Surface Of Infected Erythrocytes Research Articles

Transcription and Copy Number Variation of Plasmodium falciparum var2csa among Nonpregnant Malaria Patients in Thailand.

Placental malaria is an important cause of fetomaternal morbidity and mortality among pregnant women infected with Plasmodium falciparum. The pathogenesis involves the binding of VAR2CSA on the surface of infected erythrocytes to chondroitin sulfate proteoglycans on syncytiotrophoblasts in the intervillous space of the placenta. Anti-VAR2CSA antibodies confer protection from adverse pregnancy outcomes in falciparum malaria; therefore, VAR2CSA is a strong vaccine candidate against placental malaria. To date, little is known about transcription of var2csa among isolates from male and nonpregnant patients in low transmission areas. In this study, transcription and copy number variation of var2csa were analyzed in 55 P. falciparum isolates from nonpregnant women, men and children with symptomatic malaria in five endemic provinces of Thailand. Reverse transcription polymerase chain reaction (PCR) detected var2csa transcripts in 43 (78.2%) blood samples. Multiple copies of var2csa were identified in 16 of 55 (29.1%) isolates by quantitative real-time PCR. Copy number variation of var2csa was not associated with the patients' gender, age group, ethnicity, parasite genotypes, parasite density, and geographic origins. These data suggest that P. falciparum carrying multicopy var2csa had a wide geographic distribution in Thailand with a prevalence rate comparable to those observed in high transmission areas of Africa. The high prevalence of isolates from male and nonpregnant patients with var2csa transcription suggests that the transcription of this gene occurs naturally during blood stage infection. Whether VAR2CSA is expressed and immunogenic among nonpregnant falciparum malaria patients requires further study.

A systems serology approach to identifying key antibody correlates of protection from cerebral malaria in Malawian children

BackgroundPlasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) proteins are expressed on the surface of infected erythrocytes, mediating parasite sequestration in the vasculature. PfEMP1 is a major target of protective antibodies, but the features of the antibody response are poorly defined.MethodsIn Malawian children with cerebral or uncomplicated malaria, we characterized the antibody response to 39 recombinant PfEMP1 Duffy binding like (DBL) domains or cysteine-rich interdomain regions (CIDRs) in detail, including measures of antibody classes, subclasses, and engagement with Fcγ receptors and complement. Using elastic net regularized logistic regression, we identified a combination of seven antibody targets and Fc features that best distinguished between children with cerebral and uncomplicated malaria. To confirm the role of the selected targets and Fc features, we measured antibody-dependent neutrophil and THP-1 cell phagocytosis of intercellular adhesion molecule-1 (ICAM-1) and endothelial protein C (EPCR) co-binding infected erythrocytes.ResultsThe selected features distinguished between children with cerebral and uncomplicated malaria with 87% accuracy (median, 80–96% interquartile range) and included antibody to well-characterized DBLβ3 domains and a less well-characterized CIDRγ12 domain. The abilities of antibodies to engage C1q and FcγRIIIb, rather than levels of IgG, correlated with protection. In line with a role of FcγRIIIb binding antibodies to DBLβ3 domains, antibody-dependent neutrophil phagocytosis of ICAM-1 and EPCR co-binding IE was higher in uncomplicated malaria (15% median, 8–38% interquartile range) compared to cerebral malaria (7%, 30–15%, p < 0.001).ConclusionsAntibodies associated with protection from cerebral malaria target a subset of PfEMP1 domains. The Fc features of protective antibody response include engagement of FcγRIIIb and C1q, and ability to induce antibody-dependent neutrophil phagocytosis of infected erythrocytes. Identifying the targets and Fc features of protective immunity could facilitate the development of PfEMP1-based therapeutics for cerebral malaria.

Children with hemoglobin C or S trait have low serologic responses to a subset of malaria variant surface antigens

Children with hemoglobin AC or AS have decreased susceptibility to clinical malaria. Parasite variant surface antigen (VSA) presentation on the surface of infected erythrocytes is altered in erythrocytes with hemoglobin C (Hb AC) or sickle trait (Hb AS) mutations in vitro. The protective role of incomplete or altered VSA presentation against clinical malaria in individuals with Hb AC or AS is unclear. Using a high-throughput protein microarray, we sought to use serological responses to VSAs as a measure of host exposure to VSAs among Malian children with Hb AC, Hb AS, or wildtype hemoglobin (Hb AA). In uncomplicated malaria, when compared to Hb AA children, Hb AC children had significantly lower serological responses to extracellular Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) domains but did not differ in responses to intracellular PfEMP1 domains and other VSAs, including members of the repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR) family. Healthy children with Hb AC and Hb AS genotypes recognized fewer extracellular PfEMP1s compared to children with Hb AA, especially CD36-binding PfEMP1s. These reduced serologic responses may reflect reduced VSA presentation or lower parasite exposure in children with Hb AC or AS and provide insights into mechanisms of protection.

