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High throughput functional assays of the variant antigen PfEMP1 reveal a single domain in the 3D7 Plasmodium falciparum genome that binds ICAM1 with high affinity and is targeted by naturally acquired neutralizing antibodies.

Oleinikov AV, Amos E, Frye IT, Rossnagle E, Mutabingwa TK, Fried M, Duffy PE - PLoS Pathog. (2009)

Bottom Line: Neutralizing antibodies were uncommon in children but common in immune adults from east Africa.Inhibition of binding was much more efficient than reversal of binding, indicating a strong interaction between DBL2betaC2(PF11_0521) and ICAM1.Our high throughput approach will significantly accelerate studies of PfEMP1 binding domains and protective antibody responses.

View Article: PubMed Central - PubMed

Affiliation: Seattle Biomedical Research Institute, Seattle, Washington, USA. andrew.oleinikov@sbri.org

ABSTRACT
Plasmodium falciparum-infected erythrocytes bind endothelial receptors to sequester in vascular beds, and binding to ICAM1 has been implicated in cerebral malaria. Binding to ICAM1 may be mediated by the variant surface antigen family PfEMP1: for example, 6 of 21 DBLbetaC2 domains from the IT4 strain PfEMP1 repertoire were shown to bind ICAM1, and the PfEMP1 containing these 6 domains are all classified as Group B or C type. In this study, we surveyed binding of ICAM1 to 16 DBLbetaC2 domains of the 3D7 strain PfEMP1 repertoire, using a high throughput Bioplex assay format. Only one DBL2betaC2 domain from the Group A PfEMP1 PF11_0521 showed strong specific binding. Among these 16 domains, DBL2betaC2(PF11_0521) best preserved the residues previously identified as conserved in ICAM1-binding versus non-binding domains. Our analyses further highlighted the potential role of conserved residues within predominantly non-conserved flexible loops in adhesion, and, therefore, as targets for intervention. Our studies also suggest that the structural/functional DBLbetaC2 domain involved in ICAM1 binding includes about 80 amino acid residues upstream of the previously suggested DBLbetaC2 domain. DBL2betaC2(PF11_0521) binding to ICAM1 was inhibited by immune sera from east Africa but not by control US sera. Neutralizing antibodies were uncommon in children but common in immune adults from east Africa. Inhibition of binding was much more efficient than reversal of binding, indicating a strong interaction between DBL2betaC2(PF11_0521) and ICAM1. Our high throughput approach will significantly accelerate studies of PfEMP1 binding domains and protective antibody responses.

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Inhibition of ICAM1 binding to DBL2βC2PF11_0521 domain by sera obtained from young children living in a malaria-endemic region is infrequent.Plasma samples collected from children between 24 and 148 weeks of age were used to inhibit ICAM1 binding to DBL2βC2PF11_0521 domain (A) and to measure the reactivity of plasma IgG to DBL2βC2PF11_0521 domain (B). Levels of binding after incubation with pooled plasma from non-immune (NI) or immune adults are indicated (thickness of each line corresponds to the standard deviation margin). Different colors indicate different children donating samples at sequential time points during early life. Error bars are standard deviations of duplicate measurements for each data point. Similar results were obtained in 3 independent experiments.
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ppat-1000386-g005: Inhibition of ICAM1 binding to DBL2βC2PF11_0521 domain by sera obtained from young children living in a malaria-endemic region is infrequent.Plasma samples collected from children between 24 and 148 weeks of age were used to inhibit ICAM1 binding to DBL2βC2PF11_0521 domain (A) and to measure the reactivity of plasma IgG to DBL2βC2PF11_0521 domain (B). Levels of binding after incubation with pooled plasma from non-immune (NI) or immune adults are indicated (thickness of each line corresponds to the standard deviation margin). Different colors indicate different children donating samples at sequential time points during early life. Error bars are standard deviations of duplicate measurements for each data point. Similar results were obtained in 3 independent experiments.

Mentions: To study the acquisition of neutralizing antibodies against the ICAM1 binding interaction, we assayed plasma samples collected from infants and toddlers participating in longitudinal birth cohort studies in Tanzania. Plasma from 7 children that were collected at several time points between 24 and 148 weeks of age, were tested for inhibition of ICAM1 binding to DBL2βC2PF11_0521 domain (Figure 5A). In parallel, we tested reactivity of IgG from the same plasma to DBL2βC2PF11_0521 domain (Figure 5B). Inhibition of ICAM1 binding activity was uncommon, and appeared to be short-lived in at least one child. The IgG reactivity curves appear almost as mirror images of the ICAM1 binding-inhibition curves (with the exception of the 148 week time point for the brown line child, discussed below), suggesting that naturally acquired anti-DBL2βC2PF11_0521 domain antibodies include at least a fraction of functional antibodies as they develop in individual children.


