Open session of the standing technical committee of the EUFMD- 2004

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