computationally optimized broadly reactive antigen (cobra ......influenza viruses nature reviews...

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


Mouse Clinical Signs

Giles and Ross. 2011. Vaccine. In press

Challenged with A/Whooperswan/Monoglia/244/2005; Clade 2.2.


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




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)


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)


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)


Histopathology

Day 3 post-infection


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


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)


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


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


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


Neu

tralizati

on

Tit

er

Lo

g2

Neutralization Titers


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


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


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?

computationally optimized broadly reactive antigen (cobra ......influenza viruses nature reviews...

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