CENP-B-mediated DNA loops regulate activity and stability of human centromeres.

Chromosome inheritance depends on centromeres, epigenetically specified regions of chromosomes. While conventional human centromeres are known to be built of long tandem DNA repeats, much of their architecture remains unknown. Using single-molecule techniques such as AFM, nanopores, and optical tweezers, we find that human centromeric DNA exhibits complex DNA folds such as local hairpins. Upon binding to a specific sequence within centromeric regions, the DNA-binding protein CENP-B compacts centromeres by forming pronounced DNA loops between the repeats, which favor inter-chromosomal centromere compaction and clustering. This DNA-loop-mediated organization of centromeric chromatin participates in maintaining centromere position and integrity upon microtubule pulling during mitosis. Our findings emphasize the importance of DNA topology in centromeric regulation and stability.

A nanobody against the VWF A3 domain detects ADAMTS13-induced proteolysis in congenital and acquired VWD.

von Willebrand factor (VWF) is a multimeric protein, the size of which is regulated via ADAMTS13-mediated proteolysis within the A2 domain. We aimed to isolate nanobodies distinguishing between proteolyzed and non-proteolyzed VWF, leading to the identification of a nanobody (designated KB-VWF-D3.1) targeting the A3 domain, the epitope of which overlaps the collagen-binding site. Although KB-VWF-D3.1 binds with similar efficiency to dimeric and multimeric derivatives of VWF, binding to VWF was lost upon proteolysis by ADAMTS13, suggesting that proteolysis in the A2 domain modulates exposure of its epitope in the A3 domain. We therefore used KB-VWF-D3.1 to monitor VWF degradation in plasma samples. Spiking experiments showed that a loss of 10% intact VWF could be detected using this nanobody. By comparing plasma from volunteers to that from congenital von Willebrand disease (VWD) patients, intact-VWF levels were significantly reduced for all VWD types, and most severely in VWD type 2A-group 2, in which mutations promote ADAMTS13-mediated proteolysis. Unexpectedly, we also observed increased proteolysis in some patients with VWD type 1 and VWD type 2M. A significant correlation (r = 0.51, P < .0001) between the relative amount of high-molecular weight multimers and levels of intact VWF was observed. Reduced levels of intact VWF were further found in plasmas from patients with severe aortic stenosis and patients receiving mechanical circulatory support. KB-VWF-D3.1 is thus a nanobody that detects changes in the exposure of its epitope within the collagen-binding site of the A3 domain. In view of its unique characteristics, it has the potential to be used as a diagnostic tool to investigate whether a loss of larger multimers is due to ADAMTS13-mediated proteolysis.

Phosphorylation of the VAR2CSA extracellular region is associated with enhanced adhesive properties to the placental receptor CSA.

Plasmodium falciparum is the main cause of disease and death from malaria. P. falciparum virulence resides in the ability of infected erythrocytes (IEs) to sequester in various tissues through the interaction between members of the polymorphic P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesin family to various host receptors. Here, we investigated the effect of phosphorylation of variant surface antigen 2-CSA (VAR2CSA), a member of the PfEMP1 family associated to placental sequestration, on its capacity to adhere to chondroitin sulfate A (CSA) present on the placental syncytium. We showed that phosphatase treatment of IEs impairs cytoadhesion to CSA. MS analysis of recombinant VAR2CSA phosphosites prior to and after phosphatase treatment, as well as of native VAR2CSA expressed on IEs, identified critical phosphoresidues associated with CSA binding. Site-directed mutagenesis on recombinant VAR2CSA of 3 phosphoresidues localised within the CSA-binding region confirmed in vitro their functional importance. Furthermore, using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9), we generated a parasite line in which the phosphoresidue T934 is changed to alanine and showed that this mutation strongly impairs IEs cytoadhesion to CSA. Taken together, these results demonstrate that phosphorylation of the extracellular region of VAR2CSA plays a major role in IEs cytoadhesion to CSA and provide new molecular insights for strategies aiming to reduce the morbidity and mortality of PM.

Murine Model for Preclinical Studies of Var2CSA-Mediated Pathology Associated with Malaria in Pregnancy.

