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

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. 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.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

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

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Preclinical immunogenicity and safety of the cGMP-grade placental malaria vaccine PRIMVACResearch in context

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. Keywords: Malaria, Plasmodium, Vaccine, VAR2CSA, Placent

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

International audienceRosetting of erythrocytes infected with Plasmodium falciparum is frequently observed in children with severe malaria. This adhesion phenomenon has been linked to the DBL1␣ 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-DBL1␣ 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 maltosebinding 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-DBL1␣ 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-DBL1␣ 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-DBL1␣ 1. Thus high yields of soluble NTS-DBL1␣ 1 with native conformation have been produced, opening novel perspectives for both structure-function studies and vaccine development

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

International audienceOver 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 crossreactive 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

Competitive inhibition of recombinant DBL domains binding to decorin using various glycosaminoglycans.

<p>Recombinant proteins (A) 3D7-DBL1X-2X, (B) 3D7-DBL1X-CIDR, (C) 3D7-DBL1X-3X, (D) 3D7-DBL1X-4ε, (E) 3D7-DBL1X-5ε and (E) 3D7-DBL1X-6ε at 1 µg/mL were premixed with increasing amounts of BSA (□) or glycosaminoglycans, 0.156–100 µg/mL of CSA (×), CSC (⋄) or HS (▵) and incubated in plates previously coated with decorin.</p

Various var2CSA recombinant proteins expressed in HEK293 cells and <i>E. coli</i>.

<p>(A). Schematic view of the var2CSA domain organization and sequence limits of the recombinant domains studied (3D7-DBL1X, 3D7-DBL2X, FCR3-DBL3X, 3D7-DBL5ε, 3D7-DBL1X-2X, FCR3-DBL3X-4ε, 3D7-DBL1X-CIDR, 3D7-DBL1X-3X, 3D7-DBL1X-4ε, 3D7-DBL1X-5ε and 3D7-DBL1X-6ε). Var2CSA comprises six DBL domains (DBL1X to DBL6ε), a CIDRpam domain and three inter-domain regions (INT1-3) in the extracellular region, together with a trans-membrane segment and acidic C terminus sequence (ATS). DBL1X, DBL2X, DBL3X, DBL4ε, DBL5ε and DBL6ε are shown in green; CIDR in orange; N-terminal sequence (NTS) and inter-domain regions (INT) in grey; the trans-membrane and ATS regions in blue. The length of each bar corresponds to the domain size. (B). Purification of var2CSA derived proteins expressed in HEK293 and <i>E. coli</i>. Nu-SDS-PAGE Precast 4–12% Bis-Tris gel under nonreducing (B) and reducing (C) conditions was loaded with purified recombinant proteins. Lane 1: Marker, lane 2: 3D7-DBL1X, lane 3: 3D7-DBL2X, lane 4: FCR3-DBL3X, lane 5: 3D7-DBL5ε, lane 6: 3D7-DBL1X-2X, lane 7: FCR3-DBL3X-4ε, lane 8: 3D7-DBL1X-CIDR, lane 9: 3D7-DBL1X-3X, lane 10: 3D7-DBL1X-4ε, lane 11: 3D7-DBL1X-5ε and lane 12: 3D7-DBL1X-6ε. Proteins were visualized with Coomassie blue.</p

K_D for var2CSA recombinant proteins binding to placental CSPG.

<p>K_D values were determined from the concentration dependence of steady-state SPR response using the Biacore BIAEVALUATION 3.1 software.</p><p>*: from reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020270#pone.0020270-Srivastava1" target="_blank">[34]</a>.</p>#<p>: from reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020270#pone.0020270-Gangnard1" target="_blank">[41]</a>.</p