Full maturation of in vitro Plasmodium falciparum oocysts using the AlgiMatrix 3D culture system.

BACKGROUND: Plasmodium falciparum oocysts undergo growth and maturation in a unique setting within the mosquito midgut, firmly situated between the epithelium and the basal lamina. This location exposes them to specific nutrient exchange and metabolic processes while in direct contact with the mosquito haemolymph. The limited availability of in vitro culture systems for growth of the various P. falciparum mosquito stages hampers study of their biology and impedes progress in combatting malaria. METHODS: An artificial in vitro environment was established to mimic this distinctive setting, transitioning from a 2D culture system to a 3D model capable of generating fully mature oocysts that give rise to in vitro sporozoites. RESULTS: A two-dimensional (2D) chamber slide was employed along with an extracellular matrix composed of type IV collagen, entactin, and gamma laminin. This matrix facilitated development of the optimal medium composition for cultivating mature P. falciparum oocysts in vitro. However, the limitations of this 2D culture system in replicating the in vivo oocyst environment prompted a refinement of the approach by optimizing a three-dimensional (3D) alginate matrix culture system. This new system offered improved attachment, structural support, and nutrient exchange for the developing oocysts, leading to their maturation and the generation of sporozoites. CONCLUSIONS: This technique enables the in vitro growth of P. falciparum oocysts and sporozoites.

Accelerated prime-and-trap vaccine regimen in mice using repRNA-based CSP malaria vaccine.

Malaria, caused by Plasmodium parasites, remains one of the most devastating infectious diseases worldwide, despite control efforts to lower morbidity and mortality. Both advanced candidate vaccines, RTS,S and R21, are subunit (SU) vaccines that target a single Plasmodium falciparum (Pf) pre-erythrocytic (PE) sporozoite (spz) surface protein known as circumsporozoite (CS). These vaccines induce humoral immunity but fail to elicit CD8 + T-cell responses sufficient for long-term protection. In contrast, whole-organism (WO) vaccines, such as Radiation Attenuated Sporozoites (RAS), achieved sterile protection but require a series of intravenous doses administered in multiple clinic visits. Moreover, these WO vaccines must be produced in mosquitos, a burdensome process that severely limits their availability. To reduce reliance on WO while maintaining protection via both antibodies and Trm responses, we have developed an accelerated vaccination regimen that combines two distinct agents in a prime-and-trap strategy. The priming dose is a single dose of self-replicating RNA encoding the full-length P. yoelii CS protein, delivered via an advanced cationic nanocarrier (LIONTM). The trapping dose consists of one dose of WO RAS. Our vaccine induces a strong immune response when administered in an accelerated regimen, i.e., either 5-day or same-day immunization. Additionally, mice after same-day immunization showed a 2-day delay of blood patency with 90% sterile protection against a 3-week spz challenge. The same-day regimen also induced durable 70% sterile protection against a 2-month spz challenge. Our approach presents a clear path to late-stage preclinical and clinical testing of dose-sparing, same-day regimens that can confer sterilizing protection against malaria.

Accelerated prime-and-trap vaccine regimen in mice using repRNA-based CSP malaria vaccine.

Malaria, caused by Plasmodium parasites, remains one of the most devastating infectious diseases worldwide, despite control efforts that have lowered morbidity and mortality. The only P. falciparum vaccine candidates to show field efficacy are those targeting the asymptomatic pre-erythrocytic (PE) stages of infection. The subunit (SU) RTS,S/AS01 vaccine, the only licensed malaria vaccine to date, is only modestly effective against clinical malaria. Both RTS,S/AS01 and the SU R21 vaccine candidate target the PE sporozoite (spz) circumsporozoite (CS) protein. These candidates elicit high-titer antibodies that provide short-term protection from disease, but do not induce the liver-resident memory CD8+ T cells (Trm) that confer strong PE immunity and long-term protection. In contrast, whole-organism (WO) vaccines, employing for example radiation-attenuated spz (RAS), elicit both high antibody titers and Trm, and have achieved high levels of sterilizing protection. However, they require multiple intravenous (IV) doses, which must be administered at intervals of several weeks, complicating mass administration in the field. Moreover, the quantities of spz required present production difficulties. To reduce reliance on WO while maintaining protection via both antibodies and Trm responses, we have developed an accelerated vaccination regimen that combines two distinct agents in a prime-and-trap strategy. While the priming dose is a self-replicating RNA encoding P. yoelii CS protein, delivered via an advanced cationic nanocarrier (LION™), the trapping dose consists of WO RAS. This accelerated regime confers sterile protection in the P. yoelii mouse model of malaria. Our approach presents a clear path to late-stage preclinical and clinical testing of dose-sparing, same-day regimens that can confer sterilizing protection against malaria.

