Binding Of Infected Erythrocytes Research Articles

A conserved epitope in VAR2CSA is targeted by a cross-reactive antibody originating from Plasmodium vivax Duffy binding protein.

During Plasmodium falciparum infection in pregnancy, VAR2CSA is expressed on the surface of infected erythrocytes (IEs) and mediates their sequestration in the placenta. As a result, antibodies to VAR2CSA are largely restricted to women who were infected during pregnancy. However, we discovered that VAR2CSA antibodies can also be elicited by P. vivax Duffy binding protein (PvDBP). We proposed that infection with P. vivax in non-pregnant individuals can generate antibodies that cross-react with VAR2CSA. To better understand the specificity of these antibodies, we took advantage of a mouse monoclonal antibody (3D10) raised against PvDBP that cross-reacts with VAR2CSA and identified the epitopes targeted by this antibody. We screened two peptide arrays that span the ectodomain of VAR2CSA from the FCR3 and NF54 alleles. Based on the top epitope recognized by 3D10, we designed a 34-amino acid synthetic peptide, which we call CRP1, that maps to a highly conserved region in DBL3X. Specific lysine residues are critical for 3D10 recognition, and these same amino acids are within a previously defined chondroitin sulfate A (CSA) binding site in DBL3X. We showed by isothermal titration calorimetry that the CRP1 peptide can bind directly to CSA, and antibodies to CRP1 raised in rats significantly blocked the binding of IEs to CSA in vitro. In our Colombian cohorts of pregnant and non-pregnant individuals, at least 45% were seroreactive to CRP1. Antibody reactivities to CRP1 and the 3D10 natural epitope in PvDBP region II, subdomain 1 (SD1), were strongly correlated in both cohorts. These findings suggest that antibodies arising from PvDBP may cross-react with VAR2CSA through the epitope in CRP1 and that CRP1 could be a potential vaccine candidate to target a distinct CSA binding site in VAR2CSA.

A conformational epitope in placental malaria vaccine antigen VAR2CSA: What does it teach us?

VAR2CSA is the Plasmodium falciparum variant surface antigen that mediates binding of infected erythrocytes to chondroitin sulfate A (CSA) and their sequestration in intervillous spaces of the placenta, leading to placental malaria (PM). Relatively high polymorphism in VAR2CSA sequences has hindered development of a vaccine that induces broadly neutralizing immunity. Recent research has highlighted that a broadly reactive human monoclonal antibody, called PAM1.4, binds to multiple conserved residues of different subfragments of VAR2CSA, forming a conformational epitope. In this short perspective, we describe evidence that residues located in the interdomain-1 fragment of VAR2CSA within the PAM1.4 binding epitope might be critical to broad reactivity of the antibody. Future investigation into broadly reactive anti-VAR2CSA antibodies may be important for the following: (1) identification of similar conformation epitopes targeted by broadly neutralizing antibodies; and (2) understanding different immune evasion mechanisms used by placenta-binding parasites through VAR2CSA polymorphism in critical epitopes.

Analysis of Antibody Inhibition of PfEMP1 Binding by Competition ELISA.

Plasmodium falciparum express variant antigens on the surface of infected erythrocytes (IEs). These include proteins of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family. PfEMP1 proteins mediate binding of IEs to various human endothelial receptors and induce antibodies response during natural infections. In this chapter, we describe a competition ELISA that is an assay for antibodies neutralizing binding of PfEMP1 proteins to their cognate receptor.

Enhanced virulence of Plasmodium falciparum in blood of diabetic patients.

Rising prevalence of diabetes in sub-Saharan Africa, coupled with continued malaria transmission, has resulted more patients dealing with both communicable and non-communicable diseases. We previously reported that travelers with type 2 diabetes mellitus (T2DM) infected with Plasmodium falciparum were three times more likely to develop severe malaria than non-diabetics. Here we explore the biological basis for this by testing blood from uninfected subjects with type 1 and type 2 diabetes, ex vivo, for their effects on parasite growth and rosetting (binding of infected erythrocytes to uninfected erythrocytes). Rosetting was associated with type 2 diabetes, blood glucose and erythrocyte sedimentation rate (ESR), while parasite growth was positively associated with blood glucose, glycated hemoglobin (HbA1c), body mass index (BMI), fibrinogen and triglycerides. This study establishes a link between diabetes and malaria virulence assays, potentially explaining the protective effect of good glycemic control against severe malaria in subjects with diabetes.

