VAR2CSA Domain-Specific Analysis of Naturally Acquired Functional Antibodies to Plasmodium falciparum Placental Malaria

Abstract

Background: Placental malaria is caused by Plasmodium falciparum-infected erythrocytes (IEs) that surface-express VAR2CSA and bind chondroitin sulfate A. The inflammatory response to placenta-sequestered parasites is associated with poor pregnancy outcomes, and protection may be mediated in part by VAR2CSA antibodies that block placental IE adhesion.

Methods: In this study, we used a new approach to assess VAR2CSA domains for functional epitopes recognized by naturally acquired antibodies. Antigen-specific immunoglobulin (Ig) G targeting Duffy binding-like (DBL) domains from different alleles were sequentially purified from plasma pooled from multigravid women and then characterized using enzyme-linked immunosorbent assay, flow cytometry, and antiadhesion assays.

Results: Different DBL domain-specific IgGs could react to homologous as well as heterologous antigens and parasites, suggesting that conserved epitopes are shared between allelic variants. Homologous blocking of IE binding was observed with ID1-DBL2-ID2a-, DBL4-, and DBL5-specific IgG (range, 42%-75%), whereas partial cross-inhibition activity was observed with purified IgG specific to ID1-DBL2-ID2a and DBL4 antigens. Plasma retained broadly neutralizing activity after complete depletion of these VAR2CSA specificities.

Conclusions: Broadly neutralizing antibodies of multigravidae are not depleted on VAR2CSA recombinant antigens, and hence development of VAR2CSA vaccines based on a single construct and variant might induce antibodies with limited broadly neutralizing activity.

Keywords: DBL domains; VAR2CSA; functional antibody; malaria; multigravidae; pregnancy; vaccine.

Figure 1.

VAR2CSA antigenic constructs and specific antibodies in the pool of plasma from multigravid women (MG pool). A, Recombinant ID1-DBL2X-ID2a (no color), DBL3X (gray), DBL4ε (blue), and DBL5ε (red) proteins were constructed for VAR2CSA FCR3 variant (no pattern) and maternal isolates M466 (), M711 (), and M1010 (). Boundaries of each construct are indicated relative to FCR3 VAR2CSA (GenBank accession No. KU665624). The same residue limit was used for all DBL4ε and DBL5ε constructs [25]. Different boundaries were used for ID1-DBL2X-ID2a-1010 and ID1-DBL2X-ID2a-711 (GenBank for accession No. KU665625), and ID1-DBL2X-ID2a-FCR3 (GenBank for accession No. KU665626) recombinant constructs. B, The MG pool was screened by means of enzyme-linked immunosorbent assay (ELISA) against the VAR2CSA DBL domain recombinant and AMA1 (used as non-VAR2CSA antigen control) antigens. Data are presented as level of antibody binding to the recombinant proteins, measured by optical density (OD). Bars represent means and standard deviations of duplicate wells. Abbreviations: ATS, acidic terminal sequence; TM, transmembrane.

Figure 2.

Antigenic reactivity of the purified antigen-specific immunoglobulin (Ig) G. Purified naturally acquired antibody specific to each VAR2CSA Duffy binding–like (DBL) domain and AMA1 recombinant protein were screened by enzyme-linked immunosorbent assay (ELISA) against the panel of antigens to analyze their specificity and cross-reactivity. A–K, Antigen binding profile of each specific purified IgG following the order of depletion. Data are presented as level of antibody binding to the recombinant proteins, measured by optical density (OD). Bars represent means and standard deviations of duplicate wells.

Figure 3.

Recognition of the native VAR2CSA by the purified antigen-specific immunoglobulin (Ig) G. In flow cytometry, the ability of the purified VAR2CSA Duffy binding–like (DBL) domain-specific antibody to label the native VAR2CSA expressed on the surface of Plasmodium falciparum–infected erythrocytes by homologous (blue) and heterologous (red) parasites, was assessed on isolates FCR3 (A), M466 (B), M1010 (C), and M711 (D). Median fluorescent intensity (MFI), is shown, as well as the proportion of labeled infected erythrocytes relative to the total cells.

Figure 4.

Functional activity of the pool of plasma from multigravid women (MG pool). Inhibitory profile of the MG pool of plasma as well as different concentration of total immunoglobulin (Ig) G purified from the same pool were assessed on isolates M711 (A), M1010 (B), M736 (C) and FCR3 (D). Proportions of inhibited infected erythrocytes relative to the well without test sample are presented. Bars represent means and standard deviations of duplicate wells.

Figure 5.

Functional activity of VAR2CSA antigen-specific purified antibodies on different strains of Plasmodium falciparum. Blocking activity of the naturally acquired antibody specific to VAR2CSA Duffy binding–like (DBL) domain against the chondroitin sulfate A (CSA)–binding parasites was assessed. Purified antibodies specific to ID1-DBL2X-ID2a (A), DBL3 (B), DBL4 (C), and DBL5 (D) domain constructs from FCR3, M1010, and M711 variants were tested on the corresponding isolates, to analyze their homologous (blue histogram) and heterologous (red histogram) inhibition activity. Proportion of inhibited P. falciparum–infected erythrocytes relative to the well without test sample are presented. Bars represent means and standard deviations of duplicate wells.

Figure 6.

Characteristics of the depleted pool of plasma from multigravid women (MG pool). A, The MG pool and depleted MG pool were characterized for their inhibitory activity against laboratory strains (FCR3 and CS2) and 4 maternal isolates (M711, M736, M918, and M1010). B, Levels of antibody specific to merozoite surface protein 1 (MSP1) antigen were compared in both plasma pools. C, Their capacity to label the native VAR2CSA expressed on the surface of Plasmodium falciparum–infected erythrocytes from 3 isolates are reported, shown as median fluorescent intensity (MFI) and the proportion of labeled infected erythrocytes relative to the total cells. Bars represent means and standard deviations of duplicate wells.