Febrile Temperature Augments Ring-stage Plasmodium falciparum Adhesion to Brain Endothelial Cells.

Recent advances have identified a new paradigm for cerebral malaria pathogenesis in which endothelial protein C receptor (EPCR) is a major host receptor for sequestration of Plasmodium falciparum-infected erythrocytes (IEs) in the brain and other vital organs. The parasite adhesins that bind EPCR are members of the IE variant surface antigen family Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) containing specific adhesion domains called domain cassette (DC) 8 and DC13. The binding interaction site between PfEMP1 and EPCR has been mapped by biophysical and crystallography studies using recombinant proteins. However, studies examining the interaction of native PfEMP1 on the IE surface with EPCR are few. We aimed to study binding to EPCR by IEs expressing DC8 and DC13 PfEMP1 variants whose recombinant proteins have been used in key prior functional and structural studies. IE binding to EPCR immobilized on plastic and on human brain endothelial cells was examined in static and flow adhesion assays. Unexpectedly, we found that IEs expressing the DC13 PfEMP1 variant HB3var03 or IT4var07 did not bind to EPCR on plastic and the binding of these variants to brain endothelial cells was not dependent on EPCR. IEs expressing the DC8 variant IT4var19 did bind to EPCR, but this interaction was inhibited if normal human serum or plasma was present, raising the possibility that IE-EPCR interaction may be prevented by plasma components under physiological conditions. These data highlight a discrepancy in EPCR-binding activity between PfEMP1 recombinant proteins and IEs, and indicate the critical need for further research to understand the pathophysiological significance of the PfEMP1-EPCR interaction.

Structural Analysis of the Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) Intracellular Domain Reveals a Conserved Interaction Epitope

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a diverse family of variant surface antigens, encoded by var genes, that mediates binding of infected erythrocytes to human cells and plays a key role in parasite immune evasion and malaria pathology. The increased availability of parasite genome sequence data has revolutionised the study of PfEMP1 diversity across multiple P. falciparum isolates. However, making functional sense of genomic data relies on the ability to infer binding phenotype from var gene sequence. For P. falciparum rosetting, the binding of infected erythrocytes to uninfected erythrocytes, the analysis of var gene/PfEMP1 sequences encoding the phenotype is limited, with only eight rosette-mediating PfEMP1 variants described to date. These known rosetting PfEMP1 variants fall into two types, characterised by N-terminal domains known as "domain cassette" 11 (DC11) and DC16. Here we test the hypothesis that DC11 and DC16 are the only PfEMP1 types in the P. falciparum genome that mediate rosetting, by examining a set of thirteen recent culture-adapted Kenyan parasite lines. We first analysed the var gene/PfEMP1 repertoires of the Kenyan lines and identified an average of three DC11 or DC16 PfEMP1 variants per genotype. In vitro rosette selection of the parasite lines yielded four with a high rosette frequency, and analysis of their var gene transcription, infected erythrocyte PfEMP1 surface expression, rosette disruption and erythrocyte binding function identified four novel rosette-mediating PfEMP1 variants. Two of these were of the predicted DC11 type (one showing the dual rosetting/IgM-Fc-binding phenotype), whereas two contained DC15 (DBLα1.2-CIDRα1.5b) a PfEMP1 type not previously associated with rosetting. We also showed that a Thai parasite line expressing a DC8-like PfEMP1 binds to erythrocytes to form rosettes. Hence, these data expand current knowledge of rosetting mechanisms and emphasize that the PfEMP1 types mediating rosetting are more diverse than previously recognised.