IgG Responses to the Plasmodium falciparum Antigen VAR2CSA in Colombia Are Restricted to Pregnancy and Are Not Induced by Exposure to Plasmodium vivax.

Clinical immunity to malaria is associated with the acquisition of IgG specific for members of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family of clonally variant antigens on the surface of infected erythrocytes (IEs). The VAR2CSA subtype of PfEMP1 mediates IE binding in the placenta. VAR2CSA-specific IgG is normally acquired only after exposure to placental parasites. However, it was recently reported that men and children from Colombia often have high levels of functional VAR2CSA-specific IgG. This potentially undermines the current understanding of malaria immunity in pregnant women, and we thus conducted a study to assess further the levels of VAR2CSA-specific IgG in pregnant and nonpregnant Colombians. Plasma IgG against two full-length recombinant PfEMP1 proteins (one of the VAR2CSA type and one not) produced in baculovirus-transfected insect cells was detected frequently among Colombian men, children, and pregnant women with acute or previous malaria exposure. In contrast, IgG reactivity to a homologous full-length VAR2CSA-type protein expressed in Chinese hamster ovary (CHO) cells was low and infrequent among the Colombian plasma samples, as was reactivity to both corresponding native PfEMP1 proteins. Moreover, human and rabbit antibodies specific for Plasmodium vivax Duffy-binding protein (PvDBP), a protein with some homology to PfEMP1, did not react with VAR2CSA-type recombinant or native proteins, although the mouse monoclonal and PvDBP-specific antibody 3D10 was weakly reactive with recombinant proteins expressed in baculovirus-transfected insect cells. Our data indicate that the previously reported Colombian IgG reactivity to recombinant VAR2CSA is not malaria specific and that the acquisition of VAR2CSA-specific IgG is restricted to pregnancy, in Colombia and elsewhere.

Host factors that modify Plasmodium falciparum adhesion to endothelial receptors

P. falciparum virulence is related to adhesion and sequestration of infected erythrocytes (IE) in deep vascular beds, but the endothelial receptors involved in severe malaria remain unclear. In the largest ever study of clinical isolates, we surveyed adhesion of freshly collected IE from children under 5 years of age in Mali to identify novel vascular receptors, and examined the effects of host age, hemoglobin type, blood group and severe malaria on levels of IE adhesion to a panel of endothelial receptors. Several novel molecules, including integrin α3β1, VE-cadherin, ICAM-2, junctional adhesion molecule-B (JAM-B), laminin, and cellular fibronectin, supported binding of IE from children. Severe malaria was not significantly associated with levels of IE adhesion to any of the 19 receptors. Hemoglobin AC, which reduces severe malaria risk, reduced IE binding to the receptors CD36 and integrin α5β1, while hemoglobin AS did not modify IE adhesion to any receptors. Blood groups A, AB and B significantly reduced IE binding to ICAM-1. Severe malaria risk varies with age, but age significantly impacted the level of IE binding to only a few receptors: IE binding to JAM-B decreased with age, while binding to CD36 and integrin α5β1 significantly increased with age.

In vitro inhibition and reversal of Plasmodium falciparum cytoadherence to endothelium by monoclonal antibodies to ICAM-1 and CD36

BackgroundSequestration of parasitized red blood cells from the peripheral circulation during an infection with Plasmodium falciparum is caused by an interaction between the parasite protein PfEMP1 and receptors on the surface of host endothelial cells, known as cytoadherence. Several lines of evidence point to a link between the pathology of severe malaria and cytoadherence, therefore blocking adhesion receptors involved in this process could be a good target to inhibit pRBC sequestration and prevent disease. In a malaria endemic setting this is likely to be used as an adjunct therapy by reversing existing cytoadherence. Two well-characterized parasite lines plus three recently derived patient isolates were tested for their cytoadherence to purified receptors (CD36 and ICAM-1) as well as endothelial cells. Monoclonal antibodies against human CD36 and ICAM-1 were used to inhibit and reverse infected erythrocyte binding in static and flow-based adhesion assays.ResultsAnti-ICAM-1 and CD36 monoclonal antibodies were able to inhibit and reverse P. falciparum binding of lab and recently adapted patient isolates in vitro. However, reversal of binding was incomplete and varied in its efficiency between parasite isolates.ConclusionsThe results show that, as a proof of concept, disturbing existing ligand–receptor interactions is possible and could have potential therapeutic value for severe malaria. The variation seen in the degree of reversing existing binding with different parasite isolates and the incomplete nature of reversal, despite the use of high affinity inhibitors, suggest that anti-adhesion approaches as adjunct therapies for severe malaria may not be effective, and the focus may need to be on inhibitory approaches such as vaccines.

