1. 16TH
ANNUAL CONFERENCE ON
VACCINE RESEARCH
16TH
ANNUAL CONFERENCE ON
VACCINE RESEARCH
DEVELOPMENT OF VACCINES TOWARD THE
GLOBAL CONTROL AND ERADICATION OF
FOOT-AND-MOUTH DISEASE (FMD)
Cyril G. Gay, DVM, PhD owns stock from Pfizer Inc.

2. Development of vaccines toward the
global control and eradication of Foot-
and-Mouth Disease (FMD)Cyril Gerard Gay, DVM, PhD
Senior National Program Leader
Animal Production and Protection
Agricultural Research Service
cyril.gay@ars.usda.gov

3. Presentation Outline
1. Importance of Animal Agriculture
2. Disease Threats
3. Cost of FMD
4. FMD Eradication
5. FMD Virology and Pathogenicity
6. FMD vaccines
7. Conclusions
33

4. Importance of AnimalImportance 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

5. 2121stst
Century ChallengesCentury 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

6. Presentation Outline
1. Importance of Animal Agriculture
2. Disease Threats
3. Cost of FMD
4. FMD Eradication
5. FMD Virology and Pathogenicity
6. FMD vaccines
7. Conclusions
66

7. List of 17 Most DamagingList 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 sickness
12. 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 humans
Yellow text: FBI pathogens of Concern
E: Eradicated
H5N1 Avian Influenza Virus
Source: PHIL CDC

8. 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

9. Presentation Outline
1. Importance of Animal Agriculture
2. Disease Threats
3. Cost of FMD
4. FMD Eradication
5. FMD Virology and Pathogenicity
6. FMD vaccines
7. Conclusions
99

10. 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
1010

11. 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)

12. 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)

13. Presentation Outline
1. Importance of Animal Agriculture
2. Disease Threats
3. Cost of FMD
4. FMD Eradication
5. FMD Virology and Pathogenicity
6. FMD vaccines
7. Conclusions
1313

14. 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
®

15. 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

16. 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

17. - 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)

18. Presentation Outline
1. Importance of Animal Agriculture
2. Disease Threats
3. Cost of FMD
4. FMD Eradication
5. FMD Virology and Pathogenicity
6. FMD vaccines
7. Conclusions
1818

19. 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

20. P1 P2
poly(A)
3'UTR
3B
IRES
S
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
partialpartial
cleavagecleavage
productsproducts
P1/2A P2BC P3
3B123CD1ABC 3A
L
VP1
VP3
2C2B
3A 3B1
3B2
3B3 3C
3CD
3D

21. 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
2121

22. 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

23. 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
2323

24. Summary of FMD Early Pathogenesis in Cattle
V V
V V
V V
V V
V
V
V V
Systemic
Circulation
Aerosol Exposure
T = 0.1 HPA
T = 3 – 6 HPA
T = 12 HPA
T = 24 HPA
T = 48 HPA
24

25. FMD-Bovine
Mouth
25

26. FMD-Bovine
Hoof
26

27. Presentation Outline
1. Importance of Animal Agriculture
2. Disease Threats
3. Cost of FMD
4. FMD Eradication
5. FMD Virology and Pathogenicity
6. FMD vaccines
7. Conclusions
2727

28. 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

29. T.R. Doel / Virus Research 91 (2003) 81/99 86
Current
Vaccines
29
Inactivated
Vaccine

30. 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
3030

31. Risk of Vaccine Production withRisk of Vaccine Production with
Virulent FMDVVirulent 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
3131

32. 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
3232

33. 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

35. 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 VP
1
3C
L P1 2A 2B 2C 3A 3B 3C
Remove regions
unnecessary for
capsid formation.
3D
DHS TAD Program
Licensed by CVB-APHIS

36. 2 Negative markers: DIVA tests
3Dpol
ELISA
3B ELISA
3B23
3’NTR
IRES
S
1A 1B 1C 1D
2A
2B 2C 3A 3C 3D A
poly(C)
NON-STRUCTURALSTRUCTURAL
δ 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

37. FMD Vaccine Product Profiles :FMD Vaccine Product Profiles :
Current Inactivated versusCurrent 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

