Chondroitin sulphate A (CSA)-binding of single recombinant Duffy-binding-like domains is not restricted to Plasmodium falciparum Erythrocyte Membrane Protein 1 expressed by CSA-binding parasites

Individuals living in areas with high Plasmodium falciparum transmission acquire immunity to malaria over time and adults have a markedly reduced risk of contracting severe disease. However, pregnant women constitute an important exception. Pregnancy-associated malaria is a major cause of mother and offspring morbidity, such as severe maternal anaemia and low birth-weight, and is characterised by selective accumulation of parasite-infected erythrocytes (IE) in the placenta. A P. falciparum protein named VAR2CSA, which belongs to the large P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) family, enables the IE to bind chondroitin sulphate A (CSA) in the placenta. Knock-out studies have demonstrated the exclusive capacity of VAR2CSA to mediate IE binding to CSA, and it has been shown that four of the six Duffy-binding-like (DBL) domains of VAR2CSA have the ability to bind CSA in vitro. In this study, we confirm the CSA-binding of these DBL domains, however, the analysis of a number of DBL domains of a non-VAR2CSA origin shows that CSA-binding is not exclusively restricted to VAR2CSA DBL domains. Furthermore, we show that the VAR2CSA DBL domains as well as other DBL domains also bind heparan sulphate. These data explain a number of publications describing CSA-binding domains derived from PfEMP1 antigens not involved in placental adhesion. The data suggest that the ability of single domains to bind CSA does not predict the functional capacity of the whole PfEMP1 and raises doubt whether the CSA-binding domains of native VAR2CSA have been correctly identified.     

The Density of Knobs on Plasmodium falciparum-Infected Erythrocytes Depends on Developmental Age and Varies among Isolates

Background

The virulence of Plasmodium falciparum malaria is related to the parasite’s ability to evade host immunity through clonal antigenic variation and tissue-specific adhesion of infected erythrocytes (IEs). The P. falciparum erythrocyte membrane protein 1 (PfEMP1) family expressed on dome-shaped protrusions called knobs on the IE surface is central to both. Differences in receptor specificity and affinity of expressed PfEMP1 are important for IE adhesiveness, but it is not known whether differences in the number and size of the knobs on which the PfEMP1 proteins are expressed also play a role. Therefore, the aim of this study was to provide detailed information on isolate- and time-dependent differences in knob size and density.

Methodology/Principal Findings

We used atomic force microscopy to characterize knobs on the surface of P. falciparum-infected erythrocytes. Fourteen ex vivo isolates from Ghanaian children with malaria and 10 P. falciparum isolates selected in vitro for expression of a particular PfEMP1 protein (VAR2CSA) were examined. Knob density increased from ∼20 h to ∼35 h post-invasion, with significant variation among isolates. The knob density ex vivo, which was about five-fold higher than following long-term in vitro culture, started to decline within a few months of culture. Although knob diameter and height varied among isolates, we did not observe significant time-dependent variation in these dimensions.

Conclusions/Significance

The density of knobs on the P. falciparum-IE surface depends on time since invasion, but is also determined by the infecting isolate in a time-independent manner. This is the first study to quantitatively evaluate knob densities and dimensions on different P. falciparum isolates, to examine ex vivo isolates from humans, and to compare ex vivo and long-term in vitro-cultured isolates. Our findings contribute to the understanding of the interaction between P. falciparum parasites and the infected host.     

Longitudinal study of Plasmodium falciparum and Plasmodium vivax in a Karen population in Thailand

Background

Pregnancy malaria is caused by Plasmodium falciparum -infected erythrocytes binding the placental receptor chondroitin sulfate A (CSA). This results in accumulation of parasites in the placenta with severe clinical consequences for the mother and her unborn child. Women become resistant to placental malaria as antibodies are acquired which specifically target the surface of infected erythrocytes binding in the placenta. VAR2CSA is most likely the parasite-encoded protein which mediates binding to the placental receptor CSA. Several domains have been shown to bind CSA in vitro; and it is apparent that a VAR2CSA-based vaccine cannot accommodate all the CSA binding domains and serovariants. It is thus of high priority to define minimal ligand binding regions throughout the VAR2CSA molecule.

