scholarly journals Identification of VAR2CSA Domain-Specific Inhibitory Antibodies of the Plasmodium falciparum Erythrocyte Membrane Protein 1 Using a Novel Flow Cytometry Assay

2013 ◽  
Vol 20 (3) ◽  
pp. 433-442 ◽  
Author(s):  
Harold Obiakor ◽  
Marion Avril ◽  
Nicholas J. MacDonald ◽  
Prakash Srinivasan ◽  
Karine Reiter ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Plasmodium Falciparum ◽  
Membrane Protein ◽  
Chondroitin Sulfate ◽  
Erythrocyte Membrane ◽  
Recombinant Proteins ◽  
Binding Assay ◽  
Content Type ◽  
Erythrocyte Membrane Protein ◽  
First Time

ABSTRACTVAR2CSA, a member of thePlasmodium falciparumerythrocyte membrane protein 1 (PfEMP1) family, is a leading candidate for use in vaccines to protect first-time mothers from placental malaria (PM). VAR2CSA, which is comprised of a series of six Duffy binding-like (DBL) domains, binds chondroitin sulfate A (CSA) on placental syncytiotrophoblast. Several recombinant DBL domains have been shown to bind CSA. In order to identify and develop recombinant proteins suitable for clinical development, DBL2X and DBL3X, as well as their respective third subdomain (S3) from the FCR3 parasite clone, were expressed inEscherichia coli, refolded, and purified. All but DBL3X-S3 recombinant proteins bound to CSA expressed on Chinese hamster ovary (CHO)-K1 cells but not to CHO-pgsA745 cells, which are CSA negative as determined by flow cytometry. All but DBL3X-S3 bound to CSA on chondroitin sulfate proteoglycan (CSPG) as determined by surface plasmon resonance (SPR) analysis. Purified IgG from rats and rabbits immunized with these four recombinant proteins bound homologous and some heterologous parasite-infected erythrocytes (IE). Using a novel flow cytometry inhibition-of-binding assay (flow-IBA), antibodies against DBL3X-S3 inhibited 35% and 45% of IE binding to CSA on CHO-K1 cells compared to results for soluble CSA (sCSA) and purified multigravida (MG) IgG, respectively, from areas in Tanzania to which malaria is endemic. Antibodies generated against the other domains provided little or no inhibition of IE binding to CSA on CHO-K1 cells as determined by the flow cytometry inhibition-of-binding assay. These results demonstrate for the first time the ability to identify antibodies to VAR2CSA DBL domains and subdomains capable of inhibiting VAR2CSA parasite-IE binding to CSA by flow cytometry. The flow cytometry inhibition-of-binding assay was robust and provided an accurate, reproducible, and reliable means to identify blocking of IE binding to CSA and promises to be significant in the development of a vaccine to protect pregnant women.

scholarly journals Immunization with Recombinant Plasmodium falciparum Erythrocyte Membrane Protein 1 CIDRα1 Domains Induces Domain Subtype Inhibitory Antibodies

2018 ◽  
Vol 86 (11) ◽  
Author(s):  
Louise Turner ◽  
Thor G. Theander ◽  
Thomas Lavstsen
Keyword(s):  
Plasmodium Falciparum ◽  
Membrane Protein ◽  
Erythrocyte Membrane ◽  
Sequence Similarity ◽  
Optimal Choice ◽  
Immune Recognition ◽  
Endothelial Protein C Receptor ◽  
Content Type ◽  
Erythrocyte Membrane Protein ◽  
Inhibitory Antibodies

