Malaria during pregnancy remains a major health problem in Plasmodium falciparum endemic areas. Parasite-infected red blood cells sequester in the placenta through interaction between parasite-expressed protein VAR2CSA and the glycosaminoglycan chondroitin sulfate A (CS) abundantly present in the intervillous space. Placental malaria can have severe consequences for both mother and child by causing maternal anemia, low birth weight and stillbirth. Several VAR2CSA-based vaccines have been developed and clinically tested but they have failed to induce an antibody response that effectively inhibits placental adhesion of different genetic variants of VAR2CSA. The interaction between VAR2CSA and CS represents a unique case of an evolution-driven high-affinity interaction between a protein and an oncofetal carbohydrate. Here, we report cryo-EM structures of the VAR2CSA ectodomain up to 3.1 Å resolution revealing an overall V-shaped architecture and a complex domain organization. Notably, the surface displays a single significantly electropositive patch, compatible with binding of negatively charged CS. Using molecular docking and molecular dynamics simulations as well as comparative hydroxyl radical protein foot-printing of VAR2CSA in complex with placental CS, we identify the CS-binding groove, intersecting with the positively charged patch of the central VAR2CSA structure. We identify distinctive conserved structural features upholding the macro-molecular domain complex and CS binding capacity of VAR2CSA as well as divergent elements possibly allowing immune escape at or near the CS binding site. These observations enable rational design of second-generation placental malaria vaccines eliciting broadly VAR2CSA-reactive antibodies and novel cancer therapies.

Severe malaria is caused by the adhesion of Plasmodium falciparum-infected erythrocytes to host receptors on vascular endothelium. Specifically, binding of the parasite variant surface antigen domain CIDRa1 to host endothelial protein C receptor (EPCR) is strongly associated with severe disease. In malaria-endemic regions, children quickly develop immunity against severe malaria, indicative of the development of an effective immune response against CIDRa1 domains. Indeed, serum IgG from naturally immune individuals exhibited reactivity against a diverse panel of CIDRa1 variants. In an effort to analyze CIDRa1 antibody responses at a monoclonal level, we isolated CIDRa1-specific memory B cells from malaria-experienced individuals living in Uganda and expressed the corresponding monoclonal antibodies (mAbs). Our approach has yielded anti-CIDRa1 mAbs ranging in breadth from variant-specific to reactive with the full spectrum of CIDRa1 domain variants, irrespective of their extensive sequence diversity. The broadest antibodies can be separated into two categories, those that bind outside the EPCR binding site and display exceptional breadth with modest inhibition of EPCR binding and those that target the EPCR binding site and show potent EPCR-binding inhibition. Two EPCR-binding site-targeting mAbs isolated from two different donors showed signs of convergent evolution. Our results demonstrate that natural P. falciparum infection can induce a broad and inhibitory antibody response against CIDRa1. Current experiments are aimed at understanding the structural basis of antibody-mediated inhibition of CIDRa1-EPCR binding to inform the design of a vaccine that protects against severe malaria.