Methods

To define minimal CSA-binding regions/peptides of VAR2CSA, a phage display library based on the entire var2csa coding region was constructed. This library was screened on immobilized CSA and cells expressing CSA resulting in a limited number of CSA-binding phages. Antibodies against these peptides were affinity purified and tested for reactivity against CSA-binding infected erythrocytes.

Results

The most frequently identified phages expressed peptides residing in the parts of VAR2CSA previously defined as CSA binding. In addition, most of the binding regions mapped to surface-exposed parts of VAR2CSA. The binding of a DBL2X peptide to CSA was confirmed with a synthetic peptide. Antibodies against a CSA-binding DBL2X peptide reacted with the surface of infected erythrocytes indicating that this epitope is accessible for antibodies on native VAR2CSA on infected erythrocytes.

Conclusion

Short continuous regions of VAR2CSA with affinity for multiple types of CSA were defined. A number of these regions localize to CSA-binding domains and to surface-exposed regions within these domains and a synthetic peptide corresponding to a peptide sequence in DBL2 was shown to bind to CSA and not to CSC. It is likely that some of these epitopes are involved in native parasite CSA adhesion. However, antibodies directed against single epitopes did not inhibit parasite adhesion. This study supports phage display as a technique to identify CSA-binding regions of large proteins such as VAR2CSA.     

Malaria knowledge and agricultural practices that promote mosquito breeding in two rural farming communities in Oyo State, Nigeria

Background

Malaria caused by Plasmodium falciparum can result in several different syndromes with severe clinical consequences for the about 200 million individuals infected each year. During pregnancy, women living in endemic areas become susceptible to malaria due to lack of antibodies against a unique P. falciparum membrane protein, named VAR2CSA. This antigen is not expressed in childhood infections, since it binds chondroitin sulphate A (CSA) expressed on the intervillous space in the placenta. A vaccine appears possible because women acquire protective antibodies hindering sequestration in the placenta as a function of parity. A challenge for vaccine development is to design small constructs of this large antigen, which can induce broadly protective antibodies. It has previously been shown that one domain of VAR2CSA, DBL4-FCR3, induces parasite adhesion-blocking antibodies. In this study, it is demonstrated that other domains of VAR2CSA also can induce antibodies with inhibitory activity.

Methods

All VAR2CSA domains from the 3D7 and HB3 parasites were produced in Baculovirus-transfected insect cells. Groups of three rats per protein were immunized and anti-sera were tested for surface reactivity against infected erythrocytes expressing FCR3 VAR2CSA and for the ability to inhibit FCR3CSA parasite adhesion to CSA. The fine specificity of the immune sera was analysed by VAR2CSA peptide arrays.

Results

Inhibitory antibodies were induced by immunization with DBL3-HB3 T1 and DBL1-3D7. However, unlike the previously characterised DBL4-FCR3 response the inhibitory response against DBL1-3D7 and DBL3-HB3 T1 was poorly reproduced in the second rounds of immunizations.

Conclusion

It is possible to induce parasite adhesion-blocking antibodies when immunizing with a number of different VAR2CSA domains. This indicates that the CSA binding site in VAR2CSA is comprised of epitopes from different domains.     

Plasmodium falciparum Variability and Immune Evasion Proceed from Antigenicity of Consensus Sequences from DBL6ε; Generalization to All DBL from VAR2CSA

We studied all consensus sequences within the four least ‘variable blocks’ (VB) present in the DBL6ε domain of VAR2CSA, the protein involved in the adhesion of infected red blood cells by Plasmodium falciparum that causes the Pregnancy-Associated Malaria (PAM). Characterising consensus sequences with respect to recognition of antibodies and percentage of responders among pregnant women living in areas where P. falciparum is endemic allows the identification of the most antigenic sequences within each VB. When combining these consensus sequences among four serotypes from VB1 or VB5, the most often recognized ones are expected to induce pan-reactive antibodies recognizing VAR2CSA from all plasmodial strains. These sequences are of main interest in the design of an immunogenic molecule. Using a similar approach than for DBL6ε, we studied the five other DBL and the CIDRpam from VAR2CSA, and again identified VB segments with highly conserved consensus sequences. In addition, we identified consensus sequences in other var genes expressed by non-PAM parasites. This finding paves the way for vaccine design against other pathologies caused by P. falciparum.     

