Transfected HEK293 Cells Expressing Functional Recombinant Intercellular Adhesion Molecule 1 (ICAM-1) – A Receptor Associated with Severe Plasmodium falciparum Malaria

Intercellular adhesion molecule 1 (ICAM-1) is a membrane-bound glycoprotein expressed on endothelial cells and cells of the immune system. Human ICAM-1 mediates adhesion and migration of leucocytes, and is implicated in inflammatory pathologies, autoimmune diseases and in many cancer processes. Additionally, ICAM-1 acts as receptor for pathogens like human rhinovirus and Plasmodium falciparum malaria parasites. A group of related P. falciparum erythrocyte membrane protein 1 (PfEMP1) domains, the DBLβ, mediates ICAM-1 binding of P. falciparum-infected erythrocytes. This ICAM‑1-binding phenotype has been suggested to be involved in the development of cerebral malaria. However, more studies identifying cross-reactive antibody and ICAM-1-binding epitopes and the establishment of a clinical link between DBLβ expression and e.g. cerebral malaria are needed before the DBLβ domains can be put forward as vaccine candidates and go into clinical trials. Such studies require availability of functional recombinant ICAM-1 in large quantities. In this study, we compared recombinant ICAM-1 expressed in HEK293 and COS-7 cells with mouse myeloma NS0 ICAM-1 purchased from a commercial vendor in terms of protein purity, yield, fold, ability to bind DBLβ, and relative cost. We present a HEK293 cell-based, high-yield expression and purification scheme for producing inexpensive, functional ICAM‑1. ICAM-1 expressed in HEK293 is applicable to malaria research and can also be useful in other research fields.     

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.     

B-Cell Epitopes in NTS-DBL1α of PfEMP1 Recognized by Human Antibodies in Rosetting Plasmodium falciparum

Plasmodium falciparum is the most lethal of the human malaria parasites. The virulence is associated with the capacity of the infected red blood cell (iRBC) to sequester inside the deep microvasculature where it may cause obstruction of the blood-flow when binding is excessive. Rosetting, the adherence of the iRBC to uninfected erythrocytes, has been found associated with severe malaria and found to be mediated by the NTS-DBL1α-domain of Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1). Here we show that the reactivity of plasma of Cameroonian children with the surface of the FCR3S1.2-iRBC correlated with the capacity to disrupt rosettes and with the antibody reactivity with a recombinant PfEMP1 (NTS-DBL1α of IT4_var60) expressed by parasite FCR3S1.2. The plasma-reactivity in a microarray, consisting of 96 overlapping 15-mer long peptides covering the NTS-DBL1α domain from IT4var60 sequence, was compared with their capacity to disrupt rosettes and we identified five peptides where the reactivity were correlated. Three of the peptides were localized in subdomain-1 and 2. The other two peptide-sequences were localized in the NTS-domain and in subdomain-3. Further, principal component analysis and orthogonal partial least square analysis generated a model that supported these findings. In conclusion, human antibody reactivity with short linear-peptides of NTS-DBL1α of PfEMP1 suggests subdomains 1 and 2 to hold anti-rosetting epitopes recognized by anti-rosetting antibodies. The data suggest rosetting to be mediated by the variable areas of PfEMP1 but also to involve structurally relatively conserved areas of the molecule that may induce biologically active antibodies.     

α2-Macroglobulin Can Crosslink Multiple Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) Molecules and May Facilitate Adhesion of Parasitized Erythrocytes

