Recently, the structures of the two DBL domains (F1 and F2) of erythrocyte-binding protein (EBA)-175 and a DBL domain in Plasmodium knowlesi (Pk)α-DBL were solved [15,16]. Using the crystal structure of EBA-175 F1 (Protein Data Bank code 1ZRO) as template, a three-dimensional model of DBL3X VAR2CSA was constructed by comparative modeling on the basis of the 3D7 sequence (Figure 1C). The sequence identity between DBL3X and EBA-175 F1 was 28%. From sequence alignments of DBL3X, Pkα-DBL, and EBA-175 (Figure S1) it was apparent that a number of cysteines were conserved, and ten of these from DBL3X aligned with cysteines which form disulfide bridges in the determined structures of EBA-175 [15] and Pkα-DBL [16]. In addition, we identified 34 buried hydrophobic residues in EBA-175 F1, EBA-175 F2, and Pkα-DBL, which corresponded to hydrophobic residues buried in the DBL3X model (Figure S1). Compared to EBA-175 F1, a number of insertions were found in DBL3X, and the majority of these were predicted to have coil secondary structure (Figure 1C). One of the insertions in DBL3X (L1: R56-I63) was found to align structurally next to a region in Pkα-DBL, which is described as a flexible linker with an experimentally determined proteolytic cleavage site [16]. A second insertion (L2: N1417-E1430) was aligned in a loop region where two residues were missing structural information in the structure of EBA-175 F1 [15], which also indicates high flexibility. Thus, the alignment of DBL3X to the solved DBL domain structures seems to match characteristics such as disulfide-bridges, stabilizing hydrophobic interactions, and flexible loop regions. These findings suggest that VAR2CSA DBL3X has the same basic structure as the solved DBL structures [15–17] in spite of the extensive sequence variation. The ability of proteins to form similar structures despite marked sequence variation has also been described for the VSG molecules covering the surface of Trypanosomes [18,19].

Var2csa is a relatively conserved var gene carried by all P. falciparum genomes; however, sequence polymorphisms are present in the gene. In a previous study, it was established that var2csa is transcribed at high levels by placental parasites isolated at delivery from Senegalese women [8,20]. Using cDNA from 24 placentas from that study, the region encoding DBL3X of var2csa was cloned and sequenced. A multiple alignment of 43 sequences showed that the average nucleotide diversity was low (π = 8.48% ± 0.37%, see Materials and Methods for details) reflecting a limited inter-parasite diversity in these isolates collected from a geographically small and well-defined area of West Africa. To test whether the Senegalese placental DBL3X sequences represented a monophyletic group compared to non-Senegalese DBL3X sequences, a phylogenetic tree, which included VAR2CSA DBL3X sequences available in GenBank, was constructed (Figure 2). These non-Senegalese sequences included four lab-strain parasites with different geographic origins (DD2 from Laos, MC from Thailand, IT4 from Brazil, and 3D7 with unknown origin) and 21 sequences from a sequencing study from Malawi [21]. The four database sequences and the Malawi sequences were scattered evenly in the tree indicating that the Senegalese sequences were representative for the var2csa repertoire in general and that parasite nucleotide diversity is similar on a local and global scale. It is also apparent that there is no clear subgrouping within the DBL3X sequences. The protein alignment of the DBL3X sequences (Figure 3A) suggested that DBL3X could be divided into four relatively conserved regions (C1–4) separated by three shorter variable regions (V1–3). When the variable regions were mapped to the 3-D model (Figure 3B), V1 and V3 were part of flexible loops, whereas V2 included another flexible loop but also extended into a helical region. The length of V1 and V3 varied between sequences and the 3D7 sequence was relatively short in both regions.

