Desarrollo de nuevas estrategias de vacunación frente al virus de la fiebre aftosa basadas en péptidos sintéticos

Resumen

Foot-and-mouth disease virus (FMDV) is a causative agent of highly infectious and economically important disease of cattle, sheep, pigs and cloven-hoofed wildlife species. Despite low mortality rates, FMD severely decreases livestock production and is a major constraint to international trade in live animals and their products. The disease affects extensive areas of the world, is endemic in parts of the Middle East, Africa, Asia, and South America, and increased global trade and movement of people (legal and illegal), provide opportunities for the virus to spread. FMD control in endemic areas is implemented by regular vaccination, using chemically inactivated whole-virus vaccines, effectives to reduce the impact of FMD. However, conventional vaccines has important disadvantages, such as the requirement of a coldchain to preserve vaccine stability, the risk of viral release during vaccine production and the problems for serological distinction between infected and vaccinated animals. These limitations and the information available on the genomic organisation have favoured FMDV becoming a model system for the development of new vaccines. This doctoral thesis describes the design, immunogenicity and successful protection conferred to pigs by lineal and dendrimeric peptides, displaying FMDV T- and/or B-cell epitopes. The B-cell epitope used was located in the GH loop (site A), between positions 136-154 of capsid protein VP1. This epitope was alone or co-linearly synthesized with a T-cell epitope corresponding to NSP 3A residues 21-35. In order to enhance the immunogenicity induced by these linear peptides, we designed a multimeric structure (dendrimeric peptide), displaying in a single molecule four copies of the VP1 B-cell epitope joined to a T-cell epitope from NSP 3A, through a lysine tree plus two additional Lys residues, defining a putative cleavage site for cathepsin D. This protease has been suggested to be involved during in vivo MHC class II antigen processing. Outbred pigs were immunized with these peptides by intramuscular route and challenged by intradermal inoculation of FMDV. The peptides constructed, specifically induced significant titers of FMDV-neutralizing antibodies and activated FMDV-specific T cells. Partial protection was observed in immunized pigs with linear peptides (B and TB peptides) after challenge. Despite these peptides failed to completely protect pigs, interestingly we detected a reduction on FMDV excretion after challenge, more significant in pigs immunized with linear peptide including a specific T-cell epitope. The presentation of selected B- and T-cell epitopes in a multimeric construction, allowed the enhancement of the effectiveness conffered by linear peptides. The dendrimeric peptide constructed successfully protected completely pigs against challenge with FMDV. The pigs immunized with dendrimer did not develop significant clinical signs, neither systemic nor mucosal FMDV resplication, nor was its transmission to contact control pigs observed. Interestingly, a potent anti-FMDV IgA response (local and systemic) was observed, despite the parenteral administration of the peptide. Moreover, the dendrimeric peptide immunized pigs showed no antibodies specific of FMDV infection, which qualifies the dendrimeric peptide as a potential marker vaccine. Overall, the dendrimeric peptide used elicited an immune response comparable to that found for control FMDV-infected pigs that correlated with a solid protection against FMDV challenge. Dendrimeric designs of this type may hold sustantial promise for peptide subunit vaccine development.