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Epitope mapping and topographic analysis of VAR2CSA DBL3X involved in P. falciparum placental sequestration.

Dahlbäck M, Rask TS, Andersen PH, Nielsen MA, Ndam NT, Resende M, Turner L, Deloron P, Hviid L, Lund O, Pedersen AG, Theander TG, Salanti A - PLoS Pathog. (2006)

Bottom Line: Interestingly, surface reactive anti-VAR2CSA antibodies also target a conserved DBL3X region predicted to form an alpha-helix.Finally, we could identify DBL3X sequence motifs that were more likely to occur in parasites isolated from primi- and multigravidae, respectively.These findings strengthen the vaccine candidacy of VAR2CSA and will be important for choosing epitopes and variants of DBL3X to be included in a vaccine protecting women against pregnancy-associated malaria.

View Article: PubMed Central - PubMed

Affiliation: Centre for Medical Parasitology, University of Copenhagen and Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark.

ABSTRACT
Pregnancy-associated malaria is a major health problem, which mainly affects primigravidae living in malaria endemic areas. The syndrome is precipitated by accumulation of infected erythrocytes in placental tissue through an interaction between chondroitin sulphate A on syncytiotrophoblasts and a parasite-encoded protein on the surface of infected erythrocytes, believed to be VAR2CSA. VAR2CSA is a polymorphic protein of approximately 3,000 amino acids forming six Duffy-binding-like (DBL) domains. For vaccine development it is important to define the antigenic targets for protective antibodies and to characterize the consequences of sequence variation. In this study, we used a combination of in silico tools, peptide arrays, and structural modeling to show that sequence variation mainly occurs in regions under strong diversifying selection, predicted to form flexible loops. These regions are the main targets of naturally acquired immunoglobulin gamma and accessible for antibodies reacting with native VAR2CSA on infected erythrocytes. Interestingly, surface reactive anti-VAR2CSA antibodies also target a conserved DBL3X region predicted to form an alpha-helix. Finally, we could identify DBL3X sequence motifs that were more likely to occur in parasites isolated from primi- and multigravidae, respectively. These findings strengthen the vaccine candidacy of VAR2CSA and will be important for choosing epitopes and variants of DBL3X to be included in a vaccine protecting women against pregnancy-associated malaria.

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Sequence Differences in Infections with Different Parity(A) Kullback-Leibler sequence logo based on a multiple alignment of the DBL3X region (Figure 3). The sequences in the alignment were split into two groups of equal size: 21 sequences obtained from primigravidae and 21 sequences from multigravidae women. The x-axis shows amino acid positions in the alignment. The height of a position indicates the amount of difference between the two groups according to the symmetric Kullback-Leibler distance, and the letters indicate the amino acids contributing to this difference. The letter “O” has been chosen to signify gaps in the alignment. Polar amino acids are green, basic are blue, acidic are red, and hydrophobic amino acids are black. P-values indicating the significance of DKL based on the parity grouping compared to random groupings are shown below positions where p < 0.05.(B) DBL3X model showing the position of amino acids that were differentially found in primi-gravidae and multigravidae women. Residues in the region 135–175 of moderately significant difference are shown in orange, and highly significant residues (positions 158, 161, and 162) are shown in red.(C) The model (B) rotated 180 degrees so it is positioned similar to Figure 1C.
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ppat-0020124-g004: Sequence Differences in Infections with Different Parity(A) Kullback-Leibler sequence logo based on a multiple alignment of the DBL3X region (Figure 3). The sequences in the alignment were split into two groups of equal size: 21 sequences obtained from primigravidae and 21 sequences from multigravidae women. The x-axis shows amino acid positions in the alignment. The height of a position indicates the amount of difference between the two groups according to the symmetric Kullback-Leibler distance, and the letters indicate the amino acids contributing to this difference. The letter “O” has been chosen to signify gaps in the alignment. Polar amino acids are green, basic are blue, acidic are red, and hydrophobic amino acids are black. P-values indicating the significance of DKL based on the parity grouping compared to random groupings are shown below positions where p < 0.05.(B) DBL3X model showing the position of amino acids that were differentially found in primi-gravidae and multigravidae women. Residues in the region 135–175 of moderately significant difference are shown in orange, and highly significant residues (positions 158, 161, and 162) are shown in red.(C) The model (B) rotated 180 degrees so it is positioned similar to Figure 1C.

Mentions: 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 (DKL) between sequences from the primigravidae and the multigravidae was higher than for randomly chosen groupings of the sequences (p = 0.0075). The DKL 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 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.


