FMD_ImproCon
Publishable Final Activity Report
SSPE-CT-2003-503603
SSPE-CT-2003-503603
FMD_ImproCon
Improvement of Foot and Mouth disease control by ethically acceptable methods based
on scientifically validated assays and new knowledge on FMD vaccines, including the
impact of vaccination
Instrument: Specific Targeted Research
Thematic Priority: Policy-oriented Research
Publishable Final Activity Report
Period covered: from 01-01-2004 to 31-12-2008
Date of preparation: 22-04-2009
Start date of project: 01-01-2004
Duration: 60 months
Project coordinator:
Dr. Kris De Clercq
Centrum voor Onderzoek in Diergeneeskunde en Agrochemie (CODA)
Veterinary and Agrochemical Research Centre (VAR)
Version 01
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1 – PROJECT EXECUTION
Project objectives
There is a strong desire to reduce reliance on large-scale culling of animals to control future
outbreaks of foot-and-mouth disease (FMD) in EU Member States. Consequently, the
European Commission (EC) amended its policy and has changed its directive on FMD
control (Council Directive 2003/85/EC), making the use of emergency vaccination easier
when combined with screening for residual infection using tests for antibodies to nonstructural proteins (NSPs). In reality, this means that current contingencies must be based on
the use of existing vaccines. Therefore, this project addressed the specific gaps in our
knowledge and technological ability with respect to the implementation of a vaccinate-to-live
policy.
The availability of adequate discriminatory diagnostic tests is the keystone of the new EU
FMD control policy. The project focused on the validation of NSP-based tests to discriminate
unequivocally between infected and vaccinated animals, in order to allow the implementation
of the new policy in the immediate term. Validation of existing and new NSP-tests as
confirmatory tests were a major output of this project.
The experimental design provided expected outputs in the field of the impact of vaccination
on the carrier state and on virus dissemination, the onset of vaccinal protection, vaccine
potency in relation to emergency use, vaccine strain selection and new marker vaccines. This
project focused on marker vaccines to induce durable protection against FMD. Conventional
and marker vaccines were targeted to dendritic cells with particular attention to promote
dendritic cell mucosal homing (from parental immunisation), since mucosal immunity could
prevent FMD virus establishing local infection and the carrier status.
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Project co-ordinator and contractors
Co-ordinator
Dr. Kris De Clercq
Centrum voor Onderzoek in Diergeneeskunde en Agrochemie
CODA
Groeselenberg 99
1180 Ukkel
Belgium
Tel.: +32 2 379 04 00
Fax: +32 2 379 06 66
krdec@var.fgov.be
FMD_ImproCon
www.fmdimprocon.org
Contractors
1. CODA – Centrum voor Onderzoek in Diergeneeskunde en Agrochemie,
Groeselenberg 99, 1180 Ukkel, Belgium (Dr. Kris De Clercq, krdec@var.fgov.be)
2. IAH – Institute for Animal Health, Ash Road, Pirbright, Surrey, GU24 ONF, United
Kingdom (Dr. David Paton, david.paton@bbsrc.ac.uk)
3. CVI – Central Veterinary Institute of Wageningen UR, P.O. Box 65, 8200 AB
Lelystad, The Netherlands (Dr. Aldo Dekker, aldo.dekker@wur.nl)
4. DFVF – Danmarks Fodevareforskning, Lindholm, DK 4471 Kalvehave, Denmark
(Dr. Laurids Siig Christensen, LSI@food.dtu.dk)
5. FLI – Friedrich-Loeffler-Institut, Bundesforschungsinstitut für Tiergesundheit,
Südufer 10, 17493 Greifswald – Insel Riems, Germany (Dr. Bernd Haas,
bernd.haas@fli.bund.de)
6. INIA – Instituto Nacional de Investigácion y Tecnología Agraria y Alimentaria,
Carretera de Algete a El Casar de Talamanca, 28130 Valdeolmos, Spain (Dr. Esther
Blanco, blanco@inia.es)
7. IZSLER – Instituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia
Romagna, Via Bianchi 7/9, 25124 Brescia, Italy (Dr. Emiliana Brocchi,
emiliana.brocchi@bs.izs.it)
8. Sap – Sap Institute, PO Box 714, 06044 Ulnus-Ankara, Turkey (Dr. Fuat Ozyoruk,
fuato@sap.gov.tr)
9. AFSSA – Agence Française de Sécurité Sanitaire des Aliments, 22 Rue Pierre Curie,
94703 Maisons-Alfort, France (Dr. Stéphan Zientara, szientara@vet-alfort.fr)
10. IVI – Institute of Virology and Immunoprophylaxis, Sensemattstrasse 293, CH-3147,
Mittelhäusern,
Switzerland
(Dr.
Kenneth
McCullough,
kenneth.mccullough@ivi.admin.ch)
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Work performed, end results and degree to which the objectives were
reached
The project was divided into seven different work packages (WP) of which WP7 described
the project management structure. What is given below represents the actual scientific
progress that has been achieved in WP1, WP2, WP3, WP4, WP5 and WP6 during the entire
project period and particularly emphasises the end results and the degree to which the
objectives were reached.
WP1. Validation of existing NSP-tests
In order to evaluate the suitability of NSP tests for the purpose of helping to substantiate
FMD freedom, information is needed on both the likely extent of FMD virus dissemination
in specified population types (addressed in WP4) as well as on the sensitivity and specificity
of tests and the application of test combinations in detecting this infection. Evaluation of test
sensitivity and specificity has been addressed in WP1 as well as the application of such tests
in outbreak and post-outbreak situations.
Reference serum panels based on sera from experimental studies have been assembled for
each of the three main domestic species (cattle, sheep, pigs) and these can be used for
validation of new assays (Parida et al., 2007) and for developing secondary panels for batch
control of existing NSP tests. In addition, in collaboration with Panaftosa (Brazil) a second
panel covering different immunological and infectious statuses found in the field, was
composed (Campos et al., 2008).
An inventory of more than 3000 sera from animal experiments, involving vaccination and/or
challenge with FMD virus, was made. Several hundred of these sera were contributed to a
NSP serology workshop organised by in May 2004 (Brescia, Italy). Samples, for evaluating
NSP test sensitivity and specificity, were collected from previous animal experiments and
throughout the life of the project from a series of experimental vaccine-challenge studies
involving cattle, pigs and sheep (WP4), and in which most animals were retained until at least
one month after the period of challenge exposure, since this is the time after which
serosurveillance begins in the field. For certain sera epidemiological, virological and
serological work-up was needed to provide essential information on the vaccination and
infection status of the animals concerned. A series of logistically demanding field studies
were organised to collect and analyse samples from countries where different serotypes of
FMD virus occur and where vaccination is used targeting both naïve and exposed
populations. Cattle were sampled in the Caucasus region (Armenia, Azerbaijan and Georgia),
Zimbabwe, Botswana and Jordan, pigs in Hong Kong, sheep in Jordan, and Asian buffalo in
Vietnam. These samples were distributed to all partners evaluating (WP1) and developing
(WP2) NSP tests.
The collaborative effort of all partners involved in WP1 and the comparative testing
performed during the NSP Validation Workshop (Brescia, Italy, 2004) has enabled
calculations of the sensitivity and specificity of NSP tests used alone or in combination in
different settings and associated with different domestic species. It has also provided some
indication of the prevalence of infection in several vaccinated populations, although the
circumstances surrounding vaccination, infection and surveillance may not closely mirror
those expected in European countries. Different analyses were used to obtain sensitivity and
specificity figures, among which were the use of the conventional approach (Brocchi et al.,
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2006), the application of Bayesian analysis (Goris et al., 2007; Engel et al., 2008) and the use
of continuous data (Dekker et al., 2008) to compare six different ELISAs, three of which are
commercially available. Tables 1 and 2 provide a summary overview of the sensitivities and
specificities obtained. The sera came from cattle vaccinated and/or exposed to one of the
following six serotypes of FMDV: O, A, Asia 1, C, SAT 1, SAT 2. Although differences are
observed between the different approaches deployed, the same general trend is observed. The
World Organisation for Animal Health (OIE) index test, the NCPanaftosa-screening test,
combines a higher level of sensitivity and specificity. The same accounts for the 3ABC
trapping-ELISA (IZSLER, Italy) and the Ceditest FMDV-NS (currently known as
PrioCHECK FMDV NS). The three remaining tests were less sentive. Differences between
test specificities were usually not significant.
Following the availability of the sensitivity and specificity estimates, it was recognised that
none of the assays combine 100% sensitivity with 100% specificity which has consequences
for their applicability in outbreak situations. Hence, a study was initiated to examine the ways
in which serological testing with NSP ELISAs can be used and interpreted and the effect that
this will have on the confidence with which freedom can be demonstrated within the
guidelines specified by the OIE and the European Commision (Paton et al., 2006). It was
subsequently considered essential to disseminate this knowledge by organising three
workshops in 2007 (Tervuren, Belgium) on the design and interpretation of post FMDvaccination serosurveillance by NSP tests (Part I on dense cattle-pig populations, Part II on
the Balkan area and Part III on Scandinavian and Baltic regions).
It was concluded that:
i) the vaccination-to-live policy in conjunction with NSP-serosurveillance is a realistic and
achievable option to substantiate, not prove, freedom from infection if combined with cluster
analysis;
ii) stamping-out should also remain part of the control policy, but is not always the best
approach;
iii) where all vaccinated ruminants are tested, greater confidence in eliminating carriers can
be achieved by using a testing algorithm to maintain a high sensitivity and then slaughtering
individual reactor animals, rather than using a higher specificity and lower sensitivity
combined with slaughter of reactor herds;
iv) where multiple reactors are found then herd-based slaughter would be appropriate;
v) testing all vaccinated animals is not achievable in areas of dense pig populations and
therefore a sample scheme based on 5% prevalence and 95% confidence should be
considered;
vi) vaccination of small herds remains controversial and should be discussed further.
The full reports of the these workshops are available through the following links:
Part I: http://www.fao.org/ag/againfo/commissions/docs/Workshop_0107.pdf
Part II: http://www.var.fgov.be/pdf/FMD-NSP-2-report.pdf
Part III: http://www.fao.org/ag/againfo/commissions/docs/Workshop_1007.pdf.
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Table 1. The sensitivity estimates obtained using data collected at the NSP Validation Workshop (Brescia, Italy, 2004) using four different data
analysis approaches
Assay
Brocchi et al. (2006)
Experimental and field data
Vaccinated exposed to infection
> 28 dpi
Brocchi et al. (2006)
Experimental and field data
Vaccinated exposed to infection
Carriers
> 28 dpi
Goris et al. (2007)
Field data
Vaccinated exposed to infection
> 28 dpi
Engel et al. (2008)
Field data
Vaccinated exposed to infection
Dekker et al. (2008)
Experimental and field data
Vaccinated exposed to infection
Test specificity fixed at 99%
NCPanaftosa-screening
69.4 – 72.3
89.2 – 93.9
84.4 – 99.5
93.7 – 99.3
60.1
3ABC trapping-ELISA
63.8 – 64.8
78.4 – 86.4
80.7 – 98.2
88.2 – 97.4
68.2
Ceditest FMDV-NS
63.6 – 74.5
86.4 – 89.2
77.5 – 94.8
80.8 – 95.7
67.4
SVANOVIR FMDV
3ABC-Ab-ELISA
57.4 – 58.3
70.3 – 71.2
66.6 – 84.1
69.6 – 84.4
53.2
CHEKIT-FMD-3ABC
38.3 – 50.0
48.6 – 68.2
66.4 – 84.7
69.2 – 85.0
50.3
UBI FMDV NS ELISA
46.8 – 56.1
59.5 – 77.3
52.9 – 70.9
51.1 – 65.9
36.4
Table 2. The specificity estimates obtained using data collected at the NSP Validation Workshop (Brescia, Italy, 2004) using three different data
analysis approaches
Assay
Brocchi et al. (2006)
Goris et al. (2007)
Engel et al. (2008)
NCPanaftosa-screening
96.2 - 97.9
80.4 - 98.2
95.0 - 98.9
3ABC trapping-ELISA
96.4 - 98.0
77.2 - 97.0
95.8 - 99.9
Ceditest FMDV-NS
97.3 - 98.7
77.6 - 99.2
97.7 - 99.4
SVANOVIR FMDV
3ABC-Ab-ELISA
97.7 - 99.0
86.3 - 97.5
94.9 - 98.3
CHEKIT-FMD-3ABC
96.7 - 98.3
83.6 - 97.0
95.1 - 98.3
UBI FMDV NS ELISA
97.8 - 99.0
82.9 - 98.2
95.7 - 99.3
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WP2. Development and validation of confirmatory and new NSP tests
WP2 primarily aims at developing and validating new NSP assays as confirmatory tests or as
alternative primary assays in order to improve upon the diagnostic performance of the NSP
ELISAs validated under WP1. Table 3 summarises a list of methods investigated during the
project and according to the plan foreseen in WP2.