Regulation of PfEMP1-VAR2CSA translation by a Plasmodium translation-enhancing factor.

Pregnancy-associated malaria commonly involves the binding of Plasmodium falciparum-infected erythrocytes to placental chondroitin sulfate A (CSA) through the PfEMP1-VAR2CSA protein. VAR2CSA is translationally repressed by an upstream open reading frame. In this study, we report that the P. falciparum translation enhancing factor (PTEF) relieves upstream open reading frame repression and thereby facilitates VAR2CSA translation. VAR2CSA protein levels in var2csa-transcribing parasites are dependent on the expression level of PTEF, and the alleviation of upstream open reading frame repression requires the proteolytic processing of PTEF by PfCalpain. Cleavage generates a C-terminal domain that contains a sterile-alpha-motif-like domain. The C-terminal domain is permissive to cytoplasmic shuttling and interacts with ribosomes to facilitate translational derepression of the var2csa coding sequence. It also enhances translation in a heterologous translation system and thus represents the first non-canonical translation enhancing factor to be found in a protozoan. Our results implicate PTEF in regulating placental CSA binding of infected erythrocytes.

Comparison of functional assays used in the clinical development of a placental malaria vaccine

BackgroundMalaria in pregnancy is associated with significant morbidity in pregnant women and their offspring. Plasmodium falciparum infected erythrocytes (IE) express VAR2CSA that mediates binding to chondroitin sulphate A (CSA) in the placenta. Two VAR2CSA-based vaccines for placental malaria are in clinical development. The purpose of this study was to evaluate the robustness and comparability of binding inhibition assays used in the clinical development of placental malaria vaccines. MethodsThe ability of sera from animals immunised with different VAR2CSA constructs to inhibit IE binding to CSA was investigated in three in vitro assays using 96-well plates, petri dishes, capillary flow and an ex vivo placental perfusion assay. ResultsThe inter-assay variation was not uniform between assays and ranged from above ten-fold in the flow assay to two-fold in the perfusion assay. The intra-assay variation was highest in the petri dish assay. A positive correlation between IE binding avidity and the level of binding after antibody inhibition in the petri dish assay indicate that high avidity IE binding is more difficult to inhibit. The highest binding inhibition sensitivity was found in the 96-well and petri dish assays compared to the flow and perfusion assays where binding inhibition required higher antibody titers. ConclusionsThe inhibitory capacity of antibodies is not easily translated between assays and the high sensitivity of the 96-well and petri dish assays stresses the need for comparing serial dilutions of serum. Furthermore, IE binding avidity must be in the same range when comparing data from different days. There was an overall concordance in the capacity of antibody-mediated inhibition, when comparing the in vitro assays with the perfusion assay, which more closely represents in vivo conditions. Importantly the ID1-ID2a protein in a liposomal formulation, currently in a phase I trial, effectively induced antibodies that inhibited IE adhesion in placental tissue.

Antibody responses to the full-length VAR2CSA and its DBL domains in Cameroonian children and teenagers.

