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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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Recombinant DBL3X VAR2CSA Binds CSA and the Structure of the Domain Can Be Modeled on the Basis of the Structure of the DBL Domains of EBA-175(A) Binding assay. DBL3X binds to CSA in a protein concentration-specific manner. ELISA plates were coated with soluble CSA and binding of recombinant proteins at different concentrations was determined. DBL3X from 3D7 shows better binding compared to DBL3X from FCR3. The non-CSA binding VAR2CSA DBL4ɛ was included as a control. Results are the mean of three binding assays and the error bars indicate the standard deviation.(B) Inhibition assay. Recombinant DBL3X proteins (7 μg/ml) were preincubated with increasing amounts of soluble CSA, and binding to CSA-coated plates was determined. Binding of DBL3X is dose-dependently inhibited by soluble CSA. Results are the mean of four inhibition binding assays and error bars indicate the standard deviation.(C) Model of DBL3X using EBA-175 as template. The figure shows a superposition of the EBA-175 F1 domain in light blue and the DBL3X (PFL0030c amino acids 1,217–1,559) model in yellow with insertions highlighted in red. Arrows indicate insertions, which align with flexible loop regions in Pkα-DBL (L1) and EBA-175 F1 (L2). Side chains of glycan-binding residues in EBA-175 F1 are shown in black. Arrow 3 indicates the corresponding Pkα-DBL Duffy antigen receptor for chemokine-binding site.
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ppat-0020124-g001: Recombinant DBL3X VAR2CSA Binds CSA and the Structure of the Domain Can Be Modeled on the Basis of the Structure of the DBL Domains of EBA-175(A) Binding assay. DBL3X binds to CSA in a protein concentration-specific manner. ELISA plates were coated with soluble CSA and binding of recombinant proteins at different concentrations was determined. DBL3X from 3D7 shows better binding compared to DBL3X from FCR3. The non-CSA binding VAR2CSA DBL4ɛ was included as a control. Results are the mean of three binding assays and the error bars indicate the standard deviation.(B) Inhibition assay. Recombinant DBL3X proteins (7 μg/ml) were preincubated with increasing amounts of soluble CSA, and binding to CSA-coated plates was determined. Binding of DBL3X is dose-dependently inhibited by soluble CSA. Results are the mean of four inhibition binding assays and error bars indicate the standard deviation.(C) Model of DBL3X using EBA-175 as template. The figure shows a superposition of the EBA-175 F1 domain in light blue and the DBL3X (PFL0030c amino acids 1,217–1,559) model in yellow with insertions highlighted in red. Arrows indicate insertions, which align with flexible loop regions in Pkα-DBL (L1) and EBA-175 F1 (L2). Side chains of glycan-binding residues in EBA-175 F1 are shown in black. Arrow 3 indicates the corresponding Pkα-DBL Duffy antigen receptor for chemokine-binding site.

Mentions: It has previously been shown that DBL3X expressed on the surface of Chinese hamster ovary cells binds CSA in vitro [14]. However, it is important to test the CSA-binding properties of secreted VAR2CSA proteins produced in expression systems that could allow for larger-scale production of a vaccine. For this study, recombinant HIS-tagged proteins were produced in Baculovirus-transfected insect cells and binding to CSA was determined in an enzyme-linked immunosorbent assay (ELISA) system. It has previously been suggested that the FCR3 DBL3X domain binds CSA, whereas the 3D7 DBL3X domain does not [14]. We found that both variants had affinity to CSA and that the 3D7 variant exhibited the strongest binding. Binding of both FCR3 and 3D7 DBL3X was concentration-dependent (Figure 1A) and could be inhibited by soluble CSA in a dose-dependant manner (Figure 1B).


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)

Recombinant DBL3X VAR2CSA Binds CSA and the Structure of the Domain Can Be Modeled on the Basis of the Structure of the DBL Domains of EBA-175(A) Binding assay. DBL3X binds to CSA in a protein concentration-specific manner. ELISA plates were coated with soluble CSA and binding of recombinant proteins at different concentrations was determined. DBL3X from 3D7 shows better binding compared to DBL3X from FCR3. The non-CSA binding VAR2CSA DBL4ɛ was included as a control. Results are the mean of three binding assays and the error bars indicate the standard deviation.(B) Inhibition assay. Recombinant DBL3X proteins (7 μg/ml) were preincubated with increasing amounts of soluble CSA, and binding to CSA-coated plates was determined. Binding of DBL3X is dose-dependently inhibited by soluble CSA. Results are the mean of four inhibition binding assays and error bars indicate the standard deviation.(C) Model of DBL3X using EBA-175 as template. The figure shows a superposition of the EBA-175 F1 domain in light blue and the DBL3X (PFL0030c amino acids 1,217–1,559) model in yellow with insertions highlighted in red. Arrows indicate insertions, which align with flexible loop regions in Pkα-DBL (L1) and EBA-175 F1 (L2). Side chains of glycan-binding residues in EBA-175 F1 are shown in black. Arrow 3 indicates the corresponding Pkα-DBL Duffy antigen receptor for chemokine-binding site.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1636682&req=5

ppat-0020124-g001: Recombinant DBL3X VAR2CSA Binds CSA and the Structure of the Domain Can Be Modeled on the Basis of the Structure of the DBL Domains of EBA-175(A) Binding assay. DBL3X binds to CSA in a protein concentration-specific manner. ELISA plates were coated with soluble CSA and binding of recombinant proteins at different concentrations was determined. DBL3X from 3D7 shows better binding compared to DBL3X from FCR3. The non-CSA binding VAR2CSA DBL4ɛ was included as a control. Results are the mean of three binding assays and the error bars indicate the standard deviation.(B) Inhibition assay. Recombinant DBL3X proteins (7 μg/ml) were preincubated with increasing amounts of soluble CSA, and binding to CSA-coated plates was determined. Binding of DBL3X is dose-dependently inhibited by soluble CSA. Results are the mean of four inhibition binding assays and error bars indicate the standard deviation.(C) Model of DBL3X using EBA-175 as template. The figure shows a superposition of the EBA-175 F1 domain in light blue and the DBL3X (PFL0030c amino acids 1,217–1,559) model in yellow with insertions highlighted in red. Arrows indicate insertions, which align with flexible loop regions in Pkα-DBL (L1) and EBA-175 F1 (L2). Side chains of glycan-binding residues in EBA-175 F1 are shown in black. Arrow 3 indicates the corresponding Pkα-DBL Duffy antigen receptor for chemokine-binding site.
Mentions: It has previously been shown that DBL3X expressed on the surface of Chinese hamster ovary cells binds CSA in vitro [14]. However, it is important to test the CSA-binding properties of secreted VAR2CSA proteins produced in expression systems that could allow for larger-scale production of a vaccine. For this study, recombinant HIS-tagged proteins were produced in Baculovirus-transfected insect cells and binding to CSA was determined in an enzyme-linked immunosorbent assay (ELISA) system. It has previously been suggested that the FCR3 DBL3X domain binds CSA, whereas the 3D7 DBL3X domain does not [14]. We found that both variants had affinity to CSA and that the 3D7 variant exhibited the strongest binding. Binding of both FCR3 and 3D7 DBL3X was concentration-dependent (Figure 1A) and could be inhibited by soluble CSA in a dose-dependant manner (Figure 1B).

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