computationally optimized broadly reactive antigen (cobra ......influenza viruses nature reviews...
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Ted M. Ross*
University of Pittsburgh, Center for Vaccine Research
Computationally Optimized Broadly Reactive Antigen (COBRA):
A novel strategy for developing a broadly reactive vaccine against
emerging H5N1 influenza
Overview A goal of influenza vaccine development is the elicitation of cross-
protective immunity. It is currently impossible to predict which antigenic variants may emerge and therefore an ideal vaccine will elicit immunity to most potential variants.
H5N1 Influenza. Support from NIAID and PATH Vaccine Solutions.
Seasonal Influenza (H1N1, H3N2, and B Influenza) in partnership with Sanofi-Pasteur.
Strategies are under development using various influenza immunogens.
Hemagglutinin (HA).
Developing a cross-reactive vaccine that elicits cross-protective immunity to multiple clades may provide more promise than a vaccine that is unable to elicit immunity across current clades. Current vaccine strategies have met this goal with mixed success.
Influenza Viruses
Nature Reviews Microbiology. 2005. 3:591.
Occasionally emerge (10-50 yrs)
Genetic reassortment
Antigenic shift
World Health Organization
Criteria
• Highly pathogenic
• Novel subtype
• Efficient human to human
transmission
Influenza Pandemics
H5N1 Background
Initially emerged in poultry and humans in 1997
Diversity within subtype
• 10 phylogenetic clades
• Geographically distinct
• Human infections from
clades 0, 1, 2 and 7
Total Clade Distribution
0
1
2
3
4
5
6
7
8
9
2
1
0
Clade 2: Westward Bound
Clade 1
Clade 2.1
Clade 2.2
Clade 2.3
Clade 7
H5N1 Vaccines
High virulence complicates traditional influenza vaccine production strategies
• Wild-type viruses do not grow to high titer in eggs
• High pathogenicity mandates ABSL-3 manufacturing facilities
H5N1 HA is poorly immunogenic
• Larger doses than seasonal vaccine
• Adjuvant inclusion
Poor cross-reactivity between clades
• Current FDA approved vaccine is from clade 1
• Predict pandemic clade?
• Increase breadth of vaccines?
How do we overcome viral diversity?
Broadening Strategies
Traditional (Standard)
• Polyvalent • Mix multiple antigens
into a single formulation
• Seasonal flu, HPV, pneumococcal
• Breadth limited to that of components
New Generation
• Centralized • Utilize sequencing efforts
• Capture multiple antigenic features in single molecule
• Not naturally occurring
Centralized Sequences
Synthetic sequences that represent a given population
Three methods for generating centralized sequences
1. Ancestral: the most recent common ancestor
2. Center of the tree: the phylogenetic point that is equidistant from all input sequences
3. Consensus: the most common amino acid at each position
Nickel et al. Science. 2003
Goals
Design and characterize centralized antigen to address diversity within clade 2
• Diverse
• Prevalent
• Spreading west
Compare centralized antigen and polyvalent vaccine approaches
Evaluate protective mechanism
Antigen Design
Computationally Optimized Broadly Reactive Antigen (COBRA) • Align amino acid sequences from Clade 2 human isolates • Assemble ‘Layered’ Consensus • Limit sampling bias
Confirm presence of conserved linear epitopes • (Immune epitope database; www.immuneepitope.org)
Giles and Ross; Vaccine; 2011
COBRA Phylogeny
Giles et al. 2011. In preparation Giles and Ross. 2011. Vaccine. 29:3043-54
Recombinant Virus-like Particle Candidate
Vaccine
• Exact genetic match
• Correct 3D configuration of HA/NA
proteins
• Efficient insect cell-based production
• No safety risks associated with live virus
pPolh pPolh pPolh
NA HA M1
100 µm
HA M1 NA
Mouse Vaccine Study:
Study Design
Goals:
• Compare VLPs with COBRA HA to VLPs with Clade 2.2 HA
• Challenge with HPAI clade 2 H5N1 virus
• Morbidity/mortality
• Lung titers at D1, D3, D5 post-challenge
Group N Description HA Dose Adjuvant
1 20 COBRA 3ug Imject
2 20 Clade 2.2 3ug Imject
3 5 Mock - Imject
Week: 0 3 5 6
Procedure: Prime Boost/Bleed Bleed Challenge
Giles and Ross. 2011. Vaccine. 29:3043-54
Mouse Antibody Responses
0 3 5 6
V V B C
8-12 week
BALB/c
3ug HA + Alum
Giles and Ross. 2011. Vaccine. 29:3043-54
Mouse Clinical Signs
Giles and Ross. 2011. Vaccine. In press
Challenged with A/Whooperswan/Monoglia/244/2005; Clade 2.2.
