16TH ANNUAL CONFERENCE ON VACCINE RESEARCH

16TH ANNUAL CONFERENCE ON VACCINE RESEARCH

DEVELOPMENT OF VACCINES TOWARD THE GLOBAL CONTROL AND ERADICATION OFFOOT-AND-MOUTH DISEASE (FMD)

Cyril G. Gay, DVM, PhD owns stock from Pfizer Inc.

Development of vaccines toward the

global control and eradication of Foot-

and-Mouth Disease (FMD)Cyril Gerard Gay, DVM, PhDSenior National Program LeaderAnimal Production and Protection

Agricultural Research Servicecyril.gay@ars.usda.gov

Presentation Outline

1. Importance of Animal Agriculture2. Disease Threats3. Cost of FMD 4. FMD Eradication5. FMD Virology and Pathogenicity6. FMD vaccines7. Conclusions

33

Importance of Animal Importance of Animal AgricultureAgriculture

• FAO estimates that livestock contribute 40% of the global value of agricultural output and support the livelihoods and food security of almost 1 billion people

44

2121stst Century Challenges Century Challenges

• World population is projected to reach 9 billion

• Global food production will need to double in order to meet these food demands.

• 73% increase in consumption of animal protein

55

58% increase in consumption of dairy products

Presentation Outline

1. Importance of Animal Agriculture2. Disease Threats3. Cost of FMD 4. FMD Eradication5. FMD Virology and Pathogenicity6. FMD vaccines7. Conclusions

66

List of 17 Most Damaging List of 17 Most Damaging Animal Disease ThreatsAnimal Disease Threats

1. Highly Pathogenic AI (F) 2. Foot-and-Mouth Disease 3. Rift Valley fever (F) 4. Exotic Newcastle Disease 5. Nipah and Hendra virus

(F) 6. Classical swine fever 7. African swine fever 8. Bovine spongiform

encephalopathy (F) 9. Rinderpest (E) 10. Japanese encephalitis (F)

11. African horse sickness12. Venezuelan equine (F)

encephalitis 13. Contagious bovine

pleuropneumonia 14. Ehrlichia ruminantium

(Heartwater)15. Eastern equine

encephalitis (F)16. Coxiella burnetii (F)17. Akabane virus

F: Potentially fatal to humansYellow text: FBI pathogens of ConcernE: Eradicated

H5N1 Avian Influenza VirusSource: PHIL CDC

Emerging Diseases(and re-emerging diseases)

Human Animal• HIV/AIDS• Ebola*• Hantaan• Legionaire’s disease• BSE*• SARS*• Dengue• West Nile*• Nipah virus*• Rift Valley Fever*• Chikungunya virus• H5N1, H3N2v, H7N9*• pandemic H1N1

• BSE*• CWD• West Nile*• Foot-and-Mouth Disease• Classical Swine Fever• Blue Ear Pig Disease• Rift Valley Fever*• Avian Influenza H5N1/H7N9*• Nipah and Hendra*• Bluetongue• African Swine Fever• African Horse Sickness• pandemic H1N1**

* Zoonoses **Reverse Zoonosis

Presentation Outline

1. Importance of Animal Agriculture 2. Disease Threats3. Cost of FMD 4. FMD Eradication5. FMD Virology and Pathogenicity6. FMD vaccines7. Conclusions

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HistoryHistory

• Over 100 years of research in FMDV

• 1924-British Minister of Agriculture appointed a committee “to initiate, direct and conduct investigations into FMD… discovering means whereby the invasion of the new disease may be rendered less harmful to agriculture…”

(from B.W. Mahy, 2005)

• Successful eradication in Europe

• US free since 1929

In 1898, Freidrich Loeffler and Paul Frosch showed that a virus was responsible for foot-and-mouth disease in cattle

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The Cost of FMDThe Cost of FMD

• Total loss of 2001 outbreak in the United Kingdom was estimated to be between $12.3 and $15 billion (US$)

