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Antibodies against Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) on infected erythrocytes (IEs) play a central role in naturally acquired protection against cerebral malaria (CM), yet the determinants of effective humoral immunity remain incompletely defined. We review evidence from seroepidemiological, functional, and mechanistic studies demonstrating that antibodies to endothelial protein C receptor (EPCR)‐binding cysteine-rich interdomain regions (CIDR)α1 and Duffy binding-like (DBL)β domains associated with dual EPCR and intercellular adhesion molecule 1 (ICAM1) binding correlate with reduced risk of CM, while responses to rosetting‐associated domains (DBLα, CIDRγ) and other domains are less well characterized. We synthesize findings on antibody kinetics—early, durable responses to Group A variants versus delayed, transient responses to Groups B and C—and on effector mechanisms including opsonic phagocytosis, complement activation, and Fc glycosylation. We highlight methodological challenges in measuring PfEMP1‐specific immunity, such as antigenic switching, differences between assays using single domains and native protein on IEs, and the need for physiologically relevant 3D vascular models. Finally, we identify key research priorities: mapping immunodominant epitopes across variant repertoires; longitudinal cohort studies to track antibody maturation and post‐translational modifications; and the development of broadly inhibitory monoclonal antibodies. Addressing these gaps will be critical for designing vaccines and therapeutics that harness protective antibody functions to prevent CM.

Background Malaria remains a global public health problem responsible for 445,000 deaths in 2016. While microscopy remains the mainstay of malaria diagnosis, highly sensitive molecular methods for detection of low-grade sub-microscopic infections are needed for surveillance studies and identifying asymptomatic reservoirs of malaria transmission. Methods The Plasmodium falciparum genome sequence was analysed to identify high copy number genes that improve P. falciparum parasite detection in blood by RT-PCR. Plasmodium falciparum erythrocyte membrane protein 1 ( Pf EMP1)-specific primers were evaluated for P. falciparum detection in hospital-based microscopically positive dried blood spots and field-acquired whole blood from asymptomatic individuals from Ghana. Results Pf EMP1 outperformed the Pf 18S sequence for amplification-based P. falciparum detection. Pf EMP1 primers exhibited sevenfold higher sensitivity compared to Pf 18S primers for parasite genomic DNA. Probit analysis established a 95% detection threshold of 9.3 parasites/mL for Pf EMP1 compared to 98.2 parasites/mL for Pf 18S primers. The Pf EMP1 primers also demonstrated superior clinical sensitivity, identifying 100% (20/20) of dried blood spot samples and 70% (69/98) of asymptomatic individuals as positive versus 55% (11/20) and 54% (53/98), respectively, for Pf 18S amplification. Conclusions These results establish Pf EMP1 as a novel amplification target for highly sensitive detection of both acute infections from filter paper samples and submicroscopic asymptomatic low-grade infections.

Strong founder effects resulting from human migration out of Africa have led to geographic variation in single nucleotide polymorphisms (SNPs) and microsatellites (MS) of the malaria parasite, Plasmodium falciparum. This is particularly striking in South America where two major founder populations of P. falciparum have been identified that are presumed to have arisen from the transatlantic slave trade. Given the importance of the major variant surface antigen of the blood stages of P. falciparum as both a virulence factor and target of immunity, we decided to investigate the population genetics of the genes encoding “Plasmodium falciparum Erythrocyte Membrane Protein 1” (PfEMP1) among several countries in South America, in order to evaluate the transmission patterns of malaria in this continent. Deep sequencing of the DBLα domain of var genes from 128 P. falciparum isolates from five locations in South America was completed using a 454 high throughput sequencing protocol. Striking geographic variation in var DBLα sequences, similar to that seen for SNPs and MS markers, was observed. Colombia and French Guiana had distinct var DBLα sequences, whereas Peru and Venezuela showed an admixture. The importance of such geographic variation to herd immunity and malaria vaccination is discussed. Given the importance of the major variant surface antigen of the blood stages of Plasmodium falciparum as both a virulence factor and target of immunity, we decided to investigate the evolutionary patterns of the genes encoding “Plasmodium falciparum Erythrocyte Membrane Protein 1” (PfEMP1), in several countries of South America, in order to evaluate the transmission patterns of malaria in this continent. Deep sequencing of the DBLα domain of var genes from 128 P. falciparum isolates from five locations in South America was completed using 454 sequencing. Striking geographic variation in var DBLα types, surprisingly similar to that seen for microsatellite and SNP markers, was observed. Colombia and French Guiana had distinct var DBLα types, whereas Peru and Venezuela showed an admixture. The importance of such geographic variation to herd immunity and malaria vaccination is discussed.