Rosetting, the adhesion of Plasmodium falciparum-infected erythrocytes to uninfected erythrocytes, involves clonal variants of the parasite protein P. falciparum erythrocyte membrane protein 1 (PfEMP1) and soluble serum factors. While rosetting is a well-known phenotypic marker of parasites associated with severe malaria, the reason for this association remains unclear, as do the molecular details of the interaction between the infected erythrocyte (IE) and the adhering erythrocytes. Here, we identify for the first time a single serum factor, the abundant serum protease inhibitor α₂-macroglobulin (α₂M), which is both required and sufficient for rosetting mediated by the PfEMP1 protein HB3VAR06 and some other rosette-mediating PfEMP1 proteins. We map the α₂M binding site to the C terminal end of HB3VAR06, and demonstrate that α₂M can bind at least four HB3VAR06 proteins, plausibly augmenting their combined avidity for host receptors. IgM has previously been identified as a rosette-facilitating soluble factor that acts in a similar way, but it cannot induce rosetting on its own. This is in contrast to α₂M and probably due to the more limited cross-linking potential of IgM. Nevertheless, we show that IgM works synergistically with α₂M and markedly lowers the concentration of α₂M required for rosetting. Finally, HB3VAR06⁺ IEs share the capacity to bind α₂M with subsets of genotypically distinct P. falciparum isolates forming rosettes in vitro and of patient parasite isolates ex vivo. Together, our results are evidence that P. falciparum parasites exploit α₂M (and IgM) to expand the repertoire of host receptors available for PfEMP1-mediated IE adhesion, such as the erythrocyte carbohydrate moieties that lead to formation of rosettes. It is likely that this mechanism also affects IE adhesion to receptors on vascular endothelium. The study opens opportunities for broad-ranging immunological interventions targeting the α₂M—(and IgM-) binding domains of PfEMP1, which would be independent of the host receptor specificity of clinically important PfEMP1 antigens.     

Evolutionary analyses of the major variant surface antigen-encoding genes reveal population structure of Plasmodium falciparum within and between continents

Malaria remains a major public health problem in many countries. Unlike influenza and HIV, where diversity in immunodominant surface antigens is understood geographically to inform disease surveillance, relatively little is known about the global population structure of PfEMP1, the major variant surface antigen of the malaria parasite Plasmodium falciparum. The complexity of the var multigene family that encodes PfEMP1 and that diversifies by recombination, has so far precluded its use in malaria surveillance. Recent studies have demonstrated that cost-effective deep sequencing of the region of var genes encoding the PfEMP1 DBLα domain and subsequent classification of within host sequences at 96% identity to define unique DBLα types, can reveal structure and strain dynamics within countries. However, to date there has not been a comprehensive comparison of these DBLα types between countries. By leveraging a bioinformatic approach (jumping hidden Markov model) designed specifically for the analysis of recombination within var genes and applying it to a dataset of DBLα types from 10 countries, we are able to describe population structure of DBLα types at the global scale. The sensitivity of the approach allows for the comparison of the global dataset to ape samples of Plasmodium Laverania species. Our analyses show that the evolution of the parasite population emerging out of Africa underlies current patterns of DBLα type diversity. Most importantly, we can distinguish geographic population structure within Africa between Gabon and Ghana in West Africa and Uganda in East Africa. Our evolutionary findings have translational implications in the context of globalization. Firstly, DBLα type diversity can provide a simple diagnostic framework for geographic surveillance of the rapidly evolving transmission dynamics of P. falciparum. It can also inform efforts to understand the presence or absence of global, regional and local population immunity to major surface antigen variants. Additionally, we identify a number of highly conserved DBLα types that are present globally that may be of biological significance and warrant further characterization.     

Plasmodium falciparum Rosetting Epitopes Converge in the SD3-Loop of PfEMP1-DBL1α

The ability of Plasmodium falciparum parasitized RBC (pRBC) to form rosettes with normal RBC is linked to the virulence of the parasite and RBC polymorphisms that weaken rosetting confer protection against severe malaria. The adhesin PfEMP1 mediates the binding and specific antibodies prevent sequestration in the micro-vasculature, as seen in animal models. Here we demonstrate that epitopes targeted by rosette disrupting antibodies converge in the loop of subdomain 3 (SD3) which connects the h6 and h7 α-helices of PfEMP1-DBL1α. Both monoclonal antibodies and polyclonal IgG, that bound to epitopes in the SD3-loop, stained the surface of pRBC, disrupted rosettes and blocked direct binding of recombinant NTS-DBL1α to RBC. Depletion of polyclonal IgG raised to NTS-DBL1α on a SD3 loop-peptide removed the anti-rosetting activity. Immunizations with recombinant subdomain 1 (SD1), subdomain 2 (SD2) or SD3 all generated antibodies reacting with the pRBC-surface but only the sera of animals immunized with SD3 disrupted rosettes. SD3-sequences were found to segregate phylogenetically into two groups (A/B). Group A included rosetting sequences that were associated with two cysteine-residues present in the SD2-domain while group B included those with three or more cysteines. Our results suggest that the SD3 loop of PfEMP1-DBL1α is an important target of anti-rosetting activity, clarifying the molecular basis of the development of variant-specific rosette disrupting antibodies.     