• 36% was lost tourism , Slaughter of 6.5 million livestock• $4.2 billion paid by government in compensation to the

agriculture and food industry• Social effects, Human cost (suicides)• For some countries mass slaughter is NO LONGER an

option!! (e.g. S. Korea, 2011)

The Cost of FMDThe Cost of FMD• On the global scale FMD causes

damage and hampers development : Cost USD 5 billion per year

• Outbreaks in FMD-free countries worldwide costs USD 1 billion/year

• The world is a global village; risks for FMD-free countries will only increase

• Fighting the disease at source should be part of the prevention strategy of FMD-free countries(Rushton, 2012)

Presentation Outline

1. Importance of Animal Agriculture2. Disease Threats3. Cost of FMD 4. FMD Eradication5. FMD Virology and Pathogenicity6. FMD vaccines7. Conclusions

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Pool 7

O, A

Pool 5 O, A, SAT 1, 2

Pool 6 SAT 1, 2, 3

Pool 4 A, O, SAT 1, 2, 3

Pool 2 O, A, Asia 1

Pool 1 O, A, Asia 1

Pool 3 O, A, Asia 1

Endemic

Free with vaccination

Intermediate, sporadic

Countries with multiple zones:FMD-free, free with vaccination or not free

Free. Virus present in game parks

Free Pool positions are approximate and colours indicate that there are three principal pools, two of which can be subdivided into overlapping areas

Status of FMD showing approximate distribution of regional virus endemic pools

Institute for Animal Health-WRLFMD®

Website: http://www.fao.org/ag/againfo/commissions/eufmd/commissions/eufmd-home/progressive-control-pathway-pcp/en/

FAO/OIE Progressive Control Pathway

for the control of FMD

Within a 15-year period:

1) countries that are currently in PCP Stages 0 and 1 will have progressed at least two stages along the PCP

2) countries in PCP Stages 2 or 3 should also move up two stages, but the final objective will depend on a country’s decision based on cost-effectiveness studies

3) countries or zones that already have an OIE-recognized FMD-free status maintain this status or further improve it (i.e. go from FMD-free with vaccination to FMD-free without vaccination)

Objectives of FMD Control Strategy

- Cost of national FMD programs for 79 initial 0-2 Stage countries: 68 M- Vaccination cost for 45 initial 1-3 Stage countries (excluding India and China): 694 M- Regional level (ref. lab and epidemiology support and networks) 47 M- Global level (coordination, evaluation) 11 M

Financial implications (first 5 years)

(in USD as calculated by the World Bank)

Presentation Outline

1. Importance of Animal Agriculture 2. Disease Threats3. Cost of FMD 4. FMD Eradication5. FMD Virology and Pathogenicity6. FMD vaccines7. Conclusions

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Features of FMDV

25 nm

• Family Picornaviridae, genus aphtovirus

• Positive sense RNA Approximately 8.2 kb

• Seven serotypes: A, O, C, Asia, Sat1, Sat2, Sat3

P1 P2

poly(A)

3'UTR

3B

IRESS

5'UTR

PKs

crepoly(C)

3B1231A

Lpro 1B 1C 1D

2A

2B 2C 3A 3Cpro 3Dpol

P3

**

Protease Cleavage Sites

Lpro unknown

2A3Cpro

2 in-frame AUGs **

FMDV genome

VP0(1AB)

3AB123

structural proteins nonstructural proteins

partial partial cleavagecleavageproductsproducts

P1/2A P2BC P3

3B123CD1ABC 3A

L

VP1

VP3

2C2B

3A 3B13B23B3 3C

3CD

3D

FMDV-Key Information

• Systemic disease of domestic and wild cloven-hooved animals

• Acute disease characterized by fever, lameness, and vesicular lesions on the feet, tongue, snout, and teats