Table 3. New NSP tests developed during the whole duration of the project
Target
Level
Expression
NSP
Type of test
validation
Sp
system
Ag
notes
Sn
Reference
Partner 7;
EUFMD
Open
session
2008
Partner 7;
EUFMD
Open
session
2008
Partner 7;
EUFMD
Open
session
2008
MAb trappingindirect ELISA
Prototype;
validated on
exp. cattle sera
98.3%
I = 100%
V+I = 83%
3B
E.coli,
His.tag
Indirect ELISA
Prototype;
validated on
exp. cattle sera
99%
I = 75%
V+I = 72%
3D
E.coli,
His.tag
MAb trappingindirect ELISA
Prototype;
evaluated on
exp. cattle sera
needs
improveme
nt
needs
improveme
nt
3D
Baculovirus,
his.tag
MAb trappingindirect ELISA
Partner 9
2B
Synthetic
peptide
Indirect ELISA
Partner 2;
Inuoe et al.,
2006
3A
Baculovirus
Indirect ELISA
3B
recombinant
β-galactos.
with 3B
peptydes
3D
Baculovirus
3D
Baculovirus
3ABC
E.coli MS2
fusion
protein
3D
E.coli,
His.tag
enzymatic
assay using
chimeric
biosensors
3D competitive
ELISA (L-NS2
Mab-based)
3D competitive
ELISA (L-S18
MAb-based)
3A & 3B Mabcompetitive
ELISA
Mabcompetitive
ELISA
3A
E.coli,
His.tag
Prototype;
evaluated on
exp. sheep,
pig, cattle sera
Validated
(cattle, pigs,
sheep)
Validated
(cattle, pigs,
sheep)
Feasible :
competitors
Mabs selected
Feasible :
competitors
Mabs selected
inadequate
Similar to
3ABC tests
Partner 5
inadequate
inadequate
Partner 6
Similar to
3ABC Cedi
test
Similar to
3ABC Cedi
test
not
available
yet
high
cumulative
specificity
high
cumulative
specificity
not
available
yet
not
available
not
available
Partner 4
Partner 4
Partner 7
Partner7
V= vaccinated; I = Infected, Sp = specificity; Sn = sensitivity; Mab = monoclonal antibody
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Despite the number of different tests and approaches investigated, only the combination of
two 3D-competitive ELISAs (3D L-NS2 and 3D L-NS18) developed by partner 4 has shown
sensitivity and specificity performances at least equivalent to those of the three most best
assays validated in the framework of WP1. The 3D L-NS2 and L-NS18 ELISAs are therefore
candidates as assays for discrimination between vaccinated and infected animals (DIVA)
after emergency vaccination. With post infection sera the 3D L-NS2 ELISA in particular, but
also the 3D L-NS18 ELISA, gave reactions equal to or stronger than type specific ELISAs.
The assays reacted with all seven serotypes and may therefore be less sensitive to strain
variations than the type specific ELISAs. The results indicate that the 3D L-NS2 and L-NS18
ELISAs are candidates as type independent pan-FMDV tests.
Alternative NSP-competitive ELISAs were also designed investigating either 3D and 3ABC
as target antigens and different MAbs as competitors. These Mabs were chosen using peptide
array analyses as it represented a successful approach to identify antigenic determinants on
the 3B and 3D proteins. MAbs with the same specificity as those elicited in FMD infected
animals are ideal tools for the development of serological ELISAs based on competition.
Initial studies proved evidence of their feasibility, but further studies are needed to evaluate
diagnostic performances.
Indirect ELISAs for the profiling of antibodies against 3A, 3B and 3D were developed and
Mabs were chosen using MAbs as catching antibody for 3A and 3D and the purified 3B
directly coated to ELISA microplates. Individually, none of these indirect-ELISAs reached
the sensitivity of the best 3ABC-ELISAs validated under WP1, however, combined in an “Ab
profiling test” comprising multiple antigens, the newly developed 3A- and 3B-ELISAs may
have interesting applications as confirmatory system.
Another promising indirect ELISA is based on the detection of antibodies against 2B and
uses a synthetic 2B peptide of 13 amino acids in length, conjugated with a KLH carrier as
source of antigen (Inoue et al., 2006). The sensitivity of this test was comparable to that of
the best NSP test available commercially. After resolvin a specificity problem, the test could
be useful as a confirmatory method in combination with 3ABC protein based tests.
Other immune-assays based on new concepts, like enzymatic tests using chimeric biosensors
showed inadequate sensitivity and between-animals variability. The biosensors constructs are
based on the insertion of antigenic sites selected from NSP 3B and 3D in regions of the
enzyme -galactosidase. The use of enzymatic sensors could have important advantages
compared to the conventional diagnostic systems or those based on virus recombinant
proteins, synthetic peptides or Mabs since they allow to develop new immunoassays capable
of analysing sera from different animal species using the same test protocol (avoid the use of
specific conjugates) and can be performed much quicker (1 hour or less) than conventional
systems as ELISA. However, just one of the constructs presenting 3D epitopes seemed to
work correctly for differentiating infected and vaccinated animals. Among 3B biosensors, the
prototype assay with the recombinant proteins presenting three copies of 3B peptides
provided a moderate level of specificity mainly for pig and sheep sera (above 90%) but still a
relative low sensitivity (80%). Hence, these assays at present cannot be recommeded for use
as screening or confirmatory tests.
In conclusion, apart from the two 3D competitive ELISAs, the other new and promising NSP
tests were not yet completely validated in the laboratory of origin. Further improvements and
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evaluation, including making the tests more robust, were considered necessary before being
ready for collective and comparative analysis in an open workshop.
On the other hand, work was done on the development and evaluation of a series of
alternatives to available NSP tests for determining whether vaccinated animals have been
infected with FMD virus. These alternatives have included new tests using salivary IgA
(Parida et al., 2006) or systemic IgM.
FMD virus replicates in the oropharynx and elicits a specific mucosal IgA response that can
be detected in a variety of upper respiratory secretions including nasal and oropharyngeal
mucous. In contrast, killed vaccines are administered parenterally and mainly elicit a
systemic IgG response. Therefore, salivary IgA tests can specifically detect vaccinated
animals that have been infected, since infected vaccinates produce an IgA response.
Moreover, the use of unpurified vaccines with heavy NSP contamination, although
incompatible with NSP based DIVA tests, has no detrimental effect on IgA based DIVA
tests that detect antibodies to viral structural proteins (NSP IgA antibodies could not be
detected in saliva of infected animals). An IgA test was established to detect serotype-specific
antibodies in orally collected saliva. Initial studies on cattle found that the method had a good
sensitivity approaching that of the best NSP tests but suffered from a poor specificity with
some field samples (Parida et al., 2006). Subsequently, the test procedure was modified
resulting in a greatly improved specificity with little or no loss of sensitivity. The latest work
was presented at the EUFMD meeting in Erice in 2008 and a paper will be submitted.
IgM antibodies are usually the first to be produced in response to infection but do not usually
persist once the pathogen in question has been cleared. In the case of FMD, transient
infection or vaccination would be expected to elicit a temporary IgM response whereas
chronic infection might give rise to a persistent IgM response. On this basis, an IgM test
could be useful to determine whether seropositive animals, such as those identified by NSP
tests, had been infected recently or in the more distant past. Furthermore, the finding of IgM
in animals that had received emergency vaccination some time previously could be an
indicator of subsequent and recent infection. The circumstances which gave impetus for
developing a new IgM test were the FMD outbreaks that occurred in UK and Cyprus in 2007,
since in both of these instances it would have been useful to know whether seropositive
animals identified on some farms had been infected recently or not. A new test for detecting
IgM has now been developed and undergone preliminary evaluation with samples from cattle
and sheep. Further work is needed to fully define the kinetics of IgM development and
persistence in animals with different status with respect to vaccination, infection and viral
elimination and persistence. Nevertheless, the results obtained so far do suggest that the test
may prove useful in detailed investigations concerning the onset of infection in outbreaks and
the finding of IgM persistence in viral carrier animals may be valuable diagnostically and
in elucidating the pathogenesis of viral persistence. Since IgM does not seem to persist in
vaccinated animals, its presence in animals known to have been vaccinated a month or more
previously may indeed be an indicator of infection. Since this is precisely the time at which
post-vaccination serosurveillance is normally conducted, the assay may therefore have DIVA
potential. The work was presented at the EUFMD meeting in Erice in 2008.
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The WP further aimed at developing and validating serotype-specific and serotypeindependent immuno-assays based on the FMDV VP1 protein and on virus like particles
(VLP).
The strategy to develop serotype-specific ELISAs consisted of the expression of recombinant
FMDV VP1 protein in E coli or baculovirus expression systems. However, some anti-type O
sera did not recognise the type O VP1 recombinant antigen. The same results were observed
for FMDV VP1 type A and Asia 1 proteins. These observations demonstrate that the VP1
epitopes recognition mechanism is based on conformational epitopes. The results obtained
show that these VP1 ELISAs cannot be used to differentiate vaccinated from infected animals
since the VP1 proteins loose some of their antigenic properties.
In order to develop serotype-specific ELISAs based on VLPs, two approaches were deployed
simultaneously: (i) the production of FMDV VLPs in a new expression system (Drosophila
cells transfected by an inducible expression plasmid pMT V5) and (ii) the production of
chimeric infectious bursal disease subviral particles displaying the FMDV major antigenic
site. The first approach did not result in the establishment of a stable recombinant cell line.
Furthermore, the level of expression was too low and the number of positive cells was also
quickly decreasing upon passages. All results were presented as a poster at the FAO EUFMD
Open Session (Erice, Italy) by Rémond et al. The second approach of using an antigen
delivery system based on subviral particles (SVP) formed by self- assembly of the capsid
protein VP2 of infectious bursal disease virus (IBDV) and carrying foreign peptides at the top
of the projection domain was more successful. The FMDV immunodominant epitope was
effectively inserted in one of the four SVP external loops. The chimeric SVP reacted with
neutralising FMDV type O1 Mabs and polyclonal antibodies and elicited a neutralising
antibody response in immunized mice. Hence, these structures could be well suited to
produce vaccines and non-infectious antigens. Moreover, the VLPs have the potential for the
detection of FMDV antibodies in a competitive ELISAs for diagnostic use (Rémond et al.,
2009).
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WP3. Improved FMD virus detection
This WP has three main objectives: (i) providing a tool for validating NSP tests without
“Gold Standard”, (ii) improving the diagnostic value of FMD virus detection testing cases of
mild virus excretion and (iii) developing a mass-screening virus detection assay based on a
‘rolling-circle amplification’ (RCA) ELISA.
A tool was provided for the validation of NSP tests without “Gold Standard”. There are two
possible approaches: (i) either the development of at least four tests based on different
detection methods for the virus antigen (for instance an antigen-detection ELISA, virus
isolation, PCR and a RCA-ELISA (see below)) or (ii) the use of state-of-the-art statistical
methods such as Latent Class analysis or Bayesian analysis. Two partners have independently
developed a Bayesian methodology and have successfully applied it in WP1 (Goris et al.,
2007; Engel et al., 2008).
The reason for developing a ‘rolling-circle amplification’ (RCA) ELISA (Figure 1) consists
in the improvement of the diagnostic value of FMD virus detection tests in case of mild virus
excretion. In addition, an advanced mass-screening virus detection method could
simultaneously be developed. RCA can potentially fulfil all of these demands by means of
one generally applicable technique, basically comprising the amplification of a circular
ssDNA template using a DNA polymerase with strand displacement activity and one or two
specific primers.
Figure 1: Basic principle of rolling circle amplification
The easiest way of performing RCA-based detection of FMD viral RNA is by using padlock
probes (Figure 2).
Figure 2: RCA using padlock probes
The resulting circular template is amplified using a two-primer RCA reaction. This
ramification reaction for FMDV yielded a typical ladder pattern. Detection of viral RNA
through RCA based on padlock probes was shown to be achievable, however, false positive
results appeared very frequently.
The immunological approach (Figure 3) was taken in order to circumvent the above problem
by shifting the detection to the antibody level. Hence, RCA becomes merely a signal
amplification step and as such, multiplexing should be allowed using one ssDNA template.