Detection of naturally acquired, strain-transcending antibodies against rosetting Plasmodium falciparum strains in humans.

Strain-transcending antibodies against virulence-associated subsets of P. falciparum-infected erythrocyte surface antigens could protect children from severe malaria. However, the evidence supporting the existence of such antibodies is incomplete and inconsistent. One subset of surface antigens associated with severe malaria, rosette-mediating Plasmodium falciparum Erythrocyte Membrane Protein one (PfEMP1) variants, cause infected erythrocytes to bind to uninfected erythrocytes to form clusters of cells (rosettes) that contribute to microvascular obstruction and pathology. Here, we tested plasma from 80 individuals living in malaria-endemic regions for IgG recognition of the surface of four P. falciparum rosetting strains using flow cytometry. Broadly reactive plasma samples were then used in antibody elution experiments in which intact IgG was eluted from the surface of infected erythrocytes and transferred to heterologous rosetting strains to look for strain-transcending antibodies. We found that seroprevalence (percentage of positive plasma samples) against allopatric rosetting strains was high in adults (63%-93%) but lower in children (13%-48%). Strain-transcending antibodies were present in nine out of eleven eluted antibody experiments, with six of these recognizing multiple heterologous rosetting parasite strains. One eluate had rosette-disrupting activity against heterologous strains, suggesting PfEMP1 as the likely target of the strain-transcending antibodies. Naturally acquired strain-transcending antibodies to rosetting P. falciparum strains in humans have not been directly demonstrated previously. Their existence suggests that such antibodies could play a role in clinical protection and raises the possibility that conserved epitopes recognized by strain-transcending antibodies could be targeted therapeutically by monoclonal antibodies or vaccines.

M-O-M mediated denaturation resistant P2 tetramer on the infected erythrocyte surface of malaria parasite imports serum fatty acids

In Plasmodium falciparum, DNA replication, and asynchronous nuclear divisions precede cytokinesis during intraerythrocytic schizogony. Regulation of nuclear division through the import of serum components was largely unknown. At the trophozoite stage, P.falciparum ribosomal protein P2 (PfP2) is exported to the infected erythrocyte (IE) cytosol and the surface as a denaturation-resistant tetramer. The inaccessibility of the IE surface exposed PfP2 to its bona fide ligand led to the arrest of nuclear division. Here, we show that at the onset of schizogony, denaturation-resistant PfP2 tetramer on the IE surface imports fatty acids (FAs). Blockage of import reversibly arrested parasite schizogony. In 11Met-O-Met11 mediated denaturation resistant PfP2 tetramer, the 12/53Cys-Cys12/53 redox switch regulates the binding and release of FAs based on oxidized/reduced state of disulfide linkages. This mechanistic insight of FAs import through PfP2 tetramer reveals a unique regulation of nuclear division at the onset of schizogony.

Acquisition of antibodies that block Plasmodium falciparum adhesion to placental receptor chondroitin sulfate A with increasing gravidity in Malian women.

In malaria-endemic areas, pregnant women are more susceptible to Plasmodium falciparum infection, especially primigravidae. During pregnancy, parasites sequester in the placenta and bind to the receptor chondroitin sulfate (CSA). This unique adhesion is mediated by the parasite protein VAR2CSA expressed on the surface of infected erythrocytes (IE). Placental malaria is associated with poor pregnancy outcomes including perinatal mortality, preterm delivery, small for gestational age (SGA) and low birthweight deliveries. Over successive pregnancies, women acquire functional antibodies that inhibit IE adhesion to CSA. Here, we examine the development of anti-adhesion activity and the breadth of anti-adhesion activity as a function of number of previous pregnancies, using samples collected from pregnant women living in an area with high seasonal malaria transmission. Women reached plateau levels of anti-adhesion activity and breadth of anti-adhesion activity after 5 pregnancies. We related the level of anti-adhesion activity and reactivity with surface IE to SGA 19/232 pregnancies resulted in SGA, and report that an increase of 10% in median anti-adhesion activity reduced the odds of SGA by 13% and this relationship approached significance. Further, at an anti-adhesion activity level of 43.7%, an increase of 10% in the breadth of activity significantly reduced the odds of SGA by 21.5%. Antibodies that recognize IE surface increased over successive pregnancies, but were not associated with a reduction in SGA. These results can serve as a guideline for assessing vaccine candidates aiming to reduce poor pregnancy outcomes associated with placental malaria.

