Synthesis, isolation and characterization of Plasmodium falciparum antigenic tetrabranched peptide dendrimers obtained by thiazolidine linkages.

Different chemical alternatives were evaluated for obtaining immunogenic polypeptidic macromolecules which could then be used as vaccines. These were based on the ligation reaction between an unprotected immunogenic peptide and an unprotected multifunctional core peptide; polyantigens, designated dendrimers because their form resembles that of dendritic cells, were thus obtained. The antigen-core ligation alternatives, studied by indirect synthesis, were the formation of oxime, hydrazone and thiazolidine linkages, making use of the reaction between a weak base (acting as nucleophile) and an alkyl aldehyde. The other alternative was the formation of a thioether linkage between a sulfydryl and an alkyl halide. Finally, a multiple antigen peptide (MAP) was synthesized by direct synthesis. All reactions were monitored by SEC-HPLC and SDS-PAGE. Dendrimer molecular mass obtained was confirmed by MS MALDI-TOF. Dendrimer purification was first carried out by concentrating crude reaction products with CP-5000 centricons and (using SEC-HPLC) pure tetramers were then obtained. A 20-residue 9376 immunogenic sequence, from Plasmodium falciparum apical merozoite antigen protein (AMA-1), was used to study the best alternative for chemical ligation. It was observed that thiazolidine formation proceeded with greater yield and in less time than the others. A tetramer has been simultaneously synthesized via thiazolidine with the SPf-66 antimalarial vaccine 45-residue monomer, proving the technique's versatility. The 9376 peptide disulfide bound polymer and SPf-66 (as well as their tetrameric thiazolidine dendrimers) were inoculated in rabbits to evaluate their antibody response. It was observed that titers for tetrameric thiazolidine dendrimers were not just greater but were also sustained over time. Western blot for pre-immune and immune sera showed that dendrimer sera recognized specific Plasmodium falciparum proteins as well as disulfide-bound polymers.