Summary of PvDBP polymorphism, inhibitory epitopes, and residues impacting DARC binding.

<p>The sequence of the solved Sal1strain PvDBP variant crystal structure <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420-Batchelor1" target="_blank">[13]</a> is shown. Polymorphic amino acids from 129 PvDBP sequences <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420-Gosi1" target="_blank">[28]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420-Xainli1" target="_blank">[29]</a> are listed below the Sal1 sequence. Alpha helices in the PvDBP crystal structure <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420-Batchelor1" target="_blank">[13]</a> are indicated by “h” and labeled helix 1a to helix 9 according to convention <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420-Hodder1" target="_blank">[71]</a>. Circles above the line-up indicate important residues. N-glycosylation sites are numbered according to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat-1003420-t001" target="_blank">Table 1</a> and colored green (wild type), blue (STBP glycan), orange (P1 and Max), and red (Max). Dimer interface <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420-Batchelor1" target="_blank">[13]</a> – black circles; polymorphism – light grey (rare, <10% of sequences), dark grey (>10% of sequences); mutations that effect DARC binding from this study and others <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420-Batchelor1" target="_blank">[13]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420-Hans1" target="_blank">[17]</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420-Bolton1" target="_blank">[19]</a>, are colored blue (no effect), yellow with black shadowing (minor), orange (moderate), red (major), and black, differences between studies (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420.s007" target="_blank">Table S1</a>); linear epitopes targeted by inhibitory antibodies <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420-Chootong1" target="_blank">[20]</a> – black or grey shading (low inhibitory), blue shading (medium inhibitory), red shading (high inhibitory).</p

Glycan Masking of <i>Plasmodium vivax</i> Duffy Binding Protein for Probing Protein Binding Function and Vaccine Development

<div><p>Glycan masking is an emerging vaccine design strategy to focus antibody responses to specific epitopes, but it has mostly been evaluated on the already heavily glycosylated HIV gp120 envelope glycoprotein. Here this approach was used to investigate the binding interaction of <i>Plasmodium vivax</i> Duffy Binding Protein (PvDBP) and the Duffy Antigen Receptor for Chemokines (DARC) and to evaluate if glycan-masked PvDBPII immunogens would focus the antibody response on key interaction surfaces. Four variants of PVDBPII were generated and probed for function and immunogenicity. Whereas two PvDBPII glycosylation variants with increased glycan surface coverage distant from predicted interaction sites had equivalent binding activity to wild-type protein, one of them elicited slightly better DARC-binding-inhibitory activity than wild-type immunogen. Conversely, the addition of an N-glycosylation site adjacent to a predicted PvDBP interaction site both abolished its interaction with DARC and resulted in weaker inhibitory antibody responses. PvDBP is composed of three subdomains and is thought to function as a dimer; a meta-analysis of published PvDBP mutants and the new DBPII glycosylation variants indicates that critical DARC binding residues are concentrated at the dimer interface and along a relatively flat surface spanning portions of two subdomains. Our findings suggest that DARC-binding-inhibitory antibody epitope(s) lie close to the predicted DARC interaction site, and that addition of N-glycan sites distant from this site may augment inhibitory antibodies. Thus, glycan resurfacing is an attractive and feasible tool to investigate protein structure-function, and glycan-masked PvDBPII immunogens might contribute to <i>P. vivax</i> vaccine development.</p></div

Expression and purification of surface re-engineered PvDBPII recombinant proteins containing additional N-linked glycan residues.

<p>(A) 2 µg of PvDBPII wild type and DBPII glycosylation variants were run on SDS-PAGE gel and stained with GelCode Blue reagent. A ladder effect is seen consistent with increasing number of N-glycosylation sites present in the STBP glycan, P1 and Max hyperglycosylated variants compared to PvDBPII wild-type. (B) Western blot of 1 µg of PvDBPII and DBPII glycosylation variants probed with anti-His antibody. (C) PvDBPII wild type and DBPII glycosylation variants were either untreated (−) or digested (+) with N-glycosidase PNGaseF, run on SDS-PAGE gel and probed with anti-His antibody, except for Max variant which was probed with anti-PvDBPII serum. The P1 lanes were run separate from the other samples. Molecular mass is shown on the left.</p

Functional characterization of hyperglycosylated PvDBPII variants by COS-7 cell-RBC binding assay.