BackgroundAntigenic variation of Plasmodium falciparum erythrocyte membrane protein 1 is a key parasite mechanism for immune evasion and parasite survival. It is assumed that the number of parasites expressing the same var gene must reach high enough numbers before the host can produce detectable levels of antibodies (Ab) to the variant. VAR2CSA is a protein coded for by one of 60 var genes that is expressed on the surface of infected erythrocytes (IE) and mediates IE binding to the placenta. The idea that Ab to VAR2CSA are pregnancy-associated was challenged when VAR2CSA-specific Ab were reported in children and men. However, the frequency and conditions under which Ab to VAR2CSA are produced outside pregnancy is unclear. This study sought to determine frequency, specificity and level of Ab to VAR2CSA produced in children and whether children with hyperparasitaemia and severe malaria are more likely to produce Ab to VAR2CSA compared to healthy children.MethodsAntibody responses to a panel of recombinant proteins consisting of multiple VAR2CSA Duffy-binding-like domains (DBL) and full-length VAR2CSA (FV2) were characterized in 193 1–15 year old children from rural Cameroonian villages and 160 children with severe malaria from the city.ResultsLow Ab levels to VAR2CSA were detected in children; however, Ab levels to FV2 in teenagers were rare. Children preferentially recognized DBL2 (56–70%) and DBL4 (50–60%), while multigravidae produced high levels of IgG to DBL3, DBL5 and FV2. Sixty-seven percent of teenage girls (n = 16/24) recognized ID1–ID2a region of VAR2CSA. Children with severe forms of malaria had significantly higher IgG to merozoite antigens (all p < 0.05), but not to VAR2CSA (all p > 0.05) when compared to the healthy children.ConclusionThe study suggests that children, including teenage girls acquire Ab to VAR2CSA domains and FV2, but Ab levels are much lower than those needed to protect women from placental infections and repertoire of Ab responses to DBL domains is different from those in pregnant women. Interestingly, children with severe malaria did not have higher Ab levels to VAR2CSA compared to healthy children.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1585-y) contains supplementary material, which is available to authorized users.

Real-time measurement of Plasmodium falciparum-infected erythrocyte cytoadhesion with a quartz crystal microbalance.

BackgroundAn important virulence mechanism of the malaria parasite Plasmodium falciparum is cytoadhesion, the binding of infected erythrocytes to endothelial cells in the second half of asexual blood stage development. Conventional methods to investigate adhesion of infected erythrocytes are mostly performed under static conditions, many are based on manual or semi-automated read-outs and are, therefore, difficult to standardize. Quartz crystal microbalances (QCM) are sensitive to nanogram-scale changes in mass and biomechanical properties and are increasingly used in biomedical research. Here, the ability of QCM is explored to measure binding of P. falciparum-infected erythrocytes to two receptors: CD36 and chondroitin sulfate A (CSA) under flow conditions.MethodsBinding of late stage P. falciparum parasites is measured in comparison to uninfected erythrocytes to CD36- and CSA-coated quartzes by QCM observing frequency shifts. CD36-expressing cell membrane fragments and CSA polysaccharide were coated via poly-l-lysine to the quartz. The method was validated by microscopic counting of attached parasites and of erythrocytes to the coated quartzes.ResultsFrequency shifts indicating binding of infected erythrocytes could be observed for both receptors CD36 and CSA. The frequency shifts seen for infected and uninfected erythrocytes were strongly correlated to the microscopically counted numbers of attached cells.ConclusionsIn this proof-of-concept experiment it is shown that QCM is a promising tool to measure binding kinetics and specificity of ligand-receptor interactions using viable, parasite-infected erythrocytes. The method can improve the understanding of the virulence of P. falciparum and might be used to cross-validate other methods.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1374-7) contains supplementary material, which is available to authorized users.

The Influence of Sub-Unit Composition and Expression System on the Functional Antibody Response in the Development of a VAR2CSA Based Plasmodium falciparum Placental Malaria Vaccine.

The disease caused by Plasmodium falciparum (Pf) involves different clinical manifestations that, cumulatively, kill hundreds of thousands every year. Placental malaria (PM) is one such manifestation in which Pf infected erythrocytes (IE) bind to chondroitin sulphate A (CSA) through expression of VAR2CSA, a parasite-derived antigen. Protection against PM is mediated by antibodies that inhibit binding of IE in the placental intervillous space. VAR2CSA is a large antigen incompatible with large scale recombinant protein expression. Vaccines based on sub-units encompassing the functionally constrained receptor-binding domains may, theoretically, circumvent polymorphisms, reduce the risk of escape-mutants and induce cross-reactive antibodies. However, the sub-unit composition and small differences in the borders, may lead to exposure of novel immuno-dominant antibody epitopes that lead to non-functional antibodies, and furthermore influence the folding, stability and yield of expression. Candidate antigens from the pre-clinical development expressed in High-Five insect cells using the baculovirus expression vector system were transitioned into the Drosophila Schneider-2 cell (S2) expression-system compliant with clinical development. The functional capacity of antibodies against antigens expressed in High-Five cells or in S2 cells was equivalent. This enabled an extensive down-selection of S2 insect cell-expressed antigens primarily encompassing the minimal CSA-binding region of VAR2CSA. In general, we found differential potency of inhibitory antibodies against antigens with the same borders but of different var2csa sequences. Likewise, we found that subtle size differences in antigens of the same sequence gave varying levels of inhibitory antibodies. The study shows that induction of a functional response against recombinant subunits of the VAR2CSA antigen is unpredictable, demonstrating the need for large-scale screening in order to identify antigens that induce a broadly strain-transcending antibody response.

