Pregnancy-associated malaria (PAM) is a serious consequence of sequestration of Plasmodium falciparum-parasitized erythrocytes (PE) in the placenta through adhesion to chondroitin sulfate A (CSA) present on placental proteoglycans. Recent work implicates var2CSA, a member of the PfEMP1 family, as the mediator of placental sequestration and as a key target for PAM vaccine development. Var2CSA is a 350 kDa transmembrane protein, whose extracellular region includes six Duffy-binding-like (DBL) domains. Due to its size and high cysteine content, the full-length var2CSA extracellular region has not hitherto been expressed in heterologous systems, thus limiting investigations to individual recombinant domains. Here we report for the first time the expression of the full-length var2CSA extracellular region (domains DBL1X to DBL6ε) from the 3D7 parasite strain using the human embryonic kidney 293 cell line. We show that the recombinant extracellular var2CSA region is correctly folded and that, unlike the individual DBL domains, it binds with high affinity and specificity to CSA (K D ¼ 61 nM) and efficiently inhibits PE from binding to CSA. Structural characterization by analytical ultracentrifugation and small-angle x-ray scattering reveals a compact organization of the full-length protein, most likely governed by specific interdomain interactions, rather than an extended structure. Collectively, these data suggest that a high-affinity, CSA-specific binding site is formed by the higher-order structure of the var2CSA extracellular region. These results have important consequences for the development of an effective vaccine and therapeutic inhibitors. malaria | pregnancy | plasmodium | chondroitin | structure

The human malaria parasite Plasmodium falciparum can cause infected red blood cells (iRBC) to form rosettes with uninfected RBC, a phenotype associated with severe malaria. Rosetting is mediated by a subset of the Plasmodium falciparum membrane protein 1 (PfEMP1) variant adhesins expressed on the infected host-cell surface. Heparin and other sulfated oligosaccharides, however, can disrupt rosettes, suggesting that therapeutic approaches to this form of severe malaria are feasible. We present a structural and functional study of the N-terminal domain of PfEMP1 from the VarO variant comprising the N-terminal segment (NTS) and the first DBL domain (DBL1α 1 ), which is directly implicated in rosetting. We demonstrate that NTS-DBL1α 1 -VarO binds to RBC and that heparin inhibits this interaction in a dose-dependent manner, thus mimicking heparin-mediated rosette disruption. We have determined the crystal structure of NTS-DBL1α 1 , showing that NTS, previously thought to be a structurally independent component of PfEMP1, forms an integral part of the DBL1α domain. Using mutagenesis and docking studies, we have located the heparin-binding site, which includes NTS. NTS, unique to the DBL α-class domain, is thus an intrinsic structural and functional component of the N-terminal VarO domain. The specific interaction observed with heparin opens the way for developing antirosetting therapeutic strategies. Severe Plasmodium falciparum malaria is frequently associated with infected red blood cells (iRBC) forming rosettes with uninfected RBC (1-3). Although the relationship between rosetting and malaria pathology is not thoroughly understood, enhanced invasion of RBC (4) and microvascular obstruction caused by high rosette densities (5) are probably major contributing factors. Rosetting is mediated by a subset of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesins (2, 6, 7), a protein family involved in falciparum cyto-adhesion. PfEMP1 adhesins are expressed on the surface of the iRBC during the trophozoite and schizont phases and are clustered in knob-like structures, where they interact with diverse host receptors (8).PfEMP1 has a large N-terminal extracellular region comprising an N-terminal segment (NTS), and several Duffy binding-like (DBL) domains and cysteine-rich interdomain regions (CIDR) (8-10). DBL and CIDR domains can be assigned to a small number of sequence classes. The arrangement of these domains is modular, but most variants begin with NTS followed by DBL1α. Indeed, α-class DBL domains occur only at the N-terminal position of PfEMP1 (11). Studies on laboratory parasite strains have shown that the DBL1α 1 subclass is directly implicated in rosette formation (2, 7). In spite of a diversity of rosetting phenotypes, which interact with a variety of receptors, rosettes are frequently disrupted by sulfated glycosaminoglycans (GAG), such as heparin (6,(12)(13)(14)(15)(16). Heparin efficiently disrupts rosettes but may enhance CD36-dependent adhesion of iRBC to microvascular endothelium cells (17). The ...