ABSTRACT Plasmodium falciparum malaria pathogenesis is tied to the sequestration of parasites in the microvasculature. Parasite sequestration leading to severe malaria is mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1) binding to endothelial protein C receptor (EPCR) via its CIDRα1 domains. CIDRα1 domains are targets of naturally acquired immunity, and a vaccine eliciting antibodies inhibiting the EPCR binding of CIDRα1 could potentially prevent disease and death from malaria. CIDRα1 domains have diversified in sequence to escape immune recognition but preserved structure to maintain EPCR binding. The EPCR-binding CIDRα1 domains separate into six major sequence types predicted to form a conserved structure in which only the amino acids essential for EPCR binding are highly conserved. Here, we investigated whether antibodies elicited by vaccination with single or multiple recombinant CIDRα1 domains are able to bind and inhibit diverse CIDRα1 domains. We found that EPCR binding-inhibitory antibodies to CIDRα1 variants closely related to those used for vaccination are readily elicited, whereas antibodies binding distant CIDRα1 variants are sporadically generated and are rarely inhibitory. Despite this, sequence similarity correlated poorly with the ability of induced antibodies to inhibit across diverse variants, and no continuous sequence regions of importance for cross-inhibitory antibodies could be identified. This suggested that epitopes of cross-variant inhibitory antibodies were predominantly conformational. Vaccination with immunogens engineered to focus immune responses to specific epitopes or an optimal choice of multiple CIDRα1 variants may improve elicitation of broadly reactive and inhibitory antibody responses.

Overlapping antigenic repertoires of variant antigens expressed on the surface of erythrocytes infected by Plasmodium falciparum

Parasitology ◽  
1999 ◽  
Vol 119 (1) ◽  
pp. 7-17 ◽  
Author(s):  
H. A. GIHA ◽  
T. STAALSOE ◽  
D. DODOO ◽  
I. M. ELHASSAN ◽  
C. ROPER ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Plasmodium Falciparum ◽  
Membrane Protein ◽  
Malaria Transmission ◽  
Erythrocyte Membrane ◽  
Clonal Variation ◽  
Single Infection ◽  
Erythrocyte Membrane Protein ◽  
Degree Of Overlap ◽  
Eastern Sudan

Antibodies against variable antigens expressed on the surface of Plasmodium falciparum-infected erythrocytes are believed to be important for protection against malaria. A target for these antibodies is the P. falciparum erythrocyte membrane protein 1, PfEMP1, which is encoded by around 50 var genes and undergoes clonal variation. Using agglutination and mixed agglutination tests and flow cytometry to analyse the recognition of variant antigens on parasitized erythrocytes by plasma antibodies from individuals living in Daraweesh in eastern Sudan, an area of seasonal and unstable malaria transmission, we show that these antibodies recognize different variant antigens expressed by parasites of different genotype. Comparing the levels and acquisition of antibody to variant antigens in pairs of parasite isolates expressing different variant types, there is a correlation between the acquisition of antibodies to some combinations of variant antigens but not to others. These results indicate that (1) a single infection will induce the production of antibodies recognizing several variants of surface-expressed antigens, (2) the repertoire of variable antigens expressed by different parasites is overlapping and the degree of overlap differs between isolates, and (3) the expression of at least some variant antigens is genetically linked.

scholarly journals Neonatal and Maternal Immunological Responses to Conserved Epitopes within the DBL-γ3 Chondroitin Sulfate A-Binding Domain of Plasmodium falciparum Erythrocyte Membrane Protein 1

2005 ◽  
Vol 73 (12) ◽  
pp. 7988-7995 ◽  
Author(s):  
Kim Brustoski ◽  
Martin Kramer ◽  
Ulrike Möller ◽  
Peter G. Kremsner ◽  
Adrian J. F. Luty
Keyword(s):  
T Cells ◽  
Plasmodium Falciparum ◽  
Membrane Protein ◽  
Chondroitin Sulfate ◽  
Erythrocyte Membrane ◽  
Venous Blood ◽  
Binding Domain ◽  
Erythrocyte Membrane Protein ◽  
Conserved Regions ◽  
Chondroitin Sulfate A