• FMD is considered to be one of the most contagious infectious disease known

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FMDV-Key Information• Multiple subtypes reflect significant genetic

and antigenic variability • Some strains of the virus and some host

species show minimal or no signs of disease• The emergence of new variants of FMDV is

common• Fifty percent of infected cattle become carriers• The pathogenesis of FMDV, including

mechanisms of viral transmission and the carrier state, are not fully understood

• The early detection of FMDV is paramount to stop the spread of the virus and disease and reduce economic impact

2222

Aerosol Inoculation ModelAerosol Inoculation Model

The nebulizer consists of a commercially available aerosol delivery system that will produce an average of particles of 5 microns (Hess et al., 1996) and a large equine mask. Entire respiratory tract is exposed to virus.

Pacheco et al, 2008

Dose: FMDV 107 TCID50

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Summary of FMD Early Pathogenesis in Cattle

V V V V

V VV VV

V

V V

SystemicCirculation

Aerosol ExposureT = 0.1 HPA T = 3 – 6 HPAT = 12 HPAT = 24 HPAT = 48 HPA

24

FMD-BovineMouth

25

FMD-BovineHoof

26

Presentation Outline

1. Importance of Animal Agriculture2. Disease Threats3. Cost of FMD 4. FMD Eradication5. FMD Virology and Pathogenicity6. FMD vaccines7. Conclusions

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FMDV- Commercial Vaccines

• Conventional inactivated vaccines have been successfully used in disease eradication programs in endemic areas of Africa, South America, and Europe

• Requires adaptation of wild type virus to cell culture• Virulent virus grown on BHK cells• Production of large volume requires BSL-3 facilities• Virus yield sometimes low for hard-to-adapt viruses• Inactivated with binary ethyleneimine (BEI)• Non-structural proteins removed• Non-formulated bulk fluids are stored frozen for stockpiling• Adjuvanted with alum or oil emulsion• Vaccines provide fail to induce long lasting immunity

2828

T.R. Doel / Virus Research 91 (2003) 81/99 86

Current Vaccines

29

Inactivated Vaccine

FMDV- Vaccine Information

• FMDV Serotype O is less immunogenic• FMDV Serotype O is more prevalent in

South America • Vaccines for FMDV Serotype O need a

higher payload than Serotypes A, C, Asia, or SAT

• FMDV Serotypes SAT1, SAT2, SAT3 antigens are less stable

• FMDV Serotypes A and SAT 2 are more hypervariable than other serotypes

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Risk of Vaccine Production Risk of Vaccine Production with Virulent FMDV with Virulent FMDV

On Friday August 3, 2007 FMD was detected in a farm in Southern England located within 6 miles of the Pirbright Laboratory site Outbreak resulted in trade barriers and billions $$ loses

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Concerns with FMD Vaccines in Disease-Free Countries

• Require adaptation and growth of large volumes of wild type virus in cells

• Escape of virus from manufacturing facilities• Require banking of multiple antigen concentrates • Some antigens lack stability (low potency/short shelf

life)• Onset of protection 7-14 days• Short duration of immunity <6 months • Difficult to differentiate vaccinated from infected

animals (DIVA) due to presence of NS proteins• Vaccinated and exposed animals become carriers

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Characteristics of the “Ideal” FMD Vaccine

• Effective, rapid and long-lasting protection with one inoculation

• Prevents viral transmission• Allow differentiation of infected from vaccinated

animals (DIVA)• Produced without the need for virulent FMDV• Prevent development of carrier state• Protection against multiple serotypes• Stable antigen – long shelf life• Reasonable cost to enable eradication programs

Adenovirus-Vectored FMD Vaccine

Expressing Empty Viral Capsids

• Contains all protective epitopes present on current inactivated virus vaccine but lacks infectious viral nucleic acid and non-structural protein (NSP)

• Allows to “clearly” distinguish vaccinated from infected animals using 3D and other NSP diagnostic tests

• Can be safely produced in the United States

“Left-out” proteins can be used for DIVA tests

Processed products display

epitopes resembling

intact capsid.