Profiling the Plasmodium falciparum Erythrocyte Membrane Protein 1-Specific Immununoglobulin G Response Among Ghanaian Children With Hemoglobin S and C.

Members of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family are important targets for protective immunity. Abnormal display of PfEMP1 on the surfaces of infected erythrocytes (IEs) and reduced cytoadhesion have been demonstrated in hemoglobin (Hb) AS and HbAC, inherited blood disorders associated with protection against severe P. falciparum malaria. We found that Ghanaian children with HbAS had lower levels of immunoglobulin G against several PfEMP1 variants and that this reactivity increased more slowly with age than in their HbAA counterparts. Moreover, children with HbAS have lower total parasite biomass than those with HbAA at comparable peripheral parasitemias, suggesting impaired cytoadhesion of HbAS IEs in vivo and likely explaining the slower acquisition of PfEMP1-specific immunoglobulin G in this group. In contrast, the function of acquired antibodies was comparable among Hb groups and appears to be intact and sufficient to control parasitemia via opsonization and phagocytosis of IEs.

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.

ICAM-1-binding Plasmodium falciparum erythrocyte membrane protein 1 variants elicits opsonic-phagocytosis IgG responses in Beninese children

Members of the highly polymorphic Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family expressed on the surface of infected erythrocytes (IEs) are important virulence factors, which mediate vascular adhesion of IEs via endothelial host receptors and are targets of naturally acquired immunity. The PfEMP1 family can be divided into clinically relevant subgroups, of which some bind intercellular adhesion molecule 1 (ICAM-1). While the acquisition of IgG specific for ICAM-1-binding DBLβ domains is known to differ between PfEMP1 groups, its ability to induce antibody-dependent cellular phagocytosis (ADCP) is unclear. We therefore measured plasma levels of DBLβ-specific IgG, the ability of such IgG to inhibit PfEMP1-binding to ICAM-1, and its ability to opsonize IEs for ADCP, using plasma from Beninese children with severe (SM) or uncomplicated malaria (UM). IgG specific for DBLβ from group A and B ICAM-1-binding PfEMP1 were dominated by IgG1 and IgG3, and were similar in SM and UM. However, levels of plasma IgG inhibiting ICAM-1-binding of group A DBLβ of PFD1235w was significantly higher in children with UM than SM, and acute UM plasma induced a higher ADCP response than acute SM plasma.

The acquisition of humoral immune responses targeting Plasmodium falciparum sexual stages in controlled human malaria infections.

Individuals infected with P. falciparum develop antibody responses to intra-erythrocytic gametocyte proteins and exported gametocyte proteins present on the surface of infected erythrocytes. However, there is currently limited knowledge on the immunogenicity of gametocyte antigens and the specificity of gametocyte-induced antibody responses. In this study, we assessed antibody responses in participants of two controlled human malaria infection (CHMI) studies by ELISA, multiplexed bead-based antibody assays and protein microarray. By comparing antibody responses in participants with and without gametocyte exposure, we aimed to disentangle the antibody response induced by asexual and sexual stage parasites. We showed that after a single malaria infection, a significant anti-sexual stage humoral response is induced in malaria-naïve individuals, even after exposure to relatively low gametocyte densities (up to ~1,600 gametocytes/mL). In contrast to antibody responses to well-characterised asexual blood stage antigens that were detectable by day 21 after infection, responses to sexual stage antigens (including transmission blocking vaccine candidates Pfs48/45 and Pfs230) were only apparent at 51 days after infection. We found antigens previously associated with early gametocyte or anti-gamete immunity were highly represented among responses linked with gametocyte exposure. Our data provide detailed insights on the induction and kinetics of antibody responses to gametocytes and identify novel antigens that elicit antibody responses exclusively in individuals with gametocyte exposure. Our findings provide target identification for serological assays for surveillance of the malaria infectious reservoir, and support vaccine development by describing the antibody response to leading vaccine antigens after primary infection.

Plasmodium knowlesi Cytoadhesion Involves SICA Variant Proteins.