<p>PvDBPII wild type (A), STBP glycan mutant (B), P1 (C) and Max (D) DBPII glycosylation variants were expressed as a GFP fusion protein in COS-7 cells and incubated with 0.5% hematocrit erythrocytes. GFP-positive transfected cells with five or more RBC (rosettes) were considered positive for binding. The histogram shows the percentage surface labeling when GFP-positive transfected cells were probed with pre-immune (blue) or anti-PvDBPII plasma (red). The gating strategy for GFP-positive and anti-PvDBPII positive is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420.s003" target="_blank">Figure S3</a>. The percentage signifies GFP-positive cells that are labeled with immune plasma, indicating surface expression of PvDBPII and DBPII glycosylation variants. The shift in mean fluorescence intensities between pre-immune and immune plasma is indicated as MFI.</p

Effect of DARC phenotype on antibody binding inhibitory activity.

<p>COS-7 cells expressing PvDBPII as a GFP fusion protein were incubated with immune plasma and then RBC expressing the FyA (A) or FyB (B) Duffy blood group antigen were added. The inset shows that FyA RBCs (A) gave smaller rosettes than FyB RBCs (B) in the COS-7 cell–RBC binding assay. Statistical testing and p values as explained in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat-1003420-g006" target="_blank">Figure 6B</a>.</p

PvDBP – DARC interaction.

<p>DARC, a seven transmembrane chemokine receptor on erythrocytes, has been shown to bind to PvDBP <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420-Horuk1" target="_blank">[16]</a>. Binding has been mapped to the N-terminal 65 amino acids (illustrated as a red tail). The COS-7-RBC cytoadherance assay is based upon a multivalent interaction between PvDBPII present on surface of COS-7 cells and DARC expressed by RBC. In the yeast-PvDBPII display assay, PvDBPII present on the yeast surface interacts with dimeric recombinant DARC-Fc recombinant protein (N-terminal 65 mer region). The two assays offer different platforms to reveal inhibitory effects of antibodies using a potentially higher affinity, multimer-multimer interaction (COS-7 format) or a lower affinity multimer-dimer interaction (yeast display). Bottom of figure, polymorphisms in DARC that generate FyA/FyA^null genotype are associated with half of the number of surface DARC protein and lower <i>P. vivax</i> infection <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420-Zimmerman1" target="_blank">[4]</a>.</p

Immunogenic characterization of PvDBPII and its hyperglycosylated variants.

<p>(A) Schematic representation of immunization scheme for PvDBPII wild type and DBPII glycosylation variants. Group 1 mice received PvDBPII wild type generated in <i>E. coli</i>, Group 2 mice received PvDBPII wild type generated in HEK-293F cells. Mice in groups 3–6 received a combination of DNA and homologous protein; group 3, PvDBPII wildtype; group 4, STBP glycan; group 5, P1; group 6, Max hyperglycosylated variant. (B) The ELISA end-point antibody titers are shown for each of the immunization groups after the third DNA boost, the first protein immunization, and the second protein boost (final bleed). (C) The inhibitory activity of vaccine plasma in a COS-7-RBC binding assay. COS-7 cells expressing PvDBPII wild type protein were pre-incubated with varying dilutions of PvDBPII wild type and DBPII glycosylation variant immune plasma obtained after the third DNA boost or the second protein immunization (final bleed). The values are an average of duplicate experiments.</p

Design of surface re-engineered PvDBPII recombinant proteins containing additional N-linked glycan residues.

<p>(A–B) Subdomains and DARC binding models. Subdomain 1(red), subdomain 2 (blue), and subdomain 3 (green). Critical binding residues for model 1 are colored light blue and for model 2 are colored yellow. (C–D) Fractional Shannon entropy values <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420-Shannon1" target="_blank">[70]</a> from 0.000 (white) to 0.243 (red) for sequence polymorphism over the PvDBPII surface as compared with maximally entropic distribution over all amino acids. (E–F) Epitopes recognized by blocking antibodies <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420-Chootong1" target="_blank">[20]</a>; black (low inhibitory), blue (medium inhibitory), red (high inhibitory). (G–H) Meta-analysis of mutations that reduce or do not affect the PvDBP-DARC interaction: blue residues (no effect); yellow residues (minor); orange residues (moderate); red residues (major); black residues, differences between studies (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420.s007" target="_blank">Table S1</a>). (I–J) Location of engineered N-glycosylation sites modeled as high mannose forms; white (wild type), cyan (STBP glycan), Orange (P1 and Max), red (Max). All images modeled in PyMol on the PvDBPII dimer structure <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420-Batchelor1" target="_blank">[13]</a>, viewing opposite ends of the dimeric two-fold axis for subpanels I and II; missing density in the crystal structure has been left missing, though it contains polymorphic, inhibitory, antibody recognition and glycan-bearing sites. PvDBPII monomers are colored yellow and green in panels E, F, I, and J.</p

PvDBPII recombinant proteins.

<p>PvDBPII recombinant proteins.</p