Delineation of functional regions on Plasmodium falciparum EBA-175 by antibodies eluted from immune complexes

After several infections and drug cure, Aotus spp. monkeys are protected against homologous Plasmodium falciparum challenge. Sera from these monkeys passively transfers protection (Diggs CL, Hines F, Wellde BT. Exp Parasitol 1995;80:291-6). Antibodies in these sera agglutinate merozoites emerging from ruptured schizonts forming immune clusters and thereby presumably prevent merozoite invasion of erythroyctes (Lyon JA, Haynes JD, Diggs CL, Chulay JD, Pratt-Rossiter JM. J Immunol 1986;136:2252-8). It is reasoned that antibodies eluted from the immune clusters of merozoites must recognize exposed epitopes with functional relevance to the survival of the live, invasive merozoites. The erythrocyte binding protein, EBA-175 was used as a model to study this hypothesis. We expressed and purified nine overlapping fragments of EBA-175 as recombinant proteins and probed them in immunoblots with antibodies eluted from the immune clusters. Only the two regions of EBA-175 previously shown to be functionally relevant were recognized by the eluted antibodies. One was region II which includes the erythrocyte binding domain of this parasite ligand which binds to its receptor glycophorin A. The other was the region containing EBA-peptide 4, antibodies to which block binding of EBA-175 to erythrocytes and inhibit invasion of merozoites into erythrocytes. Immunization of mice with the region II recombinant protein induced antibodies that recognize merozoites in immunofluorescence assays, identified a 175 kDa band on unreduced immunoblots, blocked binding of EBA-175 to erythrocytes and inhibited merozoite invasion of erythrocytes. These findings corroborate the importance of EBA-175 region II and EBA-peptide 4, and suggest that antibodies eluted from immune clusters can be used to identify protective epitopes exposed on invasive merozoites from other P.falciparum proteins.