Plasmodium knowlesi poses a health threat throughout Southeast Asian communities and currently causes most cases of malaria in Malaysia. This zoonotic parasite species has been studied in Macaca mulatta (rhesus monkeys) as a model for severe malarial infections, chronicity, and antigenic variation. The phenomenon of Plasmodium antigenic variation was first recognized during rhesus monkey infections. Plasmodium-encoded variant proteins were first discovered in this species and found to be expressed at the surface of infected erythrocytes, and then named the Schizont-Infected Cell Agglutination (SICA) antigens. SICA expression was shown to be spleen dependent, as SICA expression is lost after P. knowlesi is passaged in splenectomized rhesus. Here we present data from longitudinal P. knowlesi infections in rhesus with the most comprehensive analysis to date of clinical parameters and infected red blood cell sequestration in the vasculature of tissues from 22 organs. Based on the histopathological analysis of 22 tissue types from 11 rhesus monkeys, we show a comparative distribution of parasitized erythrocytes and the degree of margination of the infected erythrocytes with the endothelium. Interestingly, there was a significantly higher burden of parasites in the gastrointestinal tissues, and extensive margination of the parasites along the endothelium, which may help explain gastrointestinal symptoms frequently reported by patients with P. knowlesi malarial infections. Moreover, this margination was not observed in splenectomized rhesus that were infected with parasites not expressing the SICA proteins. This work provides data that directly supports the view that a subpopulation of P. knowlesi parasites cytoadheres and sequesters, likely via SICA variant antigens acting as ligands. This process is akin to the cytoadhesive function of the related variant antigen proteins, namely Erythrocyte Membrane Protein-1, expressed by Plasmodium falciparum.

Meta-Analysis of Human Antibodies Against Plasmodium falciparum Variable Surface and Merozoite Stage Antigens.

Concerted efforts to fight malaria have caused significant reductions in global malaria cases and mortality. Sustaining this will be critical to avoid rebound and outbreaks of seasonal malaria. Identifying predictive attributes that define clinical malaria will be key to guide development of second-generation tools to fight malaria. Broadly reactive antibodies against variable surface antigens that are expressed on the surface of infected erythrocytes and merozoites stage antigens are targets of naturally acquired immunity and prime candidates for anti-malaria therapeutics and vaccines. However, predicting the relationship between the antigen-specific antibodies and protection from clinical malaria remains unresolved. Here, we used new datasets and multiple approaches combined with re-analysis of our previous data to assess the multi-dimensional and complex relationship between antibody responses and clinical malaria outcomes. We observed 22 antigens (17 PfEMP1 domains, 3 RIFIN family members, merozoite surface protein 3 (PF3D7_1035400), and merozoites-associated armadillo repeats protein (PF3D7_1035900) that were selected across three different clinical malaria definitions (1,000/2,500/5,000 parasites/µl plus fever). In addition, Principal Components Analysis (PCA) indicated that the first three components (Dim1, Dim2 and Dim3 with eigenvalues of 306, 48, and 29, respectively) accounted for 66.1% of the total variations seen. Specifically, the Dim1, Dim2 and Dim3 explained 52.8%, 8.2% and 5% of variability, respectively. We further observed a significant relationship between the first component scores and age with antibodies to PfEMP1 domains being the key contributing variables. This is consistent with a recent proposal suggesting that there is an ordered acquisition of antibodies targeting PfEMP1 proteins. Thus, although limited, and further work on the significance of the selected antigens will be required, these approaches may provide insights for identification of drivers of naturally acquired protective immunity as well as guide development of additional tools for malaria elimination and eradication.

Chromodomain Protein Interacts with H3K9me3 and Controls RBC Rosette Formation by Regulating the Expression of a Subset of RIFINs in the Malaria Parasite

Plasmodium falciparum expresses clonally variant proteins on the surface of infected erythrocytes to evade the host immune system. The clonally variant multigene families include var, rifin, and stevor, which express Erythrocyte Membrane Protein 1 (EMP1), Repetitive Interspersed Families of polypeptides (RIFINs), and Sub-telomeric Variable Open Reading frame (STEVOR) proteins, respectively. The rifins are the largest multigene family and are essentially involved in the RBC rosetting, the hallmark of severe malaria. The molecular regulators that control the RIFINs expression in Plasmodium spp. have not been reported so far. This study reports a chromodomain-containing protein (PfCDP) that binds to H3K9me3 modification on P. falciparum chromatin. Conditional deletion of the chromodomain (CD) gene in P. falciparum using an inducible DiCre-LoxP system leads to selective up-regulation of a subset of virulence genes, including rifins, a few var, and stevor genes. Further, we show that PfCDP conditional knockout (PfΔCDP) promotes RBC rosette formation. This study provides the first evidence of an epigenetic regulator mediated control on a subset of RIFINs expression and RBC rosetting by P. falciparum.