Giles and Ross. 2011. Vaccine. 29:3043-54
Mouse COBRA vs. Polyvalent Immunogenicity
0 3 5 6
V V B C
8-12 week
BALB/c
3ug HA + Alum
Total IgG(End Point Titers)
Cla
de 1
Cla
de 2.
1
Cla
de 2.
2
Cla
de 2.
3
100
1000
10000
100000
En
d P
oin
t T
iter
(GM
T)
Receptor Blocking Antibody(HAI Titers)
Cla
de 1
Cla
de 2.
1
Cla
de 2.
2
Cla
de 2.
3
10
20
40
80
160
320
640
1280
2560
5120COBRA
Polyvalent
Mock
HA
I T
iter
(GM
T)
*
*
**
Group N Description HA Dose Adjuvant
1 20 COBRA 3ug Imject
2 20 Clade 2.1 3ug Imject
3 20 Clade 2.2 3ug Imject
4 20 Clade 2.3 3ug Imject
5 20 Polyvalent Mix 3ug Imject
6 5 Mock - Imject
Giles et al. 2012. CVI. 19(2):128-39.
Mouse H5N1 (Clade 2.2) Challenge
Challenge virus: Clade 2.2
(A/Whooper Swan/Mongolia/244/2005)
0 3 5 6
V V B C
8-12 week
BALB/c
3ug HA + Alum
Lung Viral Titers
Day 1 Day 3 Day 5
102
103
104
105
106
107
108
109
Day Post Infection
PF
U/g
tis
su
e
COBRA
Polyvalent
Mock
Mouse Lung Viral Titers
Day 1 Day 3 Day 5
102
103
104
105
106
107
108
109
COBRA
Clade 2.1
Clade 2.2
Clade 2.3
Limit of detection
Mock
Day Post Infection
PF
U/g
tis
su
e
Giles et al. 2012. CVI. 19(2):128-39.
Mouse H5N1 (Clade 1) Challenge
Challenge virus: Clade 1 Reassortant (A/Vietnam/1203/2004)
0 3 5 6
V V B C
8-12 week BALB/c 3ug HA + Alum
Challenged with A/Vietnam/1203/2004, Clade 1
Lung Viral Titers
3 Days Post Infection
PF
U/g
tis
su
e
102
103
104
105
106
107
108
109
COBRA
Polyvalent
Mock
Weight Loss
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
70
80
90
100
110
Day Post Infection
% O
rig
inal W
eig
ht
Sickness
-1 0 1 2 3 4 5 6 7 8 9 10 1112 13140
1
2
3
4
5COBRA
Polyvalent
Mock
Day Post Infection
Rela
tive S
co
re
Giles et al. 2012. CVI. 19(2):128-39.
Ferret Immunogenicity and Protection
0 3 5 6
V V B C
3-4 month
Fitch
15ug HA + Alum
Total IgG(End Point Titers)
Cla
de 1
Cla
de 2.
1
Cla
de 2.
2
Cla
de 2.
3
100
1000
10000
100000
Test Antigen
En
d P
oin
t T
iter
(GM
T)
Receptor Blocking Antibody(HAI Titers)
Cla
de 1
Cla
de 2.
1
Cla
de 2.
2
Cla
de 2.
3
10
20
40
80
160
320
640
1280
2560
5120
10240COBRA
Polyvalent
Mock
Test AntigenH
AI T
iter
(GM
T)
*
Nasal Wash Viral Titers
Day Post Infection
PF
U/m
l
Day 1 Day 3 Day 5
100
101
102
103
104
105
106
COBRA
Polyvalent
Mock
Giles et al. 2012. CVI. 19(2):128-39.
Monkey Study
Tissues Nasal Washes RNA for Microarray Histopathology Gross Pathology Immunology
Group n=7
WS/05 VLP
COBRA-2 VLP
Unvaccinated
Cynomolgus Macaques
Route: IM
Immunogen: VLP
VLP Dose: 15ug
Adjuvant: Imject
Vaccination: Week 0, 3, 6
Challenge: WS/05; Clade 2.2
0 3 6 9
Challenge
Week
Vaccination Regimen
V V
11
Giles et al. 2012. J. Inf. Dis. 205(10):1562-70.