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Figure 3: Immuno-RCA setup
Amplification of a pre-circularised ssDNA template in the presence of an unbound antibodyoligo conjugate was successfull. A setup separating RCA and immunological steps, involving
DIG-detection generated good signal intensities. However, again significant numbers of false
positive signals appeared, which were imputed to the detection conjugate’s DNA tail.
In conclusion, research into immuno-RCA mediated detection of FMDV has encountered
many obstacles. The adaptation of the indirect sandwich ELISA for the detection of FMDV
antigen in order to develop a RCA-ELISA suffered from major problems with false positive
and false negative results as well as with reproducible problems. Consequently it was decided
to abandon further research into this topic. The “trial and error” process has been described in
a paper by Van Dessel et al. (2007).
Virus isolation: two remarkable aspects were observed with the FMDV O Netherlands 2001
isolate. First of all, the virus failed to grow at a low multiplicity of infection on secondary
porcine kidney cells and secondly, reduced clinical signs were observed in calves infected
with the FMDV isolate.
During the 2001 FMD outbreak, samples taken from vesicles from goats did not result in
cythopathogenic effects on secondary porcine kidney cells (Figure 4), whereas samples from
vesicles from cattle destroyed the monolayer within 24 hours. For diagnostic purposes, the
observation can be circumvented by using more sensitive cell systems. However, the inability
to grow on cell cultures might cause problems for growing the virus for vaccine production.
If the mechanism responsible for the inhibition is characterised, unknown viruses could be
tested beforehand or proteins could be exchanged between viruses to overcome problems in
cell culture. It was shown that the blocking of infection was transferable, indicating the
presence of cytokines, most likely interferon beta (IFN-ß). Not only growth on porcine
kidney cells, but also growth on bovine kidney cells was limited, which indicates that the
effect is not completely species specific. Further work showed that:
 Interference with virus growth is also observed in supernatant of other FMD viruses
 After ultracentrifugation and acid treatment the inhibitory effect is still present
 Experiments testing heat stability and trypsin stability indicated type I interferon
 Experiments with IFN antibodies showed no inhibition with interferon alpha, but
limited blocking with interferon beta.
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Figure 4. Immunochemical staining of monolayers of porcine kidney cells infected with FMD
virus O NET/2001: infection limited to a few cells, but without spread to other cells.
Work in other picornaviruses suggests that some proteins (2B or 3A) are involved in blocking
the protein excretion pathway. The effect observed with O Netherlands 2001 could be the
result of insufficient blocking of one of these protein excretion pathways, resulting in an
earlier release of IFN in secondary porcine kidney cells
To further characterise the proteins involved in this host virus interaction, an infectious copy
of O NET/2001 has been produced as well as a full-length clone of O1 Manisa. To identify
proteins involved in the inhibitory effect, it has been attempted to exchange parts of the
infectious clone with corresponding parts of FMD virus type O1 Manisa, which does not
show an inhibitory effect. Several chimeric DNA sequences were obtained, but infectivity
remains to be tested.
In conclusion, there are very strong indications that IFN-ß was responsible for the inhibitory
effect. No IFN production and blocking was observed on secondary lamb kidney cells, so
these cells can be used for virus isolation.
While it may appear that the availability of the real-time RT-PCR has made virus isolation
less important, vaccine strain selection procedures will require the isolation of the virus
causing the outbreak. Since primary bovine thyroid (BTY) cells, the most sensitive cells for
FMD virus isolation, are difficult to work with most diagnostic laboratories use cell lines
which are less sensitive, but more convenient to handle. A novel foetal goat tongue cell line
(ZZ-R 127) was found to be highly sensitive for FMDV and was validated as a tool for
FMDV isolation. FMDV infection could be detected visually within 18-24 hours, which is
important for decisions concerning emergency vaccination. Strains representing all seven
serotypes of FMDV could be isolated on ZZ-R 127 cells with a sensitivity that was only
slightly inferior to that of the BTY cell but consistently higher than that of BHK-21 or IB-RS2 cells. The foetal goat tongue cell line is available from the CCLV, FLI Riems to any
diagnostic laboratory.
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Within this WP, although not originally foreseen, a real-time RT-PCR system consisting of
two independent FMDV PCR protocols (plus one back up protocol) and two independent
SVD PCR protocols was established and validated. Each also contains an internal quality
control reaction. All these protocols can be run on a single plate with the same cycler
programme. The system detects all FMDV and SVDV isolates tested with two independent
primer pairs. The minimum time for a PCR run was shortened from 137 minutes to 45
minutes. As SVD is the most important differential diagnosis for FMD in pigs and published
protocols had problems to detect certain recent Italian SVD isolates, also the SVD component
of the system was improved by modification of the primers. Moreover, these and other realtime RT-PCR are being used routinely for diagnosing FMDV. Although most laboratories
determine analytical and diagnostic sensitivity and specificity, a thorough validation in terms
of establishing optimal RNA-extraction conditions, matrix effect, uncertainty of measurement
and precision is generally not performed or reported. Within WP3, different RNA-extraction
procedures were compared for two FMDV real-time RT-PCRs. Compared to cell-culture
spiked viral control samples, no matrix effect on the analytical sensitivity was found for
blood or foot epithelium. An approximate 1 log10 reduction in detection limit was noted for
faecal and tongue epithelium samples, whereas a 3 log10 decrease in detection signal was
observed for spleen samples. By testing the same dilution series in duplicate on ten different
occasions, an estimation of uncertainty of measurement and precision was obtained using
blood as matrix. Both RT-PCRs produced highly precise results emphasising their potential
to replace conventional vriological methods. Moreover, uncertainty measurement proved to
be a useful tool to evaluate the probability of making a wrong decision (Goris et al., accepted,
JVirMeth).
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WP4. Impact of vaccination on virus dissemination and the carrier state
The objectives of WP4 were i) to measure how rapidly and effectively emergency (high
potency) vaccination can protect susceptible species from direct contact virus challenge and
ii) to what extent they effect the kinetics and quantitative dynamics of virus replication and
excretion, and thereby transmission and the establishment of the carrier state.
To adhere to these objectives, a number of vaccine-challenge experiments were carried out
using O UKG 2001, with non-EC funding, to study the efficacy of FMD vaccines in cattle,
pigs and sheep. The FMD_ImproCon project provided funding for the retention of animals
until a month or more after challenge, enabling sampling and an evaluation of the status of
the animals at the time at which post-vaccination serosurveillance is normally performed in
the field.
Different FMD vaccine payloads were administered to cattle 10 or 21 days before severe
direct contact challenge. Regardless of antigen payload, emergency vaccination significantly
reduced the number of clinically infected animals, significantly reduced virus excretion
shortly after challenge, thereby reducing the possibility of transmission between animals and
herds, and significantly reduced sero-conversion for non-structural antibody, when compared
with unvaccinated cattle. Earlier challenge increased the numbers of animals showing clinical
signs of FMD and the number of vaccinated animals becoming sero-positive for nonstructural antibodies.
Increasing antigen payload 10-fold made no significant difference to non-structural antibody
sero-conversion. For 10 days vaccinated cattle, approximately half of the animals in each of
the treatment groups (vaccinated and unvaccinated) became persistently infected and there
was therefore no additional benefit provided by the 10-fold antigen payload. When the
interval was 21 days between vaccination and challenge, less animals became persistently
infected when a higher payload of vaccine was given. Although FMDV transmission
occurred from 10 day vaccinated infected cattle to similarly vaccinated cattle held in indirect
contact, no disease was induced in these animals.
Furthermore, the hypothesis was investigated that vaccine-induced reduction in virus
replication and excretion from pigs can be correlated to the severity of clinical signs of FMD
by measuring excretion of virus in natural secretions and aerosols. The other aims of this
study were to verify the existence of sub-clinical infection in vaccinated pigs, to evaluate the
correlation between this and seroconversion to FMDV NSP antibodies and to re-examine the
occurrence of FMDV persistence in the oro-pharynx of pigs.
81% of the early (10 days vaccinated) challenged pigs and 25% of the late (29 days
vaccinated) challenged pigs were clinically infected and all other vaccinated pigs were subclinically infected. Although vaccination could not provide complete clinical or virological
protection, it reduced the severity of the disease, virus excretion and production of nonstructural FMDV antibodies in vaccinated and subsequently infected pigs. RNA copies, but
no live virus was detected from the pharyngeal and soft palate tissues of a minority of
vaccinated and infected pigs beyond the acute stage of the infection (Parida et al., 2007).
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Another study has quantified the level of FMDV replication and shedding in vaccinated
sheep and correlated this to the severity of clinical signs, the induction of antibodies against
FMDV NSP and the transmission of virus to in-contact vaccinated sentinel sheep (aerosols
from O1 UKG FMDV infected pigs). Vaccinated sheep became sub-clinically infected, with
reduced virus replication and excretion compared to unvaccinated and clinically infected
sheep. Seroconversion to NSP was weak and transient in sheep in which virus replication was
of low level and short duration. Virus transmission from vaccinated sub-clinically infected
sheep to introduced vaccinated sentinels was not sufficient to cause NSP seroconversion or
significant virus shedding. 10% of 10 days and 20% of 4 days vaccinated sheep were virus
carriers compared to 38% in the unvaccinated and clinically infected sheep. These results
suggest that the low levels of virus replication are unlikely to result in the spread of infection
even under intensive management conditions if an effective vaccine is administered at least 4
days prior to challenge exposure. It may be difficult to detect this infection by
serosurveillance but the impact of missing it is likely to be low and the main value of such
testing will be to detect undisclosed clinical infection resulting from lack of observation or
from exposure to virus before or very soon after vaccination or from vaccine failure due to
maladministration or inappropriate strain selection (Parida et al., 2008).
Information derived from the animal experiments conducted in this workpackage has been
used in simple probabilistic models to quantify the effect of emergency vaccination – and
especially the time of application - on the likely number of carrier cattle and sheep in
diseased and subclinically infected animals. This reveals that the number of persistently
infected animals in a group is predominantly determined by the number of animals initially
infected on premises - the high variability of which ultimately limits the accuracy of any
predictions of carrier numbers based upon transmission models. Furthermore, results suggest
that, within a cattle herd, carrier numbers may be increased if challenge occurs shortly after
vaccination. We show that the quality of inspection is the principal factor influencing whether
or not carrier herds occur and that, by reducing clinical signs, the application of vaccination
amongst regularly checked stock also results in an increase in undetected persistently infected
animals. These predictions can be combined with information derived from WP1 on the
sensitivity and specificity of the NSP tests to look at the feasibility of detecting carriers
missed by clinical surveillance using NSP serosurveillance. Most herds containing carriers
that are not disclosed by clinical surveillance will contain only small numbers of carriers.
This low prevalence means that even if all animals are tested, serosurveillance would need to
have a sensitivity of at least 89%-92 % in order to detect infection with 95% confidence, in
the absence of any other form of detection. This is just about possible with the best available
NSP tests. In order to maximise sensitivity of testing without excessive culling of false
positive herds, we therefore recommend that animals are tested and culled on an individual
and not a herd basis. In conclusion, where effective vaccination is applied in good time and
clinical surveillance is good, there are likely to be relatively small numbers of carriers. Some
but probably not all of these can be detected by NSP serosurveillance with currently available
tests. However, the main value of NSP serosurveillance is probably as an insurance for
detection of cases that are missed because of suboptimal clinical surveillance (Shley et al.,
2009).
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A second series of transmission experiments was conducted using O NET 2001 to estimate
reproduction ratio’s in different FMD susceptible species. The reproduction ratio (R) is
defined as the average number of new infections caused by an average infectious individual
during its entire infectious period. Often the basic reproduction ratio R-naught (R0) is used,
which represents the transmission in a fully susceptible population. The reproduction ratio
has a unique trait, when the value is above 1, an infection can spread on a large scale (major
and minor outbreaks are possible), whereas when the value is below 1, the infection will fade
out (only small outbreaks can occur). The R-value can be used to estimate the efficacy of
vaccination. If R0 is above 1 and Rv (reproduction ratio after vaccination) is below 1 this
indicates that vaccination is an efficient tool to combat a disease. R can be estimated
experimentally using (small) groups of animals in which within-pen transmission is studied.
Furthermore, if Rv within-pen is below 1 then also spread of virus between groups is limited
(R < 1). Based on this concept we studied transmission between calves, milking cows, sheep
and pigs, in which we studied transmission in groups with and without vaccination.