3D

FMDV Empty Capsid Vaccine

P1 2A 2B’ 3B’ 3C

VP0 VP3 VP1

3C

L P1 2A 2B 2C 3A 3B 3C

Remove regions unnecessary for

capsid formation.

3D

DHS TAD ProgramLicensed by CVB-APHIS

2 Negative markers: DIVA tests

3Dpol ELISA

3B ELISA

3B233’NTR

IRESS

1A

1B 1C 1D

2A

2B 2C 3A 3C 3D A

poly(C)

NON-STRUCTURALSTRUCTURAL

L

5’NTR

RE1 RE2

Easy swap of capsid sequences

Vaccine seed antigens

Deletion of Leader protein (543 bp)

Attenuating factor

FMD-LL3B3D: A Safe Platform For FMD Vaccine Production With Built-In DIVA Markers Key Features

36

FMD Vaccine Product Profiles : FMD Vaccine Product Profiles : Current Inactivated versus Current Inactivated versus Inactivated FMD-LL3B3DInactivated FMD-LL3B3D

NoNoProvides cross-serotype protection

NoCompatible with “vaccinate to live” strategy

NoNoReadily deployable (ready to use)

NoNoLong duration of immunity

PossibleNoDomestic production (USA)

+/-Marked vaccine (DIVA capable)

Early onset of immunity (7 DPV)

Prevents viral transmission

MOLECULAR INACTIVATED

CURRENT INACTIVATED

PRODUCT PROFILE

Possible

Proteinase Proteinase domain/De-domain/De-ubiquitinaseubiquitinase

Topology, Topology, DNA binding, DNA binding, transcription transcription regulationregulation

Topology, Topology, interaction with interaction with phosphoproteins, phosphoproteins, signaling signaling interferenceinterference

Interaction Interaction with eIF4Gwith eIF4G

SAP FHA-likeProteinase

1 29 75 112 167 183 201

N C

Lab Lb

Bioinformatics analysis suggests the presence of multiple domains

(by Dr. James Zhu)

Disruption of the L protein SAP domain results in attenuation in vitro

SAP domains are conserved protein domains present in eukaryotic nuclear proteins involved in chromosomal organization and repression of transcription.

Double mutation of FMDV L protein SAP domain results in:

Altered L protein sub-cellular distribution: L SAP mutant localizes only to the cytoplasm of infected cells by 6 hpi while L wild type is in the cytoplasm and nucleus.

L SAP mutant is unable to cause degradation of NF-κB inducing higher levels of IFN, inflammatory cytokines and chemokines in comparison to WT.

(de los Santos et al., 2009)

00 11 22 33 44 55 66 77dpidpi

Virus intradermal inoculation in Virus intradermal inoculation in right rear foot-padright rear foot-pad

TemperatureSerumNasal Swabs

Temperature Plasma/Serum Clinical signs Nasal Swabs

1414 2121

x3x3 x3x3 x3x3xx33

x3x3

Group #1:Group #1: Group #2:Group #2: Group #3:Group #3: Group #4:Group #4: Group #5:Group #5:

FMDV A12-WT1x105 pfu/pig

FMDV A12-WT1x106 pfu/pig

FMDV A12-SAP1x105 pfu/pig

FMDV A12-SAP1x106 pfu/pig

FMDV A12-SAP1x107 pfu/pig

Serum

Does disruption of the L protein SAP Does disruption of the L protein SAP domain results in attenuation domain results in attenuation in vivo?in vivo?

