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TARGETING FMDV MINIGENES TO SLA II POSITIVE CELLS
ENHANCES THE INDUCTION OF CELLULAR RESPONSES IN SWINE
AND CONFERS PROTECTION AGAINST VIRAL CHALLENGE
B. Borrego1, J.M. Argilaguet2, E. Pérez-Martín2, A. Ezquerra3, M. Pérez-Filgueira3,4 ,
J.M. Escribano3, F. Sobrino1,5 & F. Rodríguez2
1 CISA-INIA, Valdeolmos, Madrid, Spain; 2 CReSA, UAB, Bellaterra, Barcelona, Spain; 3 Dpto de Biotecnología INIA Madrid, Spain; 4 Instituto Virología-CICVyA
INTA Buenos Aires, Argentina; 5 CBMSO, UAM Cantoblanco, Madrid, Spain
Intro
In addition to safety and economical benefits related to their production, the feasibility of manipulating DNA plasmids is one of the advantages that make DNA
vaccination a promising alternative to conventional vaccines, opening a broad range of possibilities for improvement and/or modulation of the immune responses
induced. One of the most interesting approaches is to target antigens to the extra-cellular milieu and/or driving them to antigen presenting cells (APCs). We are testing
several targeting signals by fusing them to different combinations of B- and T-cell epitopes from Foot-and-Mouth Disease Virus (FMDV). We first analysed the
VACCINE POTENTIAL of several DNA constructs in vitro and in a mice model, in order to select those most promising for an immunization/challenge experiment in
swine, a natural host for FMDV. Here we show the results of this analysis.
Expression in vitro Detection by immunodot of B-antigen in serial 4fold dilutions of
extracts of BHK cells transfected with plasmids encoding the FMDV-BTT minigenes
[antigenic sites B (aa 133-156 of VP1), T3A (11-40 of 3A) and TVP4 (20-34 of VP4),
from FMDV Cs8c1] fused to different signal peptides from porcine molecules CCL20
and CD163 showed that the CCL20 sp resulted the most efficient for in vitro
expression (fig 1).
CCL20
CD163
Immunogenicity in mice New plasmids were constructed in which the CCL20 sp was fused to different
combinations of the FMDV BTT epitopes. The BTT antigen was also fused to another target signal:
scFv, a recombinant antibody that recognizes the Class II Swine Leukocyte Antigen. After three IM
shots of 50 microg, only one mouse receiving the construct pCMV-CCL20sp-BTT was able to
generate high levels of neutralizing antibodies (fig 2a), while only in splenocytes from mice
inoculated with pCMV-scFv-BTT specific secretion of IFNg by CD4+ and CD8+ T cells was
detected by ICCS (fig 2b) ; thus these were the constructions selected for immunization of swine.
PrP
#1
100
#2
#3
#4
80
CD4+
60
IFN
gamma
40
20
CD8+
0
CMV
CCL20BTT
CCL20
BT3A
scFv
BTT
CCL20NEG
Figure 2b
Figure 2a
DNA immunization and viral challenge in pigs Groups of 4 pigs received 3 shots of 400 microg of the selected plasmids pCMV-CCL20sp-BTT and pCMV-scFv-BTT every two weeks.
A control pig (# 5) was inoculated with an irrelevant plasmid. Animals 11 and 12 received one single DNA shot on day 28. 15 days after the last DNA dose animals were needle-challenged
with infectious FMDV, then monitored daily for clinical signs of disease. Samples were taken at different time points along the experiment. At day 10 animals were euthanized.
Development of disease and other signs of infection Progression of disease in the pigs was evaluated using a
clinical score based on a semi-quantitative rating of clinical signs such as lameness, vesicle formation on each one
of the four feet, on the tongue, mouth and snout, and vesicle size, and pyrexia (rectal temperature over 40º). This
evaluation showed that animals inoculated with the scFv construction (in red) had developed a protective
response. Two animals (#8 , #12) showed no signs of disease during the 10 days monitored, and neither virus nor
antibodies to non-structural proteins NSP (indicative of viral replication) were detected in their samples. The other two
animals within this group showed a delay on the disease onset, and clinical signs were milder when compared to the
control animal, even though viral load in serum samples seemed to be equivalent. Remarkably, viral detection in
swabs from this group resulted mainly negative at the days analysed. Animals immunized with pCMV-CCL20sp-BTT
showed no significant differences when compared to the control pig.
