Immunogenicity of Duffy binding-like domains that bind chondroitin sulfate A and protection against pregnancy-associated malaria

Abstract

Sequestration of Plasmodium falciparum-infected erythrocytes in the placenta is implicated in pathological outcomes of pregnancy-associated malaria (PAM). P. falciparum isolates that sequester in the placenta primarily bind chondroitin sulfate A (CSA). Following exposure to malaria during pregnancy, women in areas of endemicity develop immunity, and so multigravid women are less susceptible to PAM than primigravidae. Protective immunity to PAM is associated with the development of antibodies that recognize diverse CSA-binding, placental P. falciparum isolates. The epitopes recognized by such protective antibodies have not been identified but are likely to lie in conserved Duffy binding-like (DBL) domains, encoded by var genes, that bind CSA. Immunization of mice with the CSA-binding DBL3gamma domain encoded by var1CSA elicits cross-reactive antibodies that recognize diverse CSA-binding P. falciparum isolates and block their binding to placental cryosections under flow. However, CSA-binding isolates primarily express var2CSA, which does not encode any DBLgamma domains. Here, we demonstrate that antibodies raised against DBL3gamma encoded by var1CSA cross-react with one of the CSA-binding domains, DBL3X, encoded by var2CSA. This explains the paradoxical observation made here and earlier that anti-rDBL3gamma sera recognize CSA-binding isolates and provides evidence for the presence of conserved, cross-reactive epitopes in diverse CSA-binding DBL domains. Such cross-reactive epitopes within CSA-binding DBL domains can form the basis for a vaccine that provides protection against PAM.

FIG. 1.

Characterization of refolded and purified DBL3γ domain (rDBL3γ) of FCR3 var1CSA. (A) Mobility of rDBLγ as determined by SDS-PAGE. Refolded rDBLγ has lower mobility in SDS-PAGE after reduction with dithiothreitol (+DTT), indicating the presence of disulfide linkages. Molecular mass markers (M) in kDa are shown. (B) Reverse-phase chromatography profile of rDBL3γ. Refolded DBL3γ elutes as a single, symmetric peak upon reverse-phase chromatography on a C₈ column, indicating that it is conformationally homogenous. AU, absorbance units (280 nm).

FIG. 2.

Binding of rDBL3γ to CSA. Binding of rDBL3γ to Bio-CSA, Bio-CSB, Bio-CSC, and Bio-HA immobilized on streptavidin-coated microwells was detected using a mouse monoclonal antibody against penta-His. Recombinant PvRII, the binding domain of PvDBP, and recombinant gC1qR/HABP1, which binds HA, were used as control ligands. rDBL3γ binds CSA but not CSB, CSC, or HA. gC1qR/HABP1 binds HA but not CSA, CSB, or CSC. PvRII does not bind any of the receptors tested. OD, optical density.

FIG. 3.

Recognition of diverse P. falciparum isolates by mouse sera raised against rDBL3γ of FCR3 var1CSA by use of flow cytometry. Mouse sera raised against rDBL3γ were tested for recognition of P. falciparum FCR3CD36 (A) and T996 (B), which do not bind CSA, and of FCR3CSA (C) and 193 (D), which bind CSA. Gray areas represent signals from preimmune sera.

FIG. 4.

Recognition of surfaces of P. falciparum IEs by anti-rDBL3γ mouse sera using L-IFA. Mouse sera raised against rDBL3γ of FCR3 var1CSA react with the IE surface of P. falciparum laboratory strain FCR3CSA and that of placental isolate 193CSA, which bind CSA, but not with that of P. falciparum laboratory strain FCR3CD36 or that of peripheral field isolate JDP8, which do not bind CSA. Parasites were stained with DNA-intercalating dye DAPI (blue) to identify IEs and with anti-rDBL3γ mouse sera followed by Alexafluor 488-conjugated chicken anti-mouse IgG (green).