RhopH2 and RhopH3 export enables assembly of the RhopH complex on P. falciparum-infected erythrocyte membranes

RhopH complexes consists of Clag3, RhopH2 and RhopH3 and are essential for growth of Plasmodium falciparum inside infected erythrocytes. Proteins are released from rhoptry organelles during merozoite invasion and trafficked to the surface of infected erythrocytes and enable uptake of nutrients. RhopH3, unlike other RhopH proteins, is required for parasite invasion, suggesting some cellular processes RhopH proteins function as single players rather than a complex. We show the RhopH complex has not formed during merozoite invasion. Clag3 is directly released into the host cell cytoplasm, whilst RhopH2 and RhopH3 are released into the nascent parasitophorous vacuole. Export of RhopH2 and RhopH3 from the parasitophorous vacuole into the infected erythrocyte cytoplasm enables assembly of Clag3/RhopH2/RhopH3 complexes and incorporation into the host cell membrane concomitant with activation of nutrient uptake. This suggests compartmentalisation prevents premature channel assembly before intact complex is assembled at the host cell membrane.

Affinity Purification of PfEMP1-Specific Antibodies from Human Blood.

Acquired immunity against Plasmodium falciparum infections relies heavily on IgG antibodies specific for PfEMP1 proteins expressed on the surface of infected erythrocytes. Purified human antibodies can be used, for example, to study the interactions between specific PfEMP1 proteins and receptors expressed by human endothelial cells, and to identify which IgG antibodies play a functional role in natural acquired immunity.This chapter describe how to affinity purify PfEMP1-specific human antibodies on an affinity column coupled with PfEMP1 protein. We include ELISA-based methods for identification of human plasma samples reactive against PfEMP1, and for testing of affinity purified IgG antibodies prior to their use in more advanced procedures.

Malaria Parasite Plasmodium falciparum Proteins on the Surface of Infected Erythrocytes as Targets for Novel Drug Discovery.

Specific adhesion (sequestration) of Plasmodium falciparum parasite-infected erythrocytes (IEs) in deep vascular beds can cause severe complications resulting in death. This review describes our work on the discovery, characterization, and optimization of novel inhibitors that specifically prevent adhesion of IEs to the host vasculature during severe malaria, especially its placental and cerebral forms. The main idea of using anti-adhesion drugs in severe malaria is to release sequestered parasites (or prevent additional sequestration) as quickly as possible. This may significantly improve the outcomes for patients with severe malaria by decreasing local and systemic inflammation associated with the disease and reestablishing the microvascular blood flow. To identify anti-malarial adhesion-inhibiting molecules, we have developed a high-throughput (HT) screening approach and found a number of promising leads that can be further developed into anti-adhesion drugs providing an efficient adjunct therapy against severe forms of malaria.

High-Throughput BioPlex Assay for the Study of Functionally Active Plasmodium Falciparum Antigens That Are Expressed on the Surface of Infected Erythrocytes.

Identification of P. falciparum infected erythrocyte surface ligands (such as PfEMP1) matched with the host receptors they interact with, as well as identification of PfEMP1 domains that are targets of protective immunity, are important for understanding of the pathophysiology of severe malaria (SM) and for design of novel vaccine candidates. In addition, identification of small-molecule drugs that can prevent or reverse receptor-ligand domain interactions could provide new tools for adjunctive therapy in SM. This protocol describes how to prepare functionally intact PfEMP1 proteins in mammalian cells (COS-7) and immobilize them on the surface of BioPlex beads. Furthermore, the protocol described how to identify PfEMP1 constructs that bind to specific host receptors or to immunoglobulins (IgG, IgM, etc.), and how to measure inhibition of the receptor binding to PfEMP1 constructs by small-molecule compounds or serum/plasma.

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.

Extraction and Immunoprecipitation of VAR2CSA, the PfEMP1 Associated with Placental Malaria.

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a key virulence factor for this human malaria parasite. During pregnancy, VAR2CSA is the only PfEMP1 variant expressed on the surface of infected erythrocytes that mediates adhesion to placenta cells and causes severe pregnancy outcomes.In this chapter, we present an optimized protocol to extract and immunoprecipitate endogenous VAR2CSA from the infected erythrocyte membrane phospholipid bilayer environment for subsequent characterization of the central role of VAR2CSA in placental malaria.

Immunomagnetic Selection of Plasmodium falciparum-Infected Erythrocytes Expressing Particular PfEMP1 Variants.

Plasmodium falciparum expresses a broad range of proteins on the surface of infected erythrocytes (IEs), including members of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family. This protocol describes an immunomagnetic selection method using PfEMP1-specific antibodies to obtain a parasite clone homogenously expressing a particular PfEMP1 protein. The expression of the corresponding PfEMP1 is later tested by flow cytometry, and the selected parasites can be used for further analysis.