ABSTRACT Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) mediates the adherence of P. falciparum-infected erythrocytes to placental syncytiotrophoblasts via interactions with chondroitin sulfate A (CSA), a characteristic of pregnancy-associated malaria. Pregnancy-associated malaria predicts increased susceptibility of newborns to malaria, and it is postulated that transplacental passage of parasite antigen induces immune regulatory activity in the neonate. We wished to examine the immune responsiveness to a CSA-binding domain of PfEMP1, the DBL-γ3 domain, in cord and maternal venous blood obtained from pregnancies with various histories of P. falciparum infection. We assessed in vitro T-cell cytokine and plasma immunoglobulin G (IgG) and IgM responses to four peptides corresponding to highly conserved regions of a DBL-γ3 domain common to central African parasite isolates. The presence of placental P. falciparum infection at delivery was associated with elevated frequencies of DBL-γ3 peptide-specific CD3+ interleukin-10-positive T cells in cord blood, while treatment and clearance of infection prior to delivery was associated with elevated frequencies of CD3+ gamma interferon-positive T cells. DBL-γ3 peptide-specific IgM antibodies were detected in 12 of 60 (20%) cord plasma samples from those born to mothers with P. falciparum infection during pregnancy. Consistent with polyclonal anti-PfEMP1 antibody responses that are associated with protection against pregnancy-associated malaria, the presence of maternal IgG antibodies with specificity for one of the DBL-γ3 peptides showed a parity-dependent profile. These data demonstrate that peptides corresponding to conserved regions of the DBL-γ3 domain of PfEMP1 are immunogenic in P. falciparum-infected mothers and their offspring.

scholarly journals The Severity of Plasmodium falciparum Infection Is Associated with Transcript Levels of var Genes Encoding Endothelial Protein C Receptor-Binding P. falciparum Erythrocyte Membrane Protein 1

2017 ◽  
Vol 85 (4) ◽  
Author(s):  
Sixbert I. Mkumbaye ◽  
Christian W. Wang ◽  
Eric Lyimo ◽  
Jakob S. Jespersen ◽  
Alphaxard Manjurano ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Membrane Protein ◽  
Severe Malaria ◽  
Erythrocyte Membrane ◽  
Protein C ◽  
Transcript Level ◽  
Endothelial Protein C Receptor ◽  
Transcript Levels ◽  
Content Type ◽  
Erythrocyte Membrane Protein

ABSTRACT By attaching infected erythrocytes to the vascular lining, Plasmodium falciparum parasites leave blood circulation and avoid splenic clearance. This sequestration is central to pathogenesis. Severe malaria is associated with parasites expressing an antigenically distinct P. falciparum erythrocyte membrane protein 1 (PfEMP1) subset mediating binding to endothelial receptors. Previous studies indicate that PfEMP1 adhesins with so-called CIDRα1 domains capable of binding endothelial protein C receptor (EPCR) constitute the PfEMP1 subset associated with severe pediatric malaria. To analyze the relative importance of different subtypes of CIDRα1 domains, we compared Pfemp1 transcript levels in children with severe malaria (including 9 fatal and 114 surviving cases), children hospitalized with uncomplicated malaria (n = 42), children with mild malaria not requiring hospitalization (n = 10), and children with parasitemia and no ongoing fever (n = 12). High levels of transcripts encoding EPCR-binding PfEMP1 were found in patients with symptomatic infections, and the abundance of these transcripts increased with disease severity. The compositions of CIDRα1 subtype transcripts varied markedly between patients, and none of the subtypes were dominant. Transcript-level analyses targeting other domain types indicated that subtypes of DBLβ or DBLζ domains might mediate binding phenomena that, in conjunction with EPCR binding, could contribute to pathogenesis. These observations strengthen the rationale for targeting the PfEMP1-EPCR interaction by vaccines and adjunctive therapies. Interventions should target EPCR binding of all CIDRα1 subtypes.

scholarly journals Multiple Plasmodium falciparum Erythrocyte Membrane Protein 1 Variants per Genome Can Bind IgM via Its Fc Fragment Fcμ

2015 ◽  
Vol 83 (10) ◽  
pp. 3972-3981 ◽  
Author(s):  
Anine Jeppesen ◽  
Sisse Bolm Ditlev ◽  
Vladyslav Soroka ◽  
Liz Stevenson ◽  
Louise Turner ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Membrane Protein ◽  
Erythrocyte Membrane ◽  
Placental Malaria ◽  
Clinical Manifestations ◽  
Content Type ◽  
Erythrocyte Membrane Protein ◽  
C Terminus ◽  
Adhesive Proteins ◽  
New Evidence

