
Anti DENV3 Neut5 0
8
512
P
32
16
DV
D
αDV4
(peptides)
(A) TV iDV (1 ug each) + nVRP adjuvant (105 IU) induced: (A) neutralizing antibodies to each serotype
after a single immunization. Two doses resulted in robust and balanced responses. (B) serotype-specific T
cells, as determined by IFNγγ ELISPOT assay at 6 days post boost. Splenocytes were stimulated with iDV1-4
or with E peptide pools representing serotypes 2, 3 and 4.
7
B
αDV2
3B
1.52 – 3.01
4
4
3A
2-5
4
Vaccine
Control
2C
20,480
0
1 week
post boost
2B
112
4 wks
post prime
50
25
2A
1.23 - 2.18
TV
TV + VRP
DV4
1-2
1024
1.0
DV3
3
64
1.5
75
DV2
4
256
2.0
100
DV1
Vaccine
128
2.5
DV-specific IFN-γ T cells
6 days post TV-boost
B
media
819
512
ug
P
ug
VR
RP
4
nV
+
PIV
ug
1
14,934
<10
Anti DENV2 Neut5 0
Anti DENV1 Neut5 0
BP
-M
BP
FNeut 50
ug
1
PIV
353
105
2.09 - 2.58
4
M
BP
-M
D4
<10
1.23 - 1.52
P
64
4
III
BP
-M
III
256
16
4
ED
2.15 - 2.96
1
2-6
MB
16
A
16384
8192
4096
2048
1024
512
256
128
64
32
16
128
Anti-VEE Neut50
64
ED
4
4-5
4
4096
1024
ED
16
C
DENV3 virus
Neut50
Neut50
64
BP
Alphavirus (VEE)-like particles
Modified RNA genome
Propagation incompetent
Target Dendritic Cells
Induce a robust innate
immune response
256
M
Neut50
256
B
DENV3 E85-VRP
*
16
256
Co
5. Depletion of EDIII binding antibodies from E85-VRP
macaque immune sera results in loss of neutralization
A
DENV3 prME-VRP
4
2
4
9. Effect of depleting serotype 3 EDIII
binding antibodies on ability of TV immune
sera to neutralize DENV1-4
n tr
o l-
III
DENV3 genotypes
RNA co-electroporation
into Vero cells
4
4
4
BP
In vitro RNA transcription
32
+n
V
iD
VR 4
V4
P
+
10
6
nV
iD
R
V4
P
iD
+
10
7
nV
V4
R
+
nV P 1
08
RP
3A
10
7
V4
V3
16
8
M
Ψ
64
IgG2a
3.0
3. nVRP adjuvant + tetravalent iDV in mice
Day 10 postchallenge
Neut50 GMT
4
1024
64
32
IV
VEE E2E1
Helper plasmid
Prechallenge
Neut50
GMT
Peak viremia
titer log10
FFU/ml
Vaccine
*
256
128
G
VEE Capsid
Helper
plasmid
Post-challenge viremia
#
Duration
monkeys
in days
w/viremia
Control
Control
128
30
Monkeys were challenged at 18 weeks post dose 2. (-) indicates below limit of detection
of 10 FFU/ml
1024
512
Neutralizing antibody titers
induced 3 weeks post dose 2 in
macaque immunized with DENV3
(GIII) E85-VRP. DENV3 strains used
in the neutralization assay were:
UNC3043 (GI), CH53489 (GII),
UNC3002 (GIII) and PR77 (GIV)
Control
A
αDV1
or
VEE replicon particles
(VRP or nVRP)
DENV4
D
V2
D
D
D
4. DENV3 E85-VRP: Broad neutralization within
serotype 3
II
Replicon RNA: adjuvant
Neut50
DENV3
V1
3
V4
D
D
V4
D
V2
D
DENV2
128
64
32
16
V2
4
V1
8
4
3
16
8
V1
16
DENV1
512
256
#
monkeys
/group
MB
32
Vaccine
group
256
IgG1
3.5
The nVRP adjuvant improves the magnitude (25 to 47 fold increase) and quality (IgG2a, Th1
isotype) of antibodies to formalin-inactivated DENV4 in mice.