Human receptor gC1qR is a 32 kD protein that mediates the cytoadherence of Plasmodium falciparum-infected erythrocytes (IEs) to human brain microvascular endothelial cells (HBMEC) and platelets. The cytoadherence of IEs to gC1qR has been associated with severe malaria symptoms. The cytoadherence to gC1qR is mediated by the Duffy binding-like β12 (DBLβ12) domain of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), PFD0020c. Here, we report the structural insights into the binding of the DBLβ12 domain of PfEMP1 with the human receptor gC1qR using computational methods. A molecular model of the DBLβ12 domain was generated and used for protein–protein docking with the host receptor gC1qR. The protein–protein docking revealed that the DBLβ12 asymmetrically interacts with two subunits of the gC1qR trimer at the solution face of gC1qR. A total of 21 amino acid residues of DBLβ12 interact with 26 amino acid residues in the gC1qR trimer through 99 nonbonding interactions and 4 hydrogen bonds. Comparative analysis of binding sites on the DBL domain fold for the two receptors gC1qR and ICAM1 showed that the two sites are distinct. This is the first study that provides structural insights into DBLβ12 binding with its receptor gC1qR and may help in designing novel antisevere malaria interventions.

Key Points In cases of severe malaria, most deaths occur within the first 24 h of hospitalization, when the characteristic features of high parasite load and obstructed microvasculature are present. Novel adjunct drugs need to be developed as it is crucial to stop the infection and restore blood flow without delay. Plasmodium falciparum exports polypeptides to the surface of infected erythrocytes; for example, to import nutrients and to bind to other erythrocytes and the host microvasculature. Binding is mediated by the adhesive polypeptides Plasmodium falciparum -encoded repetitive interspersed families of polypeptides (RIFIN), subtelomeric variant open reading frame (STEVOR) and P. falciparum erythrocyte membrane protein 1 (PfEMP1). P. falciparum has the ability to express allelic variants of hundreds of different versions of erythrocyte surface antigens. Most of these genes belong to three large multigene families, the repetitive interspersed family ( rif ) , stevor and var families, which encode RIFIN, STEVOR and PfEMP1, respectively. RIFIN, STEVOR and PfEMP1 mediate the binding of parasite-infected red blood cells (pRBCs) to the vascular endothelium (cytoadherence), to red blood cells (rosetting), and to leukocytes and serum proteins. Cytoadherence and rosetting are involved in the pathogenesis of malaria by blocking blood flow when binding is excessive, leading to a lack of oxygen in tissues, excessive production of lactate and a reduction of the pH in blood and tissues, which can culminate in respiratory distress, coma, severe anaemia or combinations thereof — the hallmarks of severe malaria. A correlation between the clinical severity of malaria and the ABO blood group of a patient suggests that the O blood group protects against severe disease, a hypothesis that is supported by genome-wide association studies. In addition, this suggestion is supported by results from various in vitro studies. Plasmodium falciparum exports several variant antigens to the surface of erythrocytes. In this Review, Wahlgren, Goel and Akhouri discuss the three best characterized of these protein families, PfEMP1, RIFIN and STEVOR, and highlight their role in the development of severe malaria. Proliferation and differentiation inside erythrocytes are important steps in the life cycle of Plasmodium spp. To achieve these, the parasites export polypeptides to the surface of infected erythrocytes; for example, to import nutrients and to bind to other erythrocytes and the host microvasculature. Binding is mediated by the adhesive polypeptides Plasmodium falciparum -encoded repetitive interspersed families of polypeptides (RIFINs), subtelomeric variant open reading frame (STEVOR) and P. falciparum erythrocyte membrane protein 1 (PfEMP1), which are encoded by multigene families to ensure antigenic variation and evasion of host immunity. These variant surface antigens are suggested to mediate the sequestration of infected erythrocytes in the microvasculature and block the blood flow when binding is excessive. In this Review, we discuss the multigene families of surface variant polypeptides and highlight their roles in causing severe malaria.

