Identification of VAR2CSA domain-specific inhibitory antibodies of the Plasmodium falciparum erythrocyte membrane protein 1 using a novel flow cytometry assay

Abstract

VAR2CSA, a member of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family, is a leading candidate for use in vaccines to protect first-time mothers from placental malaria (PM). VAR2CSA, which is comprised of a series of six Duffy binding-like (DBL) domains, binds chondroitin sulfate A (CSA) on placental syncytiotrophoblast. Several recombinant DBL domains have been shown to bind CSA. In order to identify and develop recombinant proteins suitable for clinical development, DBL2X and DBL3X, as well as their respective third subdomain (S3) from the FCR3 parasite clone, were expressed in Escherichia coli, refolded, and purified. All but DBL3X-S3 recombinant proteins bound to CSA expressed on Chinese hamster ovary (CHO)-K1 cells but not to CHO-pgsA745 cells, which are CSA negative as determined by flow cytometry. All but DBL3X-S3 bound to CSA on chondroitin sulfate proteoglycan (CSPG) as determined by surface plasmon resonance (SPR) analysis. Purified IgG from rats and rabbits immunized with these four recombinant proteins bound homologous and some heterologous parasite-infected erythrocytes (IE). Using a novel flow cytometry inhibition-of-binding assay (flow-IBA), antibodies against DBL3X-S3 inhibited 35% and 45% of IE binding to CSA on CHO-K1 cells compared to results for soluble CSA (sCSA) and purified multigravida (MG) IgG, respectively, from areas in Tanzania to which malaria is endemic. Antibodies generated against the other domains provided little or no inhibition of IE binding to CSA on CHO-K1 cells as determined by the flow cytometry inhibition-of-binding assay. These results demonstrate for the first time the ability to identify antibodies to VAR2CSA DBL domains and subdomains capable of inhibiting VAR2CSA parasite-IE binding to CSA by flow cytometry. The flow cytometry inhibition-of-binding assay was robust and provided an accurate, reproducible, and reliable means to identify blocking of IE binding to CSA and promises to be significant in the development of a vaccine to protect pregnant women.

Fig 1

Recombinant DBL2X and DBL3X and their respective subdomain 3 constructs. (A) SynEcA4-DBL2-3X gene, showing DBL2X, interdomain (ID) region, and DBL3X (GenBank accession no. AAQ73926). Below are the DBL2X fragment (residues 543 to 970), DBL2X-S3 fragment (residues 758 to 970), DBL3X fragment (residues 1220 to 1580), and DBL3X-S3 fragment (residues 1445 to 1580). All four constructs each contained a C-terminal 6×His tag. (B) Coomassie blue-stained DBL2X, DBL2X-S3, DBL3X, and DBL3X-S3 proteins under nonreduced and reduced conditions.

Fig 2

Binding interactions of DBL2X, DBL2X-S3, DBL3X, and DBL3X-S3 with CSA on CHO cells by flow cytometry. Histograms and median fluorescence intensities of the DBL2X, DBL2X-S3, DBL3X, and DBL3X-S3 recombinant proteins binding to CHO-pgsA745 cells (A) or CHO-K1 cells (B) are shown. Median fluorescence intensities were used to report the differences in binding between the two cell lines.

Fig 3

SPR kinetics analysis of DBL domains and subdomains on immobilized CSPG. Kinetics fits of DBL2X (A), DBL3X (B), DBL2X-S3 (C), or DBL3X-S3 (D) binding to CSA on GSPG are shown.

Fig 4

Flow-IBA data of homologous FCR3csa parasite-IE binding to CHO cells. (A) VFSE-stained CHO-K1 (CSA+) cells combined with CFSE-stained IE; (B) VFSE-stained CHO-pgsA745 (CSA-) cells combined with CFSE-stained IE.

Fig 5

Inhibition of FCR3csa parasite-IE binding to CSA on CHO-K1 cells by purified preabsorbed rabbit DBL domains and subdomains IgG (antibodies). Percent inhibition of FCR3csa parasite-IE binding to CSA on CHO-K1 cells using various IgG antibodies and soluble CSA. Each dot represents an independent flow cytometry inhibition-of-IE-binding experiment. Diamonds represent mean values, and error bars represent standard errors. DBL3X-S3 antibodies significantly inhibited IE binding to CSA compared to that with adjuvant control antibodies, by 35% (P < 0.0181 by Student's t test), while MG-IgG inhibited IE binding to CSA by 75% (P < 0.0210 by Student's t test).

Fig 6

(A and B) Western blot of recombinant DBL2X, DBL2X-S3, DBL3X, and DBL3X-S3 proteins probed with multigravida (MG) IgG (A) or domain-specific pooled rabbit IgG (B). (C) ELISA of recombinant DBL2X and DBL3X with serial dilutions of pooled MG-IgG. The control columns contain DBL2X- and DBL3X-coated wells without MG IgG but stained with secondary antibodies only.