Amino terminal peptides from the Plasmodium falciparum EBA-181/JESEBL protein bind specifically to erythrocytes and inhibit in vitro merozoite invasion

Several EBA-175 paralogues (EBA-140, EBA-165, EBA-175, EBA-181, and EBL-1) have been described among the Plasmodium falciparum malaria parasite proteins, which are important in the red blood cell (RBC) invasion process. EBA-181/JESEBL is a 181 kDa protein expressed in the late schizont stage and located in the micronemes; it belongs to the Plasmodium Duffy binding-like family and is able to interact with the erythrocyte surface. Here, we describe the synthesis of 78, 20-mer synthetic peptides derived from the reported EBA-181/JESEBL sequence and their ability to bind RBCs in receptor-ligand assays. Five peptides (numbered 30030, 30031, 30045, 30051, and 30060) displayed high specific binding to erythrocytes; their equilibrium binding parameters were then determined. These peptides interacted with 53 and 33 kDa receptor proteins on the erythrocyte surface, this binding being altered when RBCs were pretreated with enzymes. They were able to inhibit P. falciparum merozoite invasion of RBCs when tested in in vitro assays. According to these results, these five EBA-181/JESEBL high specific erythrocyte binding peptides, as well as the entire protein, were seen to be involved in the molecular machinery used by the parasite for invading RBCs. They are thus suggested as potential candidates in designing a multi-sub-unit vaccine able to combat the P. falciparum malaria parasite.     

Reticulocyte binding protein homologues are key adhesins during erythrocyte invasion by Plasmodium falciparum

The Apicomplexan parasite responsible for the most virulent form of malaria, Plasmodium falciparum , invades human erythrocytes through multiple ligand–receptor interactions. The P.  falciparum reticulocyte-binding protein homologue (PfRh or PfRBL) family have been implicated in the invasion process but their exact role is unknown. PfRh1 and PfRh4, members of this protein family, bind to red blood cells and function in merozoite invasion during which they undergo a series of proteolytic cleavage events before and during entry into the host cell. The ectodomain of PfRh1 and PfRh4 are processed to produce fragments consistent with cleavage in the transmembrane domain and released into the supernatant, at about the time of invasion, in a manner consistent with rhomboid protease cleavage. Processing of both PfRh1 and PfRh4, and by extrapolation all membrane-bound members of this protein family, is important for function and release of these proteins on the merozoite surface and they along with EBA-175 are important components of the tight junction, the transient structure that links the erythrocyte via receptor–ligand interactions to the actin–myosin motor in the invading merozoite.     

Plasmodium falciparum merozoite invasion is inhibited by antibodies that target the PfRh2a and b binding domains

Plasmodium falciparum, the causative agent of the most severe form of malaria in humans invades erythrocytes using multiple ligand-receptor interactions. The P. falciparum reticulocyte binding-like homologue proteins (PfRh or PfRBL) are important for entry of the invasive merozoite form of the parasite into red blood cells. We have analysed two members of this protein family, PfRh2a and PfRh2b, and show they undergo a complex series of proteolytic cleavage events before and during merozoite invasion. We show that PfRh2a undergoes a cleavage event in the transmembrane region during invasion consistent with activity of the membrane associated PfROM4 protease that would result in release of the ectodomain into the supernatant. We also show that PfRh2a and PfRh2b bind to red blood cells and have defined the erythrocyte-binding domain to a 15 kDa region at the N-terminus of each protein. Antibodies to this receptor-binding region block merozoite invasion demonstrating the important function of this domain. This region of PfRh2a and PfRh2b has potential in a combination vaccine with other erythrocyte binding ligands for induction of antibodies that would block a broad range of invasion pathways for P. falciparum into human erythrocytes.     