NHP Immunogenicity Breadth
(HAI)
Receptor Blocking Antibody(HAI Titers)
Cla
de 0
(HK/4
83/9
7)
Cla
de 1
(HK/2
13/0
3)
Cla
de 1
(VN/1
203/
04)
Cla
de 2.
1.1
(Dk/
HU/0
2)
Cla
de 2.
1.3
(IN/0
5/05
)
Cla
de 2.
2.1
(Eg/3
21/0
7)
Cla
de 2.
2.1
(Eg/3
300/
08)
Cla
de 2.
2.2
(Tk/
EG/0
7)
Cla
de 2.
2.2
(Tk/
Tk/05
)
Cla
de 2.
2.2
(WS/0
5)
Cla
de 2.
2.2
(BHG/1
/05)
Cla
de 2.
3.2
(CM
P/HK/0
7)
Cla
de 2.
3.2
(Buz/
Bul/1
0)
Cla
de 2.
3.4
(JW
E/103
8/06
)
Cla
de 2.
3.4
(AN/1
/05)
Cla
de 4
(Gs/
1175
/06)
Cla
de 7
(Ck/
VN/0
8)
Cla
de 2.
0 COBRA
3
4
5
6
7
8
9
10
Whooper Swan Clade 2.2 VLP
COBRA VLP
Mock (Alum Only)
*** p<0.001
** p<0.01
* p<0.05
0 1 2.1 2.2 2.3 4 7
** ***
***
****
****
*** **
******
**
Virus
HA
I G
MT
(L
og
2)
Giles et al. 2012. J. Inf. Dis. 205(10):1562-70.
Histopathology
Day 3 post-infection
Giles et al. 2012. J. Inf. Dis. 205(10):1562-70.
Data Summary
COBRA sequence is a functional protein
COBRA vaccine is immunogenic
COBRA vaccine protects from H5N1 viral challenge
• All vaccinated animals were completely protected from morbidity and mortality
• COBRA vaccinated animals have decreased viral replication
COBRA
Expand these results using second generation H5N1
COBRA to capture isolates.
Development of H1N1 and seasonal influenza.
Capture multiple epitopes in a single immunogen to
elicit a broadly reactive polyclonal antibody
response to HA.
Can this work for Seasonal Influenza?
Novel H1N1 CA/09 Den/57 PR/34 FM/47
Determine:
Clinical Signs,
Weight Loss,
Viral Titers,
&Transmission
3mo 3mo 3mo
Aerosol
Transmission
Contact
Transmission
CA/09 SI/06 Bris/07 NC/99 TX/91 Sing/86 CA/78
Den/57 PR/34 FM/47 1918
H1N1 Antigenic Timeline
Weiss/43
PR/34
3mo
Novel H1N1 CA/09
FM/47
3mo
Novel H1N1 CA/09
Individual Infections
Sequential Infections
Carter et al. 2013. J Virol. 87(2). 1400-10
Experimental Design
Group Number Initial Virus Infection
1 4
A/PR/8/34; A/FM/1/47;
A/Denver/1/57
2 4
A/Texas/36/91; A/NC/20/99;
A/Brisbane/59/07
3 4 A/PR/8/34
4 4 A/FM/1/47
5 4 A/Denver/1/57
6 4 A/Texas/36/91
7 4 A/NC/20/99
8 4 A/Bris/59/07
9 5 A/Cal/07/09
Ferrets will be nasal washed at Day 1, 2, 3, 5, 7 for viral load analysis
Ferrets will be bled Day 14 post infection; bled every month for 4 months
2 Ferrets from groups 3-9 were infected with A/Cal/07/09 at 4 months
• Nasal Wash at Day 1, 3, 5, 7 for viral load
• Bled at D7; 14; 28 post infection
Remaining Ferrets will be monitored until antibody titer.
2 ferrets from group 1 and 2 will be infected with next virus at 4 months
• Nasal Wash at Days 1, 3, 5, 7 for viral load
• Bled at D7; 14; 28 post infection
Remaining Ferrets will be monitored until antibody titer
Carter et al. 2013. J Virol. 87(2). 1400-10
Direct Infection
0 1 2 3 4 5 6 7 8 9 10 11 12 13 1480
85
90
95
100
105
110
115
A/FM/1/1947
Naive
A/PR/8/1934
A/Den/1/1957Historical Sequential
Days post-infection
Perc
en
t B
od
y W
eig
ht
Direct Infection
0 1 2 3 4 5 6 7 8 9 10 11 12 13 1480
85
90
95
100
105
110
A/TX/36/1991
A/NC/20/1999
Naive
A/Bris/59/2007
Modern Sequential
Days post-infection
Perc
en
t B
od
y W
eig
ht
A.