The frequently fatal outcome of cerebral malaria has been linked to the adhesion and accumulation in the cerebral microvasculature of infected erythrocytes (IEs), which express a particular type of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). This type, found in the A and B/A subsets of PfEMP1, contains a particular structural motif (DBLβmotif) and has dual affinity for the host vascular receptors ICAM-1 and EPCR. Here, we report the functional characterization of a mouse monoclonal antibody, mAb02, raised against eight different DBLβmotif domains. The antibody selectively recognizes DBLβmotif-positive PfEMP1 proteins and inhibits their binding to ICAM-1. It also recognizes IEs expressing DBLβmotif-positive PfEMP1 proteins on their surface and inhibits their adhesion to ICAM-1. The mAb02 epitope is located in an unfolded linker region of the ICAM-1-binding site of DBLβmotif and includes residues directly involved in the interaction between DBLβmotif and ICAM-1, as well as residues that are important for the positioning of the interacting residues. Our study shows that mAb02 targets a broadly conserved epitope that is found in PfEMP1 proteins binding to ICAM-1 and EPCR and implicated in the pathogenesis of cerebral malaria (CM). This suggests the potential of mAb02 in the development of monoclonal antibody-based intervention against CM and for identification of IEs with capacity to causing CM.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-18465-1.

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.

Malaria, a mosquito‐borne infectious disease caused by parasites of the genus Plasmodium continues to be a major health problem worldwide. The unicellular Plasmodium‐parasites have the unique capacity to infect and replicate within host erythrocytes. By expressing variant surface antigens Plasmodium falciparum has evolved to avoid protective immune responses; as a result in endemic areas anti‐malaria immunity develops gradually over many years of multiple and repeated infections. We are studying the role of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) expressed by asexual stages of P. falciparum responsible for the pathogenicity of severe malaria. The immunopathology of falciparum malaria has been linked to cyto‐adhesion of infected erythrocytes to specific host receptors. A greater appreciation of the PfEMP1 molecules important for the development of protective immunity and immunopathology is a prerequisite for the rational discovery and development of a safe and protective anti‐disease malaria vaccine. Here we review the role of ICAM‐1 and EPCR receptor adhering falciparum‐parasites in the development of severe malaria; we discuss our current research to understand the factors involved in the pathogenesis of cerebral malaria and the feasibility of developing a vaccine targeted specifically to prevent this disease.

The erythrocytic stage development of malaria parasites occurs within the parasitophorous vacuole inside the infected-erythrocytes, and requires transport of several parasite-encoded proteins across the parasitophorous vacuole to several locations, including the cytosol and membrane of the infected cell. These proteins are called exported proteins; and a large number of such proteins have been predicted for Plasmodium falciparum based on the presence of an N-terminal motif known as the Plasmodium export element (PEXEL) or vacuolar transport signal (VTS), which has been shown to mediate export. The majority of exported proteins contain one or more transmembrane domains at the C-terminus and one of three types of N-terminus domain architectures. (1) The majority, including the knob-associated histidine rich protein (KAHRP), contain a signal/hydrophobic sequence preceding the PEXEL/VTS motif. (2) Other exported proteins, including the P. berghei variant antigen family bir and the P. falciparum skeleton binding protein-1, do not appear to contain a PEXEL/VTS motif. (3) The P. falciparum erythrocyte membrane protein-1 (PfEMP1) family lacks a signal/hydrophobic sequence before the motif. These different domain architectures suggest the presence of multiple export pathways in malaria parasites. To determine if export pathways are conserved in plasmodia and to develop an experimental system for studying these processes, we investigated export of GFP fused with N- and C-terminus putative export domains in the rodent malaria parasite P. berghei. Export was dependent on specific N- and C-terminal domains. Constructs with a KAHRP-like or bir N-terminus, but not the PfEMP1 N-terminus, exported GFP into the erythrocyte. The C-terminus of a P. falciparum variant antigen rifin prevented GFP export by the KAHRP-like N-terminus. In contrast, GFP chimeras containing KAHRP-like N-termini and the PfEMP1 C-terminus were exported to the surface of erythrocytes. Taken together, these results suggest that proteins with KAHRP-like architecture follow a common export pathway, but that PfEMP1s utilize an alternative pathway. Functional validation of common putative export domains of malaria parasites in P. berghei provides an alternative and simpler system to investigate export mechanisms.

