STRUCTURE-FUNCTION ANALYSIS OF PLACENTAL MALARIA-ASSOCIATED ADHESIVE INTERACTIONS – STRUCT-4-PAM

Each year, 200 to 400 million clinical cases of malaria are reported globally, causing around 500 000 deaths, largely in the sub-Saharan continent. Plasmodium falciparum and, to a much lesser extent, P. vivax are the main causes of disease and death from malaria. An important difference between P. falciparum and other human malaria parasites is the way in which P. falciparum modifies the surface of the erythrocytes so that asexual parasites can adhere to host cells. Adhesion of Plasmodium falciparum-infected erythrocytes (PEs) to chondroitin-4-sulfate (CSA) present in the placental intervillous vascular spaces has been linked to the severe disease outcome of pregnancy-associated malaria (PAM). After multiple pregnancies, women acquire protective antibodies that block CSA-binding and cross-react with geographically diverse placental isolates suggesting that surface molecule(s) expressed by PAM-infected erythrocytes have conserved epitopes and that developing a PAM vaccine may be possible. Recent evidence strongly suggests that VAR2CSA, a member of the variant P. falciparum Erythrocyte Membrane protein 1 (PfEMP1) family, has an important role in PAM and immunity. Although VAR2CSA is the main candidate for a pregnancy malaria vaccine, experimental evidence suggests that antigenic polymorphism, the lack of structural information and gaps in our understanding of placental sequestration may pose a challenge for vaccine and therapeutic development. Given that VAR2CSA is mostly composed of cysteine-rich domains, structural data are needed to identify conformational regions that are conserved between the different polymorphic forms of VAR2CSA and to localize the high-affinity CSA binding site. The overall objective of this project is to (i) provide new insights on the molecular mechanisms involved in placental sequestration and (ii) to determine the 3D structure of the full length extracellular region of VAR2CSA and its individual domains in order to (iii) characterize the high affinity CSA-binding site as well as conserved epitopes that can be used for vaccine and therapeutic strategies.
To achieve these goals, we will perform a functional study as well as a structural analysis of the full-length extracellular region of VAR2CSA by single particle cryo electron microscopy (cryo-EM) and of its individual domains by X-ray crystallography. We will also seek and characterize non-CSA mediated interactions which could contribute to placental sequestration using combined mass spectrometry analysis, adhesion studies, gene deletion and siRNA approaches. Furthermore, we will study the impact of VAR2CSA phosphorylation on CSA adhesion. Preliminary data of the two partners on the crystal structure of DBL domains, on the cryo-EM structure of the full complex as well as recent evidences that VAR2CSA phosphorylation plays a major role in adhesion support the feasibility of our project. This ambitious structure-function study will unravel details on the molecular interactions involved in placental sequestration providing a structural basis for understanding PEs sequestration in the placenta during pregnancy-associated malaria. This knowledge will be very helpful in the design of novel CSA-binding PfEMP1 antigens capable of inducing broad and potent neutralising antibodies to a wide variety of strains, but also will allow the virtual screening of large compound libraries, comprising several millions derivatives to identify small molecules that prevent and possibly reverse adhesion of P. falciparum-infected erythrocytes to CSA that could be considered for therapeutic strategies. This will be a crucial step towards the structure-based design of novel vaccine and therapeutic strategies to provide protection against negative outcomes of PAM.