C.
Direct Infection
0 1 2 3 4 5 6 7 8 9 10 11100
101
102
103
104
105
106
107
108
109
A/FM/1/1947
A/PR/8/1934
Naive
A/Den/1/1957
Historical Sequential
Days post-infection
PF
U/m
l in
Nasal W
ash
Direct Infection
0 1 2 3 4 5 6 7 8 9 10 11100
101
102
103
104
105
106
107
108
109
A/TX/20/1991
A/Bris/59/2007
A/NC/20/1999
Naive
Modern Sequential
Days post-infection
PF
U/m
l in
Nasal W
ash
B.
D.
Weight and Viral Titers
Direct Infection with novel H1N1 (A/California/07/2009)
Carter et al. 2013. J Virol. 87(2). 1400-10
Fig. 7 A. B.
D. E.
G.
F.
C.
A/PR/8/34 Infected Ferrets
A/P
R/8
/34
A/F
M/1
/47
A/D
enve
r/1/
57
A/C
alifo
rnia
/1/7
8
A/S
ingap
ore/1
/86
A/T
exas
/36/
91
A/N
C/2
0/99
A/B
risb
ane/
59/0
7
A/C
alifo
rnia
/07/
09
5
10
15
Day 14
Day 84
Test Virus
HA
I T
iter
Lo
g2
A/FM/1/47 Infected Ferrets
A/P
R/8
/34
A/F
M/1
/47
A/D
enve
r/1/5
7
A/C
alifo
rnia
/1/7
8
A/S
ingap
ore/1
/86
A/T
exas
/36/
91
A/N
C/2
0/99
A/B
risb
ane/
59/0
7
A/C
alifo
rnia
/07/
09
5
10
15
Day 14Day 84
Test Virus
HA
I T
iter
log
2
A/Denver/1/57 Infected Ferrets
A/P
R/8
/34
A/F
M/1
/47
A/D
enve
r/1/
57
A/C
alifo
rnia
/1/7
8
A/S
ingap
ore/1
/86
A/T
exas
/36/
91
A/N
C/2
0/99
A/B
risb
ane/
59/0
7
A/C
alifo
rnia
/07/
09
5
10
15
Day 14Day 84
Test Virus
HA
I T
iter
Lo
g2
A/Texas/36/91 Infected Ferrets
A/P
R/8
/34
A/F
M/1
/47
A/D
enve
r/1/
57
A/C
alifo
rnia
/1/7
8
A/S
ingap
ore/1
/86
A/T
exas
/36/
91
A/N
C/2
0/99
A/B
risb
ane/
59/0
7
A/C
alifo
rnia
/07/
09
5
10
15
Day 14Day 84
Test Virus
HA
I T
iter
Lo
g2
A/NC/20/99 Infected Ferrets
A/P
R/8
/34
A/F
M/1
/47
A/D
enve
r/1/
57
A/C
alifo
rnia
/1/7
8
A/S
ingap
ore/1
/86
A/T
exas
/36/
91
A/N
C/2
0/99
A/B
risb
ane/
59/0
7
A/C
alifo
rnia
/07/
09
5
10
15
Day 14
Day 84
Test Virus
HA
I T
iter
Lo
g2
A/Brisbane/59/07 Infected Ferrets
A/P
R/8
/34
A/F
M/1
/47
A/D
enve
r/1/5
7
A/C
alifo
rnia
/1/7
8
A/S
ingap
ore/1
/86
A/T
exas
/36/
91
A/N
C/2
0/99
A/B
risb
ane/
59/0
7
A/C
alifo
rnia
/07/
09
5
10
15Day 14
Day 84
Test Virus
HA
I T
iter
Lo
g2
A/California/07/09 Infected Ferrets
A/P
R/8
/34
A/F
M/1
/47
A/D
enve
r/1/5
7
A/C
alifo
rnia
/1/7
8
A/S
ingap
ore/1
/86
A/T
exas
/36/
91
A/N
C/2
0/99
A/B
risb
ane/
59/0
7
A/C
alifo
rnia
/07/
09
5
10
15Day 7
Day 14
Day 28
Test Virus
HA
I T
iter
Lo
g2 HAI Titers
Individual Infections
Carter et al. 2013. J Virol. 87(2). 1400-10
Historical Group
0 14 84 98 168 182 252
4
6
8
10
12
14
16
A/FM/1/47
A/PR/8/34
A/Denver/1/57
A/California/07/09
Days Post Infection
HA
I T
iter
Lo
g2
Modern Group
0 14 84 98 168 182 252
4
6
8
10
12
14
16A/NC/20/99
A/Texas/36/91
A/Brisbane/59/07
A/California/07/09
Days Post InfectionH
AI T
iter
Lo
g2
Infection with
A/PR/8/34
Infection with
A/FM/1/47
Infection with
A/Denver/1/57
Infection with