The cytoplasmic domain of the Plasmodium falciparum ligand EBA-175 is essential for invasion but not protein trafficking

The invasion of host cells by the malaria parasite Plasmodium falciparum requires specific protein-protein interactions between parasite and host receptors and an intracellular translocation machinery to power the process. The transmembrane erythrocyte binding protein-175 (EBA-175) and thrombospondin-related anonymous protein (TRAP) play central roles in this process. EBA-175 binds to glycophorin A on human erythrocytes during the invasion process, linking the parasite to the surface of the host cell. In this report, we show that the cytoplasmic domain of EBA-175 encodes crucial information for its role in merozoite invasion, and that trafficking of this protein is independent of this domain. Further, we show that the cytoplasmic domain of TRAP, a protein that is not expressed in merozoites but is essential for invasion of liver cells by the sporozoite stage, can substitute for the cytoplasmic domain of EBA-175. These results show that the parasite uses the same components of its cellular machinery for invasion regardless of the host cell type and invasive form.     

A novel erythrocyte binding antigen-175 paralogue from Plasmodium falciparum defines a new trypsin-resistant receptor on human erythrocytes

The recognition and invasion of human erythrocytes by the most lethal malaria parasite Plasmodium falciparum is dependent on multiple ligand-receptor interactions. Members of the erythrocyte binding-like (ebl) family, including the erythrocyte binding antigen-175 (EBA-175), are responsible for high affinity binding to glycoproteins on the surface of the erythrocyte. Here we describe a paralogue of EBA-175 and show that this protein (EBA-181/JESEBL) binds in a sialic acid-dependent manner to erythrocytes. EBA-181 is expressed at the same time as EBA-175 and co-localizes with this protein in the microneme organelles of asexual stage parasites. The receptor binding specificity of EBA-181 to erythrocytes differs from other members of the ebl family and is trypsin-resistant and chymotrypsin-sensitive. Furthermore, using glycophorin B-deficient erythrocytes we show that binding of EBA-181 is not dependent on this sialoglycoprotein. The level of expression of EBA-181 differs among parasite lines, and the importance of this ligand for invasion appears to be strain-dependent as the EBA-181 gene can be disrupted in W2mef parasites, without affecting the invasion phenotype, but cannot be targeted in 3D7 parasites.     

Induction of biologically active antibodies in mice, rabbits, and monkeys by Plasmodium falciparum EBA-175 region II DNA vaccine

BACKGROUND: Plasmodium falciparum merozoites bind to and invade human erythrocytes via specific erythrocyte receptors. This establishes the erythrocytic stage of the parasite life cycle that causes clinical disease resulting in 2-3 million deaths per year. We tested the hypothesis that a Plasmodium falciparum ligand, EBA-175 region II (RII), which binds its erythrocyte receptor glycophorin A during invasion, can be used as an immunogen to induce antibodies that block the binding of RII to erythrocytes and thereby inhibit parasite invasion of erythrocytes. Accordingly, we immunized mice, rabbits, and monkeys with DNA plasmids that encoded the 616 amino acid RII. MATERIALS AND METHODS: DNA vaccine plasmids that targeted the secretion of recombinant RII protein with and without the universal T-cell helper epitopes P2P30 were used to immunize mice, rabbits, and Aotus monkeys. RII specific antibodies were assessed by IFA, ELISA, blocking of native [35S] labeled EBA-175 binding to human erythrocytes, and growth inhibition assays, all in vitro. RESULTS: The RII DNA plasmids were highly immunogenic as measured by ELISA and IFA. The anti-RII antibodies blocked the binding of native EBA-175 to erythrocytes, and rosetting of erythrocytes on COS-7 cells expressing RII. Most important, murine and rabbit anti-RII antibodies inhibited the invasion of merozoites into erythrocytes. We immunized nonhuman primates and showed that the RII-DNA plasmids were immunogenic and well tolerated in these monkeys. Monkeys were challenged with parasitized erythrocytes; one of three monkeys that received RII DNA plasmid was protected from fulminant disease. After challenge with live parasites, anti-RII antibody titers were boosted in the immunized monkeys. CONCLUSIONS: By proving the hypothesis that anti-RII antibodies can block merozoite invasion of erythrocytes, these studies pave the way for the clinical evaluation of EBA-175 as a receptor-blockade vaccine.     