Structure-Function-Immunogenicity Studies of PfEMP1 Domain DBL2βPF11_0521, a Malaria Parasite Ligand for ICAM-1

Plasmodium falciparum virulence has been ascribed to its ability to sequester in deep vascular beds, mediated by the variant surface antigen family PfEMP1 binding endothelial receptors like ICAM-1. We previously observed that naturally-acquired antibodies that block a PfEMP1 domain, DBL2β of PF11_0521 allele, from binding to the human ICAM1 receptor, reduce the risk of malaria hospitalization in children. Here, we find that DBL2β_{PF11_0521} binds ICAM-1 in the low nM range and relate the structure of this domain with its function and immunogenicity. We demonstrate that the interaction with ICAM-1 is not impaired by point mutations in the N-terminal subdomain or in the flexible Loop 4 of DBL2β_{PF11_0521}, although both substructures were previously implicated in binding ICAM-1. These data will help to refine the existing model of DBLβ::ICAM-1 interactions. Antibodies raised against full-length DBL2β_{PF11_0521}, but not truncated forms lacking the N terminal fragment, block its interaction with ICAM-1. Our data suggest that full length domain is optimal for displaying functional epitopes and has a broad surface of interaction with ICAM-1 that is not disrupted by individual amino acid substitutions at putative key residues. This information might be important for the future design of anti-malarial vaccines based on PfEMP1 antigens.     

Characterization of anti-var2CSA-PfEMP1 cytoadhesion inhibitory mouse monoclonal antibodies

Pregnancy-associated malaria (PAM) is associated with the massive sequestration of erythrocytes infected with CSA-binding parasites in the placenta. Natural protective immunity against PAM is acquired during the course of pregnancies, with the development of anti-PfEMP1 antibodies recognizing placental infected erythrocytes (IEs) from different geographical regions. Mouse monoclonal antibodies (mabs) were raised against Plasmodium falciparum variant surface proteins expressed by CSA-binding parasites. These mabs blocked 0-60% of CSA-binding parasite adhesion and immunoprecipitated a 350 kDa 125I-labeled PfEMP1(CSA). Two var2CSA domains expressed on the surface of CHO cells (DBL5epsilon and DBL6epsilon) were identified as the targets of three of four antibodies inhibiting CSA binding. Two of these antibodies also recognized either DBL2x or DBL3x, suggesting that some epitopes may be common to several var2CSA domains. These mabs also specifically selected CSA-binding IEs and facilitated the purification from IE extracts of the native var2CSA ligand. This purified ligand elicited antibodies in immunized mice inhibiting efficiently IE(CSA) cytoadhesion. Based on our findings, we provide the first demonstration that the parasite var2CSA surface protein can elicit inhibitory antibodies and define here the subunits of the var2CSA ligand suitable for use in vaccine development.     

Binding of Subdomains 1/2 of PfEMP1-DBL1α to Heparan Sulfate or Heparin Mediates Plasmodium falciparum Rosetting

The capacity of Plasmodium falciparum parasitized erythrocytes (pRBC) to adhere to the endothelial lining in the microvasculature and to red blood cells (RBC) is associated with the virulence of the parasite, the pathogenesis and development of severe malaria. Rosetting, the binding of uninfected RBC to pRBC, is frequently observed in individuals with severe malaria and is mediated by the N-terminal NTS-DBL1α domain of the adhesin Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) expressed at the surface of the pRBC. Heparan sulfate has been suggested to be an important receptor for the NTS-DBL1α variant IT4_var60 expressed by the parasite FCR3S1.2. Here, we have determined the binding site of NTS-DBL1α (IT4_var60) to the RBC and heparin using a set of recombinant, mutated proteins expressed in and purified from E. coli. All the variants were studied for their ability to bind to RBC, their capacities to disrupt FCR3S1.2 rosettes, their affinities for heparin and their binding to rosette-disruptive mAbs. Our results suggest that NTS-DBL1α mediates binding to RBC through a limited number of basic amino acid residues localized on the surface of subdomains 1 (SD1) and 2 (SD2). The SD2-binding site is localized in close proximity to one of two previously identified binding sites in the rosetting PfEMP1 of the parasite PaloAlto-varO. The binding site in SD2 of NTS-DBL1α could represent a template for the development of anti-rosetting drugs.     