Obligate intracellular malaria parasites reside within a vacuolar compartment generated during invasion which is the principal interface between pathogen and host. To subvert their host cell and support their metabolism, these parasites coordinate a range of transport activities at this membrane interface that are critically important to parasite survival and virulence, including nutrient import, waste efflux, effector protein export, and uptake of host cell cytosol. Here, we review our current understanding of the transport mechanisms acting at the malaria parasite vacuole during the blood and liver-stages of development with a particular focus on recent advances in our understanding of effector protein translocation into the host cell by the Plasmodium Translocon of EXported proteins (PTEX) and small molecule transport by the PTEX membrane-spanning pore EXP2. Comparison to Toxoplasma gondii and other related apicomplexans is provided to highlight how similar and divergent mechanisms are employed to fulfill analogous transport activities.

The trafficking of the virulence antigen Pf EMP1 and its presentation at the knob structures at the surface of parasite-infected RBCs are central to severe adhesion-related pathologies such as cerebral and placental malaria. This work adds to our understanding of how PfEMP1 is trafficked to the RBC membrane by defining the protein-protein interaction networks that function at the Maurer’s clefts controlling PfEMP1 loading and unloading. We characterize a protein needed for virulence protein trafficking and provide new insights into the mechanisms for host cell remodeling, parasite survival within the host, and virulence. The malaria parasite Plasmodium falciparum traffics the virulence protein P. falciparum erythrocyte membrane protein 1 ( Pf EMP1) to the surface of infected red blood cells (RBCs) via membranous organelles, known as the Maurer’s clefts. We developed a method for efficient enrichment of Maurer’s clefts and profiled the protein composition of this trafficking organelle. We identified 13 previously uncharacterized or poorly characterized Maurer’s cleft proteins. We generated transfectants expressing green fluorescent protein (GFP) fusions of 7 proteins and confirmed their Maurer’s cleft location. Using co-immunoprecipitation and mass spectrometry, we generated an interaction map of proteins at the Maurer’s clefts. We identified two key clusters that may function in the loading and unloading of Pf EMP1 into and out of the Maurer’s clefts. We focus on a putative Pf EMP1 loading complex that includes the protein GEXP07/CX3CL1-binding protein 2 (CBP2). Disruption of GEXP07 causes Maurer’s cleft fragmentation, aberrant knobs, ablation of Pf EMP1 surface expression, and loss of the Pf EMP1-mediated adhesion. ΔGEXP07 parasites have a growth advantage compared to wild-type parasites, and the infected RBCs are more deformable and more osmotically fragile. IMPORTANCE The trafficking of the virulence antigen Pf EMP1 and its presentation at the knob structures at the surface of parasite-infected RBCs are central to severe adhesion-related pathologies such as cerebral and placental malaria. This work adds to our understanding of how PfEMP1 is trafficked to the RBC membrane by defining the protein-protein interaction networks that function at the Maurer’s clefts controlling PfEMP1 loading and unloading. We characterize a protein needed for virulence protein trafficking and provide new insights into the mechanisms for host cell remodeling, parasite survival within the host, and virulence.