Accelerated prime-and-trap vaccine regimen in mice using repRNA-based CSP malaria vaccine.

Malaria, caused by Plasmodium parasites, remains one of the most devastating infectious diseases worldwide, despite control efforts that have lowered morbidity and mortality. The only P. falciparum vaccine candidates to show field efficacy are those targeting the asymptomatic pre-erythrocytic (PE) stages of infection. The subunit (SU) RTS,S/AS01 vaccine, the only licensed malaria vaccine to date, is only modestly effective against clinical malaria. Both RTS,S/AS01 and the SU R21 vaccine candidate target the PE sporozoite (spz) circumsporozoite (CS) protein. These candidates elicit high-titer antibodies that provide short-term protection from disease, but do not induce the liver-resident memory CD8+ T cells (Trm) that confer strong PE immunity and long-term protection. In contrast, whole-organism (WO) vaccines, employing for example radiation-attenuated spz (RAS), elicit both high antibody titers and Trm, and have achieved high levels of sterilizing protection. However, they require multiple intravenous (IV) doses, which must be administered at intervals of several weeks, complicating mass administration in the field. Moreover, the quantities of spz required present production difficulties. To reduce reliance on WO while maintaining protection via both antibodies and Trm responses, we have developed an accelerated vaccination regimen that combines two distinct agents in a prime-and-trap strategy. While the priming dose is a self-replicating RNA encoding P. yoelii CS protein, delivered via an advanced cationic nanocarrier (LION™), the trapping dose consists of WO RAS. This accelerated regime confers sterile protection in the P. yoelii mouse model of malaria. Our approach presents a clear path to late-stage preclinical and clinical testing of dose-sparing, same-day regimens that can confer sterilizing protection against malaria.

Genetic variations in human ATP2B4 gene alter Plasmodium falciparum in vitro growth in RBCs from Gambian adults.

BACKGROUND: Polymorphisms in ATP2B4 coding for PMCA4b, the primary regulator of erythrocyte calcium concentration, have been shown by GWAS and cross-sectional studies to protect against severe malaria but the mechanism remains unknown. METHODS: Using a recall-by-genotype design, we investigated the impact of a common haplotype variant in ATP2B4 using in vitro assays that model erythrocyte stage malaria pathogenesis. Ninety-six donors representing homozygote (carriers of the minor allele, C/C), heterozygote (T/C) and wildtype (T/T) carriers of the tagging SNP rs1541252 were selected from a cohort of over 12,000 participants in the Keneba Biobank. RESULTS: Red blood cells (RBCs) from homozygotes showed reduced PMCA4b protein expression (mean fluorescence intensities (MFI = 2428 ± 124, 3544 ± 159 and 4261 ± 283], for homozygotes, heterozygotes and wildtypes respectively, p < 0.0001) and slower rates of calcium expulsion (calcium t½ ± SD = 4.7 ± 0.5, 1.8 ± 0.3 and 1.9 ± 0.4 min, p < 0.0001). Growth of a Plasmodium falciparum laboratory strain (FCR3) and two Gambian field isolates was decreased in RBCs from homozygotes compared to heterozygotes and wildtypes (p < 0.01). Genotype group did not affect parasite adhesion in vitro or var-gene expression in malaria-infected RBCs. Parasite growth was inhibited by a known inhibitor of PMCA4b, aurintricarboxylic acid (IC50 = 122uM CI: 110-134) confirming its sensitivity to calcium channel blockade. CONCLUSION: The data support the hypothesis that this ATP2B4 genotype, common in The Gambia and other malaria-endemic areas, protects against severe malaria through the suppression of parasitaemia during an infection. Reduction in parasite density plays a pivotal role in disease outcome by minimizing all aspects of malaria pathogenesis. Follow up studies are needed to further elucidate the mechanism of protection and to determine if this ATP2B4 genotype carries a fitness cost or increases susceptibility to other human disease.