Strain-Transcendent Immune Response to Recombinant Var2CSA DBL5-ε Domain Block P. falciparum Adhesion to Placenta-Derived BeWo Cells under Flow Conditions

Background

Pregnancy-associated malaria (PAM) is a serious consequence of the adhesion to the placental receptor chondroitin sulfate A (CSA) of Plasmodium falciparum-infected erythrocytes (PE) expressing the large cysteine-rich multi-domain protein var2CSA. Women become resistant to PAM, and develop strain-transcending immunity against CSA-binding parasites. The identification of var2CSA regions that could elicit broadly neutralizing and adhesion-blocking antibodies is a key step for the design of prophylactic vaccine strategies.

Methodology

Escherichia coli expressed var2CSA DBL domains were refolded and purified prior to immunization of mice and a goat. Protein-G-purified antibodies were tested for their ability to block FCR3^CSA-infected erythrocytes binding to placental (BeWo) and monkey brain endothelial (ScC2) cell lines using a flow cytoadhesion inhibition assay mimicking closely the physiological conditions present in the placenta at shear stress of 0.05 Pa. DBL5-ε, DBL6-ε and DBL5-6-ε induced cross-reactive antibodies using Alum and Freund as adjuvants, which blocked cytoadhesion at values ranging between 40 to 96% at 0.5 mg IgG per ml. Importantly, antibodies raised against recombinant DBL5-ε from 3 distinct parasites genotypes (HB3, Dd2 and 7G8) showed strain-transcending inhibition ranging from 38 to 64% for the heterologuous FCR3^CSA.

Conclusions

Using single and double DBL domains from var2CSA and Alum as adjuvant, we identified recombinant subunits inducing an immune response in experimental animals which is able to block efficiently parasite adhesion in a flow cytoadhesion assay that mimics closely the erythrocyte flow in the placenta. These subunits show promising features for inclusion into a vaccine aiming to protect against PAM.     

Towards the Rational Design of a Candidate Vaccine against Pregnancy Associated Malaria: Conserved Sequences of the DBL6ε Domain of VAR2CSA

Background

Placental malaria is a disease linked to the sequestration of Plasmodium falciparum infected red blood cells (IRBC) in the placenta, leading to reduced materno-fetal exchanges and to local inflammation. One of the virulence factors of P. falciparum involved in cytoadherence to chondroitin sulfate A, its placental receptor, is the adhesive protein VAR2CSA. Its localisation on the surface of IRBC makes it accessible to the immune system. VAR2CSA contains six DBL domains. The DBL6ε domain is the most variable. High variability constitutes a means for the parasite to evade the host immune response. The DBL6ε domain could constitute a very attractive basis for a vaccine candidate but its reported variability necessitates, for antigenic characterisations, identifying and classifying commonalities across isolates.

Methodology/Principal Findings

Local alignment analysis of the DBL6ε domain had revealed that it is not as variable as previously described. Variability is concentrated in seven regions present on the surface of the DBL6ε domain. The main goal of our work is to classify and group variable sequences that will simplify further research to determine dominant epitopes. Firstly, variable sequences were grouped following their average percent pairwise identity (APPI). Groups comprising many variable sequences sharing low variability were found. Secondly, ELISA experiments following the IgG recognition of a recombinant DBL6ε domain, and of peptides mimicking its seven variable blocks, allowed to determine an APPI cut-off and to isolate groups represented by a single consensus sequence.

Conclusions/Significance

A new sequence approach is used to compare variable regions in sequences that have extensive segmental gene relationship. Using this approach, the VAR2CSA DBL6 domain is composed of 7 variable blocks with limited polymorphism. Each variable block is composed of a limited number of consensus types. Based on peptide based ELISA, variable blocks with 85% or greater sequence identity are expected to be recognized equally well by antibody and can be considered the same consensus type. Therefore, the analysis of the antibody response against the classified small number of sequences should be helpful to determine epitopes.     