ThePlasmodium falciparumerythrocyte membrane protein 1 (PfEMP1) adhesive proteins expressed on the surfaces of infected erythrocytes (IEs) are of key importance in the pathogenesis ofP. falciparummalaria. Several structurally and functionally defined PfEMP1 types have been associated with severe clinical manifestations, such as cerebral malaria in children and placental malaria in pregnant women. PfEMP1 that can bind the Fc part of IgM (Fcμ) characterizes one such type, although the functional significance of this IgM binding to PfEMP1 remains unclear. In this study, we report the identification and functional analysis of five IgM-binding PfEMP1 proteins encoded byP. falciparumNF54. In addition to the VAR2CSA-type PFL0030c protein, already known to bind Fcμ and to mediate chondroitin sulfate A (CSA)-specific adhesion of IEs in the placenta, we found four PfEMP1 proteins not previously known to bind IgM this way. Although they all contained Duffy binding-like ε (DBLε) domains similar to those in VAR2CSA-type PfEMP1, they did not mediate IE adhesion to CSA, and IgM binding did not shield IEs from phagocytosis of IgG-opsonized IEs. In this way, these new IgM-binding PfEMP1 proteins resemble the rosette-mediating and IgM-binding PfEMP1 HB3VAR06, but none of them mediated formation of rosettes. We could map the capacity for Fc-specific IgM binding to DBLε domains near the C terminus for three of the four PfEMP1 proteins tested. Our study provides new evidence regarding Fc-dependent binding of IgM to PfEMP1, which appears to be a common and multifunctional phenotype.

scholarly journals IgG Antibodies to Endothelial Protein C Receptor-Binding Cysteine-Rich Interdomain Region Domains of Plasmodium falciparum Erythrocyte Membrane Protein 1 Are Acquired Early in Life in Individuals Exposed to Malaria

2015 ◽  
Vol 83 (8) ◽  
pp. 3096-3103 ◽  
Author(s):  
Louise Turner ◽  
Thomas Lavstsen ◽  
Bruno P. Mmbando ◽  
Christian W. Wang ◽  
Pamela A. Magistrado ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Membrane Protein ◽  
Malaria Transmission ◽  
Severe Malaria ◽  
Erythrocyte Membrane ◽  
Protein C ◽  
Endothelial Protein C Receptor ◽  
Content Type ◽  
Erythrocyte Membrane Protein ◽  
Amino Terminal

Severe malaria syndromes are precipitated byPlasmodium falciparumparasites binding to endothelial receptors on the vascular lining. This binding is mediated by members of the highly variantP. falciparumerythrocyte membrane protein 1 (PfEMP1) family. We have previously identified a subset of PfEMP1 proteins associated with severe malaria and found that the receptor for these PfEMP1 variants is endothelial protein C receptor (EPCR). The binding is mediated through the amino-terminal cysteine-rich interdomain region (CIDR) of the subtypes α1.1 and α1.4 to α1.8. In this study, we investigated the acquisition of anti-CIDR antibodies using plasma samples collected in four study villages with different malaria transmission intensities in northeastern Tanzania during a period with a decline in malaria transmission. We show that individuals exposed to high levels of malaria transmission acquire antibodies to EPCR-binding CIDR domains early in life and that these antibodies are acquired more rapidly than antibodies to other CIDR domains. The rate by which antibodies to EPCR-binding CIDR domains are acquired in populations in areas where malaria is endemic is determined by the malaria transmission intensity, and on a population level, the antibodies are rapidly lost if transmission is interrupted. This indicates that sustained exposure is required to maintain the production of the antibodies.

scholarly journals Identification of Glycosaminoglycan Binding Domains in Plasmodium falciparum Erythrocyte Membrane Protein 1 of a Chondroitin Sulfate A-Adherent Parasite

2000 ◽  
Vol 68 (7) ◽  
pp. 3923-3926 ◽  
Author(s):  
John C. Reeder ◽  
Anthony N. Hodder ◽  
James G. Beeson ◽  
Graham V. Brown
Keyword(s):  
Plasmodium Falciparum ◽  
Membrane Protein ◽  
Chondroitin Sulfate ◽  
Erythrocyte Membrane ◽  
Binding Activity ◽  
Parasite Line ◽  
Erythrocyte Membrane Protein ◽  
Binding Domains ◽  
Nonlinear Binding ◽  
Chondroitin Sulfate A

