Stéphane Gangnard

Full-length extracellular region of the var2CSA variant of PfEMP1 is required for specific, high-affinity binding to CSA

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 (KD = 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.

Structure of a Plasmodium falciparum PfEMP1 rosetting domain reveals a role for the N-terminal segment in heparin-mediated rosette inhibition

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 (DBL1α1), which is directly implicated in rosetting. We demonstrate that NTS-DBL1α1-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-DBL1α1, 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.

Var2CSA Minimal CSA Binding Region Is Located within the N-Terminal Region

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.

Biochemical and biophysical characterisation of DBL1α1-varO, the rosetting domain of PfEMP1 from the VarO line of Plasmodium falciparum

Rosetting of erythrocytes infected with Plasmodium falciparum is frequently observed in children with severe malaria. This adhesion phenomenon has been linked to the DBL1alpha domain of P. falciparum erythrocyte membrane protein 1 (PfEMP1) in three laboratory clones: FCR3S1.2, IT4R29 and Palo Alto varO. Here, we compare the soluble recombinant NTS-DBL1alpha(1)-varO domain (NTS: N-terminal segment) obtained from E. coli, Pichia pastoris and baculovirus/insect cell expression systems. In each case, the presence of NTS was necessary for obtaining a soluble product. Successful expression in E. coli required maltose-binding protein as an N-terminal fusion partner. Each expression system produced an identical, correctly folded protein, as judged by biochemical and biophysical characterisations, and by the capacity to elicit antibodies that react with the surface of VarO-infected erythrocytes and disrupt VarO rosettes. Binding studies using surface plasmon resonance (SPR) techniques showed that NTS-DBL1alpha(1) produced in E. coli binds to heparin with micromolar affinity. IC(50) constants for other sulphated oligosaccharides were determined using SPR by measuring their competitive binding to the soluble protein in the presence of immobilized heparin. The affinity to NTS-DBL1alpha(1) was related to the degree of sulphation of the oligosaccharide, although the position of the sulphate groups on the sugar rings was also important. VarO rosettes could be disrupted by sulphated oligosaccharides with an efficacy that correlated with their binding affinity to recombinant NTS-DBL1alpha(1). Thus high yields of soluble NTS-DBL1alpha(1) with native conformation have been produced, opening novel perspectives for both structure-function studies and vaccine development.

Specific stimulation of HIV-1 replication in human placental trophoblasts by an antigen of Plasmodium falciparum.

Epidemiological data point to an increased risk of HIV-1 mother-to-child transmission in pregnant women with malaria, by unknown mechanisms. We show here that surface binding of a recombinant Plasmodium falciparum adhesin to chondroitin sulphate A proteoglycans increases HIV-1 replication in the human placental cell line BeWo, probably by a P. falciparum adhesin-induced long-terminal repeat-driven TNF-α stimulation. This suggests that placental malaria could increase the risk of HIV-1 transmission in utero.

Insight into antigenic diversity of VAR2CSA-DBL5ε domain from multiple Plasmodium falciparum placental isolates.

Background Protection against pregnancy associated malaria (PAM) is associated with high levels of anti-VAR2CSA antibodies. This protection is obtained by the parity dependent acquisition of anti-VAR2CSA antibodies. Distinct parity-associated molecular signatures have been identified in VAR2CSA domains. These two observations combined point to the importance of identifying VAR2CSA sequence variation, which facilitate parasitic evasion or subversion of host immune response. Highly conserved domains of VAR2CSA such as DBL5ε are likely to contain conserved epitopes, and therefore do constitute attractive targets for vaccine development. Methodology/Principal Findings VAR2CSA DBL5ε-domain sequences obtained from cDNA of 40 placental isolates were analysed by a combination of experimental and in silico methods. Competition ELISA assays on two DBL5ε variants, using plasma samples from women from two different areas and specific mice hyperimmune plasma, indicated that DBL5ε possess conserved and cross-reactive B cell epitopes. Peptide ELISA identified conserved areas that are recognised by naturally acquired antibodies. Specific antibodies against these peptides labelled the native proteins on the surface of placental parasites. Despite high DBL5ε sequence homology among parasite isolates, sequence analyses identified motifs in DBL5ε that discriminate parasites according to donors parity. Moreover, recombinant proteins of two VAR2CSA DBL5ε variants displayed diverse recognition patterns by plasma from malaria-exposed women, and diverse proteoglycan binding abilities. Conclusions/Significance This study provides insights into conserved and exposed B cell epitopes in DBL5ε that might be a focus for cross reactivity. The importance of sequence variation in VAR2CSA as a critical challenge for vaccine development is highlighted. VAR2CSA conformation seems to be essential to its functionality. Therefore, identification of sequence variation sites in distinct locations within VAR2CSA, affecting antigenicity and/or binding properties, is critical to the effort of developing an efficient VAR2CSA-based vaccine. Motifs associated with parasite segregation according to parity constitute one such site.