Clag9 is not essential for PfEMP1 surface expression in non-cytoadherent Plasmodium falciparum parasites with a chromosome 9 deletion

Background

The expression of the clonally variant virulence factor PfEMP1 mediates the sequestration of Plasmodium falciparum infected erythrocytes in the host vasculature and contributes to chronic infection. Non-cytoadherent parasites with a chromosome 9 deletion lack clag9, a gene linked to cytoadhesion in previous studies. Here we present new clag9 data that challenge this view and show that surface the non-cytoadherence phenotype is linked to the expression of a non-functional PfEMP1.

Methodology/Principal Findings

Loss of adhesion in P. falciparum D10, a parasite line with a large chromosome 9 deletion, was investigated. Surface iodination analysis of non-cytoadherent D10 parasites and COS-7 surface expression of the CD36-binding PfEMP1 CIDR1α domain were performed and showed that these parasites express an unusual trypsin-resistant, non-functional PfEMP1 at the erythrocyte surface. However, the CIDR1α domain of this var gene expressed in COS-7 cells showed strong binding to CD36. Atomic Force Microscopy showed a slightly modified D10 knob morphology compared to adherent parasites. Trafficking of PfEMP1 and KAHRP remained functional in D10. We link the non-cytoadherence phenotype to a chromosome 9 breakage and healing event resulting in the loss of 25 subtelomeric genes including clag9. In contrast to previous studies, knockout of the clag9 gene from 3D7 did not interfere with parasite adhesion to CD36.

Conclusions/Significance

Our data show the surface expression of non-functional PfEMP1 in D10 strongly indicating that genes other than clag9 deleted from chromosome 9 are involved in this virulence process possibly via post-translational modifications.     

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.     

School-based participatory health education for malaria control in Ghana: engaging children as health messengers

Background

Infected humans make protective antibody responses to the PfEMP1 adhesion antigens exported by Plasmodium falciparum parasites to the erythrocyte membrane, but little is known about the kinetics of this antibody-receptor binding reaction or how the topology of PfEMP1 on the parasitized erythrocyte membrane influences antibody association with, and dissociation from, its antigenic target.

Methods

A Quartz Crystal Microbalance biosensor was used to measure the association and dissociation kinetics of VAR2CSA PfEMP1 binding to human monoclonal antibodies. Immuno-fluorescence microscopy was used to visualize antibody-mediated adhesion between the surfaces of live infected erythrocytes and atomic force microscopy was used to obtain higher resolution images of the membrane knobs on the infected erythrocyte to estimate knob surface areas and model VAR2CSA packing density on the knob.

Results

Kinetic analysis indicates that antibody dissociation from the VAR2CSA PfEMP1 antigen is extremely slow when there is a high avidity interaction. High avidity binding to PfEMP1 antigens on the surface of P. falciparum-infected erythrocytes in turn requires bivalent cross-linking of epitopes positioned within the distance that can be bridged by antibody. Calculations of the surface area of the knobs and the possible densities of PfEMP1 packing on the knobs indicate that high-avidity cross-linking antibody reactions are constrained by the architecture of the knobs and the large size of PfEMP1 molecules.

Conclusions

High avidity is required to achieve the strongest binding to VAR2CSA PfEMP1, but the structures that display PfEMP1 also tend to inhibit cross-linking between PfEMP1 antigens, by holding many binding epitopes at distances beyond the 15-18 nm sweep radius of an antibody. The large size of PfEMP1 will also constrain intra-knob cross-linking interactions. This analysis indicates that effective vaccines targeting the parasite's vulnerable adhesion receptors should primarily induce strongly adhering, high avidity antibodies whose association rate constant is less important than their dissociation rate constant.     