Epitope mapping and topographic analysis of VAR2CSA DBL3X involved in P. falciparum placental sequestration.

Dahlbäck M, Rask TS, Andersen PH, Nielsen MA, Ndam NT, Resende M, Turner L, Deloron P, Hviid L, Lund O, Pedersen AG, Theander TG, Salanti A - PLoS Pathog. (2006)

Sequence Differences in Infections with Different Parity(A) Kullback-Leibler sequence logo based on a multiple alignment of the DBL3X region (Figure 3). The sequences in the alignment were split into two groups of equal size: 21 sequences obtained from primigravidae and 21 sequences from multigravidae women. The x-axis shows amino acid positions in the alignment. The height of a position indicates the amount of difference between the two groups according to the symmetric Kullback-Leibler distance, and the letters indicate the amino acids contributing to this difference. The letter “O” has been chosen to signify gaps in the alignment. Polar amino acids are green, basic are blue, acidic are red, and hydrophobic amino acids are black. P-values indicating the significance of DKL based on the parity grouping compared to random groupings are shown below positions where p < 0.05.(B) DBL3X model showing the position of amino acids that were differentially found in primi-gravidae and multigravidae women. Residues in the region 135–175 of moderately significant difference are shown in orange, and highly significant residues (positions 158, 161, and 162) are shown in red.(C) The model (B) rotated 180 degrees so it is positioned similar to Figure 1C.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636682&req=5

ppat-0020124-g004: Sequence Differences in Infections with Different Parity(A) Kullback-Leibler sequence logo based on a multiple alignment of the DBL3X region (Figure 3). The sequences in the alignment were split into two groups of equal size: 21 sequences obtained from primigravidae and 21 sequences from multigravidae women. The x-axis shows amino acid positions in the alignment. The height of a position indicates the amount of difference between the two groups according to the symmetric Kullback-Leibler distance, and the letters indicate the amino acids contributing to this difference. The letter “O” has been chosen to signify gaps in the alignment. Polar amino acids are green, basic are blue, acidic are red, and hydrophobic amino acids are black. P-values indicating the significance of DKL based on the parity grouping compared to random groupings are shown below positions where p < 0.05.(B) DBL3X model showing the position of amino acids that were differentially found in primi-gravidae and multigravidae women. Residues in the region 135–175 of moderately significant difference are shown in orange, and highly significant residues (positions 158, 161, and 162) are shown in red.(C) The model (B) rotated 180 degrees so it is positioned similar to Figure 1C.
Mentions: 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 (DKL) between sequences from the primigravidae and the multigravidae was higher than for randomly chosen groupings of the sequences (p = 0.0075). The DKL 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 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.

Bottom Line: Interestingly, surface reactive anti-VAR2CSA antibodies also target a conserved DBL3X region predicted to form an alpha-helix.Finally, we could identify DBL3X sequence motifs that were more likely to occur in parasites isolated from primi- and multigravidae, respectively.These findings strengthen the vaccine candidacy of VAR2CSA and will be important for choosing epitopes and variants of DBL3X to be included in a vaccine protecting women against pregnancy-associated malaria.

View Article: PubMed Central - PubMed

Affiliation: Centre for Medical Parasitology, University of Copenhagen and Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark.

ABSTRACT
Pregnancy-associated malaria is a major health problem, which mainly affects primigravidae living in malaria endemic areas. The syndrome is precipitated by accumulation of infected erythrocytes in placental tissue through an interaction between chondroitin sulphate A on syncytiotrophoblasts and a parasite-encoded protein on the surface of infected erythrocytes, believed to be VAR2CSA. VAR2CSA is a polymorphic protein of approximately 3,000 amino acids forming six Duffy-binding-like (DBL) domains. For vaccine development it is important to define the antigenic targets for protective antibodies and to characterize the consequences of sequence variation. In this study, we used a combination of in silico tools, peptide arrays, and structural modeling to show that sequence variation mainly occurs in regions under strong diversifying selection, predicted to form flexible loops. These regions are the main targets of naturally acquired immunoglobulin gamma and accessible for antibodies reacting with native VAR2CSA on infected erythrocytes. Interestingly, surface reactive anti-VAR2CSA antibodies also target a conserved DBL3X region predicted to form an alpha-helix. Finally, we could identify DBL3X sequence motifs that were more likely to occur in parasites isolated from primi- and multigravidae, respectively. These findings strengthen the vaccine candidacy of VAR2CSA and will be important for choosing epitopes and variants of DBL3X to be included in a vaccine protecting women against pregnancy-associated malaria.

Show MeSH
Related in: MedlinePlus