ABSTRACT Accumulation of Plasmodium falciparum-infected erythrocytes in the placenta is a key feature of maternal malaria. This process is mediated in part by the parasite ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1) at the surface of the infected erythrocyte interacting with the host receptor chondroitin sulfate A (CSA) on the placental lining. We have localized CSA binding activity to two adjacent domains in PfEMP1 of an adherent parasite line and shown the presence of at least three active glycosaminoglycan binding sites. A putative CSA binding sequence was identified in one domain, but nonlinear binding motifs are also likely to be present, since binding activity in the region was shown to be dependent on conformation. Characterization of this binding region provides an opportunity to investigate further its potential as a target for antiadhesion therapy.

scholarly journals Antibody Levels to Plasmodium falciparum Erythrocyte Membrane Protein 1-DBLγ11 and DBLδ-1 Predict Reduction in Parasite Density

mSystems ◽  
2021 ◽  
Author(s):  
Brittany N. Araj ◽  
Bruce Swihart ◽  
Robert Morrison ◽  
Patricia Gonzales Hurtado ◽  
Andrew Teo ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Membrane Protein ◽  
Erythrocyte Membrane ◽  
Infected Erythrocyte ◽  
Severe Disease ◽  
Content Type ◽  
Erythrocyte Membrane Protein ◽  
Erythrocyte Surface ◽  
Antibody Levels ◽  
Falciparum Erythrocyte Membrane Protein

Plasmodium infection causes devastating disease and high mortality in young children. Immunity develops progressively as children acquire protection against severe disease, although reinfections and recrudescences still occur throughout life in areas of endemicity, partly due to parasite immunoevasion via switching of variant proteins such as Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) expressed on the infected erythrocyte surface.

scholarly journals Rosetting Plasmodium falciparum-Infected Erythrocytes Bind to Human Brain Microvascular Endothelial CellsIn Vitro, Demonstrating a Dual Adhesion Phenotype Mediated by Distinct P. falciparum Erythrocyte Membrane Protein 1 Domains

2013 ◽  
Vol 82 (3) ◽  
pp. 949-959 ◽  
Author(s):  
Yvonne Adams ◽  
Pongsak Kuhnrae ◽  
Matthew K. Higgins ◽  
Ashfaq Ghumra ◽  
J. Alexandra Rowe
Keyword(s):  
Plasmodium Falciparum ◽  
Membrane Protein ◽  
Human Brain ◽  
Erythrocyte Membrane ◽  
Microvascular Endothelial Cell ◽  
Microvascular Endothelium ◽  
Content Type ◽  
Erythrocyte Membrane Protein ◽  
Microvascular Endothelial

ABSTRACTAdhesion interactions betweenPlasmodium falciparum-infected erythrocytes (IE) and human cells underlie the pathology of severe malaria. IE cytoadhere to microvascular endothelium or form rosettes with uninfected erythrocytes to survivein vivoby sequestering IE in the microvasculature and avoiding splenic clearance mechanisms. Both rosetting and cytoadherence are mediated by the parasite-derived IE surface protein familyPlasmodium falciparumerythrocyte membrane protein 1 (PfEMP1). Rosetting and cytoadherence have been widely studied as separate entities; however, the ability of rosettingP. falciparumstrains to cytoadhere has received little attention. Here, we show that IE of the IT/R29 strain expressing a rosette-mediating PfEMP1 variant (IT4var09) cytoadherein vitroto a human brain microvascular endothelial cell line (HBEC-5i). Cytoadherence was inhibited by heparin and by treatment of HBEC-5i with heparinase III, suggesting that the endothelial receptors for IE binding are heparan sulfate proteoglycans. Antibodies to the N-terminal regions of the IT4var09 PfEMP1 variant (NTS-DBL1α and DBL2γ domains) specifically inhibited and reversed cytoadherence down to low concentrations (<10 μg/ml of total IgG). Surface plasmon resonance experiments showed that the NTS-DBLα and DBL2γ domains bind strongly to heparin, with half-maximal binding at a concentration of ∼0.5 μM in both cases. Therefore, cytoadherence of IT/R29 IE is distinct from rosetting, which is primarily mediated by NTS-DBL1α interactions with complement receptor 1. These data show that IT4var09-expressing parasites are capable of dual interactions with both endothelial cells and uninfected erythrocytes via distinct receptor-ligand interactions.

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