Investigating the function of Fc‐specific binding of IgM to Plasmodium falciparum erythrocyte membrane protein 1 mediating erythrocyte rosetting

Acquired protection from Plasmodium falciparum malaria takes years to develop, probably reflecting the ability of the parasites to evade immunity. A recent example of this is the binding of the F_c region of IgM to VAR2CSA‐type PfEMP1. This interferes with specific IgG recognition and phagocytosis of opsonized infected erythrocytes (IEs) without compromising the placental IE adhesion mediated by this PfEMP1 type. IgM also binds via F_c to several other PfEMP1 proteins, where it has been proposed to facilitate rosetting (binding of uninfected erythrocytes to a central IE). To further dissect the functional role of F_c‐mediated IgM binding to PfEMP1, we studied the PfEMP1 protein HB3VAR06, which mediates rosetting and binds IgM. Binding of IgM to this PfEMP1 involved the F_c domains Cμ3‐Cμ4 in IgM and the penultimate DBL domain (DBLζ2) at the C‐terminus of HB3VAR06. However, IgM binding did not inhibit specific IgG labelling of HB3VAR06 or shield IgG‐opsonized IEs from phagocytosis. Instead, IgM was required for rosetting, and each pentameric IgM molecule could bind two HB3VAR06 molecules. Together, our data indicate that the primary function of F_c‐mediated IgM binding in rosetting is not to shield IE from specific IgG recognition and phagocytosis as in VAR2CSA‐type PfEMP1. Rather, the function appears to be strengthening of IE–erythrocyte interactions. In conclusion, our study provides new evidence on the molecular details and functional significance of rosetting, a long‐recognized marker of parasites that cause severe P. falciparum malaria.     

Antibodies against PfEMP1, RIFIN,MSP3 and GLURP Are Acquired during Controlled Plasmodium falciparum Malaria Infections in Na?ve Volunteers

Antibodies to polymorphic antigens expressed during the parasites erythrocytic stages are important mediators of protective immunity against P. falciparum malaria. Therefore, polymorphic blood stage antigens like MSP3, EBA-175 and GLURP and variant surface antigens PfEMP1 and RIFIN are considered vaccine candidates. However, to what extent these antibodies to blood stage antigens are acquired during naive individuals'' first infections has not been studied in depth. Using plasma samples collected from controlled experimental P. falciparum infections we show that antibodies against variant surface antigens, PfEMP1 and RIFIN as well as MSP3 and GLURP, are acquired during a single short low density P. falciparum infection in non-immune individuals including strain transcendent PfEMP1 immune responses. These data indicate that the immunogenicity of the variant surface antigens is similar to the less diverse merozoite antigens. The acquisition of a broad and strain transcendent repertoire of PfEMP1 antibodies may reflect a parasite strategy of expressing most or all PfEMP1 variants at liver release optimizing the likelihood of survival and establishment of chronic infections in the new host.     

Evasion of Immunity to Plasmodium falciparum: Rosettes of Blood Group A Impair Recognition of PfEMP1

The ABO blood group antigens are expressed on erythrocytes but also on endothelial cells, platelets and serum proteins. Notably, the ABO blood group of a malaria patient determines the development of the disease given that blood group O reduces the probability to succumb in severe malaria, compared to individuals of groups A, B or AB. P. falciparum rosetting and sequestration are mediated by PfEMP1, RIFIN and STEVOR, expressed at the surface of the parasitized red blood cell (pRBC). Antibodies to these antigens consequently modify the course of a malaria infection by preventing sequestration and promoting phagocytosis of pRBC. Here we have studied rosetting P. falciparum and present evidence of an immune evasion mechanism not previously recognized. We find the accessibility of antibodies to PfEMP1 at the surface of the pRBC to be reduced when P. falciparum forms rosettes in blood group A RBC, as compared to group O RBC. The pRBC surrounds itself with tightly bound normal RBC that makes PfEMP1 inaccessible to antibodies and clearance by the immune system. Accordingly, pRBC of in vitro cloned P. falciparum devoid of ABO blood group dependent rosetting were equally well detected by anti-PfEMP1 antibodies, independent of the blood group utilized for their propagation. The pathogenic mechanisms underlying the severe forms of malaria may in patients of blood group A depend on the ability of the parasite to mask PfEMP1 from antibody recognition, in so doing evading immune clearance.     

