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.

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.

Severe malaria is associated with parasite binding to endothelial protein C receptor.

Sequestration of Plasmodium falciparum-infected erythrocytes in host blood vessels is a key triggering event in the pathogenesis of severe childhood malaria, which is responsible for about one million deaths every year. Sequestration is mediated by specific interactions between members of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family and receptors on the endothelial lining. Severe childhood malaria is associated with expression of specific PfEMP1 subtypes containing domain cassettes (DCs) 8 and 13 (ref. 3), but the endothelial receptor for parasites expressing these proteins was unknown. Here we identify endothelial protein C receptor (EPCR), which mediates the cytoprotective effects of activated protein C, as the endothelial receptor for DC8 and DC13 PfEMP1. We show that EPCR binding is mediated through the amino-terminal cysteine-rich interdomain region (CIDRα1) of DC8 and group A PfEMP1 subfamilies, and that CIDRα1 interferes with protein C binding to EPCR. This PfEMP1 adhesive property links P. falciparum cytoadhesion to a host receptor involved in anticoagulation and endothelial cytoprotective pathways, and has implications for understanding malaria pathology and the development of new malaria interventions.

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.

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.

Diverse functional outcomes of Plasmodium falciparum ligation of EPCR: potential implications for malarial pathogenesis.

Plasmodium falciparum-infected erythrocytes (IRBC) expressing the domain cassettes (DC) 8 and 13 of the cytoadherent ligand P. falciparum erythrocyte membrane protein 1 adhere to the endothelial protein C receptor (EPCR). By interfering with EPCR anti-coagulant and pro-endothelial barrier functions, IRBC adhesion could promote coagulation and vascular permeability that contribute to the pathogenesis of cerebral malaria. In this study, we examined the adhesion of DC8- and DC13-expressing parasite lines to endothelial cells from different microvasculature, and the consequences of EPCR engagement on endothelial cell function. We found that IRBC from IT4var19 (DC8) and IT4var07 (DC13) parasite lines adhered to human brain, lung and dermal endothelial cells under shear stress. However, the relative contribution of EPCR to parasite cytoadherence on different types of endothelial cell varied. We also observed divergent functional outcomes for DC8 cysteine-rich interdomain region (CIDR)α1.1 and DC13 CIDRα1.4 domains. IT4var07 CIDRα1.4 inhibited generation of activated protein C (APC) on lung and dermal endothelial cells and blocked the APC-EPCR binding interaction on brain endothelial cells. IT4var19 CIDRα1.1 inhibited thrombin-induced endothelial barrier dysfunction in lung endothelial cells, whereas IT4var07 CIDRα1.4 inhibited the protective effect of APC on thrombin-induced permeability. Overall, these findings reveal a much greater complexity of how CIDRα1-expressing parasites may modulate malaria pathogenesis through EPCR adhesion.

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.

DC8 and DC13 var genes associated with severe malaria bind avidly to diverse endothelial cells.

During blood stage infection, Plasmodium falciparum infected erythrocytes (IE) bind to host blood vessels. This virulence determinant enables parasites to evade spleen-dependent killing mechanisms, but paradoxically in some cases may reduce parasite fitness by killing the host. Adhesion of infected erythrocytes is mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1), a family of polymorphic adhesion proteins encoded by var genes. Whereas cerebral binding and severe malaria are associated with parasites expressing DC8 and DC13 var genes, relatively little is known about the non-brain endothelial selection on severe malaria adhesive types. In this study, we selected P. falciparum-IEs on diverse endothelial cell types and demonstrate that DC8 and DC13 var genes were consistently among the major var transcripts selected on non-brain endothelial cells (lung, heart, bone marrow). To investigate the molecular basis for this avid endothelial binding activity, recombinant proteins were expressed from the predominant upregulated DC8 transcript, IT4var19. In-depth binding comparisons revealed that multiple extracellular domains from this protein bound brain and non-brain endothelial cells, and individual domains largely did not discriminate between different endothelial cell types. Additionally, we found that recombinant DC8 and DC13 CIDR1 domains exhibited a widespread endothelial binding activity and could compete for DC8-IE binding to brain endothelial cells, suggesting they may bind the same host receptor. Our findings provide new insights into the interaction of severe malaria adhesive types and host blood vessels and support the hypothesis that parasites causing severe malaria express PfEMP1 variants with a superior ability to adhere to diverse endothelial cell types, and may therefore endow these parasites with a growth and transmission advantage.