38. ProteinaseProteinase
domain/De-domain/De-
ubiquitinaseubiquitinase
Topology,Topology,
DNA binding,DNA binding,
transcriptiontranscription
regulationregulation
Topology,Topology,
interaction withinteraction with
phosphoproteins,phosphoproteins,
signalingsignaling
interferenceinterference
InteractionInteraction
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)

39. 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)

40. 00 11 22 33 44 55 66 77dpidpi
Virus intradermal inoculation inVirus intradermal inoculation in
right rear foot-padright rear foot-pad
Temperature
Serum
Nasal Swabs
Temperature Plasma/Serum
Clinical signs Nasal Swabs
1414 2121
x3x3 x3x3 x3x3xx
33
x3x3
Group #1:Group #1: Group #2:Group #2: Group #3:Group #3: Group #4:Group #4: Group #5:Group #5:
FMDV A12-WT
1x105
pfu/pig
FMDV A12-WT
1x106
pfu/pig
FMDV A12-SAP
1x105
pfu/pig
FMDV A12-SAP
1x106
pfu/pig
FMDV A12-SAP
1x107
pfu/pig
Serum
Does disruption of the L protein SAPDoes disruption of the L protein SAP
domain results in attenuationdomain results in attenuation in vivo?in vivo?

41. 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 6dpi
1.0E+00
1.0E+01
1.0E+02
1.0E+03pfu/ml
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 attenuatedA12-SAP mutant is attenuated in vivoin vivoScore(max.17)

42. Animals inoculated with A12-SAP areAnimals inoculated with A12-SAP are
completely protected when challengedcompletely 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
1x105
pfu/
pig
90 A12-WT 1x105
Neg. Neg. >3.1
91 Neg. Neg. >3.1
92 Neg. Neg. >3.1
A12-SAP
1x106
pfu/
pig
93 A12-WT 1x105
Neg. Neg. >3.1
94 Neg. Neg. >3.1
95 Neg. Neg. >3.1
A12-SAP
1x107
pfu/
pig
96 A12-WT 1x105
Neg. Neg. 3.0
97 Neg. Neg. 3.1
98 Neg. Neg. >3.1

43. x3x3
x3x3
x3x3
FMDV A12-SAP
1x106
pfu/pig
FMDV A12-SAP
1x106
pfu/pig
FMDV A12-SAP
1x106
pfu/pig
CONTROL
PBS
x3x3
x3x3
FMDV A12-SAP
1x106
pfu/pig
VACCINATION
DOSE
TIME OF
CHALLENGE
CHALLENGE
DOSE
14dpv14dpv
7dpv7dpv
4dpv4dpv
2dpv2dpv
14dpi14dpi
FMDV A12-WT
5x105
pfu/pig
FMDV A12-WT
5x105
pfu/pig
FMDV A12-WT
5x105
pfu/pig
FMDV A12-WT
5x105
pfu/pig
FMDV A12-WT
5x105
pfu/pig
SAP mutant vaccination experiment withSAP mutant vaccination experiment with
early challenge in swineearly challenge in swine

44. Inoculation with FMDV A12-SAP confersInoculation with FMDV A12-SAP confers
protection as early as 2 days postprotection as early as 2 days post
vaccinationvaccination
Virusinserumornasalswabs
(pfu/ml)
ClinicalScore
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 7dpc
0
2
4
6
8
10
12
14
16
18
14 dpv
4 dpv
7 dpv
2 dpv
control
viremia NScs

45. 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

46. Conclusions
1. Importance of Animal Agriculture
2. Disease Threats
3. Cost of FMD
4. FMD Eradication
5. FMD Virology and Pathogenicity
6. FMD vaccines
4646
www.ars.usda.gov/research/programs/programs.htm?NP_CODE=103
Publications

47. www.ars.usda.gov/GFRA/
Global Foot-and-Mouth Disease
Research Alliance
4747

48. AcknowledgementAcknowledgement
• Luis Rodriguez
• Marvin Grubman
• Jonathan Arzt
• Juan Pacheco
• Elizabeth Rieder
• James Zhu
• Teresa de los Santos
• Bill Golde
4848

49. Thank you!

50. Thank you !!!