Two different methods were used to estimate R. Both methods are based on a so-called SIR
(susceptible-infectious-removed) model. First, the final size of the experiments (using the
total number of infected animals at the end of the experimental period) was used to calculate
R by maximising the likelihood for each possible R. Secondly, virus isolation data were used
from oral pharyngeal fluid (OPF) collected daily with swabs inserted in the oral pharyngeal
cavity. Using a generalised linear model, the transmission rate (ß) was estimated which is the
average number of new infections per infectious individual per day. Based on the virus
isolation from the oro-pharyngeal swabs the duration of the virus excretion (T) was
determined, and subsequently, by multiplying ß by T, the reproduction ratio R was calculated
(Table 4).
Table 4. Reproduction ratio for FMD susceptible species following contact challenge
Species
Vaccination status
R based on final size
R based on GLM
Non-vaccinated
2.5 [1.1 ; 52]
0.67 [0.05 ; 10]
Calf
Vaccinated
0.2 [0.01 ; 1.2]
1 x 10-8 [0 ; ∞]
Non-vaccinated
∞ [1.3 ; ∞]
5.9 [2.7 ; 13]
Dairy cows
Vaccinated
0 [0 ; 3.4]
NA
Non-vaccinated
1.1 [ 0.3 ; 3]
0.6 [0.03 ; 13]
Sheep
Vaccinated
0.2 [0.01 ; 2]
0.2 [ 0.01 ; 3]
Non-vaccinated
∞ [1.3 ; ∞]
31 [11 ; 85]
Pigs
Vaccinated
2.4 [ 0.9 ; 7]
1.44 [0.2 ; 9]
Results based on Orsel et al., 2005; 2007a; 2007b; 2007c and 2008b.
For calves, dairy cows and sheep, R is reduced significantly after vaccination as compared
to non-vaccinated groups and also is < 1. For pigs, transmission is still possible after
vaccination at 14 days before infection, although the transmission is reduced significantly as
compared to non-vaccinated pigs. The results in pigs differ from the results found previously;
in which O1 Manisa or O Taiwan vaccinated pigs did not become infected at two weeks after
vaccination. But in the current experiment the pigs were challenged by 24 - 28 h exposure to
infected non-vaccinated pigs. This is a huge challenge dose and probably not representative
for the situation during an outbreak, because transport of (infected) animals will be forbidden.
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Besides the studies mentioned above, two additional experiments were performed. First, a
study to test whether the results with other serotypes would provide similar results in sheep.
Therefore, a similar experiment was performed in which sheep were challenged with FMD
type Asia-1 TUR/2000. The sheep were vaccinated with Asia-1 Shamir monovalent vaccine.
Analysing both experiments showed no difference in both the transmission rate and the
duration of virus excretion in non-vaccinated sheep compared with the type O experiment. In
the vaccinated sheep, however, the transmission rate was lower in the Asia-1 experiment and
the duration of excretion was longer. One of the remarkable findings was the long duration of
the virus excretion in the sheep. Yet, only high titres were found shortly after infection and,
therefore, it is probably not correct to multiply duration of virus excretion with the
transmission rate because the risk of transmission is most likely correlated with the amount of
virus excreted. The overall conclusion is that the results are very similar between both
strains and in both cases after vaccination the reproduction ratio Rv is below 1.
Secondly, to study whether vaccination could block transmission between pens, eight
non-vaccinated pigs were housed in separate pens approximately 70 cm away from 4 infected
non-vaccinated pigs. The former did not contract FMD (figure 5A). The experiment was
performed in two separate stables.
Figure 5: Schematic representation of the transmission experiments to study between pen
transmission. A. the first experiment in which there was approximately 70 cm distance
between the inoculated pigs and the contact pigs. B. the second and third experiment in which
the contact pens were directly linked to the pen with the inoculated pigs.
When the pens with the contact pigs were directly connected to the pen (figure 5B), 4 out of 8
contacts became infected. In the third experiment, all pigs were vaccinated two weeks before
challenge and no transmission to any of the 8 contact pigs was observed. In these experiments
the air ventilation was adjusted to a level similar to the one that can be found in commercial
holdings. The main conclusions are:
 Vaccination can prevent within pen transmission when milking cows, calves or sheep
are vaccinated 2 weeks before exposure
 Within pen transmission is not completely prevented in pigs vaccinated 2 weeks before
challenge, although transmission is reduced significantly as compared to nonvaccinated pigs. However, between pen transmission is blocked after vaccination 14
days before challenge.
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 Between farm transmission will be reduced within 2 weeks after vaccination (this is
consistent with findings during outbreaks where emergency vaccination was used).
 For calves, dairy cows, sheep and pigs, estimates of transmission parameters were
determined which can be used in mathematical models , which can for example be
used to determine the number of expected outbreaks in the high risk period of an
epidemic or can be used to evaluate which control measures can reduce transmission
to such a level that that the virus will be eradicated. The transmission parameters are
reported in the various papers.
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WP5. Improved Vaccine Strain Selection
The objectives of this workpackages can be divided into 4 main items:
1. Determination of in vivo cross-protection conferred by high potency FMD vaccines
2. Determination of in vitro correlates for cross-protection based on r-values
3. Determination of in vitro correlates for cross-protection based on antigenic profiling
4. Genetic and antigenic typing of FMDV by monitoring in an endemic situation
Because of their heterogeneity, these items will be addressed separately.
1. Determination of in vivo cross-protection conferred by high potency FMD
vaccines
To fully assess and appreciate the outcome of cross-protection vaccine-challenge trials, one
must first determine the level of between-test variability of the prescribed FMD vaccine
potency test. Although in Europe, the latter must comply with the requirements described in
Monograph 0063 of the European Pharmacopoeia (i.e. the 50% protective dose test or PD50
test), other guidelines may apply worldwide such as for instance the Protection against Podal
Generalisation (PPG) test. Hence, a series of replicate potency tests were performed with
both the PD50 and PPG tests.
Therefore, this gap in our knowledge was addressed by performing 10 identical vaccine
challenge experiments using a single vaccine batch. The results of ten replicate, individual
vaccine potency tests using a FMD virus O1 Manisa vaccine batch indicate that the obtained
potency of a vaccine with an overall PD50 value of 9.99 may vary from 4.59 to 24.25 PD50
(Table 5). Table 6 outlines the observed repeatability or VACC for each vaccine dose group
individually and the overall mean VACC for all three vaccine dose groups. The level of
VACC within the different vaccine dose groups is rather low, ranging from 63.6% to 73.7%,
and associated 95% confidence limits are wide. The overall level of VCON or inter-potency
test reproducibility is even lower and estimated to be 58.8% [95%CI: 54.8%-63.1%] (Goris et
al., 2007).
Table 5. Number of protected animals per vaccine dose group and obtained PD50 value for ten
identical FMDV O1 Manisa vaccine potency tests
Trial
Number of protected animals per vaccine dose group
2 ml
0.5 ml
0.125 ml
PD50 value
95% confidence
interval PD50 value
1
5
3
3
10.56
4.27 – 21.82
2
3
4
2
6.06
2.75 – 15.45
3
5
2
1
4.59
2.52 – 12.20
4
4
3
3
8.00
3.31 – 18.72
5
5
3
3
10.56
4.24 – 21.87
6
5
4
5
24.25
8.07 – 35.18
7
5
4
1
8.00
3.69 – 17.62
8
4
5
0
6.06
3.11 – 13.71
9
5
5
2
13.93
5.49 – 24.89
10
5
5
4
24.25
overall
46
38
24
9.99
7.99 – 35.12
7.45 – 13.27
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Table 6.Vaccine accordance and vaccine concordance of the FMD PD50 vaccine potency test
and associated 95% confidence intervals
Vaccine dose group
Vaccine accordance (%) [95% CI]
Vaccine concordance (%) [95% CI]
2 ml
73.7 [65.2 – 81.8]
68.4 [58.7 – 78.7]
0.5 ml
65.7 [58.9 – 73.1]
57.3 [51.5 – 65.6]
0.125 ml
63.6 [56.8 – 70.6]
50.6 [50.0 – 52.2]
Overall
67.6 [63.2 – 72.1]
58.8 [54.8 – 63.1]
Subsequently, a series of replicate PPG tests were performed. The objective of the stduy was
to determine in vivo measures for intra-potency test repeatability (VACC) and inter-potency
test reproducibility (VCON) for the PPG FMD vaccine potency test by using a monovalent
FMDV A24 Cruzeiro/Brazil/55 vaccine and the homologous FMDV A24 Cruzeiro/Brazil/55
challenge virus in six replicate PPG tests. The study further aimed at providing data on in
vivo cross-protection and on the precision/reliability of such in vivo data. Therefore, naïve
cattle vaccinated with the same FMDV A24 Cruzeiro/Brazil/55 vaccine as used in the
homologous replicate trials, were challenged using an intratypic heterologous FMDV A
Argentina/2001 strain. The heterologous PPG set-up was performed four times under
standard operating conditions.
The number of protected animals for the six replicate homologous PPG potency tests ranged
from 12 to all animals protected with a corresponding % PPG ranging 75.0 to 100.0%.
Overall, 88.5% PPG [95% CI: 80.7-93.5] was observed. VACC and VCON were estimated to
be 75.9% [95% CI: 64.9-86.2] and 73.7% [95% CI: 62.1-84.3] respectively (Table 7).
Table 7.Vaccine accordance and vaccine concordance of the FMD PPG vaccine potency test
and associated 95% confidence intervals
FMDV vaccine strain
FMDV challenge strain
Vaccine accordance (%)
Vaccine concordance (%)
A24 Cruzeiro (n = 6)
A24 Cruzeiro (n = 6)
75.9
[64.9-86.2]
73.7
[62.1-84.3]
A24 Cruzeiro (n = 4)
A Arg 2001 (n = 4)
65.7
[50.7-80.3]
59.2
[50.0-74.0]
The results obtained for four replicate A24 Cruzeiro PPG vaccine potency tests using
heterologous FMDV strain A Arg 2001 challenge ranged from 12.5 to 56.3% PPG. Overall,
26.6% PPG [95%CI: 17.4-38.5] was observed. VACC and VCON were estimated to be
65.7% and 59.2% respectively (Table 2) (Goris et al., 2008a).
Methods to predict whether a vaccine will protect against a heterologous challenge are
currently more or less empirical rules and need further improvement (see below). Therefore,
experimental data were produced that will help to make the decision on the use of a vaccine
in a given outbreak scenario on a sound scientific basis. Homologous and heterologous cattle
challenge experiments with FMDV strains were performed and the clinical results compared
to those of serological in vitro tests, in particular VNT titers. In a series of homologous and
heterologous challenge experiments performed according to the protocol described in the
European Pharmacopoeia monograph, it was shown in vivo that high potency vaccines
against foot-and-mouth disease (FMD) serotype A can induce protection even against
heterologous challenge infection with viruses that give low r-values with the vaccine strains
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(Brehm et. al., 2008). Three vaccines with homologous PD50 values of at least 32 showed
significant protection even against heterologous challenge with viruses showing low r-values.
In six out of eight heterologous type A challenge experiments with these vaccines, high
potency vaccines still conferred a protection of at least 6 PD50. Therefore, in a situation
when vaccination is considered, but no closely related vaccine is available, the usage of a
high potency vaccine may be justified despite low r-values. The challenge virus specific
neutralising antibody response generally correlated well with protection against serotype A.
This is important, as the results of a heterologous cattle challenge test would only be obtained
after more than a month while the decision to vaccinate usually would have to be made
within days.
2. Determination of in vitro correlates for cross-protection based on r-values
Serology is used to predict vaccine induced protection against challenge with a heterologous
strain of the same serotype of FMDV. To evaluate the accuracy of such predictions, a
collaboration with an Indian vaccine manufacturer was established to compare the protection
afforded to cattle vaccinated with the O1 Manisa strain of FMDV against challenge with
either a homologous (O1 Manisa) or a heterologous strain (O Campos). All of the animal
work was carried out in India whilst Partner 2 acted in an advisory capacity and carried out
complementary and/or confirmatory laboratory tests on samples obtained from the
experimental animals.
Serology by virus neutralisation test (VNT) using O1 Manisa antiserum predicted an
acceptable protection (r1 = 0.6) against an O1 Campos challenge. Forty eight unvaccinated
and FMDV-naive cattle were vaccinated with different pay loads (60 µg to 0.94 µg) of O1
Manisa. They were challenged with FMDV O1 Manisa or O1 Campos. Unvaccinated control
cattle were challenged with either the O1 Manisa or O1 Campos viruses. All control cattle
developed generalized FMD. The O1 Manisa vaccinated and challenged cattle were protected
from generalized FMD. In contrast, only 30% O1 Manisa vaccinated and O1 Campos
challenged cattle, were protected from generalized FMD and there was evidence of more
virus replication in the O1 Campos challenged cattle. Despite relatively good crossneutralization of O1 Campos by O1 Manisa antisera, O1 Manisa vaccinated cattle were less
well protected against challenge with O1 Campos than with homologous O1 Manisa. PD50
values calculated indirectly for the vaccine was around 29 for the vaccine/homologous
challenge combination and 2.5 for vaccine/heterologous challenge combination. Comparison
of the deduced capsid amino acid sequence of O1 Manisa and O1 Campos reveals a number of
differences that are not located at known sites of antigenic significance, except for at position
159 in site 2 of VP2. This amino acid change might be of significance in cross-protection,
something that might be tested in future, using a reverse genetics approach.