Plasmodium falciparum Parasite Lines Expressing DC8 and Group A PfEMP1 Bind to Brain, Intestinal, and Kidney Endothelial Cells.

Cytoadhesion of Plasmodium falciparum-infected red blood cells is a virulence determinant associated with microvascular obstruction and organ complications. The gastrointestinal tract is a major site of sequestration in fatal cerebral malaria cases and kidney complications are common in severe malaria, but parasite interactions with these microvascular sites are poorly characterized. To study parasite tropism for different microvascular sites, we investigated binding of parasite lines to primary human microvascular endothelial cells from intestine (HIMEC) and peritubular kidney (HKMEC) sites. Of the three major host receptors for P. falciparum, CD36 had low or negligible expression; endothelial protein C receptor (EPCR) had the broadest constitutive expression; and intercellular adhesion molecule 1 (ICAM-1) was weakly expressed on resting cells and was strongly upregulated by TNF-α on primary endothelial cells from the brain, intestine, and peritubular kidney sites. By studying parasite lines expressing var genes linked to severe malaria, we provide evidence that both the DC8 and Group A EPCR-binding subsets of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family encodes binding affinity for brain, intestinal, and peritubular kidney endothelial cells, and that DC8 parasite adhesion was partially dependent on EPCR. Collectively, these findings raise the possibility of a brain-gut-kidney binding axis contributing to multi-organ complications in severe malaria.

Genomic and functional characterization of a mucosal symbiont involved in early-stage colorectal cancer.

Colorectal cancer is a major health concern worldwide. Growing evidence for the role of the gut microbiota in the initiation of CRC has sparked interest in approaches that target these microorganisms. However, little is known about the composition and role of the microbiota associated with precancerous polyps. Here, we found distinct microbial signatures between patients with and without polyps and between polyp subtypes using sequencing and culturing techniques. We found a correlation between Bacteroides fragilis recovered and the level of inflammatory cytokines in the mucosa adjacent to the polyp. Additional analysis revealed that B. fragilis from patients with polyps are bft-negative, activate NF-κB through Toll-like receptor 4, induce a pro-inflammatory response, and are enriched in genes associated with LPS biosynthesis. This study provides fundamental insight into the microbial microenvironment of the pre-neoplastic polyp by highlighting strain-specific genomic and proteomic differences, as well as more broad compositional differences in the microbiome.

"Driver-passenger" bacteria and their metabolites in the pathogenesis of colorectal cancer.

Colorectal cancer (CRC) is a significant public health problem accounting for about 10% of all new cancer cases globally. Though genetic and epigenetic factors influence CRC, the gut microbiota acts as a significant component of the disease's etiology. Further research is still needed to clarify the specific roles and identify more bacteria related to CRC development. This review aims to provide an overview of the "driver-passenger" model of CRC. The colonization and active invasion of the "driver(s)" bacteria cause damages allowing other commensals, known as "passengers," or their by-products, i.e., metabolites, to pass through the epithelium . This review will not only focus on the species of bacteria implicated in this model but also on their biological functions implicated in the occurrence of CRC, such as forming biofilms, mucus, penetration and production of enterotoxins and genotoxins.

VAR2CSA binding phenotype has ancient origin and arose before Plasmodium falciparum crossed to humans: implications in placental malaria vaccine design.

VAR2CSA is a leading candidate for developing a placental malaria (PM) vaccine that would protect pregnant women living in malaria endemic areas against placental infections and improve birth outcomes. Two VAR2CSA-based PM vaccines are currently under clinical trials, but it is still unclear if the use of a single VAR2CSA variant will be sufficient to induce a broad enough humoral response in humans to cross-react with genetically diverse parasite populations. Additional immuno-focusing vaccine strategies may therefore be required to identify functionally conserved antibody epitopes in VAR2CSA. We explored the possibility that conserved epitopes could exist between VAR2CSA from the chimpanzee parasite Plasmodium reichenowi and Plasmodium falciparum sequences. Making use of VAR2CSA recombinant proteins originating from both species, we showed that VAR2CSA from P. reichenowi (Pr-VAR2CSA) binds to the placental receptor CSA with high specificity and affinity. Antibodies raised against Pr-VAR2CSA were able to recognize native VAR2CSA from different P. falciparum genotypes and to inhibit the interaction between CSA and P. falciparum-infected erythrocytes expressing different VAR2CSA variants. Our work revealed the existence of cross-species inhibitory epitopes in VAR2CSA and calls for pre-clinical studies assessing the efficacy of novel VAR2CSA-based cross-species boosting regimens.

