Download 61. DNA vaccines based on FMDV minigenes in a mouse model

Appendix 61
Immunogenicity and protection conferred by DNA vaccines based on FMDV minigenes in a
mouse model
Belén Borrego*1, Paloma Fernández-Pacheco1, Llilianne Ganges1, Francisco Sobrino1,2 and Fernando
Rodríguez3
1
CISA-INIA, Valdeolmos 28130 Madrid, Spain
2
CBMSO, UAM Cantoblanco 28049 Madrid, Spain
3
CreSA, 08193 Bellaterra, Barcelona, Spain
Abstract
We have studied the potential of DNA vaccines based on viral minigenes corresponding to three major
B- and T-cell FMDV epitopes coexpressed with different target signals aiming to optimize their
antigenic presentation and thus their immunogenicity. A collection of pCMV plasmids expressing the
BTT epitopes [(133-156)VP1-(11-40)3A-(20-34)VP4 from isolate Cs8c1 ] fused to different target
signals (ubiquitin, LIMP-II, a signal peptide (SP) and CTLA-4), was produced. As a first approach, we
have studied the immune response induced and the protection conferred by different vaccine
candidates in a mouse model. NIH Swiss mice (non-syngeneic) received 3 IM doses of plasmid and
neutralizing antibodies in serum after the third dose were analysed by a plaque reduction assay.
Vaccinated mice were challenged with the homologous FMDV and viremia at 48 hours post-infection
was determined.
From all mice immunized with minigene-bearing plasmids, only one of the animals immunized with
the BTT tandem epitopes fused to the signal peptide developed specific neutralizing antibodies. At day
2 post FMDV challenge, while control mice immunized with pCMV showed high titers of virus in their
blood the only animal that developed neutralizing antibodies after DNA vaccination was protected
against FMDV infection. Furthermore, 7 more animals did not show viremia at 48 h post infection,
even in the absence of detectable antibodies prior to challenge. The best vaccine candidate resulted
to be the plasmid expressing the 3 viral epitopes alone. While protection was always lower to 25% for
the rest of the plasmids, 80% of the mice immunized with pCMV-BTT were protected.
We have demonstrated the protective capacity of a DNA vaccine based on FMDV minigenes in a
mouse model. Work must be done to elucidate the mechanisms involved in protection and to
determine the protective capacity of our vaccines in natural FMDV hosts.
Introduction
Despite their immunogenicity, peptide vaccines based on a major B cell epitope (B) at the G-H loop of
VP1 FMDV capsid protein, have shown to confer partial protection to FMDV (Taboga et al., 1997).
Previous work in our laboratory has identified two major T cell epitopes, located in the VP4 structural
protein (TVP4) and in the non-structural polypeptide 3A (T3A) (Sobrino et al., 2001). An ideal vaccine
should provide a complete immune response: both humoral and cellular responses. In an attempt to
improve the immunogenicity of these epitopes after DNA vaccination in vivo, we decided to use
successful strategies previously described in our lab and in others (Boyle et al., 1997, 1998;
Rodriguez & Whitton, 2000). Thus, we fused our antigens to ubiquitin to enhance CTL responses or to
the LIMP-II target signal to improve the CD4-T cell responses. At the other hand, in an attempt to
optimize B cell responses, we targeted our epitopes to the cell membrane or to the professional
antigen presenting cells (APCs) by adding a signal peptide (sp) or by fusing them to CTLA4.
Handling of a large number of vaccine candidates (multiple plasmid constructs) exponentially increase
the number of animals to be used, a requirement hard to be afforded with FMDV natural hosts. Thus,
as a first approach, a mouse model has been developed and used to asses the immunogenicity of
minigene-based DNA vaccines. Despite mice are not natural hosts for FMDV, this species has been
shown useful to study the immune response against FMDV (Collen et al., 1989; Fernández et al.,
1986).