We have previously shown that the expression of certain var genes is associated with severe malaria in young children [22] and suggested that var gene expression is hierarchically structured. This could occur because the progeny of parasites expressing var gene products that mediate the most efficient sequestration outgrow the progeny of parasites expressing a molecule mediating less efficient binding [22–24]. A similar process could shape the expression of molecules mediating binding in the placenta. If that was the case, it would be predicted that a systematic difference between molecules mediating binding in primigravidae and multigravidae women could be detected in areas of high P. falciparum transmission where women are exposed to several parasite clones during pregnancy [25]. This was addressed by calculating the Kullback-Leibler distance between 21 VAR2CSA sequences originating from primigravidae and 21 sequences from multigravidae. The overall cumulated Kullback-Leibler distance (D_KL) between sequences from the primigravidae and the multigravidae was higher than for randomly chosen groupings of the sequences (p = 0.0075). The D_KL was also calculated for each position in the alignment to determine which polymorphisms contributed to the difference between the sequence of parasites from primi- and multigravidae women and visualized in a Kullback-Leibler sequence logo (Figure 4A). This implicated two stretches of amino acids in positions 135–175 and 233–245 located in the V2 and V3 regions, respectively. The region from position 158 to 162 was of special interest since the motif “EIEKD” was mainly found in primigravidae and the motif “GIEGE” mainly in the multigravidae (Table 1). Intermediate motifs like “(G/E)IERE” where the fourth position had changed from lysine to arginine were also found, implying that the following evolutionary pathway may have been operating: lysine (AAA or AAG) ↔ arginine (AGG) ↔ glycine (GGG). The change from glutamate and lysine/arginine, which have large charged side chains, into glycine without a side chain, could result in marked functional and antigenic changes. Thus, it was interesting that positions 1, 4, and 5 in the motif (position 158, 161, and 162) were predicted to be surface-exposed in the structural model, which was also the case for all of the other amino acid positions in V2 that differed significantly in the Kullback-Leibler sequence logo (Figure 4B and and4C).4C). The finding that parasites expressing the EIEKD motif were more prevalent in primigravidae than in multigravidae women (Table 1, Chi-square test: p < 0.001) could indicate that these parasites have a biological advantage in women experiencing their first pregnancy and that parasites expressing the other motifs (G/EIERE or GIEGE) have an advantage in women who have been pregnant before. This could arise because parasites carrying the EIEKD motif are the most efficient mediators of binding and therefore dominate in women with limited immunity against PAM. As immunity develops against these parasite forms, parasites expressing other motifs that are less efficient in binding but not serologically cross-reactive take over. Interestingly, a monoclonal Ghanaian field isolate undergoing full genome sequencing at the Sanger Institute has two copies of var2csa, one with the EIEKD motif and one with the GIEGE. Although the functional background for the observed phenomenon is presently not clear, the systematic sequence variation at positions predicted to be surface-exposed between parasites from primi- and multigravidae women strengthen the concept that VAR2CSA is the main parasite ligand for sequestration of malaria parasites in the placenta.

A recent study has suggested that sequence polymorphisms in a region of VAR2CSA upstream to DBL3X largely are due to positive natural selection pressure [13]. To further investigate the nature of sequence diversity in var2csa, the dN/dS ratios (dN, rate of non-synonymous mutations per non-synonymous site and dS, the rate of synonymous mutations per synonymous site) for DBL3X were calculated on the basis of the sequenced DBL3X domains. A dN/dS <1 indicates that the position is under negative or purifying selection pressure leading to conservation of the residue, while dN/dS >1 suggests positive or diversifying selection pressure, and suggests that amino acid changes are evolutionarily advantageous at the position. Purifying selection was mainly found in the conserved regions C1–4 and it was especially pronounced in the C2 and C4 regions, although single sites were observed to be under diversifying selection (Figure 5A). Several blocks appeared to be under strong diversifying selection and these appeared most prominently in regions V1, V2, and V3. It is interesting to note that residues under diversifying selection mainly were situated in regions predicted to be surface-exposed and concentrated on one side of the molecule (Figure 5D and and5E).5E). The two DBL domains of EBA-175 are predicted to form a reverse handshake dimer with the F1 domain of each molecule interacting with F2 of the other [15]. In this four-domain DBL structure, we replaced one of the EBA-175 F1 domains with our VAR2CSA DBL3X model (Figure 5F). It was noticeable that the largely conserved C2 and C4 regions of DBL3X were predicted to take part in the lining of the central cavity of the four-domain structure and form the region next to the cavity facing the membrane in native configuration. The model presented here is unlikely to fatefully reflect the structure of the native molecule, but the positioning of conserved DBL3X regions in the model makes biological sense, and the finding underscores the need to obtain knowledge about possible interactions between VAR2CSA DBL domains. The lack of amino acid positions under diversifying selection in the regions adjacent to the cavity might be due to the possibility that these sites are involved in ligand binding and thus functional constraint. Another explanation could be that the regions forming the predicted cavity and the area predicted to face the membrane are not accessible for antibodies in natively folded molecules.