Clinical Score

pfu

/ml

0

2

4

6

8

10

12

14

16

18

0dpc 1dpi 2dpi 3dpi 4dpi 5dpi 6dpi 7dpi

10e5 A12-SAP

10e5 A12-WT

10e6 A12-WT

10e6 A12-SAP

10e7 A12-SAP

Viremia

Nasal swabs

0dpi 1dpi 2dpi 3dpi 4dpi 5dpi 6dpi1.0E+00

1.0E+01

1.0E+02

1.0E+03p

fu/m

l

0.0E+00

4.0E+02

8.0E+02

1.2E+03

1.6E+03

0dpi 1dpi 2dpi 3dpi 4dpi 5dpi 6dpi 7dpi

A12-SAP mutant is attenuated A12-SAP mutant is attenuated in vivoin vivoS

co

re (

ma

x.

17

)

Animals inoculated with A12-SAP are Animals inoculated with A12-SAP are completely protected when challenged completely protected when challenged with WT FMDV at 21 dayswith WT FMDV at 21 days

Group Animal Challenge virus at 21dpi

Dose Viremia (dpc, day of onset, duration)

PFU in nasal swabs (dpc, day of onset,

duration)

Neutralizing antibodies PRN70

7dpc

A12-SAP 1x105pfu/pig

90 A12-WT 1x105 Neg. Neg. >3.1

91 Neg. Neg. >3.1

92 Neg. Neg. >3.1

A12-SAP 1x106pfu/pig

93 A12-WT 1x105 Neg. Neg. >3.1

94 Neg. Neg. >3.1

95 Neg. Neg. >3.1

A12-SAP1x107pfu/pig

96 A12-WT 1x105 Neg. Neg. 3.0

97 Neg. Neg. 3.1

98 Neg. Neg. >3.1

x3x3

x3x3

x3x3

FMDV A12-SAP1x106 pfu/pig

FMDV A12-SAP1x106 pfu/pig

FMDV A12-SAP1x106 pfu/pig

CONTROLPBS

x3x3

x3x3

FMDV A12-SAP1x106 pfu/pig

VACCINATION DOSE

TIME OF CHALLENGE

CHALLENGE DOSE

14dpv14dpv

7dpv7dpv

4dpv4dpv

2dpv2dpv

14dpi14dpi

FMDV A12-WT5x105 pfu/pig

FMDV A12-WT5x105 pfu/pig

FMDV A12-WT5x105 pfu/pig

FMDV A12-WT5x105 pfu/pig

FMDV A12-WT5x105 pfu/pig

SAP mutant vaccination experiment with SAP mutant vaccination experiment with early challenge in swineearly challenge in swine

Inoculation with FMDV A12-SAP confers Inoculation with FMDV A12-SAP confers protection as early as 2 days post protection as early as 2 days post vaccinationvaccination

Vir

us

in s

eru

m o

r n

asal

sw

abs

(p

fu/m

l)

Clin

ical

Sco

re

1.0E+00

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

0dpc 1dpc 2dpc 3dpc 4dpc 5dpc 6dpc 7dpc0

2

4

6

8

10

12

14

16

18

14 dpv

4 dpv

7 dpv

2 dpv

control

viremia NScs

SummarySummary

A12-SAP mutant is avirulent in swine but induces a strong neutralizing antibody response

In vivo attenuation correlates with increased levels of pro-inflammatory cytokines whose transcription

depends on NF-κB

Vaccination of swine with A12-SAP results in complete protection against homologous challenge

as early as 2 days post-inoculation, when no adaptive immune response is detectable

Conclusions

1. Importance of Animal Agriculture2. Disease Threats3. Cost of FMD 4. FMD Eradication5. FMD Virology and Pathogenicity6. FMD vaccines

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www.ars.usda.gov/research/programs/programs.htm?NP_CODE=103Publications

www.ars.usda.gov/GFRA/

Global Foot-and-Mouth Disease Research Alliance

4747

AcknowledgementAcknowledgement

• Luis Rodriguez• Marvin Grubman• Jonathan Arzt• Juan Pacheco• Elizabeth Rieder• James Zhu• Teresa de los Santos• Bill Golde

4848

Thank you!

Thank you !!!