The 2018 Open Session of the EuFMD Standing Technical Committee was held in Borgo Egnazia - Italy, 29-31 October 2018 . The session theme was on global vaccine security The European Commission for the Control of Foot-and-Mouth Disease (EuFMD), one of FAO’s oldest Commissions, came into being on the 12th June 1954, with the pledge of the sixth founding member state to the principles of a coordinated and common action against Foot-and-mouth Disease.

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IN-VITRO CORRELATES OF HETEROLOGOUS
PROTECTION USING AVIDITY AND IgG-
SUBTYPING ELISAs
1 Senior Researcher of CONICET. Instituto Nacional de Tecnologia
Agropecuaria (INTA). Institute of Virology. Buenos Aires., Argentina
2 Boyd Orr Centre for Population and Ecosystem Health, Institute of
Biodiversity, Animal Health and Comparative Medicine, College of Medical,
Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK
3 The Pirbright Institute, Ash Road, Woking, Surrey, GU24 0NF, UK
Alejandra Capozzo1, Richard Reeve2, David Paton3 and Anna Ludi3

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• Antigen: inactivated viral particles.
• No cross-protection between and in some cases, even within
serotypes.
• Challenge tests are needed to assess protection.
FMDV vaccines
O.I.E.-Manual of Diagnostic Tests and Vaccines for Terrestrial Animals
The use of animals should be avoided where possible by in vitro
alternatives.
• A serological test is considered satisfactory if there is a valid
correlation between the observed protection and the specific
antibody response.
CURRENTLY USED: Virus Neutralization Test (VNT)
Liquid Phase Blocking ELISA (LPBE)

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Is a vaccine likely to protect against a field strain?
reciprocal titer of reference serum against field virus
reciprocal titer of reference serum against vaccine virus
r1 =
r > 0.3 vaccine likely protect
r < 0.3 vaccine unlikely to protect.
CAN WE ANSWER THE QUESTION: NO
CROSS PROTECTION
Vaccine
Containing strain “A”
Field Virus
“B”
Vaccination SEROLOGY
Prediction
of
protection
CHALLENGE

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The inconsistency of “r values”
Vaccine strain
Fieldstrain
Brito e tal., Vaccine, 2014

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Antibody-mediated mechanisms against
virus infection
Neutralization Complement dependent
cytotoxicity
Antibody-dependent cellular
mechanisms of protection:
cytotoxicity, phagocytosis
CDC

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THE AMOUNT OR THE NEUTRALIZING
ACTIVITY OF ANTI-FMDV ANTIBODIES
ARE NOT THE ONLY POSSIBLE SEROLOGICAL
CORRELATES OF PROTECTION

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ASPECTS OF THE PROTECTIVE ANTIBODY RESPONSE:
isotype and avidity
• ISOTYPE - is related to the function of antibodies in
mediating protective responses.
• AVIDITY - is the functional affinity of antibodies, high
avidity antibodies are more likely to be protective.
Avidity is a parameter influenced by the amount of
antibodies, their individual affinities, the paratope
diversity and isotype of specific serum antibodies.

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HOW MANY ANTIBODIES?
WHAT CAN THEY DO
Total antibody levels
Isotype profile
Prevent virus entry
WHICH ONES
BINDING QUALITY
Functional affinity
INFORMATION PROVIDED BY SEROLOGY
LPBE
IgG1/2 ELISAs
Avidity ELISA
VNT
We developed new tests to measure other aspects of the antibody response

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Not just the immunology…
• VNT is labor intensive, not suitable for high throughput
assessments, requires live virus, cell-culture and
dedicated facilities.
• Setting up Liquid-Phase Blocking ELISA (LPBE) for new
virus strains arising in the field is time-consuming,
detector and capture antibodies need to be produced
and standardized.