Absence of Erythrocyte Sequestration and Lack of Multicopy Gene Family Expression in Plasmodium falciparum from a Splenectomized Malaria Patient

Background

To avoid spleen-dependent killing mechanisms parasite-infected erythrocytes (IE) of Plasmodium falciparum malaria patients have the capacity to bind to endothelial receptors. This binding also known as sequestration, is mediated by parasite proteins, which are targeted to the erythrocyte surface. Candidate proteins are those encoded by P. falciparum multicopy gene families, such as var, rif, stevor or PfMC-2TM. However, a direct in vivo proof of IE sequestration and expression of multicopy gene families is still lacking. Here, we report on the analysis of IE from a black African immigrant, who received the diagnosis of a malignant lymphoproliferative disorder and subsequently underwent splenectomy. Three weeks after surgery, the patient experienced clinical falciparum malaria with high parasitemia and circulating developmental parasite stages usually sequestered to the vascular endothelium such as late trophozoites, schizonts or immature gametocytes.

Methodology/Principal Findings

Initially, when isolated from the patient, the infected erythrocytes were incapable to bind to various endothelial receptors in vitro. Moreover, the parasites failed to express the multicopy gene families var, A-type rif and stevor but expression of B-type rif and PfMC-2TM genes were detected. In the course of in vitro cultivation, the parasites started to express all investigated multicopy gene families and concomitantly developed the ability to adhere to endothelial receptors such as CD36 and ICAM-1, respectively.

Conclusion/Significance

This case strongly supports the hypothesis that parasite surface proteins such as PfEMP1, A-type RIFIN or STEVOR are involved in interactions of infected erythrocytes with endothelial receptors mediating sequestration of mature asexual and immature sexual stages of P. falciparum. In contrast, multicopy gene families coding for B-type RIFIN and PfMC-2TM proteins may not be involved in sequestration, as these genes were transcribed in infected but not sequestered erythrocytes.     

Identification of a Major Dimorphic Region in the Functionally Critical N-Terminal ID1 Domain of VAR2CSA

The VAR2CSA protein of Plasmodium falciparum is transported to and expressed on the infected erythrocyte surface where it plays a key role in placental malaria (PM). It is the current leading candidate for a vaccine to prevent PM. However, the antigenic polymorphism integral to VAR2CSA poses a challenge for vaccine development. Based on detailed analysis of polymorphisms in the sequence of its ligand-binding N-terminal region, currently the main focus for vaccine development, we assessed var2csa from parasite isolates infecting pregnant women. The results reveal for the first time the presence of a major dimorphic region in the functionally critical N-terminal ID1 domain. Parasite isolates expressing VAR2CSA with particular motifs present within this domain are associated with gravidity- and parasite density-related effects. These observations are of particular interest in guiding efforts with respect to optimization of the VAR2CSA-based vaccines currently under development.     

Differential Recognition of P. falciparum VAR2CSA Domains by Naturally Acquired Antibodies in Pregnant Women from a Malaria Endemic Area

Background

Plasmodium falciparum infected red blood cells (iRBC) express variant surface antigens (VSA) of which VAR2CSA is involved in placental sequestration and causes pregnancy-associated malaria (PAM). Primigravidae are most susceptible to PAM whereas antibodies associated with protection are often present at higher levels in multigravid women. However, HIV co-infection with malaria has been shown to alter this parity-dependent acquisition of immunity, with more severe symptoms as well as more malaria episodes in HIV positive women versus HIV negative women of a similar parity.

Methods

Using VAR2CSA DBL-domains expressed on the surface of CHO-745 cells we quantified levels of DBL-domain specific IgG in sera from pregnant Malawian women by flow cytometry. Dissociations constants of DBL5ε specific antibodies were determined using a surface plasmon resonance technique, as an indication of antibody affinities.

Results

VAR2CSA DBL5ε was recognized in a gender and parity-dependent manner with anti-DBL5ε IgG correlating significantly with IgG levels to VSA-PAM on the iRBC surface. HIV positive women had lower levels of anti-DBL5ε IgG than HIV negative women of similar parity. In primigravidae, antibodies in HIV positive women also showed significantly lower affinity to VAR2CSA DBL5ε.

