PfEMP1 and var genes - Still of key importance in Plasmodium falciparum malaria pathogenesis and immunity.

The most severe form of malaria, caused by infection with Plasmodium falciparum parasites, continues to be an important cause of human suffering and poverty. The P. falciparum erythrocyte membrane protein 1 (PfEMP1) family of clonally variant antigens, which mediates the adhesion of infected erythrocytes to the vascular endothelium in various tissues and organs, is a central component of the pathogenesis of the disease and a key target of the acquired immune response to malaria. Much new knowledge has accumulated since we published a systematic overview of the PfEMP1 family almost ten years ago. In this chapter, we therefore aim to summarize research progress since 2015 on the structure, function, regulation etc. of this key protein family of arguably the most important human parasite. Recent insights regarding PfEMP1-specific immune responses and PfEMP1-specific vaccination against malaria, as well as an outlook for the coming years are also covered.

Antibodies to PfEMP1 and Variant Surface Antigens: Protection after Controlled Human Malaria Infection in Semi-immune Kenyan Adults.

OBJECTIVES: Acquisition of antibodies to Plasmodium falciparum variant surface antigens (VSA) expressed on infected red blood cells (iRBCs) is associated with naturally acquired immunity to malaria. We have previously shown that antibodies to VSA on iRBCs are associated with protection against parasite growth in the context of controlled human malaria infection (CHMI). This study explored whether antibodies to recombinant antigens derived from PfEMP1 domains were independently associated with protection during CHMI in semi-immune Kenyan adults. METHODS: We used a multiplex bead assay to measure levels of IgG antibody against a panel of 27 recombinant PfEMP1 antigens derived from the PfEMP1 repertoire of the 3D7 parasite clone. We measured IgG levels in plasma samples collected from the CHMI participants before inoculation with Sanaria® PfSPZ Challenge, on the day of diagnosis, and 35 days post-inoculation. Univariable and multivariable Cox regression analysis was used to evaluate the relationship between the levels of antibodies to the antigens and CHMI outcome. We also adjusted for previous data including antibodies to VSA on iRBCs, and we assessed the kinetics of antibody acquisition to the different PfEMP1 recombinant antigens over time. RESULTS: All study participants had detectable antibodies to multiple PfEMP1 proteins before inoculation. All PfEMP1 antigens were associated with protection against parasite growth to the threshold criteria for treatment in CHMI albeit with substantial collinearity. However, individual PfEMP1 antigens were not independently associated with protection following adjustment for breadth of reactivity to VSA on iRBCs and schizont extract. In addition, antibodies to PfEMP1 antigens derived from group B PfEMP1 were induced and sustained in the participants who could not control parasite growth. CONCLUSION: This study shows that the breadth of antibody response to VSA on iRBCs, and not to specific PfEMP1 antigens, is predictive of protection against malaria in CHMI.

Variable Surface Antigens of Plasmodium falciparum: Protein Families with Divergent Roles.

Malaria caused by Plasmodium falciparum (Pf) is an illness that contributes significantly to the global health burden. Pf makes significant alterations to the host cell to meet its metabolic demands and escape the immune response of the host. These include the export of a large number of parasite proteins to the infected Red Blood Cells (iRBC). Variable Surface Antigens (VSAs), which are highly polymorphic protein families with important roles in immune evasion, form an important component of the exported proteins. A total of five protein families constitute the VSAs, viz. PfEMP1 (Pf erythrocyte membrane protein 1), RIFIN (repetitive interspersed family), STEVOR (sub-telomeric open reading frame), SURFIN (surface-associated interspersed gene family), and PfMC-2TM (Pf Maurer's cleft two transmembrane). With orthologues present in various simian-infecting species, VSAs take up a variety of domain topologies and organizational structures while exhibiting differential expressions throughout the parasite life cycle. Their expression varies across clinical isolates and laboratory strains, which suggests their crucial role in host cell survival and defense. Members of VSAs are reported to contribute significantly to disease pathogenesis through immune evasion processes like cytoadherence, iRBC sequestration in the host vasculature, rosetting, reduced erythrocyte deformability, and direct immunosuppression. In this study, we have gathered information on various aspects of VSAs, like their orthologues, domain architecture, surface topology, functions and interactions, and three-dimensional structures, while emphasizing discoveries in the field. Considering the vast repertoire of Plasmodial VSAs with new emergent functions, a lot remains unknown about these families and, hence, malaria biology.