An automated method for determining the cytoadhesion of Plasmodium falciparum-infected erythrocytes to immobilized cells.

BackgroundPlasmodium falciparum exports antigens to the surface of infected erythrocytes causing cytoadhesion to the host vasculature. This is central in malaria pathogenesis but in vitro studies of cytoadhesion rely mainly on manual counting methods. The current study aimed at developing an automated high-throughput method for this purpose utilizing the pseudoperoxidase activity of intra-erythrocytic haemoglobin.MethodsChinese hamster ovary (CHO) cells were grown to confluence in chamber slides and microtiter plates. Cytoadhesion of co-cultured P. falciparum, selected for binding to CHO cells, was quantified by microscopy of Giemsa-stained chamber slides. In the automated assay, binding was quantified spectrophotometrically in microtiter plates after cell lysis using tetramethylbenzidine as peroxidase-catalysed substrate. The relevance of the method for binding studies was assessed using: i) binding of P. falciparum-infected erythrocytes to CHO cells over-expressing chondroitin sulfate A and ii) CHO cells transfected with CD36. Binding of infected erythrocytes including field isolates to primary endothelial cells was also performed. Data was analysed using linear regression and Bland-Altman plots.ResultsThe manual and automated quantification showed strong, positive correlation (r2 = 0.959, p <0.001) and with similar detection limit and precision. The automated assay showed the expected dose-dependent reduction in binding to CHO cells when blocking with soluble chondroitin sulfate A or anti-CD36 antibody. Quantification of binding to endothelial cells showed clear distinction between selected vs. non-selected parasite lines. Importantly, the assay was sufficiently sensitive to detect adhesion of field isolates to endothelial cells.ConclusionsThe assay is simple and in a reproducible manner quantifies erythrocyte adhesion to several types of immobilized cells.

Antibodies to Escherichia coli-Expressed C-Terminal Domains of Plasmodium falciparum Variant Surface Antigen 2-Chondroitin Sulfate A (VAR2CSA) Inhibit Binding of CSA-Adherent Parasites to Placental Tissue

Placental malaria (PM) is characterized by infected erythrocytes (IEs) that selectively bind to chondroitin sulfate A (CSA) and sequester in placental tissue. Variant surface antigen 2-CSA (VAR2CSA), a Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) protein family member, is expressed on the surface of placental IEs and mediates adherence to CSA on the surface of syncytiotrophoblasts. This transmembrane protein contains 6 Duffy binding-like (DBL) domains which might contribute to the specific adhesive properties of IEs. Here, we use laboratory isolate 3D7 VAR2CSA DBL domains expressed in Escherichia coli to generate antibodies specific for this protein. Flow cytometry results showed that antibodies generated against DBL4ε, DBL5ε, DBL6ε, and tandem double domains of DBL4-DBL5 and DBL5-DBL6 all bind to placental parasite isolates and to lab strains selected for CSA binding but do not bind to children's parasites. Antisera to DBL4ε and to DBL5ε inhibit maternal IE binding to placental tissue in a manner comparable to that for plasma collected from multigravid women. These antibodies also inhibit binding to CSA of several field isolates derived from pregnant women, while antibodies to double domains do not enhance the functional immune response. These data support DBL4ε and DBL5ε as vaccine candidates for pregnancy malaria and demonstrate that E. coli is a feasible tool for the large-scale manufacture of a vaccine based on these VAR2CSA domains.