Interplay of Plasmodium falciparum and thrombin in brain endothelial barrier disruption.

Recent concepts suggest that both Plasmodium falciparum factors and coagulation contribute to endothelial activation and dysfunction in pediatric cerebral malaria (CM) pathology. However, there is still limited understanding of how these complex inflammatory stimuli are integrated by brain endothelial cells. In this study, we examined how mature-stage P. falciparum infected erythrocytes (IE) interact with tumor necrosis factor α (TNFα) and thrombin in the activation and permeability of primary human brain microvascular endothelial cell (HBMEC) monolayers. Whereas trophozoite-stage P. falciparum-IE have limited effect on the viability of HBMEC or the secretion of pro-inflammatory cytokines or chemokines, except at super physiological parasite-host cell ratios, schizont-stage P. falciparum-IE induced low levels of cell death. Additionally, schizont-stage parasites were more barrier disruptive than trophozoite-stage P. falciparum-IE and prolonged thrombin-induced barrier disruption in both resting and TNFα-activated HBMEC monolayers. These results provide evidence that parasite products and thrombin may interact to increase brain endothelial permeability.

Binding Heterogeneity of Plasmodium falciparum to Engineered 3D Brain Microvessels Is Mediated by EPCR and ICAM-1.

Cerebral malaria is a severe neurological complication associated with sequestration of Plasmodium falciparum-infected erythrocytes (IE) in the brain microvasculature, but the specific binding interactions remain under debate. Here, we have generated an engineered three-dimensional (3D) human brain endothelial microvessel model and studied P. falciparum binding under the large range of physiological flow velocities that occur in both health and disease. Perfusion assays on 3D microvessels reveal previously unappreciated phenotypic heterogeneity in parasite binding to tumor necrosis factor alpha (TNF-α)-activated brain endothelial cells. While clonal parasite lines expressing a group B P. falciparum erythrocyte membrane protein 1 (PfEMP1) present an increase in binding to activated 3D microvessels, P. falciparum-IE expressing DC8-PfEMP1 present a decrease in binding. The differential response to endothelium activation is mediated by surface expression changes of endothelial protein C receptor (EPCR) and intercellular adhesion molecule 1 (ICAM-1). These findings demonstrate heterogeneity in parasite binding and provide evidence for a parasite strategy to adapt to a changing microvascular environment during infection. The engineered 3D human brain microvessel model provides new mechanistic insight into parasite binding and opens opportunities for further studies on malaria pathogenesis and parasite-vessel interactions.IMPORTANCE Cerebral malaria research has been hindered by the inaccessibility of the brain. Here, we have developed an engineered 3D human brain microvessel model that mimics the blood flow rates and architecture of small blood vessels to study how P. falciparum-infected human erythrocytes attach to brain endothelial cells. By studying parasite lines with different adhesive properties, we show that the malaria parasite binding rate is heterogeneous and strongly influenced by physiological differences in flow and whether the endothelium has been previously activated by TNF-α, a proinflammatory cytokine that is linked to malaria disease severity. We also show the importance of human EPCR and ICAM-1 in parasite binding. Our model sheds new light on how P. falciparum binds within brain microvessels and provides a powerful method for future investigations of recruitment of human brain pathogens to the blood vessel lining of the brain.

Antibody Targets on the Surface of Plasmodium falciparum-Infected Erythrocytes That Are Associated With Immunity to Severe Malaria in Young Children.