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.

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.

Profiles of global mutations in the human intercellular adhesion molecule-1 (ICAM-1) shed light on population-specific malaria susceptibility.

Plasmodium falciparum is responsible for malaria-related morbidity and mortality. PfEMP1 (P. falciparum erythrocyte membrane protein 1) mediates infected erythrocytes adhesion to various surface vascular receptors, including intercellular adhesion molecule-1 (ICAM-1), associating this interaction with severe malaria in several studies. Genetic variation in host ICAM-1 plays a significant role in determining susceptibility to malaria infection via clinical phenotypes such as the ICAM-1Kilifi variant which has been reported to be associated with susceptibility in populations. Our genomic and structural analysis of single nucleotide polymorphisms (SNPs) in ICAM-1 revealed 9 unique mutations each in its distinct A-type and BC-type PfEMP1 DBLß-interacting regions. These mutations are noted in only a few field isolates and mainly in the African/African American population. The ICAM-1Kilifi variant lies in a flexible loop proximal to the DBLß-interacting region. This analysis will assist in establishing functional correlations of reported global mutations via experimental and clinical studies and in the tailored design of population-specific genetic surveillance studies. Understanding host polymorphism as an evolutionary force in diverse populations can help to predict predisposition to disease severity and will contribute towards laying the framework for designing population-specific personalized medicines for severe malaria.

Natural immunity to malaria preferentially targets the endothelial protein C receptor-binding regions of PfEMP1s.

Antibody responses to variant surface antigens (VSAs) produced by the malaria parasite Plasmodium falciparum may contribute to age-related natural immunity to severe malaria. One VSA family, P. falciparum erythrocyte membrane protein-1 (PfEMP1), includes a subset of proteins that binds endothelial protein C receptor (EPCR) in human hosts and potentially disrupts the regulation of inflammatory responses, which may lead to the development of severe malaria. We probed peptide microarrays containing segments spanning five PfEMP1 EPCR-binding domain variants with sera from 10 Malian adults and 10 children to determine the differences between adult and pediatric immune responses. We defined serorecognized peptides and amino acid residues as those that elicited a significantly higher antibody response than malaria-naïve controls. We aimed to identify regions consistently serorecognized among adults but not among children across PfEMP1 variants, potentially indicating regions that drive the development of immunity to severe malaria. Adult sera consistently demonstrated broader and more intense serologic responses to constitutive PfEMP1 peptides than pediatric sera, including peptides in EPCR-binding domains. Both adults and children serorecognized a significantly higher proportion of EPCR-binding peptides than peptides that do not directly participate in receptor binding, indicating a preferential development of serologic responses at functional residues. Over the course of a single malaria transmission season, pediatric serological responses increased between the start and the peak of the season, but waned as the transmission season ended. IMPORTANCE Severe malaria and death related to malaria disproportionately affect sub-Saharan children under 5 years of age, commonly manifesting as cerebral malaria and/or severe malarial anemia. In contrast, adults in malaria-endemic regions tend to experience asymptomatic or mild disease. Our findings indicate that natural immunity to malaria targets specific regions within the EPCR-binding domain, particularly peptides containing EPCR-binding residues. Epitopes containing these residues may be promising targets for vaccines or therapeutics directed against severe malaria. Our approach provides insight into the development of natural immunity to a binding target linked to severe malaria by characterizing an "adult-like" response as recognizing a proportion of epitopes within the PfEMP1 protein, particularly regions that mediate EPCR binding. This "adult-like" response likely requires multiple years of malaria exposure, as increases in pediatric serologic response over a single malaria transmission season do not appear significant.

Novel secretory organelles of parasite origin - at the center of host-parasite interaction.