Methods
Plasmid Generation. A plasmid had been previously constructed carrying in tandem the epitopes BTVP4, including a ClaI restriction site between both epitopes (B-epitope corresponds to VP1 137-156
and T corresponds to VP4 20-34 residues of the type C FMDV isolate Cs8-c1) (Domenech et al.,
unpublished results). The sequence corresponding to epitope T3A (residues 11-40 of 3A) was
amplified by PCR using a plasmid carrying the whole 3A protein as template, and primers including a
ClaI restriction site. This restriction site was used to clone the fragment amplified into the ClaI
restriction site between epitopes B and T, to obtain the BTT construct. These amplicons included
proper restriction sites at their ends to facilitate their cloning alone or fused to different target signals
in the pCMV plasmid (Clontech) under the control of an eukaryotic promoter. Thus, the ORFs were
cloned in the following plasmids: pCMV (to express the epitopes alone), pCMV-LIMP II and pCMVUbiquitin in which FMDV epitopes were expressed as fusions with LIMP II (Class II targeting) or
ubiquitin (Class I targeting), respectively. Furthermore, FMDV epitopes were cloned in plasmids
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pCMV-SP (signal peptide) and pCMV-CTLA4 to drive the antigens to the membrane and to APCs,
respectively. The sequence of the diverse constructs was confirmed by automatic sequencing and the
plasmids were produced free of endotoxins in a large scale (QIAGEN kits) and used to inoculate mice.
In Vitro Expression. Monolayers of BHK-21 cells were transfected by using the lipofectamine-Plus
reagent (GIBCO-BRL). At 48 h after transfection, cells were immunoperoxidase-stained using a MAb
against the FMDV B epitope.
Immunization And Infection. All experiments were done using the NIH Swiss strain of mice. For DNA
immunization, 3 doses of 100 µg each of plasmid were administrated intramuscularly. Two control
groups were included: animals vaccinated with 2 doses of 100
g of synthetic peptide A24,
corresponding to positions (138-156) VP1 of FMDV Cs8c1; and animals vaccinated with 2 doses of 106
pfu, BEI-inactivated, of FMDV C-S8c1 (Sáiz et al., 1992). These antigens were IP inoculated as 1:1
emulsion in complete (1st injection) or incomplete (second) Freund’s adjuvant. For challenge, 103 pfu
of FMDV Cs8c1 were inoculated in the footpad.
Virus titration. Viremia was followed by infecting monolayers of IBRS-2 cells in M96 plates with serial
dilutions of serum and monitoring of the cytopathic effect at 72 hpi. Titre was defined as the
reciprocal of the serum dilution (log10) causing cytopathic effect (cpe) in 50% of the wells. Titers
lower than 1.3 were considered as negative, since this was the value corresponding to the first
dilution tested (1/20).
Immune responses. FMDV specific antibodies were detected both by a trapping ELISA against
unpurified CS8c1 virus captured using a polyclonal antiserum and by a plaque-reduction
neutralization assay (Mateu et al, 1987). Neutralization titres are expressed as the reciprocal of the
serum dilution (log10) that caused 50% of plaque reduction.
For the ICCS assay, spleen cells from mice infected 5 days in advance with FMDV, were stimulated
directly ex vivo O/N with different viral stimuli. Three hours before incubating the cells with specific,
labeled antibodies, Brefeldin A (10 µg/ml) was added to the cultures to avoid cytokine secretion. Cells
were surface stained with an anti-CD4 antibody, permeabilized and labeled with an anti-IFN gamma
antibody. Double positive cells (CD4+ and IFN +) were plotted by using a Flux Cytometer.
Results
Construction of Plasmids Expressing Viral Epitopes
We first generated different versions of plasmid pCMV expressing the B cell epitope alone (B) and in
tandem with TVP4 (BT) or with TVP4 and T3A (BTT) (Table 1). Before testing the plasmids in vivo we
checked their expression after transfection in BHK-21 cells by immunostaining with a specific
monoclonal antibody against the B epitope. Only cells transfected with pCMV-BTT, expressing the
three epitopes, showed a specific signal (Table 1 and Fig 1a). Conversely, no expression was detected
upon transfection with plasmids pCMV-B and pCMV-BT. Thus, BTT was selected to characterize the
potential of minigene-based DNA vaccine to elicit protective immune responses to FMDV.
In order to explore the effect of directing the expression of the BTT tandem minigene to different cell
compartments, new plasmids were constructed in which BTT was fused to different target signals. The
expression of the B cell epitope was detectable in all cases (figure 1, panel e). Relative to what
observed with plasmid pCMV-BTT, a higher number of cells were positive when a signal peptide was
fused to the BTT (pCMV-sp-BTT). This increase was not detected when BTT was co-expressed with
Ubiquitin, LII or CTLA4 (Fig 1, panels b,c,d).