Conclusions

Pregnant women from a malaria-endemic area had increased levels of anti-DBL5ε IgG by parity, indicating this domain of VAR2CSA to be a promising vaccine candidate against PAM. However, it is important to consider co-infection with HIV, as this seems to change the properties of antibody response against malaria. Understanding the characteristics of antibody response against VAR2CSA is undoubtedly imperative in order to design a functional and efficient vaccine against PAM.     

Immunisation with recombinant PfEMP1 domains elicits functional rosette-inhibiting and phagocytosis-inducing antibodies to Plasmodium falciparum

Background

Rosetting is a Plasmodium falciparum virulence factor implicated in the pathogenesis of life-threatening malaria. Rosetting occurs when parasite–derived P. falciparum Erythrocyte Membrane Protein One (PfEMP1) on the surface of infected erythrocytes binds to human receptors on uninfected erythrocytes. PfEMP1 is a possible target for a vaccine to induce antibodies to inhibit rosetting and prevent severe malaria.

Methodology/Findings

We examined the vaccine potential of the six extracellular domains of a rosette-mediating PfEMP1 variant (ITvar9/R29var1 from the R29 parasite strain) by immunizing rabbits with recombinant proteins expressed in E. coli. Antibodies raised to each domain were tested for surface fluorescence with live infected erythrocytes, rosette inhibition and phagocytosis-induction. Antibodies to all PfEMP1 domains recognized the surface of live infected erythrocytes down to low concentrations (0.02–1.56 µg/ml of total IgG). Antibodies to all PfEMP1 domains except for the second Duffy-Binding-Like region inhibited rosetting (50% inhibitory concentration 0.04–4 µg/ml) and were able to opsonize and induce phagocytosis of infected erythrocytes at low concentrations (1.56–6.25 µg/ml). Antibodies to the N-terminal region (NTS-DBL1α) were the most effective in all assays. All antibodies were specific for the R29 parasite strain, and showed no functional activity against five other rosetting strains.

Conclusions/Significance

These results are encouraging for vaccine development as they show that potent antibodies can be generated to recombinant PfEMP1 domains that will inhibit rosetting and induce phagocytosis of infected erythrocytes. However, further work is needed on rosetting mechanisms and cross-reactivity in field isolates to define a set of PfEMP1 variants that could induce functional antibodies against a broad range of P. falciparum rosetting parasites.     

Full-Length Recombinant Plasmodium falciparum VAR2CSA Binds Specifically to CSPG and Induces Potent Parasite Adhesion-Blocking Antibodies

Plasmodium falciparum malaria remains one of the world's leading causes of human suffering and poverty. Each year, the disease takes 1-3 million lives, mainly in sub-Saharan Africa. The adhesion of infected erythrocytes (IEs) to vascular endothelium or placenta is the key event in the pathogenesis of severe P. falciparum infection. In pregnant women, the parasites express a single and unique member of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family named VAR2CSA, which is associated with the ability of the IEs to adhere specifically to chondroitin sulphate A (CSA) in the placenta. Several Duffy-binding-like domains from VAR2CSA molecules have been shown in vitro to bind to CSA, but it has also been demonstrated that Duffy-binding-like domains from PfEMP1 proteins other than VAR2CSA can bind CSA. In addition, the specificity of the binding of VAR2CSA domains to glycosaminoglycans does not match that of VAR2CSA-expressing IEs. This has led to speculation that the domains of native VAR2CSA need to come together to form a specific binding site or that VAR2CSA might bind to CSA through a bridging molecule. Here, we describe the expression and purification of the complete extracellular region of VAR2CSA secreted at high yields from insect cells. Using surface plasmon resonance, we demonstrate that VAR2CSA alone binds with nanomolar affinity to human chondroitin sulphate proteoglycan and with significantly weaker affinity to other glycosaminoglycans, showing a specificity similar to that observed for IEs. Antibodies raised against full-length VAR2CSA completely inhibit recombinant VAR2CSA binding, as well as parasite binding to chondroitin sulphate proteoglycan. This is the first study to describe the successful production and functionality of a full-length PfEMP1. The specificity of the binding and anti-adhesion potency of induced IgG, together with high-yield production, encourages the use of full-length PfEMP1 in vaccine development strategies.