Structural Basis for the ABO Blood-Group Dependence of Plasmodium falciparum Rosetting

The ABO blood group influences susceptibility to severe Plasmodium falciparum malaria. Recent evidence indicates that the protective effect of group O operates by virtue of reduced rosetting of infected red blood cells (iRBCs) with uninfected RBCs. Rosetting is mediated by a subgroup of PfEMP1 adhesins, with RBC binding being assigned to the N-terminal DBL1α₁ domain. Here, we identify the ABO blood group as the main receptor for VarO rosetting, with a marked preference for group A over group B, which in turn is preferred to group O RBCs. We show that recombinant NTS-DBL1α₁ and NTS-DBL1α₁-CIDR1γ reproduce the VarO-iRBC blood group preference and document direct binding to blood group trisaccharides by surface plasmon resonance. More detailed RBC subgroup analysis showed preferred binding to group A₁, weaker binding to groups A₂ and B, and least binding to groups A_x and O. The 2.8 Å resolution crystal structure of the PfEMP1-VarO Head region, NTS-DBL1α₁-CIDR1γ, reveals extensive contacts between the DBL1α₁ and CIDR1γ and shows that the NTS-DBL1α₁ hinge region is essential for RBC binding. Computer docking of the blood group trisaccharides and subsequent site-directed mutagenesis localized the RBC-binding site to the face opposite to the heparin-binding site of NTS-DBLα₁. RBC binding involves residues that are conserved between rosette-forming PfEMP1 adhesins, opening novel opportunities for intervention against severe malaria. By deciphering the structural basis of blood group preferences in rosetting, we provide a link between ABO blood grouppolymorphisms and rosette-forming adhesins, consistent with the selective role of falciparum malaria on human genetic makeup.     

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.     

Recovery of adhesion to chondroitin-4-sulphate in Plasmodium falciparum varCSA disruption mutants by antigenically similar PfEMP1 variants

Protection against maternal malaria has been associated with the acquisition of a specific antibody response that prevents adhesion of Plasmodium falciparum-infected erythrocytes to the glycosaminoglycan chondroitin-4-sulphate (CSA), which is present in the placental intervillous space. These antibodies are directed against variant forms of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) that mediate binding to CSA. We have generated insertional disruption mutants of the gene encoding the CSA-binding phenotype in the P. falciparum clone FCR3 (varCSA) to test the hypothesis that strategies targeting the parasite's determinant for this adhesive phenotype may prevent sequestration of infected erythrocytes in the placenta and hence the development of maternal malaria. The varCSA-disruption mutants were initially unable to adhere to CSA; however, they could recover the phenotype after repeated selection over CSA. We show that recovery of CSA binding is varCSA independent and mediated by the activation of a novel var variant. Importantly, the corresponding PfEMP1 protein reacts with a monoclonal antibody recognizing the DBL3 gamma domain of the varCSA gene product, indicating that the DBL3 gamma CSA-binding domains are conserved between these PfEMP1-binding variants. Our data support strategies exploring these conserved epitopes as vaccine candidates against maternal malaria.     

Allelic diversity of the Plasmodium falciparum erythrocyte membrane protein 1 entails variant-specific red cell surface epitopes

The clonally variant Plasmodium falciparum PfEMP1 adhesin is a virulence factor and a prime target of humoral immunity. It is encoded by a repertoire of functionally differentiated var genes, which display architectural diversity and allelic polymorphism. Their serological relationship is key to understanding the evolutionary constraints on this gene family and rational vaccine design. Here, we investigated the Palo Alto/VarO and IT4/R29 and 3D7/PF13_003 parasites lines. VarO and R29 form rosettes with uninfected erythrocytes, a phenotype associated with severe malaria. They express an allelic Cys2/group A NTS-DBL1α(1) PfEMP1 domain implicated in rosetting, whose 3D7 ortholog is encoded by PF13_0003. Using these three recombinant NTS-DBL1α(1) domains, we elicited antibodies in mice that were used to develop monovariant cultures by panning selection. The 3D7/PF13_0003 parasites formed rosettes, revealing a correlation between sequence identity and virulence phenotype. The antibodies cross-reacted with the allelic domains in ELISA but only minimally with the Cys4/group B/C PFL1955w NTS-DBL1α. By contrast, they were variant-specific in surface seroreactivity of the monovariant-infected red cells by FACS analysis and in rosette-disruption assays. Thus, while ELISA can differentiate serogroups, surface reactivity assays define the more restrictive serotypes. Irrespective of cumulated exposure to infection, antibodies acquired by humans living in a malaria-endemic area also displayed a variant-specific surface reactivity. Although seroprevalence exceeded 90% for each rosetting line, the kinetics of acquisition of surface-reactive antibodies differed in the younger age groups. These data indicate that humans acquire an antibody repertoire to non-overlapping serotypes within a serogroup, consistent with an antibody-driven diversification pressure at the population level. In addition, the data provide important information for vaccine design, as production of a vaccine targeting rosetting PfEMP1 adhesins will require engineering to induce variant-transcending responses or combining multiple serotypes to elicit a broad spectrum of immunity.     

