Evidence for Globally Shared, Cross-Reacting Polymorphic Epitopes in the Pregnancy-Associated Malaria Vaccine Candidate VAR2CSA

Abstract

Pregnancy-associated malaria (PAM) is characterized by the placental sequestration of Plasmodium falciparum-infected erythrocytes (IEs) with the ability to bind to chondroitin sulfate A (CSA). VAR2CSA is a leading candidate for a pregnancy malaria vaccine, but its large size (ϳ350 kDa) and extensive polymorphism may pose a challenge to vaccine development. In this study, rabbits were immunized with individual VAR2CSA Duffy binding-like (DBL) domains expressed in Pichia pastoris or var2csa plasmid DNA and sera were screened on different CSA-binding parasite lines. Rabbit antibodies to three recombinant proteins (DBL1, DBL3, and DBL6) and four plasmid DNAs (DBL1, DBL3, DBL5, and DBL6) reacted with homologous FCR3-CSA IEs. By comparison, antibodies to the DBL4 domain were unable to react with native VAR2CSA protein unless it was first partially proteolyzed with trypsin or chymotrypsin. To investigate the antigenic relationship of geographically diverse CSA-binding isolates, rabbit immune sera were screened on four heterologous CSA-binding lines from different continental origins. Antibodies did not target conserved epitopes exposed in all VAR2CSA alleles; however, antisera to several DBL domains cross-reacted on parasite isolates that had polymorphic loops in common with the homologous immunogen. This study demonstrates that VAR2CSA contains common polymorphic epitopes that are shared between geographically diverse CSA-binding lines.

Identification of VAR2CSA Domain-Specific Inhibitory Antibodies of the Plasmodium falciparum Erythrocyte Membrane Protein 1 Using a Novel Flow Cytometry Assay

“…Sequences for FCR3/IT4, 3D7/NF54, and HB3 were already available (13). The DBL3X domain portion of Pf2004-CSA and Pf2006-CSA VAR2CSA was recently published (29).…”

“…P. falciparum parasites were grown in RPMI 1640 supplemented with 10% (vol/vol) heat-inactivated human serum, 25 mM/liter sodium bicarbonate, 0.125 mg/ml gentamicin (Invitrogen), and 5% hematocrit of human group O ϩ erythrocytes. CSA binding laboratory isolates, FCR3/IT4-CSA (24), 7G8-CSA, HB3-CSA allele A and HB3-CSA allele B, and NF54-CSA (29), and Pf2004-CSA and Pf2006-CSA (35), were maintained by periodic selection on CSA. For non-CSA binding controls, A4ultra (CD36 binder expressing the IT4var14 gene) was used.…”

“…Genotyping of parasites was done using MSP1/MSP2 primers according to published approaches (36). var gene transcription was assessed by quantitative reverse transcription-PCR (qRT-PCR) using universal primers as previously described (29). The DBL2X domain portion of var2csa was sequenced for the isolates Pf2004-CSA and Pf2006-CSA.…”

“…Initially, the homologous FCR3 VAR2CSA-expressing parasite line was evaluated by flow cytometry, followed by a panel of five CSA-binding isolates from diverse geographical locations. A CD36-binding control parasite line, A4ultra, which does not express VAR2CSA (29), was included as a specificity control. All VAR2CSA-expressing parasite lines bound with high affinity to CSA, except the low-affinity-CSA-binding 3D7/NF54 line.…”

“…Whereas full-length VAR2CSA recombinant proteins are highly immunogenic, the adhesion blocking activity they elicited was strain specific (19,27). Conversely, most single-domain antigens, with the exception of DBL4ε and DBL5ε, have elicited limited adhesion blocking activity (28,29,30,31,32). More recently, larger, multidomain VAR2CSA constructs incorporating the "core" binding region (DBL2 plus flanking regions) was shown to elicit adhesion blocking antibodies that were partially cross-inhibitory on placental isolates (23,33).…”