Induction of adhesion-inhibitory antibodies against placental Plasmodium falciparum parasites by using single domains of VAR2CSA

Abstract

In areas of endemicity pregnancy-associated malaria is an important cause of maternal anemia, stillbirth, and delivery of low-birth-weight children. The syndrome is precipitated by the accumulation of Plasmodium falciparum-infected erythrocytes in the placenta, mediated through an interaction between a parasite protein expressed on erythrocytes named variant surface antigen 2-chondroitin sulfate A (VAR2CSA) and CSA on syncytiotrophoblasts. VAR2CSA is a large polymorphic protein consisting of six Duffy binding-like (DBL), domains and with current constraints on recombinant protein production it is not possible to produce entire VAR2CSA recombinant proteins. Furthermore, the presence of polymorphisms has raised the question of whether it is feasible to define VAR2CSA antigens eliciting broadly protective antibodies. Thus, the challenge for vaccine development is to define smaller parts of the molecule which induce antibodies that inhibit CSA binding of different parasite strains. In this study, we produced a large panel of VAR2CSA proteins and raised antibodies against these antigens. We show that antibodies against the DBL4 domain effectively inhibit parasite binding. As the inhibition was not limited to homologous parasite strains, it seems feasible to base a protective malaria vaccine on a single VAR2CSA DBL domain.

FIG. 1.

Functional capacity of the anti-VAR2CSA antibodies. (A) Reactivity measured by flow cytometry of antibodies against native VAR2CSA expressed on the surface of erythrocytes infected with an FCR3 parasite line selected for CSA binding. Each column shows the reactivity in rats immunized with the indicated VAR2CSA constructs based on FCR3 DNA. Groups of three rats were immunized with the same constructs, and 10 μl pooled rat serum was used to stain 2 × 10⁵ IE. Samples without primary antibody (blank), prebleed sera, and DBL3g-VAR1 and DBL2b-VAR5 immunizations were used as controls. Bars represent the fluorescein isothiocyanate mean fluorescence intensity from 5000 IE. (B) Ability of immune serum to inhibit binding to CSA by VAR2CSA-expressing FCR3-IE. A high-throughput plate assay was used due to the number of serum samples. Fifteen microliters of pooled rat serum was used in a total volume of 120 μl containing 2 × 10⁵ IE. Statistically significant adhesion inhibitory capacity compared to sera prior to immunization is represented by an asterisk (P < 0.01, t test). Bars represent the mean percent binding compared to binding without serum. Error bars are standard deviations of triplicate measurements. (C) Consecutive bleeds of rats immunized with DBL4 at 3-week intervals. Bars represent the mean proportion of binding compared to binding without serum. Error bars are standard deviations of triplicate measurements. The circles indicate the proportion of mean fluorescence intensity values compared to a negative control serum. All binding assays were performed multiple times with similar results.

FIG. 2.

Adhesion-inhibitory antibodies are cross-reactive (A) Ability of antiserum to inhibit the CSA binding of VAR2CSA-expressing erythrocytes infected with FCR3, 7G8, or PL1 parasites. A pool of preimmune and a pool of control-immunized sera were used as controls. (B) Inhibition of adhesion by pools of Tanzanian human serum and Danish nonimmune serum (DK) at different concentrations. All bars represent the mean percentage of binding compared to binding without serum. Error bars are standard deviations of triplicate measurements. The assays were performed multiple times with similar results.

FIG. 3.

Inhibition of CSA binding using purified IgG from DBL4-VAR2CSA and DBL3g-VAR1 specific sera. All assays were performed using FCR3-IE expressing VAR2CSA in a petri dish assay. Bars are mean numbers of IE per mm². Error bars are standard deviations from four replicates. The assay was performed multiple times with similar results.