Malaria pathology is driven by the accumulation of Plasmodium falciparum -infected erythrocytes in microvessels 1 . This process is mediated by the polymorphic erythrocyte membrane protein 1 (PfEMP1) adhesion proteins of the parasite. A subset of PfEMP1 variants that bind to human endothelial protein C receptor (EPCR) through their CIDRα1 domains is responsible for severe malaria pathogenesis 2 . A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP1 variants. Here we describe two broadly reactive and inhibitory human monoclonal antibodies to CIDRα1. The antibodies isolated from two different individuals exhibited similar and consistent EPCR-binding inhibition of diverse CIDRα1 domains, representing five of the six subclasses of CIDRα1. Both antibodies inhibited EPCR binding of both recombinant full-length and native PfEMP1 proteins, as well as parasite sequestration in bioengineered 3D human brain microvessels under physiologically relevant flow conditions. Structural analyses of the two antibodies in complex with three different CIDRα1 antigen variants reveal similar binding mechanisms that depend on interactions with three highly conserved amino acid residues of the EPCR-binding site in CIDRα1. These broadly reactive antibodies are likely to represent a common mechanism of acquired immunity to severe malaria and offer novel insights for the design of a vaccine or treatment targeting severe malaria. Two broadly reactive and inhibitory human monoclonal antibodies against the malaria parasite Plasmodium falciparum have been characterized, providing insights into immunity, prevention and treatment of severe malaria.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family members mediate receptor- and tissue-specific sequestration of infected erythrocytes (IEs) in malaria. Antibody responses are a central component of naturally acquired malaria immunity. PfEMP1-specific IgG likely protects by inhibiting IE sequestration and through IgG-Fc Receptor (FcγR) mediated phagocytosis and killing of antibody-opsonized IEs. The affinity of afucosylated IgG to FcγRIIIa is up to 40-fold higher than fucosylated IgG, resulting in enhanced antibody-dependent cellular cytotoxicity. Most IgG in plasma is fully fucosylated, but afucosylated IgG is elicited in response to enveloped viruses and to paternal alloantigens during pregnancy. Here we show that naturally acquired PfEMP1-specific IgG is strongly afucosylated in a stable and exposure-dependent manner, and efficiently induces FcγRIIIa-dependent natural killer (NK) cell degranulation. In contrast, immunization with a subunit PfEMP1 (VAR2CSA) vaccine results in fully fucosylated specific IgG. These results have implications for understanding protective natural- and vaccine-induced immunity to malaria. Here, Larsen et al. describe differences in Fc fucosylation of P. falciparum PfEMP1-specific IgG produced in response to natural infection versus VAR2CSA-type subunit vaccination, which leads to differences in the ability to induce FcγRIIIa-dependent natural killer cell degranulation.

Sequestration of Plasmodium falciparum ‐infected erythrocytes (IE) within the brain microvasculature is a hallmark of cerebral malaria (CM). Using a microchannel flow adhesion assay with TNF‐activated primary human microvascular endothelial cells, we demonstrate that IE isolated from Malawian paediatric CM cases showed increased binding to brain microvascular endothelial cells compared to IE from uncomplicated malaria (UM) cases. Further, UM isolates showed significantly greater adhesion to dermal than to brain microvascular endothelial cells. The major mediator of parasite adhesion is P. falciparum erythrocyte membrane protein 1, encoded by var genes. Higher levels of var gene transcripts predicted to bind host endothelial protein C receptor (EPCR) and ICAM‐1 were detected in CM isolates. These data provide further evidence for differential tissue binding in severe and uncomplicated malaria syndromes, and give additional support to the hypothesis that CM pathology is based on increased cytoadherence of IE in the brain microvasculature. Synopsis Cytoadherence of Plasmodium falciparum ‐infected erythrocytes (IE) to the endothelial cells lining brain vessels is a hallmark of cerebral malaria (CM). This study shows that the ability of IE to cytoadhere in the brain of patients with CM and uncomplicated malaria is associated with the disease. IE from children with uncomplicated malaria do not bind well to brain endothelial cells, whereas IE from CM patients show high levels of binding. Significant associations in IE binding to brain endothelial cells were seen for both ICAM‐1 and EPCR. PfEMP1 variants containing EPCR‐binding motifs were associated with cerebral malaria. Graphical Abstract Cytoadherence of Plasmodium falciparum ‐infected erythrocytes (IE) to the endothelial cells lining brain vessels is a hallmark of cerebral malaria (CM). This study shows that the ability of IE to cytoadhere in the brain of patients with CM and uncomplicated malaria is associated with the disease.