Plasmodium falciparum infection during pregnancy leads to abortions, stillbirth, low birth weight, and maternal mortality. Infected erythrocytes (IEs) accumulate in the placenta by adhering to chondroitin sulfate A (CSA) via var2CSA protein exposed on the P. falciparum IE membrane. Plasmodium berghei IE infection in pregnant BALB/c mice is a model for severe placental malaria (PM). Here, we describe a transgenic P. berghei parasite expressing the full-length var2CSA extracellular region (domains DBL1X to DBL6ε) fused to a P. berghei exported protein (EMAP1) and characterize a var2CSA-based mouse model of PM. BALB/c mice were infected at midgestation with different doses of P. berghei-var2CSA (P. berghei-VAR) or P. berghei wild-type IEs. Infection with 10(4) P. berghei-VAR IEs induced a higher incidence of stillbirth and lower fetal weight than P. berghei At doses of 10(5) and 10(6) IEs, P. berghei-VAR-infected mice showed increased maternal mortality during pregnancy and fetal loss, respectively. Parasite loads in infected placentas were similar between parasite lines despite differences in maternal outcomes. Fetal weight loss normalized for parasitemia was higher in P. berghei-VAR-infected mice than in P. berghei-infected mice. In vitro assays showed that higher numbers of P. berghei-VAR IEs than P. berghei IEs adhered to placental tissue. Immunization of mice with P. berghei-VAR elicited IgG antibodies reactive to DBL1-6 recombinant protein, indicating that the topology of immunogenic epitopes is maintained between DBL1-6-EMAP1 on P. berghei-VAR and recombinant DBL1-6 (recDBL1-6). Our data suggested that impairments in pregnancy caused by P. berghei-VAR infection were attributable to var2CSA expression. This model provides a tool for preclinical evaluation of protection against PM induced by approaches that target var2CSA.

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.

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.

Beninese children with cerebral malaria do not develop humoral immunity against the IT4-VAR19-DC8 PfEMP1 variant linked to EPCR and brain endothelial binding.

BACKGROUND: Malaria is still one of the most prevalent infectious diseases in the world. Sequestration of infected erythrocytes (IEs) is the prime mediator of disease. Cytoadhesion of IEs is mediated by members of the highly diverse Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). A restricted sub-set of var genes encoding for PfEMP1s possessing the domain cassettes DC8 and DC13 were found to bind to the endothelial protein C receptor (EPCR). These var genes were shown to be highly expressed by parasites from patients with severe malaria clinical outcomes compared to those from patients with uncomplicated symptoms. METHODS: In order to further study the molecular mechanisms underlying DC8/DC13 expressing IEs adhesion to EPCR, a method was developed to produce highly pure recombinant EPCR. The IT4 parasite strain was selected on either anti-IT4-VAR19 purified IgG, EPCR or human brain endothelial cell line and their var gene expression profiles as well as their binding phenotypes were compared. The N-terminal region of IT4-VAR19 comprising a full-length DC8 cassette as well as the single EPCR binding CIDRα1.1 domain were also produced, and their immune recognition (IgG) was assessed using plasma samples from Beninese children presenting acute mild malaria, severe malaria or cerebral malaria at the time of their admission to the clinic, and from convalescent-phase plasma collected 30 days after anti-malarial treatment. RESULTS: The multi-domain VAR19-NTS-DBLγ6 binds to EPCR with a greater affinity than the CIDRα1.1 domain alone and this study also demonstrates that VAR19-NTS-DBLγ6 binding to the EPCR-expressing endothelial cell line (HBEC5i) is more pronounced than that of the CIDRα1.1 domain alone. IT4-VAR19 represents the preferentially expressed-PfEMP1 when FCR3-IEs are selected based on their capability to bind EPCR. Notably, no significant difference in the levels of antibodies towards IT4-VAR19 antigens was observed within all clinical groups between plasma samples collected during the acute malaria phase compared to samples collected 30 days after anti-malaria treatment. CONCLUSIONS: These data indicate that even being the preferentially selected IT4-EPCR-binding variant, the IT4-VAR19-DC8 region does not appear to be associated with the acquisition of antibodies during a single severe paediatric malaria episode in Benin.

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 (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.

V(H)H (nanobody) directed against human glycophorin A: a tool for autologous red cell agglutination assays.

The preparation of a V(H)H (nanobody) named IH4 that recognizes human glycophorin A (GPA) is described. IH4 was isolated by screening a library prepared from the lymphocytes of a dromedary immunized by human blood transfusion. Phage display and panning against GPA as the immobilized antigen allowed isolating this V(H)H. IH4, representing 67% of the retrieved V(H)H sequences, was expressed as a soluble correctly folded protein in SHuffle Escherichia coli cells, routinely yielding approximately 100 mg/L fermentation medium. Because IH4 recognizes GPA independently of the blood group antigens, it recognizes red cells of all humans with the possible exception of those with some extremely rare genetic background. The targeted linear epitope comprises the GPA Y52PPE55 sequence. Based on surface plasmon resonance results, the dissociation constant of the IH4-GPA equilibrium is 33 nM. IH4 is a stable protein with a transition melting temperature of 75.8 °C (measured by differential scanning calorimetry). As proof of concept, we fused HIV p24 to IH4 and used the purified construct expressed in E. coli to show that IH4 was amenable to the preparation of autologous erythrocyte agglutination reagents: reconstituted blood prepared with serum from an HIV-positive patient was readily agglutinated by the addition of the bifunctional reagent.