BackgroundDuring the erythrocytic cycle, Plasmodium falciparum malaria parasites express P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) that anchor the infected erythrocytes (IE) to the vascular lining of the host. The CIDRα1 domain of PfEMP1 is responsible for binding host endothelial protein C receptor (EPCR), and increasing evidence support that this interaction triggers severe malaria, accounting for the majority of malaria-related deaths. In high transmission regions, children develop immunity to severe malaria after the first few infections. This immunity is believed to be mediated by antibodies targeting and inhibiting PfEMP1, causing infected erythrocytes to circulate and be cleared in the spleen. The development of immunity to malaria coincides with acquisition of broad antibody reactivity across the CIDRα1 protein family. Altogether, this identifies CIDRα1 as an important vaccine target. However, the antigenic diversity of the CIDRα1 domain family is a challenge for vaccine development.MethodsImmune responses in mice vaccinated with Virus-Like Particles (VLP) presenting CIDRα1 antigens were investigated. Antibody reactivity was tested to a panel of recombinant CIDRα1 domains, and the antibodies ability to inhibit EPCR binding by the recombinant CIDRα1 domains was tested in Luminex-based multiplex assays.ResultsVLP-presented CIDRα1.4 antigens induced a rapid and strong IgG response capable of inhibiting EPCR-binding of multiple CIDRα1 domains mainly within the group A CIDRα1.4–7 subgroups.ConclusionsThe study observations mirror those from previous CIDRα1 vaccine studies using other vaccine constructs and platforms. This suggests that broad CIDRα1 antibody reactivity may be achieved through vaccination with a limited number of CIDRα1 variants. In addition, this study suggest that this may be achieved through vaccination with a human compatible VLP vaccine platform.

Plasmodium falciparum malaria still places a major health burden upon the developing world. The high virulence of this parasite is linked to the variegated expression of single P. falciparum erythrocyte membrane protein 1 (PfEMP1) variants that are encoded by the var gene family. Most vars and other virulence genes implicated in host-parasite interactions are found within transcriptionally inert chromatin domains at the nuclear periphery and are associated with heterochromatin protein 1 (PfHP1). Mutually exclusive gene activation relies on the escape of a single var locus from this repressive environment into a transcriptionally competent perinuclear zone and non-coding elements play an essential role in this process. The exact mechanisms involved in singular gene choice, however, remain elusive. Using a transfection-based approach we identified cis-acting regulatory promoter elements, namely an upstream activating sequence and a mutual exclusion element (MEE), which are essential for var gene induction and singular locus recognition, respectively. Interestingly, in absence of the MEE, active var promoters are no longer introduced into the mutual exclusion programme. A yet unknown nuclear factor binds specifically to the MEE and I consider this interaction to be a prime candidate for the regulation of singular var activity. In addition, the MEE regulates PfEMP1 expression post-transcriptionally. On the mRNA level, this element is able to drastically reduce translation in an autonomous manner. In an independent project, I generated a conditional PfHP1 loss-of-function mutant to functionally characterise this epigenetic regulator. After inducing PfHP1 depletion during early intra-erythrocytic development, parasites progress normally through schizogony but do not enter mitosis in the subsequent generation. Detailed analysis of these PfHP1-deprived parasites revealed four striking phenotypes: (1) they show a massive de-repression of PfHP1-controlled virulence genes, including the entire var repertoire; (2) over 50% of the cells in the parasite population represent viable early stage gametocytes that complete sexual development in absence of PfHP1; (3) this high conversion rate is linked to the induction of an apiap2 TF family member during schizogony of the previous cell cycle; (4) the non-gametocyte cells represent asexual parasites that are reversibly arrested prior to enter S-phase and mitosis. We could thus identify essential roles for PfHP1 in gene silencing, cell cycle progression and showed for the first time that the mechanism of sexual conversion involves a strong epigenetic component.