BACKGROUND: Sequestration of Plasmodium falciparum-infected erythrocytes (IEs) in the microvasculature contributes to pathogenesis of severe malaria in children. This mechanism is mediated by antigens expressed on the IE surface. However, knowledge of specific targets and functions of antibodies to IE surface antigens that protect against severe malaria is limited. METHODS: Antibodies to IE surface antigens were examined in a case-control study of young children in Papua New Guinea presenting with severe or uncomplicated malaria (n = 448), using isolates with a virulent phenotype associated with severe malaria, and functional opsonic phagocytosis assays. We used genetically modified isolates and recombinant P. falciparum erythrocyte membrane protein 1 (PfEMP1) domains to quantify PfEMP1 as a target of antibodies associated with disease severity. RESULTS: Antibodies to the IE surface and recombinant PfEMP1 domains were significantly higher in uncomplicated vs severe malaria and were boosted following infection. The use of genetically modified P. falciparum revealed that PfEMP1 was a major target of antibodies and that PfEMP1-specific antibodies were associated with reduced odds of severe malaria. Furthermore, antibodies promoting the opsonic phagocytosis of IEs by monocytes were lower in those with severe malaria. CONCLUSIONS: Findings suggest that PfEMP1 is a dominant target of antibodies associated with reduced risk of severe malaria, and function in part by promoting opsonic phagocytosis.

Declining Malaria Transmission Differentially Impacts the Maintenance of Humoral Immunity to Plasmodium falciparum in Children.

Background: We investigated the poorly understood impact of declining malaria transmission on maintenance of antibodies to Plasmodium falciparum merozoite antigens and infected erythrocytes (IEs), including functional immunity. Methods: In a 3-year longitudinal cohort of 300 Kenyan children, antibodies to different AMA1 and MSP2 alleles of merozoites, IE surface antigens, and antibody functional activities were quantified. Results: Over a period in which malaria transmission declined markedly, AMA1 and MSP2 antibodies decreased substantially; estimated half-lives of antibody duration were 0.8 year and 1-3 years, respectively. However, 69%-74% of children maintained their seropositivity to AMA1 alleles and 42%-52% to MSP2 alleles. Levels and prevalence of antimerozoite antibodies were consistently associated with increasing age and concurrent parasitemia. Antibodies promoting opsonic phagocytosis of merozoites declined rapidly (half-life, 0.15 years). In contrast, complement-fixing antibodies to merozoites did not decline and antibodies to IE surface antigens expressing virulent phenotypes were much better maintained (half-life, 4-10 years). Conclusions: A decline in malaria transmission is associated with reduction in naturally acquired immunity. However, loss of immunity is not universal; some key functional responses and antibodies to IEs were better maintained and these may continue to provide some protection. Findings have implications for malaria surveillance and control measures and informing vaccine development.

Pathogenicity Determinants of the Human Malaria Parasite Plasmodium falciparum Have Ancient Origins.

Plasmodium falciparum, the most deadly of the human malaria parasites, is a member of the Laverania subgenus that also infects African Great Apes. The virulence of P. falciparum is related to cytoadhesion of infected erythrocytes in microvasculature, but the origin of dangerous parasite adhesion traits is poorly understood. To investigate the evolutionary history of the P. falciparum cytoadhesion pathogenicity determinant, we studied adhesion domains from the chimpanzee malaria parasite P. reichenowi. We demonstrate that the P. reichenowi var gene repertoire encodes cysteine-rich interdomain region (CIDR) domains which bind human CD36 and endothelial protein C receptor (EPCR) with the same levels of affinity and at binding sites similar to those bound by P. falciparum. Moreover, P. reichenowi domains interfere with the protective function of the activated protein C-EPCR pathway on endothelial cells, a presumptive virulence trait in humans. These findings provide evidence for ancient evolutionary origins of two key cytoadhesion properties of P. falciparum that contribute to human infection and pathogenicity. IMPORTANCE Cytoadhesion of P. falciparum-infected erythrocytes in the microcirculation is a major virulence determinant. P. falciparum is descended from a subgenus of parasites that also infect chimpanzees and gorillas and exhibits strict host species specificity. Despite their high genetic similarity to P. falciparum, it is unknown whether ape parasites encode adhesion properties similar to those of P. falciparum or are as virulent in their natural hosts. Consequently, it has been unclear when virulent adhesion traits arose in P. falciparum and how long they have been present in the parasite population. It is also unknown whether cytoadhesive interactions pose a barrier to cross-species transmission. We show that parasite domains from the chimpanzee malaria parasite P. reichenowi bind human receptors with specificity similar to that of P. falciparum. Our findings suggest that parasite adhesion traits associated with both mild and severe malaria have much earlier origins than previously appreciated and have important implications for virulence evolution in a major human pathogen.

