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

Different chemical alternatives were evaluated for obtaining immunogenic po lypeptidic macromolecules which could then be used as vaccines. These were based on the ligation reaction between an unprotected immunogenic peptide a nd an unprotected multifunctional core peptide; polyantigens, designated de ndrimers because their form resembles that of dendritic cells, were thus ob tained. The antigen-core ligation alternatives, studied by indirect synthes is, were the formation of oxime, hydrazone and thiazolidine linkages, makin g use of the reaction between a weak base (acting as nucleophile) and an al kyl aldehyde. The other alternative was the formation of a thioether linkag e between a sulfydryl and an alkyl halide. Finally, a multiple antigen pept ide (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 concentrat ing crude reaction products with CP-5000 centricons and (using SEC-HPLC) pu re tetramers were then obtained. A 20-residue 9376 immunogenic sequence, fr om 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 t he others. A tetramer has been simultaneously synthesized via thiazolidine with the SPf-66 antimalarial vaccine 45-residue monomer, proving the techni que's versatility. The 9376 peptide disulfide bound polymer and SPf-66 (as well as their tetrameric thiazolidine dendrimers) were-inoculated in rabbit s to evaluate their antibody response. It was observed that titers for tetr americ thiazolidine dendrimers were not just greater but were also sustaine d overtime. Western blot for pre-immune and immune sera showed that dendrim er sera recognized specific Plasmodium falciparum proteins as well as disul fide-bound polymers.