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 DBL6epsilon) 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) = 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.

BMFPs, a versatile therapeutic tool for redirecting a preexisting Epstein-Barr virus antibody response toward defined target cells.

Industrial production of therapeutic monoclonal antibodies is mostly performed in eukaryotic-based systems, allowing posttranslational modifications mandatory for their functional activity. The resulting elevated product cost limits therapy access to some patients. To address this limitation, we conceptualized a novel immunotherapeutic approach to redirect a preexisting polyclonal antibody response against Epstein-Barr virus (EBV) toward defined target cells. We engineered and expressed in bacteria bimodular fusion proteins (BMFPs) comprising an Fc-deficient binding moiety targeting an antigen expressed at the surface of a target cell, fused to the EBV-P18 antigen, which recruits circulating endogenous anti-P18 IgG in EBV+ individuals. Opsonization of BMFP-coated targets efficiently triggered antibody-mediated clearing effector mechanisms. When assessed in a P18-primed mouse tumor model, therapy performed with an anti-huCD20 BMFP significantly led to increased survival and total cancer remission in some animals. These results indicate that BMFPs could represent potent and useful therapeutic molecules to treat a number of diseases.

VAR2CSA binding phenotype has ancient origin and arose before Plasmodium falciparum crossed to humans: implications in placental malaria vaccine design.

VAR2CSA is a leading candidate for developing a placental malaria (PM) vaccine that would protect pregnant women living in malaria endemic areas against placental infections and improve birth outcomes. Two VAR2CSA-based PM vaccines are currently under clinical trials, but it is still unclear if the use of a single VAR2CSA variant will be sufficient to induce a broad enough humoral response in humans to cross-react with genetically diverse parasite populations. Additional immuno-focusing vaccine strategies may therefore be required to identify functionally conserved antibody epitopes in VAR2CSA. We explored the possibility that conserved epitopes could exist between VAR2CSA from the chimpanzee parasite Plasmodium reichenowi and Plasmodium falciparum sequences. Making use of VAR2CSA recombinant proteins originating from both species, we showed that VAR2CSA from P. reichenowi (Pr-VAR2CSA) binds to the placental receptor CSA with high specificity and affinity. Antibodies raised against Pr-VAR2CSA were able to recognize native VAR2CSA from different P. falciparum genotypes and to inhibit the interaction between CSA and P. falciparum-infected erythrocytes expressing different VAR2CSA variants. Our work revealed the existence of cross-species inhibitory epitopes in VAR2CSA and calls for pre-clinical studies assessing the efficacy of novel VAR2CSA-based cross-species boosting regimens.

Preclinical immunogenicity and safety of the cGMP-grade placental malaria vaccine PRIMVAC.

BACKGROUND: VAR2CSA is the lead antigen for developing a vaccine that would protect pregnant women against placental malaria. A multi-system feasibility study has identified E. coli as a suitable bacterial expression platform allowing the production of recombinant VAR2CSA-DBL1x-2x (PRIMVAC) to envisage a prompt transition to current Good Manufacturing Practice (cGMP) vaccine production. METHODS: Extensive process developments were undertaken to produce cGMP grade PRIMVAC to permit early phase clinical trials. PRIMVAC stability upon storage was assessed over up to 3 years. A broad toxicology investigation was carried out in rats allowing meanwhile the analysis of PRIMVAC immunogenicity. FINDINGS: We describe the successful cGMP production of 4. 65 g of PRIMVAC. PRIMVAC drug product was stable and potent for up to 3 years upon storage at -20 °C and showed an absence of toxicity in rats. PRIMVAC adjuvanted with Alhydrogel® or GLA-SE was able to generate antibodies able to recognize VAR2CSA expressed at the surface of erythrocytes infected with different strains. These antibodies also inhibit the interaction of the homologous NF54-CSA strain and to a lower extend of heterologous strains to CSA. INTERPRETATION: This work paved the way for the clinical development of an easily scalable low cost effective vaccine that could protect against placental malaria and prevent an estimated 10,000 maternal and 200,000 infant deaths annually. FUND: This work was supported by a grant from the Bundesministerium für Bildung und Forschung (BMBF), Germany through Kreditanstalt für Wiederaufbau (KfW) (Reference No: 202060457) and through funding from Irish Aid, Department of Foreign Affairs and Trade, Ireland.

Down-selection of the VAR2CSA DBL1-2 expressed in E. coli as a lead antigen for placental malaria vaccine development.

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.

Structure of the DBL3X-DBL4ε region of the VAR2CSA placental malaria vaccine candidate: insight into DBL domain interactions.

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 Šof 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.

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.

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.

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.

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 donor's 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.