Primary structure of the 175K Plasmodium falciparum erythrocyte binding antigen and identification of a peptide which elicits antibodies that inhibit malaria merozoite invasion

The Plasmodium falciparum gene encoding erythrocyte binding antigen-175 (EBA-175), a putative receptor for red cell invasion (Camus, D., and T. J. Hadley. 1985. Science (Wash. DC). 230:553-556.), has been isolated and characterized. DNA sequencing demonstrated a single open reading frame encoding a translation product of 1,435 amino acid residues. Peptides corresponding to regions on the deduced amino acid sequence predicted to be B cell epitopes were assessed for immunogenicity. Immunization of mice and rabbits with EBA-peptide 4, a synthetic peptide encompassing amino acid residues 1,062-1,103, produced antibodies that recognized P. falciparum merozoites in an indirect fluorescent antibody assay. When compared to sera from rabbits immunized with the same adjuvant and carrier protein, sera from rabbits immunized with EBA-peptide 4 inhibited merozoite invasion of erythrocytes in vitro by 80% at a 1:5 dilution. Furthermore, these sera inhibited the binding of purified, authentic EBA-175 to erythrocytes, suggesting that their activity in inhibiting merozoite invasion of erythrocytes is mediated by blocking the binding of EBA-175 to erythrocytes. Since the nucleotide sequence of EBA-peptide 4 is conserved among seven strains of P. falciparum from throughout the world (Sim, B. K. L. 1990. Mol. Biochem. Parasitol. 41:293-296.), these data identify a region of the protein that should be a focus of vaccine development efforts.     

A conserved region in the EBL proteins is implicated in microneme targeting of the malaria parasite Plasmodium falciparum

The proliferation of the malaria parasite Plasmodium falciparum within the human host is dependent upon invasion of erythrocytes. This process is accomplished by the merozoite, a highly specialized form of the parasite. Secretory organelles including micronemes and rhoptries play a pivotal role in the invasion process by storing and releasing parasite proteins. The mechanism of protein sorting to these compartments is unclear. Using a transgenic approach we show that trafficking of the most abundant micronemal proteins (members of the EBL-family: EBA-175, EBA-140/BAEBL, and EBA-181/JSEBL) is independent of their cytoplasmic and transmembrane domains, respectively. To identify the minimal sequence requirements for microneme trafficking, we generated parasites expressing EBA-GFP chimeric proteins and analyzed their distribution within the infected erythrocyte. This revealed that: (i) a conserved cysteine-rich region in the ectodomain is necessary for protein trafficking to the micronemes and (ii) correct sorting is dependent on accurate timing of expression.     

Delineation of stage specific expression of Plasmodium falciparum EBA-175 by biologically functional region II monoclonal antibodies

Background

The malaria parasite Plasmodium falciparum EBA-175 binds its receptor sialic acids on glycophorin A when invading erythrocytes. The receptor-binding region (RII) contains two cysteine-rich domains with similar cysteine motifs (F1 and F2). Functional relationships between F1 and F2 domains and characterization of EBA-175 were studied using specific monoclonal antibodies (mAbs) against these domains.

Methods and Findings

Five mAbs specific for F1 or F2 were generated. Three mAbs specific for F2 potently blocked binding of EBA-175 to erythrocytes, and merozoite invasion of erythrocytes (IC₅₀ 10 to 100 µg/ml IgG in growth inhibition assays). A mAb specific for F1 blocked EBA-175 binding and merozoite invasion less effectively. The difference observed between the IC₅₀ of F1 and F2 mAbs was not due to differing association and disassociation rates as determined by surface plasmon resonance. Four of the mAbs recognized conformation-dependent epitopes within F1 or F2. Used in combination, F1 and F2 mAbs blocked the binding of native EBA-175 to erythrocytes and inhibited parasite invasion synergistically in vitro. MAb R217, the most potent, did not recognize sporozoites, 3-day hepatocyte stage parasites, nor rings, trophozoites, gametocytes, retorts, ookinetes, and oocysts but recognized 6-day hepatocyte stage parasites, and schizonts. Even though efficient at blocking binding to erythrocytes and inhibiting invasion into erythrocytes, MAb R217 did not inhibit sporozoite invasion and development in hepatocytes in vitro.