n tr
o l-
64
DV
32
D
64
Neut50
128
Neut50
128
2048
1024
27
8. TV E85-VRP protects from viremia upon
challenge with DV3 and DV4; partial
protection against DV1 and DV2
Challenge
virus
(105 PFU)
Virus
4096
256
24
Co
E85-VRP
512
256
21
512
Neut50
All monkeys were challenged with
PFU of DENV3
at 14 weeks after last immunization. (-) indicates
below limit of detection of 10 FFU/ml
1024
iD
15,306
2048
iD
V4
6,097
106
105
18
3wpb
D
12
-
15
After 2 immunizations, 16 out of 16 monkeys seroconverted to all 4 serotypes (seroconversion: Neut50 > = 10)
DENV3 E85-VRP
3. Replicon Packaging
Ψ
2.94 –
3.17
0
512
D
Genome RNA
Antigen
26S
2
0
Serotype-specific and cross-reactive neutralizing antibody titers at 3
weeks after second dose immunization with DENV3 prME-VRP or E85-VRP,
and 4 weeks after DENV3 virus infection.
Replicon RNA: antigen expression
VEE replicon plasmid
2
6
G
VEE NSP 1-4
6
DENV3
E85-VRP
G
Ψ
DENV3
prME-VRP
12
weeks post prime
Fold increase anti DENV titers
45
9
BP
40
6
I- M
35
I
VEE NSP 1-4
3.00 – 3.89
G
Ψ
2-6
Neut50
C E2 E1
4
3
D II
30
DV3 challenge
DV
Neut50
• Family: Togaviridae
VEE NSP 1-4
0
3E
25
170
4
log10 FFU/ml
DV
20
<20
Peak titer
P
15
28 days
postchallenge
DV3 Neut50
GMT
Duration
days
controls
prME-VRP
26S
8
MB
10
Prechallenge
DV3 Neut50
GMT
#
monkey
s
3. E85-VRP induces serotype specific neutralization
in contrast to prME-VRP and virus infection
INTRODUCTION
2. VEE genome and VEE replicon genomes
16
ELISA
IgG
iDV4
+/D4
PIV
+/-nVRP
nulltiters
VRP
3 weeks post boost
4096
32
Co
5
Immunizations
Ψ
Post-challenge Viremia
#
Monkeys
/group
Unvaccinated
4
Weeks post prime
Genus: Alphavirus
+ ssRNA genome
Enveloped
Structural: C, EI, E2
Non-structural: 1-4
64
BP
Vaccine
group
16
8
1. Venezuelan equine encephalitis virus (VEE)
128
64
32
Neutralizing antibody titers
D4
Post-challenge viremia and anamnestic responses
128
256
nVRP
105 IU
D4 PIV +/- null VRP
4wpp
4 weeks
post prime
PIV
D3ME VRP
D3E85 VRP
256
512
+
4096
2048
1024
512
256
128
64
32
16
8
D4
Neutralization titers
512
Inactivated DENV4
(iDV4)
1-8 ug
DENV1
DENV2
DENV3
DENV4
1024
Neutralization titers
DENV3
E85-VRP
108 IU
2. DENV3 E85-VRP is more immunogenic and protective
0
•
•
•
•
•
7. TV E85-VRP induces balanced neutralizing
antibody titers
VEE replicon RNA
DENV3
PrME-VRP
108 IU
2. nVRP adjuvant + monovalent iDV4 in mice:
Neut50
(Secreted E dimers)
VRPE85
DV4
2x108 IU
V1
D
V2
D
V3
D
V4
D
V1
D
V2
D
V3
D
V4
E85
+
VRPE85
DV3
108 IU
Neut50
(Secreted subviral particles)
+
VRPE85
DV2
108 IU
iD
V4
i
+n DV
iD
VR 4
V4
P
+
10
6
nV
iD
RP
V4
10
iD
+
7
nV
V4
R
+
nV P 1
08
R
P3
A
10
PrME
GOALS
1. Pre-clinical development of next-generation dengue vaccines based on
alphavirus replicon particle technology.
2. Characterize in depth the quality of vaccine-induced immune responses.
3. Identify the best alphavirus replicon approach and the best dengue antigen
configuration to be considered for further testing in clinical trials.
+
VRPE85
DV1
108IU
1. Rationale
The nVRP adjuvant has been used successfully to enhance antibodies, T cells and protection to other
pathogens in mice and NHP models. Efficacy was demonstrated with multivalent vaccines, and significant
dose-sparing potential was shown (LoBue 2006, 2009, Thompson2006, 2008, Tonkin 2010, Carroll 2011).