Reorganization of cell organelle-deprived host red blood cells by the apicomplexan malaria parasite Plasmodium falciparum enables their cytoadherence to endothelial cells that line the microvasculature. This increases the time red blood cells infected with mature developmental stages remain within selected organs such as the brain to avoid the spleen passage, which can lead to severe complications and cumulate in patient death. The Maurer's clefts are a novel secretory organelle of parasite origin established by the parasite in the cytoplasm of the host red blood cell in order to facilitate the establishment of cytoadherence by conducting the trafficking of immunovariant adhesins to the host cell surface. Another important function of the organelle is the sorting of other proteins the parasite traffics into its host cell. Although the organelle is of high importance for the pathology of malaria, additional putative functions, structure, and genesis remain shrouded in mystery more than a century after its discovery. In this review, we highlight our current knowledge about the Maurer's clefts and other novel secretory organelles established within the host cell cytoplasm by human-pathogenic malaria parasites and other parasites that reside within human red blood cells.

Knobs, Adhesion, and Severe Falciparum Malaria.

Plasmodium falciparum can cause a severe disease with high mortality. A major factor contributing to the increased virulence of P. falciparum, as compared to other human malarial parasites, is the sequestration of infected erythrocytes in the capillary beds of organs and tissues. This sequestration is due to the cytoadherence of infected erythrocytes to endothelial cells. Cytoadherence is primarily mediated by a parasite protein expressed on the surface of the infected erythrocyte called P. falciparum erythrocyte membrane protein-1 (PfEMP1). PfEMP1 is embedded in electron-dense protuberances on the surface of the infected erythrocytes called knobs. These knobs are assembled on the erythrocyte membrane via exported parasite proteins, and the knobs function as focal points for the cytoadherence of infected erythrocytes to endothelial cells. PfEMP1 is a member of the var gene family, and there are approximately 60 antigenically distinct PfEMP1 alleles per parasite genome. Var gene expression exhibits allelic exclusion, with only a single allele being expressed by an individual parasite. This results in sequential waves of antigenically distinct infected erythrocytes and this antigenic variation allows the parasite to establish long-term chronic infections. A wide range of endothelial cell receptors can bind to the various PfEMP1 alleles, and thus, antigenic variation also results in a change in the cytoadherence phenotype. The cytoadherence phenotype may result in infected erythrocytes sequestering in different tissues and this difference in sequestration may explain the wide range of possible clinical manifestations associated with severe falciparum malaria.

Antibody to Plasmodium falciparum Variant Surface Antigens, var Gene Transcription, and ABO Blood Group in Children With Severe or Uncomplicated Malaria.

BACKGROUND: Antibodies to variant surface antigens (VSAs) such as Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) may vary with malaria severity. The influence of ABO blood group on antibody development is not understood. METHODS: Immunoglobulin G antibodies to VSAs in Papua New Guinean children with severe (n = 41) or uncomplicated (n = 30) malaria were measured by flow cytometry using homologous P falciparum isolates. Isolates were incubated with ABO-matched homologous and heterologous acute and convalescent plasma. RNA was used to assess var gene transcription. RESULTS: Antibodies to homologous, but not heterologous, isolates were boosted in convalescence. The relationship between antibody and severity varied by blood group. Antibodies to VSAs were similar in severe and uncomplicated malaria at presentation, higher in severe than uncomplicated malaria in convalescence, and higher in children with blood group O than other children. Six var gene transcripts best distinguished severe from uncomplicated malaria, including UpsA and 2 CIDRα1 domains. CONCLUSIONS: ABO blood group may influence antibody acquisition to VSAs and susceptibility to severe malaria. Children in Papua New Guinea showed little evidence of acquisition of cross-reactive antibodies following malaria. Var gene transcripts in Papua New Guinean children with severe malaria were similar to those reported from Africa.

Structural and genomic analysis of single nucleotide polymorphisms in human host factor endothelial protein C receptor (EPCR) reveals complex interplay with malaria parasites.