Conclusions

The role of the F1 and F2 domains in erythrocyte invasion and binding was elucidated with mAbs. These mAbs interfere with native EBA-175 binding to erythrocyte in a synergistic fashion. The stage specific expression of EBA-175 showed that the primary focus of activity was the merozoite stage. A recombinant RII protein vaccine consisting of both F1 and F2 domains that could induce synergistic activity should be optimal for induction of antibody responses that interfere with merozoite invasion of erythrocytes.     

Two Plasmodium rhomboid proteases preferentially cleave different adhesins implicated in all invasive stages of malaria

Invasion of host cells by the malaria pathogen Plasmodium relies on parasite transmembrane adhesins that engage host-cell receptors. Adhesins must be released by cleavage before the parasite can enter the cell, but the processing enzymes have remained elusive. Recent work indicates that the Toxoplasma rhomboid intramembrane protease TgROM5 catalyzes this essential cleavage. However, Plasmodium does not encode a direct TgROM5 homolog. We examined processing of the 14 Plasmodium falciparum adhesins currently thought to be involved in invasion by both model and Plasmodium rhomboid proteases in a heterologous assay. While most adhesins contain aromatic transmembrane residues and could not be cleaved by nonparasite rhomboid proteins, including Drosophila Rhomboid-1, Plasmodium falciparum rhomboid protein (PfROM)4 (PFE0340c) was able to process these adhesins efficiently and displayed novel substrate specificity. Conversely, PfROM1 (PF11_0150) shared specificity with rhomboid proteases from other organisms and was the only PfROM able to cleave apical membrane antigen 1 (AMA1). PfROM 1 and/or 4 was thus able to cleave diverse adhesins including TRAP, CTRP, MTRAP, PFF0800c, EBA-175, BAEBL, JESEBL, MAEBL, AMA1, Rh1, Rh2a, Rh2b, and Rh4, but not PTRAMP, and cleavage relied on the adhesin transmembrane domains. Swapping transmembrane regions between BAEBL and AMA1 switched the relative preferences of PfROMs 1 and 4 for these two substrates. Our analysis indicates that PfROMs 1 and 4 function with different substrate specificities that together constitute the specificity of TgROM5 to cleave diverse adhesins. This is the first enzymatic analysis of Plasmodium rhomboid proteases and suggests an involvement of PfROMs in all invasive stages of the malaria lifecycle, in both the vertebrate host and the mosquito vector.     

Basic fibroblast growth factor-induced activation of latent transforming growth factor beta in endothelial cells: regulation of plasminogen activator activity

Plasmodium falciparum malaria parasites invade human erythrocytes by means of a parasite receptor for erythrocytes, the 175-kD erythrocyte binding antigen (EBA-175). Similar to invasion efficiency, binding requires N-acetylneuraminic acid (Neu5Ac) on human erythrocytes, specifically the glycophorins. EBA-175 bound to erythrocytes with receptor-like specificity and was saturable. The specificity of EBA-175 binding was studied to determine if its binding is influenced either by simple electrostatic interaction with the negatively charged Neu5Ac (on the erythrocyte surface); or if Neu5Ac indirectly affected the conformation of an unknown ligand, or if Neu5Ac itself in specific linkage and carbohydrate composition was the primary ligand for EBA-175 as demonstrated for hemagglutinins of influenza viruses. Most Neu5Ac on human erythrocytes is linked to galactose by alpha 2-3 and alpha 2-6 linkages on glycophorin A. Soluble Neu5Ac by itself in solution did not competitively inhibit the binding of EBA-175 to erythrocytes, suggesting that linkage to an underlying sugar is required for binding in contrast to charge alone. Binding was competitively inhibited only by Neu5Ac(alpha 2-3)Gal-containing oligosaccharides. Similar oligosaccharides containing Neu5Ac(alpha 2-6)Gal-linkages had only slight inhibitory effects. Binding inhibition assays with modified sialic acids and other saccharides confirmed that oligosaccharide composition and linkage were primary factors for efficient binding. EBA-175 bound tightly enough to glycophorin A that the complex could be precipitated with an anti-glycophorin A monoclonal antibody. Selective cleavage of O-linked tetrasaccharides clustered at the NH2 terminus of glycophorin A markedly reduced binding in inhibition studies. We conclude that the Neu5Ac(a2,3)-Gal- determinant on O-linked tetrasaccharides of glycophorin A appear to be the preferential erythrocyte ligand for EBA-175.     

