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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 385 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 386 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 387 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). References Blanco E, McCullough K, Summerfield A, Fiorini J, Andreu D, Chiva C, Borras E, Barnett P & Sobrino F. (2000). Interspecies major histocompatibility complex-restricted Th cell epitope on footand-mouth disease virus capsid protein VP4. J Virol. 74(10), 4902-7. Blanco E, Garcia-Briones M, Sanz-Parra A, Gomes P, De Oliveira E, Valero ML, Andreu D, Ley V & Sobrino F. (2001). Identification of T-cell epitopes in nonstructural proteins of foot-and-mouth disease virus. J Virol. 75(7), 3164-74. Boyle JS, Koniaras C & Lew AM. (1997). Influence of cellular location of expressed antigen on the efficacy of DNA vaccination: cytotoxic T lymphocyte and antibody responses are suboptimal when antigen is cytoplasmic after intramuscular DNA immunization. Int. Immunol. 12, 1897-1906 . Boyle JS, Brady JL & Lew AM (1998). Enhanced responses to a DNA vaccine encoding a fusion antigen that is directed to sites of immune induction. Nature 392, 408-411. Collen T, Pullen L & Doel TR. (1989). T. Cell dependent induction of antibody against foot-andmouth disease virus in a mouse model. J. Gen. Virol. 70, 395-403. Fernandez FM, Borca MV, Sadir AM, Fondevila N, Mayo J & Schudel AA. (1986) Foot-andmouth disease virus (FMDV) experimental infection: susceptibility and immune response of adult mice. Vet. Microb.12, 15-24 . Mateu MG, Rocha E, Vicente O, Vayreda F, Navalpotro C, Andreu D, Pedroso E, Giralt E, Enjuanes L & Domingo E. (1987) Reactivity with monoclonal antibodies of viruses from an episode of foot-and-mouth disease. Virus Res. 8, 261-274 . Rodriguez F & Whitton JL. (2000). Enhancing DNA Immunization. Virology 268, 233-238 . Sobrino F, Saiz M, Jimenez-Clavero MA, Nunez JI, Rosas MF, Baranowski E & Ley V. (2001). Foot-and-mouth disease virus: a long known virus, but a current threat. Vet Res. 32(1), 1-30. Review. Sáiz JC, Rodríguez A, González M., Alonso F & Sobrino F. (1992). Heterotypic lymphoproliferative response in pigs vaccinated with foot-and-mouth disease virus. Involvement of isolated capsid proteins. J. Gen. Virol. 73, 2601-2607. Taboga O, Tami C, Carrillo E, Nunez JI, Rodriguez A, Saiz JC, Blanco E, Valero ML, Roig X, Camarero JA, Andreu D, Mateu MG, Giralt E, Domingo E, Sobrino F & Palma EL. (1997). A large-scale evaluation of peptide vaccines against foot-and-mouth disease: lack of solid protection in cattle and isolation of escape mutants. J. Virol. 71 (4), 2606-2614 . 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) 388 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. 389 Figure 3. Virus in blood at 48 hpi. Figure 4. Cell response (ICCS) at 5 days post FMDV challenge . 390