Var2CSA, a key molecule linked with pregnancy-associated malaria (PAM), causes sequestration of Plasmodium falciparum infected erythrocytes (PEs) in the placenta by adhesion to chondroitin sulfate A (CSA). Var2CSA possesses a 300 kDa extracellular region composed of six Duffy-binding like (DBL) domains and a cysteine-rich interdomain region (CIDRpam) module. Although initial studies implicated several individual var2CSA DBL domains as important for adhesion of PEs to CSA, new studies revealed that these individual domains lack both the affinity and specificity displayed by the full-length extracellular region. Indeed, recent evidence suggests the presence of a single CSA-binding site formed by a higher-order domain organization rather than several independent binding sites located on the different domains. Here, we search for the minimal binding region within var2CSA that maintains high affinity and specificity for CSA binding, a characteristic feature of the full-length extracellular region. Accordingly, truncated recombinant var2CSA proteins comprising different domain combinations were expressed and their binding characteristics assessed against different sulfated glycosaminoglycans (GAGs). Our results indicate that the smallest region within var2CSA with similar binding properties to those of the full-length var2CSA is DBL1X-3X. We also demonstrate that inhibitory antibodies raised in rabbit against the full-length DBL1X-6ε target principally DBL3X and, to a lesser extent, DBL5ε. Taken together, our results indicate that efforts should focus on the DBL1X-3X region for developing vaccine and therapeutic strategies aimed at combating PAM.

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Over 50 million women are exposed to the risk of malaria during pregnancy every year. Malaria during pregnancy is a leading global cause of maternal morbidity and adverse pregnancy outcomes. Adhesion of Plasmodium falciparum-infected erythrocytes to placental chondroitin-4-sulfate (CSA) has been linked to the severe disease outcome of placental malaria. Accumulated evidence strongly supports VAR2CSA as the leading placental malaria vaccine candidate. Recombinant proteins encompassing the VAR2CSA high affinity CSA binding site have been generated, and their activity as immunogens that elicit functional (inhibitory) and cross-reactive antibodies against CSA-binding parasites assessed. The expression of His-tagged proteins was compared in four different expression systems and their capacity to bind specifically to CSA was analyzed. CHO cells and E. coli SHuffle cells were the two expression systems able to express some of the recombinant proteins in reasonable amounts. Larger analytical scale production of DBL1x-2× (3D7) and DBL3x-4ε (FCR3) best expressed in CHO and E. coli SHuffle cells were performed. Purified proteins were administered to rats either alone or adjuvanted with human approved adjuvants. Analysis of the functionality and cross-reactivity of the induced antibodies allowed us to down-select the DBL1x-2(3D7) expressed in E. coli SHuffle cells as the best antigen to be transitioned to further clinical development in order to protect future pregnant women living in malaria endemic areas against the severe clinical outcomes of placental malaria.

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The human malaria parasite, Plasmodium falciparum, is able to evade spleen-mediated clearing from blood stream by sequestering in peripheral organs. This is due to the adhesive properties conferred by the P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) family exported by the parasite to the surface of infected erythrocytes. Expression of the VAR2CSA variant of PfEMP1 leads to pregnancy-associated malaria, which occurs when infected erythrocytes massively sequester in the placenta by binding to low-sulfated Chondroitin Sulfate A (CSA) present in the intervillous spaces. VAR2CSA is a 350 kDa protein that carries six Duffy-Binding Like (DBL) domains, one Cysteine-rich Inter-Domain Regions (CIDR) and several inter-domain regions. In the present paper, we report for the first time the crystal structure at 2.9 Å of a VAR2CSA double domain, DBL3X-DBL4ε, from the FCR3 strain. DBL3X and DBL4ε share a large contact interface formed by residues that are invariant or highly conserved in VAR2CSA variants, which suggests that these two central DBL domains (DBL3X-DBL4ε) contribute significantly to the structuring of the functional VAR2CSA extracellular region. We have also examined the antigenicity of peptides corresponding to exposed loop regions of the DBL4ε structure.