The possibility of replacing or supplementing the polyclonal antibody (PAb) based method
for vaccine selection with one based on use of monoclonal antibodies (Mab) was also
investigated. Panels of Mabs raised against two serotype O vaccine strains were examined for
reactivity with 22 field viruses. Prediction of antigenic match based on Mab reactivity did not
correlate closely with the results of a PAb-based “gold-standard” method and it is concluded
that a wider panel of Mabs is needed that recognise all protective epitopes present on the
surface of FMD virus and/or a better understanding of which epitopes engender significant
protection.
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3. Determination of in vitro correlates for cross-protection based on antigenic
profiling
In case of a FMD epidemic with a new strain it is essential to match the vaccine strains as
closely as possible to the field strains. It is therefore necessary to continuously monitor the
prevailing FMD viruses to ensure prompt identification of new variants. Typing can be done
by means of antigen profiling. Monoclonal Antibodies (MAbs) are strategic resources to
study the antigenic profile of field isolates of FMDV and their antigenic relationships with
reference and vaccine strains. However, in order to better interpret and correlate MAbs
profiles with expected cross-protection, it is beneficial to use well characterised MAbs,
directed against known antigenic sites. The development and characterisation of Mabs panels
against vaccine strains and representative field isolates was imperative to design assays for
virus antigenic profiling. Although the FMDV serotypes O and A were judged priorities, also
other circulating serotypes were considered for the production of Mabs panels suited for
antigenic profiling. An inventory of available Mabs with their characterisation status has been
prepared. It includes Mabs specific for types O, A, C, Asia 1, SAT1 and SAT2. Most of Mabs
were produced by partner 7 and are stored at its facilities. In summary, the yield of available
Mabs panels, the majority of them produced and/or characterised within the project is
summarised below.


Three panels of type O Mabs produced against:
- O Lausanne vaccine strain, including Mabs mapping to neutralising sites 1, 2 and 3;
- O1 Manisa vaccine strain, including Mabs mapping to neutralising sites 2 and 3;
- O UK2001 field isolate, including Mabs mapping to neutralising sites 2, 3 and 4.
Six panels of partially characterised type A Mabs, raised against:
- A5, A 22 Iraq, A24 Cruzeiro, A 15 Tahiland 16/60, A Iran 96 vaccine strains
- A Malaysia 16/97 field isolate

One panel of fully characterised Mabs raised against type Asia 1, including Mabs
mapping to antigenic sites 1, 2, 4 and to another new site, identified as n. 5

One panel of fully characterised Mabs raised against type SAT 1, including Mabs
mapping to known antigenic sites 1, 2 and other identifying new sites called 6,7,8

One panel of fully characterised Mabs raised against type SAT 2, including Mabs
mapping to antigenic sites 1 and to another correlated site.
Using above Mabs panels, antigenic profiles of relevant isolates, with major focus for
types O and A, have been produced. A simple trapping ELISA was used to this purpose and
results are expressed as percentage reactivity of each isolate with respect to the homologous
strain. The results of antigenic profiling of 24 type A isolates, obtained with six panels of
type A specific Mabs, raised against five different vaccine strains and one field isolate, are
shown in Figure 6. Results were presented as a poster at at the FAO EUFMD Open Session
(Erice, Italy) by Brocchi et al.
Wide panels of Mabs were obtained against each of five strains of FMDV type A. The variety
of Mabs obtained, their various profiles of intra-type reactivity confirmed the high level of
antigenic variability within type A. Moreover, the diverse reactivity in Western Blot and
VNT were indicative that these Mabs actually cover different antigenic sites and may be
useful for the creation of panels suitable for antigenic profiling.
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Cross-protection between vaccine and field strains should mainly rely on neutralising sites;
then, antigenic profiling obtained with neutralising Mabs is more likely to predict the level of
cross-protection. In order to complete the characterisation of the new Mabs and verify
whether they sufficiently cover the main antigenic sites, mapping of epitopes target of the
neutralising Mabs is in progress. However, heterogeneous profiles of intra-type reactivity
were observed with both neutralising and non neutralising Mabs, confirming the high level of
antigenic variability within type A and indicating that also non neutralising Mabs have
potential use for these purposes.
The pattern of reactivity of 29 type O isolates with selected, neutralising type O Mabs is
shown in Figure 7. On the basis of the different profiles of reactivity, Mabs can be clustered
in separate groups: some groups include Mabs broadly reactive with all isolates (conserved
sites), other groups include Mabs showing reactivity with subgroups of isolates or almost
exclusively with the homologous one. These patterns allow to define antigenic lineages, as
regards to which any new isolate can be classified.
When interpreting results of Mab profiling to generate estimates of antigenic homology, the
composition of the Mabs panel and how it is representative of the capsid antigenic spectrum
should be taken into account. Previous evidence as well as data obtained in this project
showed that several partially overlapping epitopes are present within some antigenic sites
(particularly for the more complex ones, like site 1 and 2). Consistently, antigenic profiles of
field isolates obtained within WP5 proved that the variations occurring in the field may
differently affect the reactivity of Mabs directed against the same site, with some Mabs
maintaining and some other loosing their reactivity with the isolates. Conclusion: it is
necessary to include more than one Mab for each antigenic site in the panels used for
antigenic profiling.
To complete the above-mentioned panels, Mabs were produced and characterised against
FMDV type A vaccine strains or field isolates specific for Turkey and not covered by the
existing Mabs panels (e.g. A Iran 96) and targeted to obtain antigenic profiles of reference
and field strains, using ELISA assays based on Mabs. The first attempt was not successful
and almost all subsequent attempts also failed. Therefore, contributions were made by
different partners to extend the Turkish Mab library. A profiling experiment was conducted
with 95 isolates of the A Iran 2005 genotype collected in Turkey between 2005-2006 using
Mabs raised against the A Iraq 22 vaccine strain. Twelve out of 95 isolates showed low
profile with at least 3 out of 5 neutralising Mabs. Epidemiological meaning of these strains
were not resolved by phylogenic analysis based on VP1 sequences. Moreover, the
neutralising Pan-FMD MAb 4F6 was reactive with all the isolates suggesting a good
scientific tool for the quality assessment of vaccine antigens.
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Figure 6. Results of antigenic profiling for serotype A
O JPN 1/00
O UK 33/01
O TUR 5/00
O TAW 11/97
O BUL 1/96
O PHI 3/95
O TUR 2/00
O TUR 7/00
O GRE 22/96
O1 Manisa
O 1 Svizzera
100
100
64
100
100
100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100
100
100
100
100
100
100 100 100 100
100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100
70
100
95
100 100
75
74
60
60
80
100 100
80
100
95
100 100
90
96
80
83
100 100
100
100
100
100
100
90
100 100
87
83
100
100
100
77
100
100
100 100 100 100 100 100 100 100 100 100 100 100 100
97
80
85
100 100
87
100 100
100
100
100
100
100
72
85
3H4
100 100
0
0
4
site 3
3C8 100 100 62 100
0
0
0
100
72
5
site 2
4C9
0
0
0
0
78
93
47
0
0
60
60
40
60
86
6
site 2
4C6
76
100
70
80
0
0
0
0
0
0
0
60
0
0
0
0
0
22
0
0
0
30
0
61
0
0
21
64
55
0
0
0
0
20
0
36
0
0
0
26
17
0
0
0
0
0
0
56
0
0
17
28
19
0
0
0
0
0
41
0
0
0
22
0
0
0
0
0
0
48
0
0
0
0
0
0
0
0
0
0
49
0
0
0
0
0
0
0
0
0
41
0
0
0
0
0
0
0
0
0
0
24
17
0
0
0
100 100
93
64
2B4 0
1D7 23
3H7 0
site 2 1C12 0
7
site2,3 2A10 0
4D11 0
4E1 0
site 2 3B12 0
1F3
1A6
4G5
2E9
2G6
0
site 2
10 site 2
8
9
90
80
\
site 3
0
100
85
81
3
0
98
100 100 100 100 100
100 100 100
70
O ISR 1/96
86
71
O BUL 1/91
56
95
O IRN 15/97
O KUW 3/88
43
100
100
72
O 93 R 1148
O SAU8/88
O93 R 1111
100
100
O BUL 1/93
100
86
O GRE 2/94
82
70
O LIB 3/94
52
68
O TUR 3/94
49
100
O TUR 3/87
100 100
75
O GRE 21/94
61
60
O CAM1/98
61
72
O HKN 1/96
65
97
O SAU 72/94
100 100
81
72
site2,3 2F10 73 100 61 100 100
site 2 2H6 57 100 100 100 100
2
4B7 80 96 90 88 100
7E1 94 100 100 100 100
HOM
51
O BRA 4/94
B2
D9
O VIT 2/97
site 1
O KW 4/97
1
O VIT.7/97
MAb
FMDV O TYPE FIELD ISOLATES
100 100 100 100 100 100 100 100 100 100 100
100 100 100
88
100 100 100 100
0
0
0
0
0
0
0
100
10
32
62
48
67
88
0
0
0
0
0
0
0
0
100
0
41
60
48
60
45
47
57
0
0
0
100
100
100 100 100 100 100 100 100 100 100 100 100 100
100
100
100
100 100
0
100 100 100 100 100 100 100 100 100 100 100 100
100
100
100
100
0
100 100
92
85
87
90
100
91
93
98
80
90
100
96
100
100 100
0
100
78
100
58
83
93
65
76
100
89
74
83
90
92
62
100 100
0
0
100
81
100
62
82
100
94
84
100
75
76
85
94
100
83
100 100
0
0
100
73
100
57
79
100
73
77
100
58
68
76
93
97
60
100 100
0
0
100
93
100
57
51
100 100
75
100
55
70
67
84
83
68
100 100
0
0
0
100
88
100
56
44
100 100
84
100
54
73
76
86
75
77
100
10
0
0
0
100
74
100
58
38
100 100
53
100
51
52
57
73
70
50
100
10
100 100
90
64
84
21
11
52
41
34
23
0
0
0
0
0
0
0
100
10
100 100 100 100 100 100 100
47
45
100 100 100
100
0
0
0
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0
76
70
53
33
71
74
47
33
54
0
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26
18
17
0
0
0
0
0
0
0
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100
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10
10
11
10
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45
36
32
20
44
42
28
30
19
12
0
13
12
11
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10
0
0
10
0
10
100
20
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0
0
0
0
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77
66
69
38
78
74
47
31
37
0
0
19
0
0
0
0
0
0
0
0
0
0
100
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0
0
0
0
0
0
0
77
61
52
21
65
65
33
26
27
0
0
0
0
0
0
0
0
0
0
0
0
0
100
50
6C3
0
0
0
0
76
0
100
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
100
1C6
0
0
0
0
0
0
0
23
0
0
0
0
0
0
0
26
0
28
100
0
0
0
0
0
0
0
0
0
0
0
100
Figure 7. Results of antigenic profiling for serotype O
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In general, Mab antigenic profiling data is a very effective tool for monitoring how
effectively a vaccine strains protects against field viruses. However, mapping data combined
with full capsid sequences of field strains would provide more information which helps to
resolve the crucial epitopes like 2G5 and development of vaccine strategies focusing these
epitopes. Moreover, further research is required to better understand the contribution of
different viral epitopes to cross-protection and thereby to improve predictive methods of
vaccine efficacy.
Attempts have been made to improve the antigenic profiling ELISA system by using an
universal capture ligand i.e. alpha V beta 6 integrin which is well known as a receptor for
FMD virus (poster presented at the EUFMD Open session in 2008).