The Epitope of Monoclonal Antibodies Blocking Erythrocyte Invasion by Plasmodium falciparum Map to The Dimerization and Receptor Glycan Binding Sites of EBA-175

The malaria parasite, Plasmodium falciparum, and related parasites use a variety of proteins with Duffy-Binding Like (DBL) domains to bind glycoproteins on the surface of host cells. Among these proteins, the 175 kDa erythrocyte binding antigen, EBA-175, specifically binds to glycophorin A on the surface of human erythrocytes during the process of merozoite invasion. The domain responsible for glycophorin A binding was identified as region II (RII) which contains two DBL domains, F1 and F2. The crystal structure of this region revealed a dimer that is presumed to represent the glycophorin A binding conformation as sialic acid binding sites and large cavities are observed at the dimer interface. The dimer interface is largely composed of two loops from within each monomer, identified as the F1 and F2 β-fingers that contact depressions in the opposing monomers in a similar manner. Previous studies have identified a panel of five monoclonal antibodies (mAbs) termed R215 to R218 and R256 that bind to RII and inhibit invasion of erythrocytes to varying extents. In this study, we predict the F2 β-finger region as the conformational epitope for mAbs, R215, R217, and R256, and confirm binding for the most effective blocking mAb R217 and R215 to a synthetic peptide mimic of the F2 β-finger. Localization of the epitope to the dimerization and glycan binding sites of EBA-175 RII and site-directed mutagenesis within the predicted epitope are consistent with R215 and R217 blocking erythrocyte invasion by Plasmodium falciparum by preventing formation of the EBA-175– glycophorin A complex.     

Evolutionary relationships of conserved cysteine-rich motifs in adhesive molecules of malaria parasites

Malaria parasites invade erythrocytes in a process mediated by a series of molecular interactions. Invasion of human erythrocytes by Plasmodium vivax is dependent upon the presence of a single receptor, but P. falciparum, as well as some other species, exhibits the ability to utilize multiple alternative invasion pathways. Conserved cysteine-rich domains play important roles at critical times during this invasion process and at other stages in the life cycle of malaria parasites. Duffy-binding-like (DBL) domains, expressed as a part of the erythrocyte-binding proteins (DBL-EBP), are such essential cysteine-rich ligands that recognize specific host cell surface receptors. DBL-EBP, which are products of the erythrocyte-binding-like (ebl) gene family, act as critical determinants of erythrocyte specificity and are the best-defined ligands from invasive stages of malaria parasites. The ebl genes include the P. falciparum erythrocyte-binding antigen-175 (EBA-175) and P. vivax Duffy-binding protein. DBL domains also mediate cytoadherence as a part of the variant erythrocytic membrane protein-1 (PfEMP-1) antigens expressed from var genes on the surface of P. falciparum-infected erythrocytes. A paralogue of the ebl family is the malarial ligand MAEBL, which has a chimeric structure where the DBL domain is functionally replaced with a distinct cysteine-rich erythrocyte-binding domain with similarity to the apical membrane antigen-1 (AMA-1) ligand domain. The Plasmodium AMA-1 ligand domain, which encompasses the extracellular cysteine domains 1 and 2 and is well conserved in a Toxoplasma gondii AMA-1, has erythrocyte-binding activity distinct from that of MAEBL. These important families of Plasmodium molecules (DBL-EBP, PfEMP-1, MAEBL, AMA-1) are interrelated through the MAEBL. Because MAEBL and the other ebl products have the characteristics expected of homologous ligands involved in equivalent alternative invasion pathways to each other, we sought to better understand their roles during invasion by determining their relative origins in the Plasmodium genome. An analysis of their multiple cysteine-rich domains permitted a unique insight into the evolutionary development of PLASMODIUM: Our data indicate that maebl, ama-1, and ebl genes have ancient origins which predate Plasmodium speciation. The maebl evolved as a single locus, including its unique chimeric structure, in each Plasmodium species, in parallel with the ama-1 and the ebl genes families. The ancient character of maebl, along with its different expression characteristics suggests that MAEBL is unique and does not play an alternative role in invasion to ebl products such as EBA-175. The multiple P. falciparum ebl paralogues that express DBL domains, which have occurred by duplication and diversification, potentially do provide multiple functionally equivalent ligands to EBA-175 for alternative invasion pathways.     