A Mouse Model For Fmdv Immunization And Challenge
An initial experiment was performed to characterize the viremia and the humoral immune response
induced upon infection of different strains of mice with FMDV C-S8-c1. In outbred NIH-Swiss mice
infected with 103 pfu, the virus was detected in blood as soon as at 24 hpi, reaching a peak at 48 hpi
and being the virus completely cleared at 72 hpi (figure 2a). 10-20% of the infected animals died
between days 3 and 10 p.i. Viral clearance in blood at 3 dpi correlated with the detection of
neutralizing antibodies in sera. The titers of neutralizing antibodies reached a peak as early as at 8
dpi, which remained for months (figure 2b).
To characterize the level of protection developed by convalescent mice, animals were challenged with
the same dose of FMDV C-S8c1 and both viremia and antibody response were followed at different
times post-infection. None of the pre-infected animals showed virus in blood at any time after the reinfection, while control naïve mice showed a typical viremia, as described previously. These results
clearly demonstrate that preimmunization of mice with a sub lethal dose of virus confers total
protection against a subsequent challenge with the homologous virus. Mice immunized with a single
dose of a BEI-inactivated FMDV (C-S8c1) vaccine also induced neutralization titres equivalent to
those reached in infected animals (see table 2), and a 100% protection when animals were
challenged after two doses of vaccine (see figure 3).
DNA Vaccination
Groups of NIH Swiss mice received 3 im injections of each of the minigene-bearing plasmids, as
indicated in Table 2. As controls, 8 mice were injected with the empty plasmid (pCMV) and 4 animals
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were infected with 103 pfu of FMDV C-S8c1. An additional group of mice was immunized twice with a
synthetic peptide corresponding to the B epitope included in the minigene constructs. All mice were
bled 10 days after the last inoculation and the sera were used to detect FMDV specific antibodies by
ELISA and by a neutralization assay. As expected, pCMV injected mice did not develop specific
antibodies, while 100% of the surviving virus-infected mice developed high titres of neutralizing
antibodies (>2.4) (table 2). However, from the 30 mice immunized with minigene-bearing plasmids,
only one, belonging to the group of mice immunized with the BTT fused to the signal peptide (pCMVsp-BTT), developed specific neutralizing antibody titers (1.6). One of the five animals immunized with
peptide A24 also developed neutralizing antibodies (titer 2.0).
Protection Of Immunized Animals Against Viral Challenge
To evaluate the protective capacity of the minigene-bearing plasmids, all mice were challenged with
103 pfu of the homologous FMDV C-S8 at least two moths after the last antigen dose. Animals were
bled at 48 h post-infection. As expected, the animal that developed neutralizing antibodies after
inoculation with pCMV-sp-BTT was fully protected against FMDV infection, as estimated by the lack of
viremia at day 2. Interestingly, 7 out of the 30 animals immunized with minigene-bearing plasmids
did not show viremia at 48 h post infection, in spite of not having developed detectable neutralizing
antibodies prior to challenge (figure 3). The higher protection, in 4 of the 5 animals analysed, was
conferred by plasmid pCMV-BTT that expressed the BTT minigene alone. Unexpectedly, protection
was lower to 25% for the rest of the plasmids designed to drive the antigens to different antigen
presenting pathways, while 50% of the mice immunized with peptide A24 were protected. As
expected, control mice immunized with pCMV (empty plasmid) exhibited high titers of virus in their
blood at day 2 post FMDV challenge (figure 3). The protection observed by the pCMV-BTT vaccine
seemed to correlate with the induction of specific CD4-T cell responses against FMDV. At the time of
sacrifice it was possible to detect CD4 T cells that specifically secrete IFN in response to ex vivo
stimulation with the specific FMDV peptides in at least two of the pCMV-BTT protected mice (figure 4).
Discussion
Our previous results demonstrated the capacity of sequences corresponding to T cell epitopes TVP4 or
T3A to provide in vitro T help leading to the production of neutralizing antibodies, when presented as
fusion peptides with the major B cell site from capsid protein VP1(Blanco et al., 2000; 2001). These
results opened the possibility of designing subunit vaccines including B and T cell epitopes relevant
for the induction of protection against FMDV. As the efficient synthesis of long peptides is still an
unsolved problem, the possibility of expressing these minigene constructs in DNA expression vectors
is an interesting possibility to test its immunogenicity in animal models. Here, we have used this
approach in combination with a mouse model system for FMDV immunization and challenge that
allows the analysis of a considerable number of variables, using statistically significant numbers of
animals.