SIGNS OF
VIRAL INFECTION
CLINICAL SCORE
4.0
neg
1.786
2
4.5
neg
1.682
4
3
4.0
neg
0.742
5
4
4.0
neg
1.665
5
PCR POS
2.5
1.52
7
1.0
3.0
1.069
3
spCCL20-BTT
7
2
control
8
d9
scFv-BTT
d1
0
d8
d7
d6
d4
de
sa
fi
d3
12
d2
0
o
11
d1
1
Day 2
Day 3
Seroconversion
to NSPc
1
2
3
RT-PCR
on swab samplesb
Day 3
5
4
Viral isolation
and/or RT-PCR
on serum samplesa
Day 10
8
PCR NEG
PCR NEG
0.035
11
Neg
2.0
1.234
12
PCR NEG
PCR NEG
0.234
days post challenge
a.
b.
c.
Immune response after vaccination The immune response induced
by DNA inoculation, both humoral and cellular responses, was
analysed in samples collected at day 43 (prior to challenge) by a
neutralization assay, by ELISPOT and by lymphoproliferation. The
only assay in which a positive response was detected was IFNgamma ELISPOT, in which animals inoculated with the scFvconstruction showed a significant number of IFN-g producing
cells that specifically responded to BEI inactivated virus.
Infectious virus
BEI inactivated virus
Medium
35,0
Day 2
1
6
Conclusions
CCL20BTVP4
Samples were inoculated onto IBRS cells monolayers and monitored for cpe (for 3 freeze/thaw cycles). Results
expressed as TCID50/10 microl (log10). Some of the negative samples were also assayed by RT-PCR.
Results expressed as: black cell: both swab samples (nasal and pharyngeal) positive; grey cell, only one
sample positive; white cell: both samples negative.
Results expressed as OD at day 10 - OD at day 0 in a 3ABC-ELISA.
nr spots per 10^6 cls
Results
Figure 1
CCL20B
30,0
25,0
20,0
15,0
10,0
5,0
0,0
1
2
3
spCCL20-BTT
4
7
8
11
12
scFv-BTT
The DNA vaccine in which the FMDV BTT antigen was fused to the signal peptide from the porcine chemokine CCL20 failed in inducing specific antibodies in swine and
after viral challenge animals developed typical clinical signs of disease. In contrast, immunization with a construction in which antigen was fused to a scFv that
specifically recognize SLAII molecules induced a significant cellular response that, even in the absence of anti-FMDV antibodies, resulted in protection for 50% of the
animals, in which no signs of disease or viral replication were observed.
Thesepreliminary
preliminaryresults
resultsprovide
providesome
someinteresting
interestingobservations:
observations:
These
i)
cellular
responses
alone
may
play
crucialrole
rolein
inprotection
protectionagainst
againstFMD
FMD
i) cellular responses alone may play aacrucial
ii)targeting
targetingof
ofantigens
antigensto
toAPCs
APCsseems
seemsto
tobe
beaavery
veryeffective
effectiveapproach
approachto
toinduce
inducesuch
suchprotective
protectivecellular
cellularresponses
responses
ii)
iii)immune
immuneresponses
responseselicited
elicitedagainst
againsthighly
highlyconserved
conservedFMDV
FMDVT-cell
T-cellepitopes
epitopescould
couldovercome
overcomethe
thevariability
variabilityof
ofFMDV
FMDVstrains
strains
iii)
We are
are currently
currently extending
extending and
and completing
completing these
these results
results in
in order
order to
to elucidate
elucidate the
the mechanisms
mechanisms responsible
responsible for
for the
the protection
protection afforded
afforded by
byour
our DNA
DNAvaccine
vaccine
We
and
to
analyse
the
possibility
of
using
these
vaccines
to
confer
protection
against
heterologous
FMDV
strains.
and to analyse the possibility of using these vaccines to confer protection against heterologous FMDV strains.
We thank M.G.Esguevillas & N.de la Losa for excellent technical assistance.