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.     

Multiple var2csa-Type PfEMP1 Genes Located at Different Chromosomal Loci Occur in Many Plasmodium falciparum Isolates

Background

The var2csa gene encodes a Plasmodium falciparum adhesion receptor which binds chondroitin sulfate A (CSA). This var gene is more conserved than other PfEMP1/var genes and is found in all P. falciparum isolates. In isolates 3D7, FCR3/It4 and HB3, var2csa is transcribed from a sub-telomeric position on the left arm of chromosome 12, but it is not known if this location is conserved in all parasites. Genome sequencing indicates that the var2csa gene is duplicated in HB3, but whether this is true in natural populations is uncertain.

Methodology/Principal Findings

To assess global variation in the VAR2CSA protein, sequence variation in the DBL2X region of var2csa genes in 54 P.falciparum samples was analyzed. Chromosome mapping of var2csa loci was carried out and a quantitative PCR assay was developed to estimate the number of var2csa genes in P.falciparum isolates from the placenta of pregnant women and from the peripheral circulation of other malaria patients. Sequence analysis, gene mapping and copy number quantitation in P.falciparum isolates indicate that there are at least two loci and that both var2csa-like genes can be transcribed. All VAR2CSA DBL2X domains fall into one of two distinct phylogenetic groups possessing one or the other variant of a large (∼26 amino acid) dimorphic motif, but whether either motif variant is linked to a specific locus is not known.

Conclusions/Significance

Two or more related but distinct var2csa-type PfEMP1/var genes exist in many P. falciparum isolates. One gene is on chromosome 12 but additional var2csa-type genes are on different chromosomes in different isolates. Multiplicity of var2csa genes appears more common in infected placentae than in samples from non-pregnant donors indicating a possible advantage of this genotype in pregnancy associated malaria.     

Structural basis for binding of Plasmodium falciparum erythrocyte membrane protein 1 to chondroitin sulfate and placental tissue and the influence of protein polymorphisms on binding specificity

Chondroitin sulfate (CS) A is a key receptor for adhesion of Plasmodium falciparum-infected erythrocytes (IEs) in the placenta and can also mediate adhesion to microvascular endothelial cells. IEs that adhere to CSA express var2csa-type genes, which encode specific variants of the IE surface antigen P. falciparum erythrocyte membrane protein 1 (PfEMP1). We report direct binding of native PfEMP1, isolated from IEs and encoded by var2csa, to immobilized CSA. Binding of PfEMP1 was dependent on 4-O-sulfated disaccharides and glucuronic acid rather than iduronic acid, consistent with the specificity of intact IEs. Using immobilized CS oligosaccharides as neoglycolipid probes, the minimum chain length for direct binding of PfEMP1 was eight monosaccharide units. Similarly for IE adhesion to placental tissue there was a requirement for 4-O-sulfated GalNAc and glucuronic acid mixed with non-sulfated disaccharides; 6-O-sulfation interfered with the interaction between placental CSA and IEs. The minimum chain length for maximal inhibition of adhesion was 10 monosaccharide residues. Partially 4-O-sulfated CS oligosaccharides (45-55% sulfation) were highly effective inhibitors of placental adhesion (IC(50), 0.15 microg/ml) and may have potential for therapeutic development. We used defined P. falciparum isolates expressing different variants of var2csa in adhesion assays and found that there were isolate-specific differences in the preferred structural motifs for adhesion to CSA that correlated with polymorphisms in PfEMP1 encoded by var2csa-type genes. This may influence sites of IE sequestration or parasite virulence. These findings have significant implications for understanding the pathogenesis and biology of malaria, particularly during pregnancy, and the development of targeted interventions.