Thrombin Cleavage of Plasmodium falciparum Erythrocyte Membrane Protein 1 Inhibits Cytoadherence.

UNLABELLED: Plasmodium falciparum malaria remains one of the most deadly infections worldwide. The pathogenesis of the infection results from the sequestration of infected erythrocytes (IRBC) in vital organs, including the brain, with resulting impairment of blood flow, hypoxia, and lactic acidosis. Sequestration occurs through the adhesion of IRBC to host receptors on microvascular endothelium by Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), a large family of variant surface antigens, each with up to seven extracellular domains that can bind to multiple host receptors. Consequently, antiadhesive therapies directed at single endothelial adhesion molecules may not be effective. In this study, we demonstrated that the serine protease thrombin, which is pivotal in the activation of the coagulation cascade, cleaved the major parasite adhesin on the surface of IRBC. As a result, adhesion under flow was dramatically reduced, and already adherent IRBC were detached. Thrombin cleavage sites were mapped to the Duffy binding-like δ1 (DBLδ1) domain and interdomains 1 and 2 in the PfEMP1 of the parasite line IT4var19. Furthermore, we observed an inverse correlation between the presence of thrombin and IRBC in cerebral malaria autopsies of children. We investigated a modified (R67A) thrombin and thrombin inhibitor, hirugen, both of which inhibit the binding of substrates to exosite I, thereby reducing its proinflammatory properties. Both approaches reduced the barrier dysfunction induced by thrombin without affecting its proteolytic activity on PfEMP1, raising the possibility that thrombin cleavage of variant PfEMP1 may be exploited as a broadly inhibitory antiadhesive therapy. IMPORTANCE: Plasmodium falciparum malaria is the third leading cause of mortality due to a pathogen, with 214 million people infected and 438,000 deaths annually. The adhesion of Plasmodium falciparum-infected erythrocytes (IRBC) to microvascular endothelium is a major pathological process in severe malaria. While the recent implementation of artemisinin-based antimalarial therapy for severe malaria improves patient survival by targeting all parasite stages, antiparasite drugs alone may not immediately reverse pathophysiological processes in occluded vessels. Here we show that thrombin, an enzyme intimately involved in the clotting process, cleaves the main parasite adhesin expressed on the surface of IRBC, thereby preventing and reversing the binding of IRBC to endothelial cells. This beneficial effect of thrombin can be achieved by modified thrombins that cause significantly less clotting and vessel leakage while preserving the ability to cleave the parasite protein. Our results provide the basis for using modified thrombins as adjunctive therapy in severe malaria.

Interaction between Endothelial Protein C Receptor and Intercellular Adhesion Molecule 1 to Mediate Binding of Plasmodium falciparum-Infected Erythrocytes to Endothelial Cells.

UNLABELLED: Intercellular adhesion molecule 1 (ICAM-1) and the endothelial protein C receptor (EPCR) are candidate receptors for the deadly complication cerebral malaria. However, it remains unclear if Plasmodium falciparum parasites with dual binding specificity are involved in cytoadhesion or different parasite subpopulations bind in brain microvessels. Here, we investigated this issue by studying different subtypes of ICAM-1-binding parasite lines. We show that two parasite lines expressing domain cassette 13 (DC13) of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family have dual binding specificity for EPCR and ICAM-1 and further mapped ICAM-1 binding to the first DBLß domain following the PfEMP1 head structure in both proteins. As PfEMP1 head structures have diverged between group A (EPCR binders) and groups B and C (CD36 binders), we also investigated how ICAM-1-binding parasites with different coreceptor binding traits influence P. falciparum-infected erythrocyte binding to endothelial cells. Whereas levels of binding to tumor necrosis factor alpha (TNF-α)-stimulated endothelial cells from the lung and brain by all ICAM-1-binding parasite lines increased, group A (EPCR and ICAM-1) was less dependent than group B (CD36 and ICAM-1) on ICAM-1 upregulation. Furthermore, both group A DC13 parasite lines had higher binding levels to brain endothelial cells (a microvascular niche with limited CD36 expression). This study shows that ICAM-1 is a coreceptor for a subset of EPCR-binding parasites and provides the first evidence of how EPCR and ICAM-1 interact to mediate parasite binding to both resting and TNF-α-activated primary brain and lung endothelial cells. IMPORTANCE: Cerebral malaria is a severe neurological complication of P. falciparum infection associated with infected erythrocyte (IE) binding in cerebral vessels. Yet little is known about the mechanisms by which parasites adhere in the brain or other microvascular sites. Here, we studied parasite lines expressing group A DC13-containing PfEMP1 variants, a subset that has previously been shown to have high brain cell- and other endothelial cell-binding activities. We show that DC13-containing PfEMP1 variants have dual EPCR- and ICAM-1-binding activities and that both receptors are involved in parasite adherence to lung and brain endothelial cells. As both EPCR and ICAM-1 are implicated in cerebral malaria, these findings suggest the possibility that parasites with dual binding activities are involved in parasite sequestration to microvascular beds with low CD36 expression, such as the brain, and we urge more research into the multiadhesive properties of PfEMP1 variants.