A restricted subset of var genes mediates adherence of Plasmodium falciparum-infected erythrocytes to brain endothelial cells.

Cerebral malaria (CM) is a deadly complication of Plasmodium falciparum infection, but specific interactions involved in cerebral homing of infected erythrocytes (IEs) are poorly understood. In this study, P. falciparum-IEs were characterized for binding to primary human brain microvascular endothelial cells (HBMECs). Before selection, CD36 or ICAM-1-binding parasites exhibited punctate binding to a subpopulation of HBMECs and binding was CD36 dependent. Panning of IEs on HBMECs led to a more dispersed binding phenotype and the selection of three var genes, including two that encode the tandem domain cassette 8 (DC8) and were non-CD36 binders. Multiple domains in the DC8 cassette bound to brain endothelium and the cysteine-rich interdomain region 1 inhibited binding of P. falciparum-IEs by 50%, highlighting a key role for the DC8 cassette in cerebral binding. It is mysterious how deadly binding variants are maintained in the parasite population. Clonal parasite lines expressing the two brain-adherent DC8-var genes did not bind to any of the known microvascular receptors, indicating unique receptors are involved in cerebral binding. They could also adhere to brain, lung, dermis, and heart endothelial cells, suggesting cerebral binding variants may have alternative sequestration sites. Furthermore, young African children with CM or nonsevere control cases had antibodies to HBMEC-selected parasites, indicating they had been exposed to related variants during childhood infections. This analysis shows that specific P. falciparum erythrocyte membrane protein 1 types are linked to cerebral binding and suggests a potential mechanism by which individuals may build up immunity to severe disease, in the absence of CM.

Clonal variants of Plasmodium falciparum exhibit a narrow range of rolling velocities to host receptor CD36 under dynamic flow conditions.

Cytoadhesion of Plasmodium falciparum parasitized red blood cells (pRBCs) has been implicated in the virulence of malaria infection. Cytoadhesive interactions are mediated by the protein family of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). The PfEMP1 family is under strong antibody and binding selection, resulting in extensive sequence and size variation of the extracellular domains. Here, we investigated cytoadhesion of pRBCs to CD36, a common receptor of P. falciparum field isolates, under dynamic flow conditions. Isogeneic parasites, predominantly expressing single PfEMP1 variants, were evaluated for binding to recombinant CD36 under dynamic flow conditions using microfluidic devices. We tested if PfEMP1 size (number of extracellular domains) or sequence variation affected the pRBC-CD36 interaction. Our analysis showed that clonal parasite variants varied ∼5-fold in CD36 rolling velocity despite extensive PfEMP1 sequence polymorphism. In addition, adherent pRBCs exhibited a characteristic hysteresis in rolling velocity at microvascular flow rates, which was accompanied by changes in pRBC shape and may represent important adaptations that favor stable binding.

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.

Multilaboratory approach to preclinical evaluation of vaccine immunogens for placental malaria.

Pregnancy malaria is caused by Plasmodium falciparum-infected erythrocytes that adhere to the placental receptor chondroitin sulfate A (CSA) and sequester in the placenta; women become resistant to pregnancy malaria as they acquire antiadhesion antibodies that target surface proteins of placental parasites. VAR2CSA, a member of the P. falciparum EMP1 variant surface antigen family, is the leading candidate for a pregnancy malaria vaccine. Because VAR2CSA is a high-molecular-weight protein, a vaccine based on the full-length protein may not be feasible. An alternative approach has been to develop a vaccine targeting individual Duffy binding-like (DBL) domains. In this study, a consortium of laboratories under the Pregnancy Malaria Initiative compared the functional activity of antiadhesion antibodies elicited by different VAR2CSA domains and variants produced in prokaryotic and eukaryotic expression systems. Antisera were initially tested against laboratory lines of maternal parasites, and the most promising reagents were evaluated in the field against fresh placental parasite samples. Recombinant proteins expressed in Escherichia coli elicited antibody levels similar to those expressed in eukaryotic systems, as did the two allelic forms of the DBL4 and DBL5 domains. The procedures developed for this head-to-head comparison will be useful for future evaluation and down-selection of malaria vaccine immunogens.

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.

High levels of antibodies to multiple domains and strains of VAR2CSA correlate with the absence of placental malaria in Cameroonian women living in an area of high Plasmodium falciparum transmission.