The lack of expression in cells transfected with pCMV-BT and pCMV-B might be due, among other
possibilities, to the instability of these short peptides (39 amino acids the longer) in the cytoplasm of
transfected cells. The detection of expression when the T3A epitope was included (pCMV-BTT) could
be related to an improvement in the stability of the tandem peptide expressed (69 amino acid).
When driving the BTT polypeptide to different cell compartments, expression of the B cell epitope was
detectable in all cases (figure 1). When compared to pCMV-BTT, the only construct resulting in a
higher number of positive cells was pCMV-sp-BTT in which the presence of a signal peptide seems to
improve the stability of the BTT polypeptide (Fig 1, panel e). The lower number of positive cells
obtained after transfection with the rest of the plasmids might be explained by the rapid degradation
(pCMV-BTT-LII, pCMV-Ubq-BTT; panels b and c respectively) or by the fast secretion of the BTT
epitopes to the milieu (pCMV-CTLA4BTT, panel d).
The contribution of the humoral response to the in vivo protection against FMDV has been clearly
established along the years. In particular, a strong correlation is found in convalescent and
conventionally vaccinated animals between neutralizing activity in sera and protection against FMDV
challenge (reviewed in Sobrino et al., 2001). The analysis of the inhibition of viremia upon FMDV
challenge in the mouse model developed has provided interesting information on the protective
immune responses elicited by the pCMV derivatives expressing the different versions of BTT epitopes
studied. As expected, the only animal that developed neutralizing antibodies after immunization with
pCMV-sp-BTT was solidly protected against FMDV infection. However other animals were also
protected in the absence of detectable neutralizing antibodies prior to challenge (table 2 and figure
3). Thus, 4 of the 5 mice that received pCMV-BTT were able to clear the virus upon challenge. Driving
BTT to the MHC I and MHC II presenting pathways does not improve either the specific antibody
production or the protection conferred. However, fusing the epitopes to a strong signal peptide
improved both their in vitro expression and the induction of protective neutralizing antibodies.
Interestingly, a correlation is observed between protection to challenge and the induction of FMDV
specific CD4-T cell responses in mice immunized with pCMV-BTT. We are currently working in the
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characterization of the T cell responses elicited by these plasmids as well as assessing the efficacy of
these DNA immunization strategies in the pig, an important natural host for FMDV.
Acknowledgements
Work supported by UE project “Optimizing DNA based vaccination against FMDV in sheep and pigs”
(QLK2-CT2002-01304).
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Table 1. Plasmids expressing FMDV minigenes used in this study
PLASMID
pCMV
pCMV-B
pCMV-BT
pCMV-BTT
VIRAL EPITOPES
(FMDV C-S8c1)
none
(133-156)VP1
(133-156)VP1-(20-34)VP4
(133-156)VP1 - (11-40) 3A - (20-34)VP4
EXPRESSION
Negative
Negative
Negative
Positive (fig.1)
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Table 2. Neutralizing antibodies after inoculation of mice
SN positivea / total
Inoculum
SN titre
mice
pCMV
0/8
--pCMV-BTT
0/5
--pCMV-BTT-L II
0/5
--pCMV-Ubq-BTT
0/5
--pCMV-CTLA4-BTT
0/5
--pCMV-sp-BTT
1/4
1.6
Plasmid mix
0/6
--Peptide A24
1/5
2.0
BEI-inactivated virus
5/5
> 2.3
C-S8c1 virus
4/4
> 2.4
a
values < 1.0 were considered as negative.
Figure 1. Expression of viral epitopes in BHK-cells transfected with plasmids coding for the
BTT epitopes fused to different target signals
Figure 2. Viremia (a) and development of neutralizing antibodies (b) in Swiss mice
infected with FMDV. Black, naïve animal; white, preimmunized animals.
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Figure 3. Virus in blood at 48 hpi.
Figure 4. Cell response (ICCS) at 5 days post FMDV challenge .
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