Previous studies have reported that frequent recombination events generate sequence diversity in the PfEMP1 family [26–28]. To determine the role of recombination for DBL3X sequence variation, we estimated the population recombination rate ρ defined for partially inbreeding haploid species by the compound parameter 2N_er(1− f), where N_e represents the effective population size, r is the per-generation cross-over recombination rate per base pair (bp), and f is the inbreeding coefficient. Variations in ρ across DBL3X correspond to variations in the recombination rate r, as N_e and f are constant for the dataset. Two recombinational hotspots were observed at bp positions 138–178 and 704–730 (Figure 5B). The two best defined recombination breakpoints were present in the C1/V1 borderline at bp 177–179 and within V3 at bp 728–730. Both the V1 and V3 region harbored major deletions/insertions in several of the sequences, which may have arisen as a consequence of unequal cross-over during recombination at the hotspots. Unequal cross-over results in either deletions or insertions of variable number of tandem repeats (VNTR), and the DBL3X V1 region did indeed contain a high amount of VNTR. The loop region of V2 contained a small VNTR insert in some sequences, while VNTR were less obvious in the V3 region. In a recent study of the P. falciparum Chromosome 3, high recombination rates were found in sub-telomeric regions, and African P. falciparum strains showed a much higher population recombination rate than strains from other regions of the world [28]. The overall population recombination rate for the DBL3X region was estimated to ρ = 0.54 per bp (95% CI: 0.41–0.90). This is in accordance with a recent report for a VAR2CSA region upstream to DBL3X around interdomain 1, where the rate was estimated to 0.71 per bp [13], and in agreement with recombination rates in Chromosome 3 of African parasite lines summarized as ρ > 0.1 per bp [28]. The high population recombination rate in DBL3X combined with the observed sequence variation adjacent to the detected recombination hotspots, suggests that recombination is an important factor in generating sequence variation. The V1 region that seems to be most strongly affected by recombination is predicted to form a structurally unrestricted flexible loop allowing for sequence variation, and it is possible that the whole V1 region is under diversifying selection pressure exerted by the immune system, even though this could not be predicted by the dN/dS method due to gaps. This notion is supported by the findings discussed below, showing that this region is part of a major B cell epitope.

VAR2CSA epitopes exposed on the surface of the protein and accessible for immunoglobulin gamma (IgG) binding could be under diversifying selection resulting in escape mutations and high dN/dS ratios, whereas residues involved in protein folding, stability, and anchoring could be under purifying selection with low dN/dS ratios. To predict linear B cell epitopes, the 3D7 sequence was submitted to the BepiPred server [29] and seven epitopes were predicted within the DBL3X sequence (Figure 5C). Some of these epitopes were located in areas with high dN/dS ratios and there was a weak but statistically significant association between the BepiPred score and the dN/dS ratio (Pearson's r = 0.18 and p = 2.5*10⁻⁹). The reason for this weak association could be that antibody epitopes are situated in regions that are functionally constrained or that positive selection pressure is also driven by other forces like MHC-2 binding and T helper cell activation as found for HIV-1 [30]. Furthermore, the BepiPred algorithm predicts linear epitopes, and some of these could be located in parts of the molecule that are not accessible to antibodies in the native folded molecule. Nevertheless, most of the predicted epitopes (Figure 5G) were situated in surface-exposed loop regions of the model, and one of the highest scoring epitopes was in the V1 region. Residues that align directly to the glycan-binding residues of EBA-175 F1 and to the Duffy antigen receptor for chemokines (DARC) binding site in Pkα-DBL were not predicted to be part of epitopes. However, a part of the V2 region in proximity to the putative glycan-binding loop was predicted to be targeted by antibodies and had high dN/dS values.