4. Genetic and antigenic typing of FMDV by monitoring in an endemic situation
The exploitation of the quasi-species structure of FMDV strains can be used for molecular
high-resultion strain identification. Results obtained during this project have proven
valuable in this respect. Sequence similarities are valuable markers of strain identity, but
similarity is always a relative characteristic in terms of strain identity. Similarities of straininherent diversities (multiple isolates collected from an animal, an outbreak or a geographical
region) is a much stronger indication of strain identity and is most useful to reveal the
multiplicity of strains emerging and/or circulating during outbreaks. This was illustrated by
the analysis of the 1982-3 epidemic of FMD in Denmark revealing 3 concurrent introductions
to Denmark (Christensen et al., 2005a; 2005b) and during studies in Turkey surprisingly
revealing that few introductions to Turkey rapidly became disseminated to the most distant
parts of Turkey, suggesting comprehensive transportation of live animals (Christensen et al.,
2006).
Building on the expertise gained by the high-resultion sequencing, sequence information on
215 FMDV isolates collected in Turkey since 1998 to 2007 were gathered by full-length
sequencing of VP1 and phylogenetic analysis (Klein et al., 2006; Parlak et al., 2007). The
analysis of FMD type A strains revealed the circulation of the genotypes A Iran 96 (A96), A
Iran 99 (A99) and A Iran 2005 (A05) and a sub-lineage of A Iran 2005, ARD-07. A96, A05
and A96 differed from each other by approximately 20% whereas ARD-07 differed from its
ancestor A05 by 5%. A96 and A99 genotypes were found to co-circulate during 1999-2005
while A05 and ARD-07 circulated during 2005-2007 and from 2007 onwards, respectively.
Due to this rapid evolution and the strain replacements, the choice of vaccine strains has
had to be changed twice with emergence of A05 and ARD-07 causing a significant challenge
to diagnosticians and vaccine producers who are supposed to select and produce matching
vaccine strain on time (Parlak et al., 2007; Knowles et al., 2008).
For serotype O, two genotypes were identified (PanAsia and Iran2001-CI&CII), three
pandemic strains (Iran2001-CI, PanAsia and PanAsia II) and nine distinct sub-lineages
belonging to two genotypes in 2004-2007 were observed (Christensen et al., 2006; Ozyoruk
et al., 2008). Unlike type A strains, type O strains were in the past generally covered in
vaccine matching test by a single vaccine strain, being O Manisa. Field observations during
the PanAsia II epidemic suggesting poor protection in vaccinated animals was also supported
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by the preliminary data obtained from cross-challenge animal experiments and poor in vitro
matching results.
To summarise, there are still two obstacles for the quick response to emerging FMD
epidemics by vaccination, which are (i) selecting the correct vaccine strain in antigen banks
and (ii) industrial adaptation of the homologous strain. Because vaccine matching tests are
not well established and depended on many variables such as quantification of field virus or
cell culture sensitivity, bioinformatics algorithms evaluating the cross-reactivity between
field and vaccine strains should be developed in order to reduce the number of variables of
existing tests. Because satisfactory adaptation of a new strain to cell culture for large scale
vaccine production is an unpredictable process, either genetic basis of cell culture adaptation
has to be understood or chimeric constructs mimicking the antigenicity of field virus have to
be developed in the near future.
Epidemiological studies of FMD were also initiated in Uganda with support from the Danish
Ministry of Foreign Affairs. The initial purpose of this project was to enhance the diagnostic
and research capacities in Uganda. This project is currently ongoing, but no longer with
participation of FMD-ImproCon partners.
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Other achievements: Alternative vaccine potency tests
Vaccine manufacturers evaluate the efficiency of their vaccine according to the method
which is defined in the European Pharmacopoeia. For FMD vaccines there are some
difficulties to find animals for potency tests in the countries like Turkey where FMD is
endemic and vaccine campaigns are carried out. In addition, potency tests must be carried out
in containments having high biosecurity levels. There are many publications indicating a
correlation between protection from virus challenge and neutralising antibody response.
However, up to now, none of the suggested method has been found valid.
Therefore, based on the sera obtained from the ten replicate O1 Manisa PD50 vaccine potency
tests performed in the framework of this project, a procedure was set-up to correlate
serology to protection. Serology was performed by four different laboratories (CODA, FLI,
Bayer HealthCare and FGI-ARRIAH) on serological in 9 different assays (VNT, LPBE,
SPCE, Ceditest type O ELISA). In total, 9 generalised linear models based on logistic
regression were built using the log10 transformed serum titres and binary protection statuses
of the 150 vaccinated animals, each representing a different serological assay performed by
the participating laboratories. The best association between log10 serum titre and probability
of protection is observed for models built for 3 different serological assays, namely the BHC
VNT, the VAR SPCE and the VAR LPBE with a Somers’D rank correlation ranging from
0.801 to 0.765. This was further confirmed by poorer Akaike Information Criterion for model
fit (AIC) values for the latter logistic models compared to the BHC VNT, VAR SPCE and
VAR LPBE models. This indicates an inferior degree of model fit to the in vivo data.
In order to determine a suitable antibody or log10 serum titre pass-level, a Receiver
Operating Chracteristics (ROC) analysis was performed. For instance, a SPCE log10 serum
titre of 1.45 (i.e. tC/O) corresponds to a probability of protection of 0.795 [95% CI: 0.7010.865] and a highest achievable level of accuracy (ACC) of 80.2%. The same ROC analysis
was performed for the remaining models. Three models reach ACC levels superior to 80.0%,
namely the BHC VNT, the VAR SPCE and the VAR LPBE model indicating the model’s
suitability for use as an indirect vaccine potency test.
The validity of the VAR SPCE logistic model was challenged and demonstrated externally
using a tC/O of 1.45. Using this value as antibody pass-level for protection means that primovaccinated animals with a log10 serum titre of at least 1.45 at 21 dpv are regarded as protected
against live virus challenge. Based on 1:2 serially titrated serum samples obtained at 21 dpv
from a Ph.Eur. FMDV O1 Manisa vaccine potency test performed in 2004, which was
unrelated to the one used to build up the model, log10 serum titres were determined for all 15
vaccinated animals and serologically predicted binary protection statuses were assigned.
These SPCE titrations were performed on 10 different occasions to assess VACC and VCON
of the proposed indirect FMD vaccine potency test, which were estimated to be 65.8% [95%
CI: 57.4-74.3] and 60.7% [95% CI: 52.3-69.2], respectively (Goris et al., 2008b). In other
words, a statistical correlation between serology and protection has been provided and
the applied methodology proved valid.
Similar analysis was made of data from potency tests on four batches O1 Manisa and two
batches of Asia-1 Tur 73 monovalent oil adjuvanted FMD vaccines. Regression were
calculated for the relation of protection from virus challenge versus antigenic load (Log
146S) and neutralising antibody response (Log SN50), versus only Log 146S, versus only
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Log SN50. For the relation of protection from virus challenge versus Log 146S and Log
SN50, R square was determined as 0,809 for O1 Manisa vaccines, 0,866 for Asia-1 Tur 73
vaccines. In addition, the amount of required antigen for % 50 protection in O1 Manisa and
Asia-1 Tur 73 vaccines was found 1,15 µg and 0,75 µg respectively (Alkan et al., 2008)
Within WP5, a study was initiated to standardise the association between antibody level for
FMD type O1 Manisa and protection in two different ways, first by using a standardised
commercial type PrioCHECK FMDV Type O ELISA and secondly by the inclusion of a
standard post vaccination serum, from a cow vaccinated with Cedivac® O1 Manisa FMD
vaccine, in both the ELISA and the VNT. Sera were available from 6 O1 Manisa potency
tests performed by partner 3, 10 O1 Manisa potency by partner 1, and 1 set of sera from a
potency test performed by partner 2. Sera were titrated for this study in the laboratory that
performed the potency tests, both in the PrioCHECK FMDV Type O ELISA and the VNT. In
each test a titration of the standard serum was included. Serological responses were fitted by
logistic regression. Significant differences were found in the titre of the control serum
obtained in the various laboratories, in both the ELISA and the VNT. Only a small difference
in the mean titre was found in protected and non-protected cattle. In both the ELISA and the
VNT, a highly significant (p<0.01) influence of the location was found on the relation
between antibodies titres, but also between standardised titres, and protection. The slope of
the relation between antibodies and protection was the same in each laboratory. The slope of
the relation between antibodies and protection was steeper when analysing the results
obtained in the VNT in comparison with the results obtained in the ELISA. Based on
previous studies to standardise serological tests, it was thought that standardisation by using a
standard serum would remove the variation between laboratories in the serological test. This
indicates that the relation between amount of antibodies and protection is not the same in the
different vaccines used in the different laboratories. Conclusion: a vaccine producer has to
determine the relation between antibody response and protection for his own vaccine and that
this relation cannot be extrapolated to other vaccine producers.
As shown above, it is well known that neutralising antibody titres are important in protecting
against FMDV infection. However, it has often been shown that the humoral antibody titre is
not always fully predictive of vaccine-induced protection against FMD. Therefore, a
correlation between cell mediated immune responses, humoral immune responses and
post-vaccination protection against FMDV infection was investigated. Samples were
collected from 5 vaccine challenge experiments conducted at Pirbright, UK. Blood samples
from FMDV vaccinated, non-vaccinated and vaccinated-and-challenged cattle were restimulated overnight with inactivated FMDV antigen and the level of induced IFN-γ was
measured. Humoral antibody levels were measured by virus neutralisation test. A positive
correlation was found between humoral antibody response, IFN-γ response and protection
against the clinical disease. It is concluded that T cell stimulation assays such as the whole
blood IFN-γ assay along with VNT are potential candidates for vaccine evaluation and could
reduce the need for in vivo challenge in the future.
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WP6. Development of a marker vaccine
An overall aim of this workpackage was to evaluate the marker vaccine potential of
intertypic chimera FMD viruses in which the protein coat of the virus contains elements of
both the original serotype as well as a part that has been substituted from another serotype.
Initial attempts to produce new chimeras based on an O1 Manisa vaccine virus backbone
were unsuccessful so the study used instead chimera viruses that had been produced
previously in the USA, by genetic engineering through removal of the immunodominant GHloop region of the virus capsid and substitution with the equivalent region from another
serotype of FMDV. These chimera viruses that had been made in the USA by Dr Elizabeth
Rieder and co-workers in the 1990’s were designed in an attempt to develop a bivalent FMD
vaccine that would protect against two different serotypes by producing stable, fully
infectious FMDV chimeric viruses in which the immunodominant VP1 G-H loop of the viral
capsid had been substituted with that of another FMDV serotype. Swine inoculated with this
chimeric vaccine, demonstrated neutralising antibodies to both serotypes, which conferred
protective immunity to the backbone serotype and partial protection against the inserted VP1
G-H loop serotype. The failure of the foreign loop to fully protect against its own serotype
meant that these chimeras were not pursued as commercial products. Furthermore, no studies
were carried out in cattle, which are the principal target species for FMD vaccination, and
which have been found more difficult than pigs to protect with certain vaccine types.
Recently, it was recognised that this type of chimera virus construct might be used as a
monovalent marker vaccine in which the substitution of the loop would provide a negative
marker for infection. For example, a type A vaccine with a GH loop substitution from type O
might be used to protect against an outbreak of type A. The lack of any GH loop specific type
A antibodies in the vaccinated population could differentiate such animals from vaccinated
and infected animals, as the latter would be expected to generate type A specific GH loop
antibodies.
Therefore, a vaccine challenge study was conducted to see if a vaccine made from the
chimera virus could (1) protect cattle against FMD, and (2) induce an immune response that
was more readily distinguishable from infection than that induced by conventional
vaccination. For this purpose cattle were vaccinated with either parental type A vaccine or
with one of two chimeric type A vaccines in which the GH loop had been substituted with
loops from type O or type C FMDV. Thereafter, the cattle were challenged with virulent type
A FMDV. Both of the chimeras were fully effective at clinically protecting cattle against type
A challenge, despite the lack of an homologous type A GH loop. Furthermore, virological
and NSP testing only identified two vaccinated animals that showed evidence of subclinical
infection. However, more work is required to demonstrate that loss of the homologous G-H
loop has no detrimental effect on vaccine-induced protection when compared at different
doses to the efficacy of an unsubstituted vaccine. A titration of the parental and chimera virus
vaccines prior to challenge would be needed to establish the relative potency of the parental
and chimera virus vaccines but was not attempted, due to the large number of animals that
would be needed to show this conclusively.
In order to determine whether these chimeric FMDV constructs could be used as novel
marker vaccines, samples from challenged cattle were tested in two novel serological
differentiation assays developed specifically for this study to detect antibodies to GH loop
peptides. Despite the apparently low level of virus replication indicated by the NSP serology
and virological tests, sub-clinical infection was able to be identified by both novel peptide
assays (an indirect and a monoclonal antibody –MAb– based competition assay). The un-
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vaccinated cattle were clearly positive in the tests from day 5 or 7 after challenge using the
indirect assay, and from day 14 or 21 using the MAb based assay. More importantly, animal
VQ86 which was shown to be sub-clinically infected using the well characterised VI, RTPCR and PrioCHECK FMDV NS test was also shown to be positive by both novel assays and
remained positive in the MAb based assay until the last day of sampling. In addition, a
response to challenge was noted and suggested that both these assays are potentially more
sensitive than the PrioCHECK FMDV NS.