High throughput functional assays of the variant antigen PfEMP1 reveal a single domain in the 3D7 Plasmodium falciparum genome that binds ICAM1 with high affinity and is targeted by naturally acquired neutralizing antibodies.

Oleinikov AV, Amos E, Frye IT, Rossnagle E, Mutabingwa TK, Fried M, Duffy PE - PLoS Pathog. (2009)

Inhibition of ICAM1 binding to DBL2βC2PF11_0521 domain by sera obtained from young children living in a malaria-endemic region is infrequent.Plasma samples collected from children between 24 and 148 weeks of age were used to inhibit ICAM1 binding to DBL2βC2PF11_0521 domain (A) and to measure the reactivity of plasma IgG to DBL2βC2PF11_0521 domain (B). Levels of binding after incubation with pooled plasma from non-immune (NI) or immune adults are indicated (thickness of each line corresponds to the standard deviation margin). Different colors indicate different children donating samples at sequential time points during early life. Error bars are standard deviations of duplicate measurements for each data point. Similar results were obtained in 3 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2663049&req=5

ppat-1000386-g005: Inhibition of ICAM1 binding to DBL2βC2PF11_0521 domain by sera obtained from young children living in a malaria-endemic region is infrequent.Plasma samples collected from children between 24 and 148 weeks of age were used to inhibit ICAM1 binding to DBL2βC2PF11_0521 domain (A) and to measure the reactivity of plasma IgG to DBL2βC2PF11_0521 domain (B). Levels of binding after incubation with pooled plasma from non-immune (NI) or immune adults are indicated (thickness of each line corresponds to the standard deviation margin). Different colors indicate different children donating samples at sequential time points during early life. Error bars are standard deviations of duplicate measurements for each data point. Similar results were obtained in 3 independent experiments.
Mentions: To study the acquisition of neutralizing antibodies against the ICAM1 binding interaction, we assayed plasma samples collected from infants and toddlers participating in longitudinal birth cohort studies in Tanzania. Plasma from 7 children that were collected at several time points between 24 and 148 weeks of age, were tested for inhibition of ICAM1 binding to DBL2βC2PF11_0521 domain (Figure 5A). In parallel, we tested reactivity of IgG from the same plasma to DBL2βC2PF11_0521 domain (Figure 5B). Inhibition of ICAM1 binding activity was uncommon, and appeared to be short-lived in at least one child. The IgG reactivity curves appear almost as mirror images of the ICAM1 binding-inhibition curves (with the exception of the 148 week time point for the brown line child, discussed below), suggesting that naturally acquired anti-DBL2βC2PF11_0521 domain antibodies include at least a fraction of functional antibodies as they develop in individual children.

Bottom Line: Neutralizing antibodies were uncommon in children but common in immune adults from east Africa.Inhibition of binding was much more efficient than reversal of binding, indicating a strong interaction between DBL2betaC2(PF11_0521) and ICAM1.Our high throughput approach will significantly accelerate studies of PfEMP1 binding domains and protective antibody responses.

View Article: PubMed Central - PubMed

Affiliation: Seattle Biomedical Research Institute, Seattle, Washington, USA. andrew.oleinikov@sbri.org

ABSTRACT
Plasmodium falciparum-infected erythrocytes bind endothelial receptors to sequester in vascular beds, and binding to ICAM1 has been implicated in cerebral malaria. Binding to ICAM1 may be mediated by the variant surface antigen family PfEMP1: for example, 6 of 21 DBLbetaC2 domains from the IT4 strain PfEMP1 repertoire were shown to bind ICAM1, and the PfEMP1 containing these 6 domains are all classified as Group B or C type. In this study, we surveyed binding of ICAM1 to 16 DBLbetaC2 domains of the 3D7 strain PfEMP1 repertoire, using a high throughput Bioplex assay format. Only one DBL2betaC2 domain from the Group A PfEMP1 PF11_0521 showed strong specific binding. Among these 16 domains, DBL2betaC2(PF11_0521) best preserved the residues previously identified as conserved in ICAM1-binding versus non-binding domains. Our analyses further highlighted the potential role of conserved residues within predominantly non-conserved flexible loops in adhesion, and, therefore, as targets for intervention. Our studies also suggest that the structural/functional DBLbetaC2 domain involved in ICAM1 binding includes about 80 amino acid residues upstream of the previously suggested DBLbetaC2 domain. DBL2betaC2(PF11_0521) binding to ICAM1 was inhibited by immune sera from east Africa but not by control US sera. Neutralizing antibodies were uncommon in children but common in immune adults from east Africa. Inhibition of binding was much more efficient than reversal of binding, indicating a strong interaction between DBL2betaC2(PF11_0521) and ICAM1. Our high throughput approach will significantly accelerate studies of PfEMP1 binding domains and protective antibody responses.

Show MeSH
Related in: MedlinePlus