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. 2013;8(2):e56326.
doi: 10.1371/journal.pone.0056326. Epub 2013 Feb 15.

The epitope of monoclonal antibodies blocking erythrocyte invasion by Plasmodium falciparum map to the dimerization and receptor glycan binding sites of EBA-175

Xavier Ambroggio  1 , Lubin Jiang, Joan Aebig, Harold Obiakor, Jan Lukszo, David L Narum
Affiliations

The epitope of monoclonal antibodies blocking erythrocyte invasion by Plasmodium falciparum map to the dimerization and receptor glycan binding sites of EBA-175

Xavier Ambroggio et al. PLoS One. 2013.

Abstract

The malaria parasite, Plasmodium falciparum, and related parasites use a variety of proteins with Duffy-Binding Like (DBL) domains to bind glycoproteins on the surface of host cells. Among these proteins, the 175 kDa erythrocyte binding antigen, EBA-175, specifically binds to glycophorin A on the surface of human erythrocytes during the process of merozoite invasion. The domain responsible for glycophorin A binding was identified as region II (RII) which contains two DBL domains, F1 and F2. The crystal structure of this region revealed a dimer that is presumed to represent the glycophorin A binding conformation as sialic acid binding sites and large cavities are observed at the dimer interface. The dimer interface is largely composed of two loops from within each monomer, identified as the F1 and F2 β-fingers that contact depressions in the opposing monomers in a similar manner. Previous studies have identified a panel of five monoclonal antibodies (mAbs) termed R215 to R218 and R256 that bind to RII and inhibit invasion of erythrocytes to varying extents. In this study, we predict the F2 β-finger region as the conformational epitope for mAbs, R215, R217, and R256, and confirm binding for the most effective blocking mAb R217 and R215 to a synthetic peptide mimic of the F2 β-finger. Localization of the epitope to the dimerization and glycan binding sites of EBA-175 RII and site-directed mutagenesis within the predicted epitope are consistent with R215 and R217 blocking erythrocyte invasion by Plasmodium falciparum by preventing formation of the EBA-175- glycophorin A complex.

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Conflict of interest statement

Competing Interests: The authors have read the journal’s policy and have the following conflicts: DNarum has a competing interest declared below: Narum, D.L., Sim, B.K.L. U.S. Patent Number: US 7,025,961, Anti-Plasmodium Compositions and Methods of Use. Apr. 11, 2006. Abstract: Compositions that inhibit the binding of Plasmodium falciparum to erythrocytes are provided. More particularly, antibodies specific for Plasmodium falciparum binding proteins and blocking peptides that prevent the binding of Plasmodium falciparum are included in the present invention. The methods provided utilize the antibody and peptide compositions provided herein and include methods for the diagnosis, prevention, and treatment of Plasmodium falciparum diseases such as malaria as well as methods for the detection of Plasmodium falciparum in biological samples and culture media. Narum, D.L., Liang, H., Fuhrmann, S., Sim, B.K.L. Patent Number: U.S. Patent Number: US 7,078,507, Synthetic Genes for Malarial Proteins and Methods of Use. Jul. 18, 2006. Abstract: Synthetic gene sequences encoding erythrocyte binding protein of a malaria pathogen for the expression of the erythrocyte binding protein. The codon composition of the synthetic gene sequences approximates the mammalian codon composition. The synthetic gene sequences are useful for incorporation into the DNA vaccine vectors, for the incorporation into various expression vectors for production of malaria proteins, or both. The synthetic genes may be modified to avoid post-translational modification of the encoded protein in hosts. Administration of the synthetic gene sequences, or the encoded protein, as an immunization agent is useful for induction of immunity against malaria, treatment of malaria, or both. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Figures

Figure 1
Figure 1. Mapping of epitope predictions to the rEBA-175 RII crystal structure.
(A) rEBA-175 RII crystal structure as a dimer of two RII molecules (cyan and grey), the region corresponding to the F2βf peptide is highlighted in yellow and R422 is highlighted in magenta. The amino- and carboxy- terminal residues are colored in blue and red respectively. The white box represents the region highlighted in panel B. (B) The F2 domain of one monomer in the rEBA-175 RII crystal structure is shown as a ribbon diagram and residues are colored by the number of times they were predicted by either PepSurf and Mapitope to be part of the epitope for mAbs R215, R217, or R256 from never (cyan) to most often (magenta; see SOM for raw data). Residues discussed in the text are labeled according to rEBA-175 RII numbering (EBA-175 3D7 sequence number –144) and shown in stick representation.
Figure 2
Figure 2. Binding characteristics of EBA-175 RII specific mAbs R215, R217 and R218 to recombinant EBA-175 RII (A) or disulfide-constrained F2βf peptide (B) by ELISA.
Figure 3
Figure 3. F2βf peptide competes with mAbs R215 (A) and R217 (B) but not R218 (C) for binding to recombinant EBA-175 RII by Surface Plasmon Resonance studies.
Monoclonal antibody sensograms show binding to immobilized EBA-175 RII with the concentrations of mAb and cyclic F2βf peptide as indicated. The concentrations of cyclic F2βf peptide were added to a fixed concentration of mAb.
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