Differential Induction of Functional IgG Using the Plasmodium falciparum Placental Malaria Vaccine Candidate VAR2CSA

BackgroundIn Plasmodium falciparum malaria endemic areas placental malaria (PM) is an important complication of malaria. The recurrence of malaria in primigravidae women irrespective of acquired protection during childhood is caused by the interaction between the parasite-expressed VAR2CSA antigen and chondroitin sulfate A (CSA) in the placental intervillous space and lack of protective antibodies. PM impairs fetal development mainly by excessive inflammation processes. After infections during pregnancy women acquire immunity to PM conferred by antibodies against VAR2CSA. Ideally, a vaccine against PM will induce antibody-mediated immune responses that block the adhesion of infected erythrocytes (IE) in the placenta.Principal FindingsWe have previously shown that antibodies raised in rat against individual domains of VAR2CSA can block IE binding to CSA. In this study we have immunized mice, rats and rabbits with each individual domain and the full-length protein corresponding to the FCR3 VAR2CSA variant. We found there is an inherently higher immunogenicity of C-terminal domains compared to N-terminally located domains. This was irrespective of whether antibodies were induced against single domains or the full-length protein. Species-specific antibody responses were also found, these were mainly directed against single domains and not the full-length VAR2CSA protein.Conclusions/SignificanceBinding inhibitory antibodies appeared to be against conformational B-cell epitopes. Non-binding inhibitory antibodies reacted highly against the C-terminal end of the VAR2CSA molecule especially the highly polymorphic DBL6ε domain. Differential species-specific induction of antibody responses may allow for more direct analysis of functional versus non-functional B-cell epitopes.

Chondroitin Sulfate A-Adhering Plasmodium falciparum-Infected Erythrocytes Express Functionally Important Antibody Epitopes Shared by Multiple Variants

Acquired protection from Plasmodium falciparum placental malaria, a major cause of maternal, fetal, and infant morbidity, is mediated by IgG specific for the P. falciparum erythrocyte membrane protein 1 variant VAR2CSA. This protein enables adhesion of P. falciparum-infected erythrocytes to chondroitin sulfate A in the intervillous space. Although interclonal variation of the var2csa gene is lower than that among var genes in general, VAR2CSA-specific Abs appear to target mainly polymorphic epitopes. This has raised doubts about the feasibility of VAR2CSA-based vaccines. We used eight human monoclonal IgG Abs from affinity-matured memory B cells of P. falciparum-exposed women to study interclonal variation and functional importance of Ab epitopes among placental and peripheral parasites from East and West Africa. Most placental P. falciparum isolates were labeled by several mAbs, whereas peripheral isolates from children were essentially nonreactive. The mAb reactivity of peripheral isolates from pregnant women indicated that some were placental, whereas others had alternative sequestration foci. Most of the mAbs were comparable in their reactivity with bound infected erythrocytes (IEs) and recombinant VAR2CSA and interfered with IE and/or VAR2CSA binding to chondroitin sulfate A. Pair-wise mAb combinations were more inhibitory than single mAbs, and all of the mAbs together was the most efficient combination. Each mAb could opsonize IEs for phagocytosis, and a combination of the eight mAbs caused phagocytosis similar to that of plasma IgG-opsonized IEs. We conclude that functionally important Ab epitopes are shared by the majority of polymorphic VAR2CSA variants, which supports the feasibility of VAR2CSA-based vaccines against placental malaria.

High Levels of Plasmodium falciparum Rosetting in All Clinical Forms of Severe Malaria in African Children

Plasmodium falciparum rosetting (the spontaneous binding of infected erythrocytes to uninfected erythrocytes) is a well-recognized parasite virulence factor. However, it is currently unclear whether rosetting is associated with all clinical forms of severe malaria, or only with specific syndromes such as cerebral malaria. We investigated the relationship between rosetting and clinical malaria in 209 Malian children enrolled in a case-control study of severe malaria. Rosetting was significantly higher in parasite isolates from severe malaria cases compared with non-severe hyperparasitemia and uncomplicated malaria controls (F(2,117) = 8.15, P < 0.001). Analysis of sub-categories of severe malaria (unrousable coma, severe anemia, non-comatose neurological impairment, repeated seizures or a small heterogeneous group with signs of renal failure or jaundice) showed high levels of rosetting in all sub-categories, and no statistically significant differences in rosetting between sub-categories (F(4,67) = 1.28, P = 0.28). Thus rosetting may contribute to the pathogenesis of all severe malaria syndromes in African children, and interventions to disrupt rosetting could be potential adjunctive therapies for all forms of severe malaria in Africa.