This work clearly indicates that VP1 G-H loop substituted chimera vaccines can not only
confer clinical protection in cattle but can greatly reduce sub-clinical virus replication.
Although the marker potential of the chimera vaccines could only be partially evaluated as a
result of the almost ‘sterile’ immunity generated from these vaccines, significantly, the results
indicated that animals identified by existing DIVA tests such as the PrioCHECK FMDV NS
can also be detected using these two new assay approaches based on detecting regions
contained within structural proteins. In addition, this work indicates that the VP1 G-H loop
may not be required for a protective immune response. Further studies should be undertaken
to investigate whether a vaccine without the VP1 G-H loop can also protect species such as
cattle from foot-and-mouth disease (Fowler et al., 2008).
A type A vaccine virus has been discovered at IAH that was a mixture of two G-H loop
phenotypes. One variant included a full G-H loop and the other was a spontaneously
occurring deletion mutant that lacks most of the G-H-loop. Groups of five cattle were
immunised with vaccines formulated with one or other of these viruses and antisera collected
from the two groups were compared for their ability to cross-react with a range of
antigenically diverse serotype A viruses. This revealed that loss of the G-H loop had little
impact on cross-reactivity, indicating that a vaccine of this phenotype should elicit similar
cross-protection as a conventional vaccine that has the G-H loop region. A report of these
studies was presented at the FAO EUFMD Open Meeting (Erice, Italy) in 2008. The marker
potential will also be further explored by analysis of sera from these vaccinated and
challenged cattle.
The WP further aimed at targeting and enhancing mucosal immune responses through
vaccination. Dendritic cells (DC), essential for inducing and regulating immune defences
and responses, represent the critical target for vaccines against pathogens such as FMDV.
Consequently, the overall aim of this work was to specifically target DC, which would be
involved in promoting systemic plus mucosal immune responses. This represents an
important scientific basis for improving FMDV vaccines. The approach selected was based
on the use of particular immuno-modulating and immuno-stimulating factors in combination
with conventional FMD vaccines. These were first screened in vitro using DC and
DC/lymphocyte co-cultures and then tested in animal models including mouse and pigs.
The first objective was to characterise how FMDV vaccine antigen interacts with DC. DC
internalise FMDV, with no evidence for virus replication. In general, DC internalisation of
FMDV was most efficient for vaccine virus with heparin sulphate binding capacity, but this
was not an exclusive requirement. Also both non-heparin sulphate binding virus and
infectious RNA interacting with DC induced specific immune responses, albeit less
efficiently compared with heparin sulphate binding virus (Harwood et al., 2008). When
FMDV antigen and a second antigen (ovalbumin) were simultaneously internalised by DC,
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there was an observable delay in the processing of the latter. This is indicative of the DC
being activated by the presence of the FMDV, probably entering into the early stages of their
maturation pathway, which is essential for antigen processing to occur. Following
internalisation of FMDV antigen, these DC were efficient antigen presenting cells, observed
in terms of their ability to stimulate specific lymphocyte proliferation and virus-specific
antibody production. Taken together, these results are advantageous for conventional FMD
vaccines, which will be composed cell-culture adapted HS-binding variants of FMDV.
The second objective was the identification of immuno-modulatory factors targeting DC
for promotion of antibody and mucosal immune responses. The approach used was based
on the fact that DC are not only central in inducing immune reactions but also play pivotal
roles in controlling mucosal immune responses. This is by conferring the expression of
tissue-specific homing receptors to lymphocytes, and by secretion of cytokines able to
promote mucosal immune responses. Lymphocyte expression of mucosa-specific receptors is
pivotal for effector cell homing to mucosal compartments and the establishment of mucosal
immunity. We have established an in vitro screening system consisting of co-cultures of
lymphocytes and DC pre-treated with FMDV antigen and candidate factors including
vitamins, enterotoxins and toll-like receptor (TLR) ligands. Measurement of lymphocyte
proliferation and the production of antigen-specific immunoglobulin (Ig) including IgG and
IgA isotypes were chosen as read-out for the overall potency of the adjuvant factors. Several
readouts were used to assess the capacity of adjuvant factors to promote the induction of
mucosal responses, including the expression levels of the mucosal homing receptors CCR9
and the integrin 47, cytokine profile including TGF secretion and pro-inflammatory
versus anti-inflammatory responses. In these studies, the vitamin A derivative all-transretinoic acid was identified as a novel potential mucosal adjuvant. Retinoic acid-treated DC
were potent in promoting mucosal homing receptor expression on co-cultured lymphocytes,
secreted high levels of TGF and IL-6, promoted IgG responses and IgA isotype switching
(Saurer et al., 2007). An E. coli-derived heat-labile enterotoxin (LT) has also been
identified as potentially alternative factor with immunostimulating properties and the capacity
to promote mucosal immune responses. DC treated with LT undergo phenotypic and
functional maturation in terms of their T-cell stimulatory capacity. In the presence of LT high
anti-FMDV antibody and T-cell responses can be induced in vitro. Intradermal vaccination
with FMDV antigen formulated with LT was highly immunogenic in terms of serum
antibody responses but did not induce mucosal antibodies. On the other hand, it was possible
to obtain mucosal antibodies directed against LT but not FMDV in the saliva of vaccinated
mice when LT was combined with CpG immunostimulation and FMDV antigen. These
results indicate that it is principally feasible to obtain mucosal antibody responses after
intradermal vaccination.
The use of TLR ligands as additional immunostimulating molecules to promote systemic and
mucosal immune responses was characterised in vitro and in vivo. Retinoic acid acts
synergistically with TLR ligands in activating DC to produce cytokines, including IL-1, IL6, and TGF-, but not IL-12 and TNF- which is indicating a Th2-type of modulation.
Accordingly, in vivo addition of a TLR ligand to a vaccine formulation promoted both
systemic and mucosal antibody responses. Based on these results, retinoic acid is proposed as
a potential factor promoting mucosal immunity. In two independent immunisation
experiments using ovalbumin as antigen formulated in a retinoic acid containing vaccine used
in a mice model, the proof-of-concept was demonstrated in one of the trials. Taken all data
together, there is evidence that retinoic acid applied parenterally has the potential to promote
mucosal immune responses. Nevertheless, with some animals this was not observable
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indicating that the formulation and/or type of delivery of such a vaccine needs further
optimization before testing it in the FMD context and a large animal such as the pig.
Another aim was to test the utility of TLR-based immunostimulation to promote early
immunity against FMDV. To this end, a challenge model was established in pigs using a
CpG-adjuvanted anti-FMDV vaccine. This was tested for its capacity to induce early
protection against FMDV. The transcription level of Mx1, OAS and IRF-7 were identified as
sensitive measurements of CpG-induced activation of innate antiviral defence. Elevated
mRNA levels for these genes were detectable 8-96h after injection of CpG formulated in
Emulsigen. Despite this, pigs vaccinated with a combination of conventional vaccine (O1
Manisa) and CpG formulated in Emulsigen two days before challenge (O UK/2001) were
not protected and even had a more acute disease development compared to controls. Applied
at 7 days before challenge, CpG did not influence the protective value of the vaccine.
Conclusion: while our results demonstrate the potency of conventional FMD vaccines in a
severe heterologous challenge infection of pigs, the study also shows that caution is required
when translating findings from mouse models to a natural host of FMD (Summerfield et al.,
2008a; 2008b).
In addition to the workplan foreseen at the start of the project, a third approach to develop
new marker vaccines was established. Dendrimers are branched, synthetic polymers with
layered architectures that show promise in several biomedial applications, incluiding drug
delivery, gene transfection, imaging and others. The high level of control possible over the
architectural design of dendrimers; their size, shape, branching length, etc, clearly
distinguishes these structures as unique and optimum carriers in those applications. Recent
successes in simplifying and optimising the synthesis of dendrimers provide a large variety of
structures while at the same time reducing the cost of their production. The reflections on
biomedical applications of dendrimers clearly demonstrate the potential of this new fourth
major class of polymer architecture and indeed substantiate the high hopes for the future of
dendrimers. Numerous reports on the in vitro efficacy of dendrimers have been published but
only a few in vivo therapeutic studies exist. Therefore, it was decided to explore the potential
of dendrimer peptides to induce specific immune response capable to protect against FMDV.
Consequently, four pigs were immunised with a a dendrimer peptide containing the
immunodominant T-cell peptide 3 A [21-35] (dendrimeric core) branching to three-four
copies of the B-cell epitope VP1 [137-156]. Challenge and immunisation experiments carried
out in pigs showed the efficiency of such vaccines. The administration of two doses of
dendrimeric peptides conferred full protection against FMDV. The mechanism of immune
response were probably related to the activation of mucosa immune response, since a
correlation between anti-FMDV IgA serology and protection was found. Experiments
performed in mice model demonstrated that the immunogenicity of these constructions
correlate with the number of branches displayed in the dendrimer. Meanwhile constructions
expressing just one copy of peptide requires the administration of two doses of vaccine before
to detect significant titers of neutralising antibodies, the immunisation with dendrimers
displying three or four copies allow to reach high titers of antibodies and more rapid than
whit lineal constructions (Cubillos et al., 2008). In addition to these results, the crossreactivity of antibodies generated in response to dendrimer was evaluated, against different
subtypes of FMDV. The results obtained showed that the immunisation with dendrimeric
peptides provide r values greater than 3, suggestion a wide protection of this kind of vaccine
against not homologous FMDV isolates.
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Impact of the project on foot-and-mouth disease industry, control policy
and research sector
The project brought together key players in the field of FMD diagnostics and vaccinology.
All involved FMD experts belong to internationally recognised reference laboratories, are
often consulted by DG SANCO, FAO and OIE, have internationally recognised publications
and most of them are members of the FAO EUFMD Research Group. Much of their work is
published in Vaccine and The Journal of Virological Methods. The same level of scientific
dissemination was accomlished during the course of this project as illustrated by the 35 stateof-the-art publications resulting from this project (see publication list below) and the four
international workshops organised.
WP 1 of the project has contributed significantly to the availability of validated assays able to
discriminate unequivocally between vaccinated and infected animals. Knowledge
(advantages and limitations) on their sensitivity and specificity is essential in light of the
requirements laid down in the new Council Directive on Community Measures for the
Control of FMD (Council Directive 2003/85/EC) and their application to substantiate
freedom from FMD has been discussed. The work performed in WP2 was aimed at kit
production opportunities with a view to facilitate the distribution of the product to all
interested countries in Europe and the rest of the world. Some of the developed prototype
assays have already been taken up by kit manufacturers and other might in the future. The
animal experiments performed in WP4 and WP5 provided indispensable serum sample
collections from vaccinated and/or infected animals that enabled validation and development
of existing and new DIVA test. These sera can readily be shared with other institutes and
organisations, especially since reference serum panels have been developed for the purpose
of assessing FMD DIVA tests.
Improving on existing FMD detection methods in WP3 has sometimes proven difficult, but
by better understanding the basic principles of FMD growth on cell culture progress has been
made. Furthermore, a new goat cell line has been identified to enhance FMD virus detection
that also enables a quicker diagnosis.
WP4 provided new insights in virus transmission and the impact of vaccination, which will
prove crucial when faced with new FMD outbreaks. The value of vaccination in reducing
virus transmission has been demonstrated in different FMD susceptible species (cattle, sheep
and pigs) with different FMDV strains and serotypes. However, the results of WP4 also
clearly show that vaccination will not be able to prevent infection when it occurs within a
week following the administration of the vaccine. Nevertheless, these experiments were
successful in measuring the efficacy of vaccines, the dynamics of NSP seroconersion and its
relationship to virus replication, persistence and clinical signs. Mathematical analysis of the
experimental data has been used to predict carrier numbers in cattle herds and sheep flocks
after emergency vaccination, as an aid to determining the feasibility of using NSP
serosurveillance to substantiate FMD freedom.
The data provided on vaccine strain selection in WP5 will help decision makers in their
difficult choice of vaccine use and in identifying relevant strains for inclusion in FMDV
antigen banks. The advantage of using high potency vaccines when no matching vaccines are
readily available in antigen reserves has been shown. High potency vaccines enhance the
probability of achieving adequate levels of cross-protection even when faced with low rvalues and sequence homology. Furthermore, the limits of the existing in vivo FMD vaccine
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potency tests have been demonstrated (high level of between test variability) which point
towards more international acceptance of alternative in vitro approaches avoiding live viral
challenge (e.g. serology, interferon-gamma responses and antigen payload).
