Plasmodium falciparum: further characterization of a functionally active region of the merozoite invasion ligand EBA-175

Abstract

A 42 amino acid peptide, Pf EBA-175 (1062-1103), also called EBA-peptide 4 of the 175-kDa Plasmodium falciparum sialic acid binding protein, a putative merozoite invasion ligand, has been shown to be a target of parasite growth inhibitory antibodies. We expressed and purified a recombinant protein, NS1-Pf EBA-175 (946-1133) which included the 42 amino acid peptide, and compared antibodies induced by immunization with the protein to antibodies raised against the 42 amino acid peptide. Sera from rabbits immunized with the recombinant protein and the synthetic peptide immunoprecipitated authentic EBA-175, and had comparable ELISA titers against peptide Pf EBA-175 (1062-1103). However, IFAT titers against infected erythrocytes and growth inhibitory activity were substantially higher in sera from animals immunized with the 42 amino acid synthetic peptide. Epitope mapping of the 42 amino acid peptide identified a 19 amino acid peptide, Pf EBA-175 (1069-1087), which blocked the ability of antibodies against the 42 amino acid peptide to (1) immunoprecipitate EBA-175, (2) bind to the 42 amino acid peptide in an ELISA, and (3) recognize infected parasites in an IFAT. Sera from rabbits immunized with the 19 amino acid peptide conjugated to KLH had excellent parasite growth inhibitory activity (at 1:5 serum dilution, 49.9 +/- 7.4%, mean +/- SD of three separate assays), but the activity was lower in each of the three assays than that of sera from rabbits immunized with the 42 amino acid peptide (67.8 +/- 24.8%). These data indicate that the activity of antibodies raised against the linear 42 amino acid peptide, Pf EBA-175 (1062-1103) are primarily, if not exclusively, directed against 19 of the 42 amino acids, and identify this region of Pf EBA 175 as a target for vaccine development.