The optimal dose of VRP as an adjuvant will be much lower that that for expressing optimal amounts of
antigen. Therefore, we anticipate that lower doses (<108 IU) of nVRP mixed with soluble DENV antigens, will
be safe, immunogenic and protective with a significant reduction in cost.
D4
Dengue proteins
Ψ
nVRP AS ADJUVANT
6. Tetravalent (TV) E85-VRP vaccine
1. DENV antigens: prME vs. E85
Anti DV3 Neut50
Two tetravalent vaccines for dengue fever virus (DENV) are being tested in mice
and macaques. The first is based on a VEE replicon particle (VRP) expression
system. Each VRP in the tetravalent cocktail expresses a truncated form of the DEN
E protein (E85) derived from one of the four DEN virus serotypes (DENV1-4). These
single cycle VRP infect dendritic cells (DC) of the draining lymph node and express
the DENV1-4 E85. In macaques, 9/16 animals seroconverted to all 4 serotypes after
only 6 weeks after the first inoculation, and 100% of macaques seroconverted after
a second inoculation 6 weeks after the first. The geometric mean neutralization
titers were 283, 446, 202 and 284, for DENV1-4 respectively. Groups of vaccinated
animals were challenged with each of the DENV serotypes independently at 24
weeks after initiating the vaccination series, and protection against each challenge
serotype was observed as determined by little or no viremia post-challenge.
The second vaccine consists of formalin-inactivated DENV virions mixed with an
alphavirus adjuvant. The adjuvant consists of VRP having only the 5’ and 3’ ends of
the genome flanking the replicase cassette. When this VRP enters a DC, the RNA is
translated into an RNA replication machine and a powerful inducer of the innate
immune response. Adaptive immunity to co-administered soluble and killed virus
immunogens results, including enhanced antibodies and T-cells in both systemic
and mucosal compartments. Two inoculations of a monovalent DENV4 + 106 or 108
IU of the adjuvant, given 6 wks apart, induced geometric mean neutralization titers
of 266 and 368 respectively, both significantly > 13 for the unadjuvanted control but
not significantly different from each other. In each adjuvanted group, 100%
seroconversion was evident as soon as 3 wks after the first inoculation. These
animals will be challenged at week 24, and assessment of a tetravalent inactivated,
adjuvanted vaccine in mice and monkeys is in progress.
n tr
o l-
ABSTRACT
A Not-For-Profit
Vaccine Company
15
10
5
0
8
16
32
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This study is the first to map antigenic domains and specificities targeted by vaccination vs. natural infection,
revealing that unlike prME-VRP and live virus, E85-VRP induced only serotype specific antibodies, which
targeted predominantly EDIII. This suggests a protective mechanism different from that induced by live virus
and possibly live attenuated vaccines. We hypothesize that immunodominance of antibody responses to
specific domains/epitopes on E is influenced by the physical organization of the antigen, and how epitopes are
presented during immunization.
2. A tetravalent E85-VRP dengue vaccine induced a simultaneous and protective
response after 2 doses given 6 weeks apart.
3. nVRP is a potent adjuvant for inactivated dengue virus in mice and NHP.
20
4
CONCLUSIONS
1. E85-VRP induced neutralizing antibodies faster, to higher titers and with
improved protective efficacy than prME-VRP in NHP.
128 256 512 1024
Anti VEE Neut50
(A) Anti-VEE neutralizing antibody titers at 7 wks post prime
and 6 wks post boost. (B) For each monkey, anti VEE titer at
the time of boost is plotted against the fold-increase of antiDENV3 titers (DENV titer post boost/DENV titer pre-boost).
Robust neutralization after a single immunization, with average fold increases of 8X after the prime and 47 X
after the boost in mice, and 5X after the prime and 27X after the boost in NHP.
Qualitative shift from predominantly Th2 to a balance of Th2 and Th1 IgG isotypes.
Significant T cell responses to the E protein in mice.
Macaque effective dose is 106 IU or less, suggesting significant dose sparing potential.
We estimate that the cost of using VRP as an adjuvant in a tetravalent dengue vaccine will be reduced by at
least 2 orders of magnitude relative to the VRP expression system.
This work was supported by Public Health Service grant 5UO1-AI078060 Awarded
by NIAID, NIH.