Placental malaria, caused by sequestration of Plasmodium falciparum-infected erythrocytes in the placenta, is associated with increased risk of maternal morbidity and poor birth outcomes. The parasite antigen VAR2CSA (variant surface antigen 2-chondroitin sulfate A) is expressed on infected erythrocytes and mediates binding to chondroitin sulfate A, initiating inflammation and disrupting homeostasis at the maternal-fetal interface. Although antibodies can prevent sequestration, it is unclear whether parasite clearance is due to antibodies to a single Duffy binding-like (DBL) domain or to an extensive repertoire of antibodies to multiple DBL domains and allelic variants. Accordingly, plasma samples collected longitudinally from pregnant women were screened for naturally acquired antibodies against an extensive panel of VAR2CSA proteins, including 2 to 3 allelic variants for each of 5 different DBL domains. Analyses were performed on plasma samples collected from 3 to 9 months of pregnancy from women living in areas in Cameroon with high and low malaria transmission. The results demonstrate that high antibody levels to multiple VAR2CSA domains, rather than a single domain, were associated with the absence of placental malaria when antibodies were present from early in the second trimester until term. Absence of placental malaria was associated with increasing antibody breadth to different DBL domains and allelic variants in multigravid women. Furthermore, the antibody responses of women in the lower-transmission site had both lower magnitude and lesser breadth than those in the high-transmission site. These data suggest that immunity to placental malaria results from high antibody levels to multiple VAR2CSA domains and allelic variants and that antibody breadth is influenced by malaria transmission intensity.

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.

Identification of a role for the PfEMP1 semi-conserved head structure in protein trafficking to the surface of Plasmodium falciparum infected red blood cells.

Transport of Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) variants to the red blood cell (RBC) surface enables malarial parasite evasion of host immunity by modifying the antigenic and adhesive properties of infected RBCs. In this study, we applied the Bxb1 integrase system to integrate transgenes encoding truncated PfEMP1-GFP fusions into cytoadherent A4 parasites and characterize their surface transport requirements. Our studies revealed that the semi-conserved head structure of PfEMP1 proteins, in combination with the predicted transmembrane region and cytoplasmic tail, encodes sufficient information for RBC surface display. In contrast, miniPfEMP1 proteins with truncated head structures were exported to the RBC cytoplasm but were not detected at the RBC surface by flow cytometry or immuno-electron microscopy. We demonstrated the absence of a mechanistic barrier to having native and miniPfEMP1 proteins displayed simultaneously at the RBC surface. However, surface-exposed miniPfEMP1 proteins did not convey cytoadherence properties to their host cells, implicating potential steric considerations in host-receptor interactions or the need for multiple domains to mediate cell binding. This study establishes a new system to investigate PfEMP1 transport and demonstrates that the PfEMP1 semi-conserved head structure is under selection for protein transport, in addition to its known roles in adhesion.

Induction of strain-transcendent antibodies to placental-type isolates with VAR2CSA DBL3 or DBL5 recombinant proteins.

BACKGROUND: Pregnancy associated malaria is a severe clinical syndrome associated with sequestration of Plasmodium falciparum-infected erythrocytes in the placenta. Placental binding is mediated by VAR2CSA, which adheres to chondroitin sulphate A (CSA). VAR2CSA is a large and polymorphic protein that has six Duffy binding-like (DBL) domains. There is still limited understanding as to how effective individual VAR2CSA domains are at generating inhibitory antibodies or the number of domain variants needed for universal vaccine coverage. METHODS: To investigate the immunogenic properties of single domain VAR2CSA recombinant proteins, rats or rabbits were immunized with five of the six VAR2CSA domains produced in Pichia pastoris. Immune plasma was analysed against a geographically diverse panel of CSA-binding lab lines to assess antibody breadth and inhibitory activity. RESULTS: Of the five domains, DBL3, and to a lesser extent DBL5, induced antibodies that cross-reacted on five diverse CSA-binding parasite lines by flow cytometry. By comparison, anti-DBL6 antibodies were highly strain-specific and anti-DBL1 and anti-DBL4 antibodies were poorly reactive by flow cytometry. From this series of recombinant proteins, adhesion-blocking activity was restricted to a single rat immunized against a DBL4 recombinant protein. CONCLUSIONS: Single domain VAR2CSA recombinant proteins produced in P. pastoris had limited efficacy in eliciting adhesion blocking antibody responses, but VAR2CSA DBL3 and DBL5 domains contain strain-transcendent epitopes that can be targeted by vaccination and may have application for vaccine development.

"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.