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a key virulence factor for this species of human malarial parasite. PfEMP1 is expressed on the surface of infected erythrocytes (IEs) and directly mediates adhesion to a variety of host cells. A number of other parasite-encoded proteins are similarly exported to the IE plasma membrane and play an indirect role in this adhesion process through the modification of the erythrocyte cytoskeleton and the formation of electron dense knobs into which PfEMP1 is anchored. Analysis of the specific contribution of knob-associated proteins to adhesion is difficult due to rapid PfEMP1 switching during in vitro culture. Furthermore, these studies typically assume that the level and distribution of PfEMP1 exposed in knobby (K(+)) and knobless (K(-)) IEs is unaltered, an assumption not yet supported with data. We describe here the preparation and characterisation of a panel of isogenic K(+) and K(-) parasite clones that express one of two defined PfEMP1 variants. Analysis of the cytoadhesive properties of these clones shows that both static and flow adhesion is reduced in all the K(-) clones and, further, that this correlates with an approximately 50% reduction in PfEMP1 displayed on the IE surface. However, despite this reduction, the gross distribution of PfEMP1 in K(-) IEs appears unaltered. These data impact on our current interpretation of the role of knobs in adhesion and the mechanism of trafficking PfEMP1 to the IE surface.

Parasite adhesion and immune evasion in placental malaria.

Plasmodium falciparum Erythrocyte Membrane Protein-1 Specifically Suppresses Early Production of Host Interferon-γ

To investigate the involvement of oxidative tissue damage in the pathogenesis of murine cerebral malaria (CM), brain levels of protein carbonyls, 3,4-dihydroxyphenylalanine (DOPA), o-tyrosine, and dityrosine were measured during Plasmodium berghei ANKA (PbA) and P. berghei K173 (PbK) infections. During PbA infection in a CM model, brain levels of the substances were similar to those in uninfected mice. The role of phagocyte-derived reactive oxygen species in the pathogenesis of CM was examined in gp91phox gene knockout mice. The course of CM in these mice was the same as in their wild type counterparts. To examine whether superoxide production in the central nervous system could have occurred via increased xanthine oxidase activity, brain concentrations of urate were measured in CM mice and in mice infected with PbK (which does not cause CM). Brain urate concentration increased significantly in both groups of mice, suggesting that purine breakdown is not specific to CM. These results indicate that reactive oxygen species probably do not contribute to the pathogenesis of murine CM.

Haemoglobin C, which carries a glutamate-to-lysine mutation in the beta-globin chain, protects West African children against Plasmodium falciparum malaria. Mechanisms of protection are not established for the heterozygous (haemoglobin AC) or homozygous (haemoglobin CC) states. Here we report a marked effect of haemoglobin C on the cell-surface properties of P. falciparum-infected erythrocytes involved in pathogenesis. Relative to parasite-infected normal erythrocytes (haemoglobin AA), parasitized AC and CC erythrocytes show reduced adhesion to endothelial monolayers expressing CD36 and intercellular adhesion molecule-1 (ICAM-1). They also show impaired rosetting interactions with non-parasitized erythrocytes, and reduced agglutination in the presence of pooled sera from malaria-immune adults. Abnormal cell-surface display of the main variable cytoadherence ligand, PfEMP-1 (P. falciparum erythrocyte membrane protein-1), correlates with these findings. The abnormalities in PfEMP-1 display are associated with markers of erythrocyte senescence, and are greater in CC than in AC erythrocytes. Haemoglobin C might protect against malaria by reducing PfEMP-1-mediated adherence of parasitized erythrocytes, thereby mitigating the effects of their sequestration in the microvasculature.

The virulence of Plasmodium falciparum is closely linked to P. falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by a diverse var gene family. PfEMP1 mediates parasite immune evasion and vascular adhesion of infected red blood cells, contributing to severe disease. While expression of group A or domain cassettes (DC8 and DC13)-containing var genes have been associated with severe malaria, comparisons between the severe malaria syndromes remain limited. Furthermore, interactions between specific var gene expression and the rosetting phenotype, a known marker of severe malaria, are incompletely understood across severe malaria syndromes. We analyzed parasites and clinical data from 712 Kenyan children with non-severe and severe malaria syndromes collected over 18 years in Kilifi, Kenya. We used RT-qPCR and DBLα-tag sequencing to quantify var gene expression. We show that parasites expressing var genes containing the cys2 MFK+REY−motif or encoding DC8 domains were associated with impaired consciousness (IC), while rosetting was associated with respiratory distress (RD) and severe malarial anaemia (SMA), but not IC. These findings demonstrate that distinct PfEMP1 variants are preferentially expressed in specific severe malaria syndromes, highlighting potential targets for variant-specific future therapeutic and diagnostic strategies.