INDUCTION OF PROTECTIVE IMMUNE-RESPONSES BY IMMUNIZATION WITH LINEAR MULTIEPITOPE PEPTIDES BASED ON CONSERVED SEQUENCES FROM PLASMODIUM-FALCIPARUM ANTIGENS

A cysteine-containing peptide motif, EWSPCSVTCG, is found highly conse rved in the circumsporozoite protein (CSP) and the thrombospondin-rela ted anonymous protein (TRAP) of all the Plasmodium species analyzed so far and has been shown to be crucially involved in the sporozoite inv asion of hepatocytes. We have recently shown that peptide sequences co ntaining this moth, and also the antibodies raised against the motif, inhibit the merozoite invasion of erythrocytes. However, during natura l infection, and upon immunization with recombinant CSP, this motif re presents a cryptic epitope. Here we present the results of immunizatio n studies with two linear multiepitopic constructs, a 60-residue (P60) and a 32-residue (P32) peptide, containing the conserved motif sequen ce. Both the peptides per se generated high levels of specific antibod ies in BALB/c mice. P32 was found to be genetically restricted to H-2( d) and H-2(b) haplotypes of mice, whereas P60 was found to be immunoge nic in five different strains of mice. The antibody response was predo minantly targeted to the otherwise cryptic, conserved motif sequence i n P60. Anti-P60 antibodies specifically stained the asexual blood stag es of Plasmodium falciparum and Plasmodium yoelii in an immunofluoresc ence assay, recognized a 60- to 65-kDa parasite protein in an immunobl ot assay, and blocked P. falciparum merozoite invasion of erythrocytes in a dose-dependent manner. Immunization with P60 also induced signif icant levels of the cytokines interleukin-2 (IL-2), IL-4, and gamma in terferon in BALB/c mice. Moreover, >60% of mice immunized with P60 sur vived a heterologous challenge infection with a lethal strain of P. yo elii. These results indicate that appropriate medium-sized synthetic p eptides might prove useful in generating specific immune responses to an otherwise cryptic but critical and putatively protective epitope in an antigen and could form part of a multicomponent malaria vaccine.