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Purified 140S
particles
Serum samples
(serial twofold
dilution)
Anti-bovine IgG1
or IgG2 conjugate
Readout:
Ab. titer
FMDV-IgG1 and IgG2 ELISAs

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Purified 140S particles
bound to the wells
DUPLICATE WELLS
Serum samples
(single dilution)
Anti-bovine
conjugate/
chromogen
Readout: AVIDITY
INDEX
PBS WASH
FMDV AVIDITY ELISA
UREA WASH to
remove weak
binders

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2012
2014

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 HETEREOLOGOUS VIRUS challenge study performed in 2006 (Mattion et al., 2009)
n=64 –
 A24/Cruzeiro vaccinated animals
 A/Argentina/2001 challenge strain
PPG test
47 animals had symptoms (POS)
17 animals were protected (NEG)
Assessment of serology as a tool for predicting
cross-protection

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Specificity
0.81 – 1.00
Sensitivity
0.12 – 0.7
All the assays are specific, but have low sensitivity
Only IgG1/IgG2 ratio yielded acceptable sensitivity
Sensitivity and specificity of the different serological
assays for predicting cross-protection
Lavoria et.al Vaccine 2012.
Brito et al., Vaccine, 2014

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• All tests were highly specific but had low
sensitivity
• Increase sensitivity- CONDITIONAL TESTING
In this approach, a first test (T1) is applied; for
samples not detected as “protected”, a second
test (T2) is applied
- if “protected” is the result of T1 or T2, then the
animal is classified as protected.
Brito etal., Vaccine, 2014

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TEST 1 TEST 2 Sensitivity Specificity Accuracy
IgG1 (A/Arg/01) Avidity (A/Arg/01) 0.87 0.88 0.88
Avidity (A/Arg/01) IgG1/IgG2 (A/Arg/01) 0.88 0.95 0.93
IgG1/IgG2 (A/Arg/01) VNT (A/Arg/01) 0.88 0.98 0.95
VNT (A/Arg/01) IgG1/IgG2 (A/Arg/01) 0.88 0.98 0.95
Results of conditional testing
Combining AVIDITY and ISOTYPE measurements gives an
accurate prediction of cross-protection
Brito etal., Vaccine, 2014

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Combining different assays improves the
accuracy of serology for predicting
cross-protection

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Application of current and novel serological tests in a
heterologous protection study
Experimental
vaccine
Antigen
payload per dose
Vaccination
schedule
Protected
animals/total
(% of protected animals)
1- A24/Cruzeiro 10 μg 1-dose 3 / 5 (60%)
2- A24/Cruzeiro 40 μg 1-dose 4 / 5 (80%)
3- A24/Cruzeiro 10 μg
2-doses
(15 days apart)
5 / 5 (100%)
4- A24/Cruzeiro
C3/Indaial
O1/Campos
40 μg
(10+10+20)
1-dose
5 / 5 (100%)
5- PBS 0 / 2 (0%)
Di Giacomo et al. Manuscript in preparation
N=5 animals per group. Challenge virus: A/Arg/01

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Di Giacomo et al. Manuscript in preparation
Protection was not predicted by VNT or LPBE
Neutralizing
antibodies
Total
antibodies

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Di Giacomo et al. Manuscript in preparation
Avidity predicted protection against
heterologous challenge
Non-protected Protected
0
10
20
30
40
50
60
70
80
90
100
Avidity (A/Arg 2001)
*** (p<0.0001)
Challenged with A/Arg/2001
AvidityIndex(%)
105104
84
86
82
92106102
96108
97
91
85100
93101
83
87323103
0
10
20
30
40
50
60
70
80
90
100
Avidity (A/Arg 2001)
AvidityIndex(%)
100
Avidity (A24/Cruzeiro)
** (p=0.0011) 100
Avidity (A24/Cruzeiro)
p<0.001
NON PROTECTED ANIMALS HAD
LOW AVIDITY ANTIBODIES
AGAINST THE FIELD VIRUS

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Di Giacomo et al. Manuscript in preparation
The capacity of avidity to discriminate between protected and
not-protected animals is not restricted to the challenge strainNon-protected Protected
0
10
20
30
40
50
60
70
80
Challenged with A/Arg/2001
AvidityIndex(%)
105104
84
86
82
92106102
96108
97
91
85100
93101
83
87323103
0
10
20
30
40
50
60
70
80
AvidityIndex(%)
Non-protected Protected
0
10
20
30
40
50
60
70
80
90
100
Avidity (A24/Cruzeiro)
** (p=0.0011)
Challenged with A/Arg/2001
AvidityIndex(%)
105104
84
86
82
97108
91
96100102106
93
92
85
87103323101
83
0
10
20
30
40
50
60
70
80
90
100
Avidity (A24/Cruzeiro)
AvidityIndex(%)
Non-protected Protected
0
10
20
30
40
50
60
70
80
90
100
Avidity (O1/Campos)
** (p=0.0018)
Challenged with A/Arg/2001
AvidityIndex(%)
105104
83
84
86
82
97
96106102108
92
91
85
87100
93323103101
0
10
20
30
40
50
60
70
80
90
100
Avidity (O1/Campos)
AvidityIndex(%)
NON PROTECTED ANIMALS HAVE LOW
AVIDITY ANTIBODIES AGAINST THE
VACCINE VIRUSAvidity of
antibodies
against the
vaccine
strain
Avidity of
antibodies
against
another
serotype