A/Texas/36/91
Infection with
A/NC/20/99
Infection with
A/Brisbane/59/07
A. B.
HAI Titers
Sequential Infections
Carter et al. 2013. J Virol. 87(2). 1400-10
A. B.
A/P
R/8
/193
4
A/F
M/1
/194
7
A/D
en/1
/195
7
A/T
X/36/
1991
A/N
C/2
0/19
99
A/B
ris/5
9/20
07
A/C
A/0
7/20
09
5
10
15
Sera from Infected Ferrets
Neu
tralizati
on
Tit
er
Lo
g2
First
His
torica
l
Second H
isto
rica
l
Third H
isto
rica
l
First
Moder
n
Second M
odern
Third M
odern
A/C
A/0
7/09
5
10
15
Sera from Infected Ferrets
Neu
tralizati
on
Tit
er
Lo
g2
Neutralization Titers
Carter et al. 2013. J Virol. 87(2). 1400-10
1918
A/P
R/8
/34
A/F
M/1
/47
A/D
env/
1/57
A/T
exas
/1/9
1
A/N
C/2
0/99
A/B
ris/
59/0
7
A/C
A/0
7/09
3
4
5
6
7
8
9
10Modern Mixed Sera
Historical Mixed Sera
Virus
HA
I T
iter
Lo
g2
His
toric
al Mix
Moder
n Mix
A/C
A/0
7/09 x c v b
5
10
15
Sera from Infected Ferrets
Neu
traliz
ati
on
Tit
er
Lo
g2
B. A.
A/P
R/8
/34
A/F
M/1
/47
A/D
en/1
/57
A/T
exas
/1/9
1
A/N
C/2
0/99
A/B
ris/
59/0
7
A/C
A/0
7/09
3
4
5
6
7
8
9
10PR/34-Bris/07
PR/34-Den/57
Virus
HA
I T
iter
Lo
g2
C.
HAI and Neutralization Titers
Mixing Individual Serum Samples
Carter et al. 2013. J Virol. 87(2). 1400-10
Acknowledgements University of Pittsburgh
• Corey Crevar
• Donald Carter
• Franklin Toapanta
• Dilhari DeAlmeida
• Kirsten Schneider-Orhum
• Brendan Giles
• Nitin Bhardwaj
• Hermancia Eugene
• Xian-Chun Tang
• Heather Weirback
• Hai-Rong Lu
• Brooke Pierce
• Joshua Roy
Center Modeling Pulmonary Immunity
Penny Morel
Joanne Flynn
Jerry Nau
Russell Salter
Sholmo Ta’asan
Gilles Clermont
Takis Benos
Department of NeuroPathology Clayton Wiley Stephanie Bissel Department of Immunology Olja Finn Karen Norris Department of MMG Mickey Corb Center for Vaccine Research Simon Barratt-Boyes Elodie Ghedin Jared Evans Ernesto Marques Cristian Apetrei Ivona Pandrea Douglas Reed Kate Ryman William Klimstra Charles Scanga UPMC Shanta Zimmer Rick Zimmerman Sally Wetzel Bruce Lee Kerry Empey
Department of IDM Phalguni Gupta Todd Reinhart
Influenza Projects Supported by:
NIH/NIAID
NSF
DoD
PATH Vaccine Solutions
Sanofi-Pasteur
Rick Bright-HHS-BARDA
David Lipmam & Joshua Cherry-NCBI
Terry Tumpey, Thomas Rowe, Ruben Donis-CDC
Annie DeGroot-Univ. Rhode Island/Epivax
Jay Kolls-LSU
Shabaana Khader-CHP
Nikolai Petrovsky-Flinders Med Ctr/Vaxxine
David Kelvin-UHN Toronto
Hana Golding-FDA/CBER
John Hiscott-VGTI-FL
Dave Wentworth-JCVI
James Smith, Janet McNichols-CDC
Mark Heise-Univ. North Carolina
Robert Doms-Univ. Pennsylvania
David Weiner-Univ. Pennsylvania
William Wilson-USDA-ABADRU/CGAHR
Michael Diamond-Washington Univ.
Shan Lu-UMass
National & International Collaborations
Influenza-Related Non-Influenza-Related
Questions?