Plasmodium parasites responsible for malaria follow a complex life cycle of which half takes place inside the human host. Parasites present diverse antigens at different stages of their life cycle and interact with many surface molecules to attach to and enter host cells. The CIDRα1 domain of Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) in infected erythrocytes adheres to one such vascular receptor endothelial protein C receptor (EPCR). EPCR is implicated in the pathogenesis of severe malaria as preferential binding of CIDRα1 to endothelium results in widespread sequestration of infected erythrocytes leading to endothelium inflammation and severe disease. A single EPCR variant S219G is clinically reported to provide protection from severe malaria. In this work, we have collated all single nucleotide polymorphisms (SNPs) in EPCR from dbSNP. We structurally mapped the SNPs on the three-dimensional complex of EPCR and PfEMP1 CIDRα1. Analysis shows that most EPCR mutations lie on the receptor surface and are non-conservative. Of the 11 mutations in the CIDRα1-interaction region of EPCR, S88P, L96V/I, and R98L/H/P/C are seen with comparably higher occurrences in diverse populations. Our structural analysis details a framework of the interactions between the parasite ligand and host factor EPCR. These structural glimpses provide a blueprint for designing both field-based variant sequencing studies and vaccine development.

The Rapid and Spontaneous Postpartum Clearance of Plasmodium falciparum Is Related to Expulsion of the Placenta.

Parasitemia among pregnant women with protective immunity to Plasmodium falciparum malaria is often dominated by VAR2CSA-positive infected erythrocytes (IEs). VAR2CSA mediates sequestration of IEs in the placenta. We hypothesized that the previously observed spontaneous postpartum clearance of parasitemia in such women is related to the expulsion of the placenta, which removes the sequestration focus of VAR2CSA-positive IEs. We assessed parasitemias and gene transcription before and shortly after delivery in 17 Ghanaian women. The precipitous decline in parasitemia postpartum was accompanied by selective reduction in transcription of the gene encoding VAR2CSA. Our findings provide a mechanistic explanation for the earlier observation.

Global gene expression of human malaria parasite liver stages throughout intrahepatocytic development.

Plasmodium falciparum (Pf) is causing the greatest malaria burden, yet the liver stages (LS) of this most important parasite species have remained poorly studied. Here, we used a human liver-chimeric mouse model in combination with a novel fluorescent PfNF54 parasite line (PfNF54cspGFP) to isolate PfLS-infected hepatocytes and generate transcriptomes that cover the major LS developmental phases in human hepatocytes. RNA-seq analysis of early Pf LS trophozoites two days after infection, revealed a central role of translational regulation in the transformation of the extracellular invasive sporozoite into intracellular LS. The developmental time course gene expression analysis indicated that fatty acid biosynthesis, isoprenoid biosynthesis and iron metabolism are sustaining LS development along with amino acid metabolism and biosynthesis. Countering oxidative stress appears to play an important role during intrahepatic LS development. Furthermore, we observed expression of the variant PfEMP1 antigen-encoding var genes, and we confirmed expression of PfEMP1 protein during LS development. Transcriptome comparison of the late Pf liver stage schizonts with P. vivax (Pv) late liver stages revealed highly conserved gene expression profiles among orthologous genes. A notable difference however was the expression of genes regulating sexual stage commitment. While Pv schizonts expressed markers of sexual commitment, the Pf LS parasites were not sexually committed and showed expression of gametocytogenesis repression factors. Our results provide the first comprehensive gene expression profile of the human malaria parasite Pf LS isolated during in vivo intrahepatocytic development. This data will inform biological studies and the search for effective intervention strategies that can prevent infection.

Leukocyte and IgM Responses to Immunization with the CIDR1α-PfEMP1 Recombinant Protein in the Wistar Rat.