To verify the above bioinformatical predictions, we evaluated the fine specificity of naturally acquired human antibodies to VAR2CSA DBL3X in a peptide array consisting of 442 overlapping 31mer peptides covering exon 1 of VAR2CSA of the 3D7 sequence. Antibody reactivity to individual amino acids was assigned on the basis of an algorithm based on the observation that a major part of antibody binding motifs in a set of conformational epitopes are from two to seven amino acids long, containing either two or three defined residues spaced by undefined amino acids [31]. The VAR2CSA of 3D7 contains 31,149 such motifs and each was assigned an average PepScan value by adding the measured reactivity from the 31mers in which the motif was present, and dividing with the number of times the motif occurred. The method, described in Materials and Methods, was validated by testing serum from rabbits immunized with a VAR2CSA construct, which showed that both the measurements based on individual peptide readings and the algorithm described above, mapped antibody responses to regions present in the antigen used for immunization (Figure S2). Furthermore, there was a high concurrence between antibody peaks defined by the reactivity of the individual peptides and the peaks defined by the algorithm defining single amino acid scores (Figure S2). Plasma from individuals not exposed to malaria did not react with any of the peptides in the array (unpublished data). The IgG reactivity in the plasma of eight Ghanaian women with a known history of a placental malaria infection was analyzed and the peptide array data was visualized on the DBL3X model (Figure 6). The regions with the highest reactivity were on the side of the domain where glycan-binding is found in EBA-175 F1 (Figure 6A and and6B)6B) and therefore IgG reactivity was visualized on a model positioned with this side in the front (Figure 6A and and6C–I).6C–I). It was clear that the majority of the individuals had specific IgG against the variable regions, V1 or V2. V3 is partly deleted in 3D7, and IgG reactivity could thus not be measured in the peptide array based on the 3D7 sequence. Remarkably, a short α-helix (Figure 6A, arrow 1) in proximity to the loop for glycan-binding in EBA-175 F1 showed the highest antibody reactivity in all serum samples, despite the fact that this region had very low dN/dS ratios (Figure 5A, positions 120–132). Another α-helix was also often recognized by antibodies (Figure 6A, arrow 2). This helix was predicted to contain a B cell epitope by the BepiPred algorithm and had polymorphic residues (Figure 5A and and5C,5C, positions 251–281). The region corresponding to the loops containing the EBA-175 F1 glycan-binding sites was not recognized by any of the serum samples. Conserved regions (Figure 6A, arrow 1) targeted by naturally acquired IgG are of considerable interest in the search for vaccine constructs which could elicit a broad protective immune response.

Binding to CSA (C9819, Sigma-Aldrich, http://www.sigmaaldrich.com) was determined in an ELISA system. ELISA plates (Falcon 351172) were coated overnight with CSA (50 μg/ml) in PBS at 4 °C. Coating with 1% BSA in PBS (blocking buffer) was used as negative control. Plates were incubated with blocking buffer for 1 h at room temperature (RT) to inhibit non-specific adsorption to the plate. The VAR2CSA proteins were diluted in blocking buffer (1–10 μg/ml protein), added to the wells, and incubated for 1 h at RT. For the inhibition assays, proteins (7 μg/ml) were pre-incubated with different concentrations of soluble CSA for 30 min. Plates were washed four times in PBS between the different steps. Specific binding was visualized by adding an HRP-conjugated antibody (R960–25, Invitrogen, http://www.invitrogen.com) targeting the V5 epitope of the constructs. Plates were incubated for 1 h with the anti-V5 antibody diluted 1:3000 in blocking buffer. The color reactions were developed for 15 min by the addition of o-phenylenediamine substrate and stopped by adding 2.5 M H₂SO₄. The optical density was measured at 490 nm.

The alignment of 43 placental and four database VAR2CSA DBL3X sequences, covering the 3D7 amino acid positions 1256–1549, was constructed using the software RevTrans [36], and subsequently manually corrected for errors. To cover more of the DBL3X domain, an alignment of 17 database sequences was constructed in the same way, covering the 3D7 positions 1217–1255. For the phylogenetic tree, 21 Malawian sequences [21] were aligned with the sequences mentioned above, again using RevTrans and manual correction, resulting in a 609-bp alignment. The program MrModeltest version 2.2 [37] was used to find the most appropriate nucleotide substitution model based on the Akaike information criterion [38]. Phylogenetic trees based on the above alignments were constructed by Bayesian inference using the program MrBayes version 3.1.1 [39]. In all cases Markov chain Monte Carlo (MCMC) sampling was performed for 10,000,000 generations with eight chains. Convergence was confirmed by comparing the results of two independent runs. Burn-in was determined using Tracer [40] and 50% majority rule consensus trees were constructed.