Effect of arginine mutation on EBA-175 RII binding to human erythrocytes.

a<p>the rosette number was an average counted from two biological repeats in each experiment.</p>b<p>erythrocyte-binding assay was performed with normal erythrocytes in two independent experiments.</p>c<p>erythrocyte-binding assay was performed with neuraminidase-treated erythrocytes Nm, neuraminidase.</p>*<p>Residue 566 of EBA-175 corresponds to residue 422 of rEBA-175 RII.</p

The Epitope of Monoclonal Antibodies Blocking Erythrocyte Invasion by <em>Plasmodium falciparum</em> Map to The Dimerization and Receptor Glycan Binding Sites of EBA-175

<div><p>The malaria parasite, <em>Plasmodium falciparum</em>, 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 <em>Plasmodium falciparum</em> by preventing formation of the EBA-175– glycophorin A complex.</p> </div

Deduced amino acid sequences of clones enriched for each EBA-175 RII specific mAb.

<p>Deduced amino acid sequences of clones enriched for each EBA-175 RII specific mAb.</p

Mapping of epitope predictions to the rEBA-175 RII crystal structure.

<p>(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.</p

Summary of binding properties of EBA-175 RII specific mAbs.

<p>SPR: surface plasmon resonance. ND: not determined.</p>*<p>Does not inhibit parasite growth <i>in vitro</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056326#pone.0056326-Sim2" target="_blank">[21]</a>.</p>**<p>None of the mAbs demonstrated binding to linear F2βf.</p>c<p>These mAbs were shown to compete against each other for binding by competition ELISA.</p

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.

<p>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.</p

Phase 1 Study in Malaria Naïve Adults of BSAM2/Alhydrogel®+CPG 7909, a Blood Stage Vaccine against <em>P. falciparum</em> Malaria

<div><p>A Phase 1 dose escalating study was conducted in malaria naïve adults to assess the safety, reactogenicity, and immunogenicity of the blood stage malaria vaccine BSAM2/Alhydrogel®+ CPG 7909. BSAM2 is a combination of the FVO and 3D7 alleles of recombinant AMA1 and MSP1₄₂, with equal amounts by weight of each of the four proteins mixed, bound to Alhydrogel®, and administered with the adjuvant CPG 7909. Thirty (30) volunteers were enrolled in two dose groups, with 15 volunteers receiving up to three doses of 40 µg total protein at Days 0, 56, and 180, and 15 volunteers receiving up to three doses of 160 µg protein on the same schedule. Most related adverse events were mild or moderate, but 4 volunteers experienced severe systemic reactions and two were withdrawn from vaccinations due to adverse events. Geometric mean antibody levels after two vaccinations with the high dose formulation were 136 µg/ml for AMA1 and 78 µg/ml for MSP1₄₂. Antibody responses were not significantly different in the high dose versus low dose groups and did not further increase after third vaccination. <em>In vitro</em> growth inhibition was demonstrated and was closely correlated with anti-AMA1 antibody responses. A Phase 1b trial in malaria-exposed adults is being conducted.</p> <h3>Trial Registration</h3><p>Clinicaltrials.gov <a href="http://clinicaltrials.gov/ct2/show/NCT00889616">NCT00889616</a></p> </div

Participants flow sheet.

<p>Thirty (30) participants were enrolled, 24 participants completed vaccinations, and 24 completed follow-up to Day 360.</p

Local (injection site pain, tenderness, erythema, swelling, induration) and related systemic adverse events after first, second and third vaccinations in the low dose group.

<p>The x-axis shows the number of participants experiencing adverse events, with the highest severity event for each participant after each vaccination shown.</p

Antibody responses shown are the arithmetic mean of the FVO and 3D7 responses for each antigen for all volunteers who received all 3 vaccines and were not excluded per protocol (n = 23).

<p>Thicker lines show the geometric mean response; arrows indicate vaccinations. Mann-Whitney tests with Hodges-Lehmann confidence intervals were done to compare responses in low versus high dose groups and 2 weeks after 2^nd and 3^rd vaccinations (days 70 and 194); although the 160 µg group had slightly higher geometric means (AMA1 D70: Fold Change = 1.49 [95% CI 0.78, 2.92] p = 0.28; AMA1 D194: FC = 1.30 [0.68, 2.49] p = 0.61;MSP1 D70: FC = 1.18 [0.57,2.49] p = 0.70; MSP1 D 194: FC = 1.30 [0.85, 1.91]) p = 0.21; differences were not significant at the 0.05 level.</p