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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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Phylogenetic Relationship between Different VAR2CSA DBL3X SequencesBayesian inference tree of 43 placental cDNA sequences from Senegal (blue), 21 Malawian sequences (orange), and database sequences from four isolates (green) of different geographic origin. The posterior probability of the clades is shown at the branches.
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ppat-0020124-g002: Phylogenetic Relationship between Different VAR2CSA DBL3X SequencesBayesian inference tree of 43 placental cDNA sequences from Senegal (blue), 21 Malawian sequences (orange), and database sequences from four isolates (green) of different geographic origin. The posterior probability of the clades is shown at the branches.

Mentions: 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.


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)

Phylogenetic Relationship between Different VAR2CSA DBL3X SequencesBayesian inference tree of 43 placental cDNA sequences from Senegal (blue), 21 Malawian sequences (orange), and database sequences from four isolates (green) of different geographic origin. The posterior probability of the clades is shown at the branches.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-0020124-g002: Phylogenetic Relationship between Different VAR2CSA DBL3X SequencesBayesian inference tree of 43 placental cDNA sequences from Senegal (blue), 21 Malawian sequences (orange), and database sequences from four isolates (green) of different geographic origin. The posterior probability of the clades is shown at the branches.
Mentions: 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.

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