Abstract
BackgroundThe malaria parasite Plasmodium falciparum EBA-175 binds its receptor sialic acids on glycophorin A when invading erythrocytes. The receptor-binding region (RII) contains two cysteine-rich domains with similar cysteine motifs (F1 and F2). Functional relationships between F1 and F2 domains and characterization of EBA-175 were studied using specific monoclonal antibodies (mAbs) against these domains.Methods and FindingsFive mAbs specific for F1 or F2 were generated. Three mAbs specific for F2 potently blocked binding of EBA-175 to erythrocytes, and merozoite invasion of erythrocytes (IC50 10 to 100 µg/ml IgG in growth inhibition assays). A mAb specific for F1 blocked EBA-175 binding and merozoite invasion less effectively. The difference observed between the IC50 of F1 and F2 mAbs was not due to differing association and disassociation rates as determined by surface plasmon resonance. Four of the mAbs recognized conformation-dependent epitopes within F1 or F2. Used in combination, F1 and F2 mAbs blocked the binding of native EBA-175 to erythrocytes and inhibited parasite invasion synergistically in vitro. MAb R217, the most potent, did not recognize sporozoites, 3-day hepatocyte stage parasites, nor rings, trophozoites, gametocytes, retorts, ookinetes, and oocysts but recognized 6-day hepatocyte stage parasites, and schizonts. Even though efficient at blocking binding to erythrocytes and inhibiting invasion into erythrocytes, MAb R217 did not inhibit sporozoite invasion and development in hepatocytes in vitro.ConclusionsThe role of the F1 and F2 domains in erythrocyte invasion and binding was elucidated with mAbs. These mAbs interfere with native EBA-175 binding to erythrocyte in a synergistic fashion. The stage specific expression of EBA-175 showed that the primary focus of activity was the merozoite stage. A recombinant RII protein vaccine consisting of both F1 and F2 domains that could induce synergistic activity should be optimal for induction of antibody responses that interfere with merozoite invasion of erythrocytes.
Highlights
The invasion of erythrocytes by malaria parasites is mediated by specific molecular interactions between erythrocyte receptors and parasite ligands
The role of the F1 and F2 domains in erythrocyte invasion and binding was elucidated with monoclonal antibodies (mAbs)
A recombinant region II (RII) protein vaccine consisting of both F1 and F2 domains that could induce synergistic activity should be optimal for induction of antibody responses that interfere with merozoite invasion of erythrocytes
Summary
The invasion of erythrocytes by malaria parasites is mediated by specific molecular interactions between erythrocyte receptors and parasite ligands. The presence of one or two DBL domains and other elements including a C-terminal cysteine-rich region and a type I transmembrane domain classifies EBA-175 as a member of the erythrocyte binding-like (EBL) superfamily of proteins [2]. This EBL superfamily includes BAEBL/EBA-140/ EBP2 [10,11,12] MAEBL [13], EBA-181/JESEBL [14]and var genes, a large family of genes regulated by chromatin modification [15,16] that encode antigenically variant proteins including PfEMP-1 [5,7]. Functional relationships between F1 and F2 domains and characterization of EBA-175 were studied using specific monoclonal antibodies (mAbs) against these domains
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