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We now have simple tools to
predict protective responses.
They need to be validated

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Technology transfer has already started.
The assays have been deployed to:
• OVI. South Africa. Katherine Scott and Francois Marée.
• CSIRO, Australia. Nagendra Singanallur, Jacquelin
Horsington and Wilna Vosloo.
• SENASA- Argentina. Ongoing. Andrea Pedemonte and
Sabrina Galdo-Novo.
• WRL- The Pirbright Institute, UK. Anna Ludi & team.
Who´s next?

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INTA
• María de los Angeles Lavoria
• Sebastian Di Giacomo
• Juan Manuel Sala
• Myrian Trotta
• Florencia Mansilla
• Danilo Bucafusco
• Mariano Pérez Filgueira
Biogenesis. Bago
SENASA
Collaborators
• Andrés Pérez
• Bárbara Brito
UK
• Richard Reeve
• David Paton
• Anna Ludi

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FAO HQ Expert Group on FMD vaccine matching

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The Applied Veterinary Immunology Lab.
Institute of Virology, INTA. Buenos Aires.
THANK YOU

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ASSESSMENT OF EXISTING AND FUTURE
VACCINE SELECTION TECHNIQUES –
MOVING FORWARD
Richard Reeve1, David Paton2, Anna Ludi2 and Alejandra Capozzo3
1 Boyd Orr Centre for Population and Ecosystem Health, Institute of
Biodiversity, Animal Health and Comparative Medicine, College of Medical,
Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK.
2 The Pirbright Institute, Ash Road, Woking, Surrey, GU24 0NF, UK.
3 Senior Researcher of CONICET, Instituto Nacional de Tecnologia
Agropecuaria (INTA), Institute of Virology, Buenos Aires., Argentina.

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OIE Manual
of
Diagnostic
Tests

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Vaccine
selection:
the gold
standard

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Vaccine
selection:
the gold
standard

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Vaccine
selection:
the gold
standard

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Vaccine selection?
Vaccines with
high potency
Vaccines
that match
circulating
strains
Available or
affordable
vaccines
?

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Vaccine selection
Vaccines with
high potency
Available or
affordable
vaccines
Vaccines
that match
circulating
strains

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Vaccine selection
Vaccines with
high potency
Available or
affordable
vaccines
Vaccines
that match
circulating
strains
*

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Vaccine selection
Vaccines with
high potency
Available or
affordable
vaccines
Vaccines
that match
circulating
strains

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Vaccine selection
Vaccines that
are potent
enough against
circulating
strains

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Vaccine selection

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Vaccine selection

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Shortcomings of VNT and ELISA?
0.20.40.60.81.0
Unprotected Protected
●
●
Predictedprobofprotection
Testing
with good
quality,
high
potency
vaccines…

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0.00.20.40.60.81.0
Unprotected Protected
●
●Predictedprobofprotection
Shortcomings of VNT and ELISA?

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0.00.20.40.60.81.0
Unprotected Protected
●
●Predictedprobofprotection
Shortcomings of VNT and ELISA?

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New vaccine
selection
tools

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So how do
IgG1/IgG2
and the
avidity index
stack up?

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So how do
IgG1/IgG2
and the
avidity index
stack up?

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Conclusions
• r1 values – whether VNT or ELISA – are disastrous
tools for vaccine matching
• IgG1/IgG2 isotype ELISA ratios look very promising
for vaccine selection
• All of these tests can predict match / help selection
better in combination than individually
• We need more sera from experimentally
challenged animals to validate and help refine
these tests!
• Especially from heterologous challenges…

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Acknowledgements
Samples and lab work:
• Argentina: INTA, SENASA, Biogénesis Bagó
• UK: Pirbright Institute
• South Africa: Onderstepoort Veterinary Institute
• Germany: FLI
• Belgium: VAR / Sciensano
• You ?

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INTA
University of Glasgow
The Pirbright Institute
Funders:
EuFMD, DEFRA, EUFP7