The malaria vaccine is an important strategy for the global malaria elimination program, but the complexity of the Plasmodium antigen is a major hurdle in malaria vaccine development. The cysteine-rich interdomain region 1α (CIDR1α) of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is crucial in malaria pathogenesis, making it a vaccine candidate. This study investigated the leukocyte and IgM response generated after administering a CIDR1α-PfEMP1 recombinant protein injection in Wistar rats. The rats were divided into a control group, who received a physiological saline solution (PSS), and a treatment group, who were subcutaneously injected with 150 µg of purified CIDR1α-PfEMP1 protein three times at the 3-week interval. Blood samples were collected every week after each injection. The number of leukocytes were counted using a Neubauer chamber, and the IgM concentration was determined using an enzyme-linked immunosorbent assay (ELISA). Data were analyzed using an independent, paired-T test, a Mann−Whitney test, and a Wilcoxon test, based on the distribution of the data. The total number of leukocytes notably increased on day 29 (p < 0.05). The percentage of neutrophils decreased, especially on day 8 (p < 0.05), whereas the percentages of monocytes and lymphocytes increased, primarily on day 14 (p < 0.05). The IgM concentration increased on day 14 (p < 0.05). In conclusion, the CIDR1α-PfEMP1 recombinant protein may induce leukocyte and IgM responses, making it a potential malaria vaccine candidate.

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.

Single-Cell Sorting of Plasmodium falciparum-Infected Erythrocytes Expressing Particular PfEMP1 Variants.

Cultures of Plasmodium falciparum often contain a heterogeneous parasite population. However, several studies require analysis of single infected erythrocytes (IEs) or a clonal parasite population derived from a single parasite. This protocol describes an efficient method for cloning by using fluorescence-activated cell sorting (FACS). For this, an antibody for a particular IEs surface protein it is added to the cell mixture to separate positive and negative IEs for that marker. After the separation, the viable homogeneous population can be used to grow in culture or for molecular analysis.

Analysis of var Gene Transcript Patterns by Quantitative Real-Time PCR.

Quantitative real-time PCR (qPCR) is a simple and sensitive method for determining the amount of a specific target DNA sequence present in a sample. Compared to RNA-seq, reverse transcription qPCR (RT-qPCR) is fast, requires only low input material and is easy to analyze. Therefore, qPCR is widely used to analyze gene expression in P. falciparum, including analyses of the multicopy gene families encoding variant surface antigens (VSAs), whose expression is clonally variant and prone to changes over time. In the recent years, several P. falciparum genomes of culture-adapted strains have been sequenced, providing the knowledge to design variable gene family-specific qPCR primers for each P. falciparum genetic background. Here, we describe the required materials, methods and key factors to perform RT-qPCR experiments to determine VSA transcript abundances in the P. falciparum clones 3D7/NF54, IT4, HB3, and 7G8.

Analysis of var Gene Transcription Pattern Using DBLα Tags.

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigens, which are encoded by a multigene family called var genes, are exported and inserted onto the surface of the infected erythrocytes. PfEMP1 plays a key role in the pathogenesis of severe malaria and are major targets of naturally acquired immunity. Studying the expression pattern of var genes in P. falciparum clinical isolates is crucial for understanding disease mechanism and immunity to malaria. However, var genes are highly variable, which makes it difficult to study their expression in clinical isolates obtained directly from malaria patients. In this chapter, we describe an approach for analysis of var gene expression that targets a region referred to as DBLα tag, which is relatively conserved in all var genes.

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.

Expression of Single-Domain Soluble and Disulfide-Folded PfEMP1 Antigens in the Escherichia coli SHuffle Expression System.

The genome of Plasmodium falciparum has an A/T content of around 81%. This, together with a high cysteine content and the high molecular weight of several proteins, make the expression of recombinant parasite proteins in heterologous systems challenging. P. falciparum erythrocyte membrane protein 1 (PfEMP1) is a family of proteins composed of several Duffy-binding like (DBL) and cysteine-rich inter-domain region (CIDR) domains involved in cytoadhesion to human host receptors and development of severe malaria. Expression of correctly folded single- and multiple-domain PfEMP1 fragment regions containing cysteines forming disulfide bonds, remains particularly difficult. Nevertheless, expression of single DBL and CIDR domains has been successful and this protocol describes the expression and purification of single-domain soluble PfEMP1 fragments using the Escherichia coli SHuffle expression system.