Plasmodium falciparum adhesive protein that mediates sequestration of parasite‐infected red blood in human placenta and cause pregnancy specific malaria

Malaria caused by Plasmodium falciparum results in millions of deaths every year. In endemic areas, although immunity to malaria develops during childhood years, women become susceptible to malaria during pregnancy. This is due to the sequestration of P. falciparum infected erythrocytes (IEs) in the placenta by binding to host chondroitin 4‐sulfate (C4S). A parasite protein called var2CSA, a member of antigenically diverse family of 60 parasite proteins expressed on the erythrocyte membrane is involved in IE adherence to C4S. Var2‐CSA consists of several Duffy binding like (DBL) domains and how exactly they bind C4S remain unclear. We expressed various DBL domains on yeast cell surface and analyzed cell adherence to C4S. None of the DBL domains could support binding of yeast to C4S, suggesting that individual DBL domains cannot support IRBC binding. It is possible that a conformational structure involving two or more domains is involved in IRBC binding. Alternatively, var2CSA alone may not be sufficient for IE binding, instead a multiprotein complex is involved. To test this hypothesis, we performed affinity binding of parasite ligand from the lysates of 35S‐labeled IEs using C4S‐conjugated beads. In addition to var2CSA, several other proteins could specifically bind to C4S‐beads. Together these data suggest that a complex of var2CSA and other proteins is involved in IE binding. Funded by AI45086 from NIAID, NIH.

CXCR3 determines strain susceptibility to murine cerebral malaria by mediating T lymphocyte migration toward IFN‐γ‐induced chemokines

Cerebral malaria (CM) results from the binding of infected erythrocytes and leukocytes to brain endothelia. The precise mechanisms underlying lymphocyte recruitment and activation in CM remain unclear. Therefore, the expression of various chemokines was quantified in brains of mice infected with Plasmodium berghei ANKA (PbA). Several chemokines attracting monocytes and activated T-lymphocytes were expressed at high levels. Their expression was almost completely abrogated in IFN-gamma ligand and receptor KO mice, indicating that IFN-gamma is an essential chemokine inducer in vivo. Surprisingly, the expression levels of chemokines, IFN-gamma and also adhesion molecules in the brain were not lower in CM-resistant Balb/c and DBA/2 mice compared to CM-sensitive C57BL/6 and DBA/1 mice, although T lymphocyte sequestration in the brain was significantly less in CM-resistant than in CM-sensitive mice. This difference correlated with a higher up-regulation of the CXC chemokine receptor (CXCR)-3 on splenic T cells and a higher chemotactic response to IFN-gamma-inducible protein-10 (IP-10) in C57BL/6 compared to Balb/c mice. In conclusion, parasite-induced IFN-gamma in the brain results in high local expression levels of specific chemokines for monocytes and lymphocytes. The strain-dependent susceptibility to develop CM is more related to the expression of CXCR3 in circulating leukocytes than to the chemokine expression levels in the brain.

Mapping a common interaction site used by Plasmodium falciparum Duffy binding‐like domains to bind diverse host receptors

The Duffy binding-like (DBL) domain is a key adhesive module in Plasmodium falciparum, present in both erythrocyte invasion ligands (EBLs) and the large and diverse P. falciparum erythrocyte membrane protein 1 (PfEMP1) family of cytoadherence receptors. DBL domains bind a variety of different host receptors, including intercellular adhesion molecule 1 (ICAM-1), a receptor interaction that may have a role in infected erythrocyte binding to cerebral blood vessels and cerebral malaria. In this study, we expressed the nearly full complement of DBLbeta-C2 domains from the IT4/25/5 (IT4) parasite isolate and showed that ICAM-1-binding domains (DBLbeta-C2(ICAM-1)) were confined to group B and group C PfEMP1 proteins and were not present in group A, suggesting that ICAM-1 selection pressure differs between PfEMP1 groups. To further dissect the molecular determinants of binding, we modelled a DBLbeta-C2(ICAM-1) domain on a solved DBL structure and created alanine substitution mutants in two DBLbeta-C2(ICAM-1) domains. This analysis indicates that the DBLbeta-C2::ICAM-1 interaction maps to the equivalent glycan binding region of EBLs, and suggests a general model for how DBL domains evolve under dual selection for host receptor binding and immune evasion.