Functional and immunological characterization of the var2CSA-DBL5ɛ domain of a placental Plasmodium falciparum isolate

Pregnancy-associated malaria (PAM) arises from sequestration of Plasmodium falciparum-parasitized erythrocytes (PE) in the placenta, leading to chronic symptoms in the expectant mother and serious consequences for fetal development. Placental sequestration has been linked to binding of chondroitin sulphate A (CSA) by the var2CSA variant of PfEMP1 expressed on the PE surface, and a substantial body of evidence shows that the immune response to var2CSA gives an effective protection against PAM. We have expressed the var2CSA-DBL5epsilon domain, derived from a placental isolate from Senegal, as soluble product in Escherichia coli and have shown using different criteria that the recombinant protein is obtained with the native conformation. Using surface plasmon resonance techniques, we have examined binding of DBL5epsilon to placental chondroitin sulphate proteoglycan and CSA; however, the recombinant protein also binds to other sulphated oligosaccharides, with higher affinity in some cases, indicating that the single domain lacks the specificity for CSA shown by the complete extra-cellular region of var2CSA and placental parasites. Recombinant DBL5epsilon was specifically recognized by sera from malaria-exposed Senegalese women in a parity-dependent manner but by sera not from children or males from the same endemic region. Conversely, DBL5epsilon induced antibodies in mice that recognized placental isolates from Benin but not isolates from children. The presence of universal epitopes thus supports DBL5epsilon as an interesting component of var2CSA to be considered for vaccine development.

Llama immunization with full-length VAR2CSA generates cross-reactive and inhibitory single-domain antibodies against the DBL1X domain

VAR2CSA stands today as the leading vaccine candidate aiming to protect future pregnant women living in malaria endemic areas against the severe clinical outcomes of pregnancy associated malaria (PAM). The rational design of an efficient VAR2CSA-based vaccine relies on a profound understanding of the molecular interactions associated with P. falciparum infected erythrocyte sequestration in the placenta. Following immunization of a llama with the full-length VAR2CSA recombinant protein, we have expressed and characterized a panel of 19 nanobodies able to recognize the recombinant VAR2CSA as well as the surface of erythrocytes infected with parasites originating from different parts of the world. Domain mapping revealed that a large majority of nanobodies targeted DBL1X whereas a few of them were directed towards DBL4ε, DBL5ε and DBL6ε. One nanobody targeting the DBL1X was able to recognize the native VAR2CSA protein of the three parasite lines tested. Furthermore, four nanobodies targeting DBL1X reproducibly inhibited CSA adhesion of erythrocytes infected with the homologous NF54-CSA parasite strain, providing evidences that DBL1X domain is part or close to the CSA binding site. These nanobodies could serve as useful tools to identify conserved epitopes shared between different variants and to characterize the interactions between VAR2CSA and CSA.

Parity-Dependent Recognition of DBL1X-3X Suggests an Important Role of the VAR2CSA High-Affinity CSA-Binding Region in the Development of the Humoral Response against Placental Malaria

ABSTRACT Plasmodium falciparum multidomain protein VAR2CSA stands today as the leading vaccine candidate against pregnancy-associated malaria (PAM). Most of the studies aiming to decrypt how naturally acquired immunity develops have assessed the immune recognition of individual VAR2CSA Duffy-binding-like (DBL) domains, thus overlooking the presence of conformational epitopes resulting from the overall folding of the full-length protein. In order to characterize the development of humoral immunity toward VAR2CSA, we made use of a large cohort of 293 Senegalese pregnant women to assess the level of recognition by plasma IgG of the full-length VAR2CSA protein of the 3D7 parasite strain (3D7-VAR2CSA), the CSA-binding multidomains 3D7-DBL1X to -DBL3X (3D7-DBL1X-3X), and the CSA nonbinding multidomains 3D7-DBL4ε to -DBL6ε (3D7-DBL4ε-6ε), as well as individual 3D7-DBL domains. Our results revealed a parity-dependent recognition of the full-length 3D7-VAR2CSA and of the CSA-binding region, 3D7-DBL1X-3X. Indeed, multigravid women possess significantly higher levels of antibodies directed against these constructs than primigravidae. Our results suggest an important role of antibodies targeting the CSA-binding region in the development of immunity against PAM, therefore providing new insights on how natural protection might be acquired and further information for the design of VAR2CSA-based vaccines.

Structural and immunological correlations between the variable blocks of the VAR2CSA domain DBL6ε from two Plasmodium falciparum parasite lines.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), a family of adhesins of the falciparum species of the malaria parasite, is exposed on the surface of the infected erythrocyte. In general, only one PfEMP1 variant is expressed at a time but switching between variants occurs, changing both host-cell receptor specificity and serotype. The PfEMP1 variant VAR2CSA causes sequestration of infected erythrocytes in the intervillous spaces of the placenta via the glycosaminoglycan chondroitin sulfate A. This leads to pregnancy-associated malaria, which has severe consequences for the fetus and mother. The extracellular region of VAR2CSA comprises six DBL (Duffy-binding-like) domains and a single CIDR (cysteine-rich inter-domain region) domain. The C-terminal domain DBL6ε, the most polymorphic domain of VAR2CSA, has seven regions of high variability termed variable blocks (VBs). Here we have determined the crystal structure of DBL6ε from the FCR3 parasite line and have compared it with the previously determined structure of that from the 3D7 line. We found significant differences particularly in the N-terminal region, which contains the first VB (VB1). Although DBL6ε is the most variable VAR2CSA domain, DBL6ε-FCR3 and DBL6ε-3D7 react with IgG purified from immune sera of pregnant women. Furthermore, IgG purified on one domain cross-reacts with the other, confirming the presence of cross-reactive epitopes. We also examined reactivity of immune sera to the four least variable VB (VB1, VB2, VB4 and VB5) using peptides with the consensus sequence closest, in turn, to the FCR3 or 3D7 domain. These results provide new molecular insights into immune escape by parasites expressing the VAR2CSA variant.