Severe adult malaria is associated with specific PfEMP1 adhesion types and high parasite biomass.

The interplay between cellular and molecular determinants that lead to severe malaria in adults is unexplored. Here, we analyzed parasite virulence factors in an infected adult population in India and investigated whether severe malaria isolates impair endothelial protein C receptor (EPCR), a protein involved in coagulation and endothelial barrier permeability. Severe malaria isolates overexpressed specific members of the Plasmodium falciparum var gene/PfEMP1 (P. falciparum erythrocyte membrane protein 1) family that bind EPCR, including DC8 var genes that have previously been linked to severe pediatric malaria. Machine learning analysis revealed that DC6- and DC8-encoding var transcripts in combination with high parasite biomass were the strongest indicators of patient hospitalization and disease severity. We found that DC8 CIDRα1 domains from severe malaria isolates had substantial differences in EPCR binding affinity and blockade activity for its ligand activated protein C. Additionally, even a low level of inhibition exhibited by domains from two cerebral malaria isolates was sufficient to interfere with activated protein C-barrier protective activities in human brain endothelial cells. Our findings demonstrate an interplay between parasite biomass and specific PfEMP1 adhesion types in the development of adult severe malaria, and indicate that low impairment of EPCR function may contribute to parasite virulence.

Inhibition of Infected Red Blood Cell Binding to the Vascular Endothelium.

Plasmodium falciparum-infected red blood cells (IRBC) adhere to the endothelium via receptors expressed on the surface of vascular endothelial cells (EC) and sequester in the microvasculature of several organs. Sequestration is the primary step leading to complications related to the severity of malaria. In order to study this cytoadhesion phenomenon, IRBC in vitro binding assays have been developed using a monolayer of primary or transformed endothelial cells. Here we describe the methodology of an assay to inhibit the binding of IRBC on vascular endothelial cells under static adhesion conditions. Similar techniques could be used for conducting a binding inhibition assay under flow assay conditions using an appropriate device.

Plasmodium falciparum adhesion domains linked to severe malaria differ in blockade of endothelial protein C receptor.

Cytoadhesion of Plasmodium falciparum-infected erythrocytes to endothelial protein C receptor (EPCR) is associated with severe malaria. It has been postulated that parasite binding could exacerbate microvascular coagulation and endothelial dysfunction in cerebral malaria by impairing the protein C-EPCR interaction, but the extent of binding inhibition has not been fully determined. Here we expressed the cysteine-rich interdomain region (CIDRα1) domain from a variety of domain cassette (DC) 8 and DC13 P. falciparum erythrocyte membrane protein 1 proteins and show they interact in a distinct manner with EPCR resulting in weak, moderate and strong inhibition of the activated protein C (APC)-EPCR interaction. Overall, there was a positive correlation between CIDRα1-EPCR binding activity and APC blockade activity. In addition, our analysis from a combination of mutagenesis and blocking antibodies finds that an Arg81 (R81) in EPCR plays a pivotal role in CIDRα1 binding, but domains with weak and strong APC blockade activity were distinguished by their sensitivity to inhibition by anti-EPCR mAb 1535, implying subtle differences in their binding footprints. These data reveal a previously unknown functional heterogeneity in the interaction between P. falciparum and EPCR and have major implications for understanding the distinct clinical pathologies of cerebral malaria and developing new treatment strategies.