Progress has been made in WP6 to enhance mucosal immunity. Moreover, a new generation
vaccine based on a serotype A chimera vaccine in which the GH-loop region was replaced
with that of another serotype proved fully protective in cattle against challenge from the
unsubstituted parental serotype A virus. Furthermore, the presence of a heterologous GHloop could be exploited to discriminate serologically between vaccinated and vaccinated-andchallenged cattle. Subsequently, a spontaneous loop-deleted mutant vaccine virus was
discovered and shown to elict an antibody response that was similarly cross-reactive
compared to antibodies elicited by a loop-undeleted vaccine virus. Follow-up funding has
been granted to further pursue the marker vaccine potential of this virus.
New diagnostic experience and new knowledge about the epidemiology and virus properties
were transferred in different ways. Results open to the public were placed on the website
(www.fmdimprocon.org) and presented not only at scientific meetings of the project group,
but also at the annual meeting of the EU reference laboratories for vesicular diseases and at
the open sessions of the FAO-EUFMD RG that brings together, every 2 years, 32 European
countries and reference laboratory representatives from all other continents.
Workshops have been organised in collaboration with DG RTD and DG SANCO for the EU
reference laboratories, in collaboration with other FP6 projects (EPIZONE and CA
CSF&FMD), FAO EUFMD, OIE and TAIEX for candidate Member States.
Results will further be discussed with DG-RTD and DG-SANCO. If results are deemed
appropriate to change the control policy, they could be presented to the EC Scientific
Committee. Through close collaboration between some of the partners and the EU, FAOEUFMD and the OIE, possible changes can be communicated to these organisations.
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2 – DISSEMINATION AND USE
WP1. Validation of existing NSP-tests
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Brocchi E, Bergmann I, Dekker A, Paton DJ, Sammin DJ, Greiner M, Grazioli S, De Simone F,
Yadin H, Haas B, Bulut N, Malirat V, Neitzert E, Goris N, Parida S, Sorensen K, De Clercq K.
2006. Comparative evaluation of six ELISAs for the detection of antibodies to the non-structural
proteins of foot-and-mouth disease virus. Vaccine, 24(47-48):6966-6979.
Campos Rde M, Malirat V, Neitzert E, Grazioli S, Brocchi E, Sanchez C, Falczuk AJ, Ortiz S,
Rebello MA, Bergmann IE. 2008. Development and characterization of a bovine serum evaluation
panel as a standard for immunoassays based on detection of antibodies against foot-and-mouth disease
viral non-capsid proteins. J. Virol. Methods. 2008 151: 15-23.
Dekker A, Sammin D, Greiner M, Bergmann I, Paton D, Grazioli S, De Clercq K, Brocchi E.
2008. Use of continuous results to compare ELISAs for the detection of antibodies to non-structural
proteins of foot-and-mouth disease virus. Vaccine, 26 :2723-2732.
Engel B, Buist W, Orsel K, Dekker A, De Clercq K, Grazioli S, van Roermund H. 2008. A
Bayesian evaluation of six diagnostic tests for foot-and-mouth disease for vaccinated and nonvaccinated cattle. Preventive Veterinary Medicine, 86 :124-138.
Goris N, Praet N, Sammin D, Yadin H, Paton D, Brocchi E, Berkvens D, DeClercq K. 2007. Footand-mouth disease non-structural protein serology in cattle: use of a Bayesian framework to estimate
diagnostic sensitivity and specificity of six ELISA tests and true prevalence in the field. Vaccine,
25:7177-7196.
Parida S, Fleming L, Gibson D, Hamblin PA, Grazioli S, Brocchi E, Paton DJ. 2007. Bovine
serum panel for evaluation of FMDV non structural protein antibody tests. J. Vet. Diagn. Invest.,
19(5):539-44.
Paton DJ, De Clercq K, Greiner M, Dekker A, Brocchi E, Bergmann I, Sammin D, Gubbins S,
Parida S. 2006. Application of non-structural protein antibody tests in substantiating freedom from
foot-and-mouth disease infection after emergency vaccination. Vaccine, 24:6503-6512.
WP2. Development and validation of new immunoassays such as NSP-tests as primary and
confirmatory tests
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Inoue T, Parida S, Paton DJ, Linchongsubongkoch W, Mackay D, Oh Y, Aunpomma D, Gubbins
S, Saeki T. 2006. Development and evaluation of an indirect enzyme-linked immunosorbent assay for
detection of foot-and-mouth disease virus non structural protein antibody using a chemically
synthesized 2B peptide as antigen. Journal of Veterinary Diagnostic Investigation, 18:545-552.
Parida S, Anderson J, Cox SJ, Barnett PV, Paton DJ. 2006. Secretory IgA as an indicator of oropharyngeal foot-and-mouth disease virus replication and as a tool for post vaccination surveillance.
Vaccine, 24:1107-1116
Perkins J, Parida S, Clavijo A. 2007. Use of a standardized bovine serum panel to evaluate a
multiplexed non-structural protein antibody assay for serological surveillance of food-and-mouth
disease. Clin. Vaccine Immunol., 14(11):1472-82.
Rémond M, Da Costa B, Riffault S, Parida S, Breard E, Lebreton F, Zientara S, Delmas B. 2009.
Infectious bursal disease subviral particles displaying the foot-and-mouth disease virus major antigenic
site. Vaccine, 27:93-98.
Sanchez, MT, Rosas MF, Ferraz RM, Delgui,L, Blanco E, Villaverde A and Sobrino F.
Discriminating Foot-and-mouth disease virus-infected and vaccinated animals through betagalactosidase allosteric biosensors. Submitted to J.Clin.Microbiology January 2009.
WP3. Improved FMD virus detection
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Goris N, Vandenbussche F, Herr C, Villers J, Van der Stede Y, De Clercq K. submitted.
Validation of two real-time RT-PCR methods for foot-and-mouth disease diagnosis: RNA-extraction,
matrix effect, uncertainty of measurement and precision. Journal of Virological Methods, submitted.
Van Dessel W, Vandenbussche F, Staes M, Goris N, De Clercq K. 2008. Assessment of the
diagnostic potential of immuno-RCA in 96-well ELISA plates for foot-and-mouth disease virus.
Journal of Virological Methods, 117:151-156.
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WP4. Impact of vaccination on virus dissemination and the carrier state
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Orsel K, Dekker A, Bouma A, Stegeman JA, de Jong MCM. 2005. Vaccination against Foot and
Mouth Disease reduces virus transmission in groups of calves. Vaccine, 23(41):4887-4894.
Orsel K, de Jong MCM, Bouma A, Stegeman JA, Dekker A. 2007a. The effect of vaccination on
foot and mouth disease virus transmission among dairy cows. Vaccine, 25(2):327-35.
Orsel K, Dekker A, Bouma A, Stegeman JA, de Jong MC. 2007b. Quantification of foot and mouth
disease virus excretion and transmission within groups of lambs with and without vaccination. Vaccine,
25(14):2673-2679.
Orsel K, de Jong MC, Bouma A, Stegeman JA, Dekker A. 2007c. Foot and mouth disease virus
transmission among vaccinated pigs after exposure to virus shedding pigs. Vaccine, 25(34):6381-6391.
Orsel K, Roest HIJ, Elzinga-Bril EM, Van Hemert-Kluitenberg F, Dekker A. 2008. Detection of
foot-and-mouth disease virus in infected pigs by RT-PCR four weeks after challenge. Veterinary
Record, 162:753-754.
Orsel K, Bouma A, Dekker A, Stegeman JA, de Jong MCM. 2009. Foot and mouth disease virus
transmission during the incubation period of the disease in piglets, lambs, calves and dairy cows.
Preventive Veterinary Medicine 88, 158-163.
Parida S, Fleming L, Oh Y, Mahapatra M, Hamblin P, Gloster J, Doel C, Gubbins S, Paton DJ.
2007. Reduction of foot-and-mouth disease (FMD) virus load in nasal excretions, saliva and exhaled air
of vaccinated pigs following direct contact challenge. Vaccine, 25(45):7806-17.
Parida S, Fleming L, Oh Y, Mahapatra M, Hamblin P, Gloster J, Paton DJ. 2008. Emergency
vaccination of sheep against foot-and-mouth disease: Significance and detection of subsequent subclinical infection. Vaccine, 26:3469-3479.
Schley D, Paton DJ, Cox SJ, Parida S, Gubbins S. 2009. The effect of vaccination on undetected
persistence of foot-and-mouth disease virus in cattle herds and sheep flocks. Epidemiology and
Infection.
WP5. Improved vaccine strain selection
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Brehm KE, Kumar N, Thulke H-H, Haas B. 2008. High potency vaccines induce protection against
heterologous challenge with foot-and-mouth disease. Vaccine, 26:1681-1687.
Christensen LS, Normann P, Thykier-Nielsen S, Sorensen JH, de Stricker K, Rosenorn S. 2005a.
Analysis of the epidemiological dynamics during the 1982-1983 epidemic of foot-and-mouth disease
(FMD) in Denmark based on molecular high-resolution strain identification. Journal of General
Virology, 86:2577-2584.
Christensen LS, Normann P, Thykier-Nielsen S, Sorensen JH, de Stricker K, Rosenorn S. 2005b.
En revurdeing af den danske mund- og klovesygeepidemi i 1982-1983 baseret pa stammaidentifikation
ved sekvensbestemmelse. Dansk Veterinaertidskrift, 88:16-20.
Goris N, Merkelbach-Peters P, Diev VI, Verloo D, Zakharov VM, Kraft H-P, De Clercq K. 2007.
European Pharmacopoeia foot-and-mouth disease vaccine potency testing in cattle: Between test
variability and its consequences. Vaccine, 25:3373-3379.
Goris N, Maradei E, D’Aloia R, Fondevila N, Mattion N, Perez A, Smitsaart E, Nauwynck HJ,
La Torre J, Palma E, De Clercq K. 2008a. Foot-and-mouth disease vaccine potency testing in cattle
using homologous and heterologous challenge strains: Precision of the “Protection against podal
generalisation test”. Vaccine, 26:3432-3437.
Goris N, Willems T, Diev VI, Merkelbach-Peters P, Vanbinst T, Van der Stede Y, Kraft H-P,
Zakharov VM, Borisov VV, Nauwynck HJ, Haas B, De Clercq K. 2008b. Indirect foot-and-mouth
disease vaccine potency testing based on a serological alternative. Vaccine, 26:3870-3879.
Klein J, Parlak U, Ozyoruk F, Christensen LS. 2006. The molecular epidemiology of foot-andmouth disease virus serotypes A and O from 1998 to 2004 in Turkey. BMC Veterinary Research, 2:35.
Parlak U, Ozyoruk F, Knowles NJ, Armstrong RM, Aktas S, Alkan F, Cokcaliskan C,
Christensen LS. 2007. Characterisation of foot-and-mouth disease virus strains circulating in Turkey
during 1996-2004. Archives of Virology, 152(6):1175-1185.
WP6. Development of a new marker vaccine
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Cubillos C, de la Torre BG, Jakab A, Clementi G, Borras E, Barcena J, Andreu D, Sobrino F,
Blanco E. 2008. Enhanced mucosal immunoglobulin A response and solid protection against foot-andmouth disease virus challenge induced by a novel dendrimeric peptide. Journal of Virology, 82:72237230.
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Fowler VL, Paton DJ, Rieder E, Barnett PV. 2008. Chimeric foot-and-mouth disease viruses:
Evaluation of their efficacy as potential marker vaccines in cattle. Vaccine, 26:1982-1989.
Harwood JL, Gerber H, Sobrino F, Summerfield A, McCullough KC. 2008. Dendritic cell
internalization of foot-and-mouth disease virus: influence of heparan sulfate binding on virus uptake
and induction of the immune response. Journal of Virology, 82:6379-6394.
Saurer L, McCullough KC, Summerfield A. 2007. Modulation of dendritic cells to imprint T cell
mucosal homing receptor expression in vitro. J. Immunol., 79(6):3504-35.
Summerfield A, McCullough KC. 2008a. The porcine dendritic cell family. Developmental and
Comparative Immunology, 33:299-309.
Summerfield A, Guzylack-Piriou L, Harwood L, McCullough KC. 2008b. Innate immune
responses against foot-and-mouth disease virus: current understanding and future directions. Vet.
Immunol. Immunopathol., Epub ahead of press.
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