Malarial EBA-175 region VI crystallographic structure reveals a KIX-like binding interface

The malaria parasite proliferates in the bloodstream of its vertebrate host by invading and replicating within erythrocytes. To achieve successful invasion, a number of discrete and essential events need to take place at the parasite-host cell interface. Erythrocyte-binding antigen 175 (EBA-175) is a member of a family of Plasmodium falciparum erythrocyte-binding proteins involved in the formation of a tight junction, a necessary step in invasion. Here we present the crystal structure of EBA-175 region VI (rVI), a cysteine-rich domain that is highly conserved within the protein family and is essential for EBA-175 trafficking. The structure was solved by selenomethionine single-wavelength anomalous dispersion at 1.8 Å resolution. It reveals a homodimer, containing in each subunit a compact five-α-helix core that is stabilized by four conserved disulfide bridges. rVI adopts a novel fold that is likely conserved across the protein family, indicating a conserved function. It shows no similarity to the Duffy-binding-like domains of EBA-175 involved in erythrocyte binding, indicating a distinct role. Remarkably, rVI possesses structural features related to the KIX-binding domain of the coactivator CREB-binding protein, supporting the binding and trafficking roles that have been ascribed to it and providing a rational basis for further experimental investigation of its function.     

The Baculovirus-Expressed Binding Region of Plasmodium falciparum EBA-140 Ligand and Its Glycophorin C Binding Specificity

The erythrocyte binding ligand 140 (EBA-140) is a member of the Plasmodium falciparum DBL family of erythrocyte binding proteins, which are considered as prospective candidates for malaria vaccine development. The EBA-140 ligand is a paralogue of the well-characterized P. falciparum EBA-175 protein. They share homology of domain structure, including Region II, which consists of two homologous F1 and F2 domains and is responsible for ligand-erythrocyte receptor interaction during invasion. In this report we describe, for the first time, the glycophorin C specificity of the recombinant, baculovirus-expressed binding region (Region II) of P. falciparum EBA-140 ligand. It was found that the recombinant EBA-140 Region II binds to the endogenous and recombinant glycophorin C, but does not bind to Gerbich-type glycophorin C, neither normal nor recombinant, which lacks amino acid residues 36–63 of its polypeptide chain. Our results emphasize the crucial role of this glycophorin C region in EBA-140 ligand binding. Moreover, the EBA-140 Region II did not bind either to glycophorin D, the truncated form of glycophorin C lacking the N-glycan or to desialylated GPC. These results draw attention to the role of glycophorin C glycans in EBA-140 binding. The full identification of the EBA-140 binding site on glycophorin C molecule, consisting most likely of its glycans and peptide backbone, may help to design therapeutics or vaccines that target the erythrocyte binding merozoite ligands.     

A novel ligand from Plasmodium falciparum that binds to a sialic acid-containing receptor on the surface of human erythrocytes

Invasion of the merozoite form of Plasmodium falciparum into human erythrocytes involves multiple receptor-ligand interactions. The EBA175 protein of P. falciparum has been shown to be the ligand that binds to a sialic acid-dependent site on glycophorin A. We have identified a novel P. falciparum ligand, termed erythrocyte-binding antigen 140 (EBA140), that shares structural features and homology with EBA175. Subcellular localization of EBA140 suggests that it is located in the micronemes, the same localization as EBA175. EBA140 binds to a sialic acid-dependent receptor on the surface of human erythrocytes. Binding of EBA140 to this erythrocyte receptor is sensitive to neuraminidase and resistant to trypsin, proteinase K and pronase. The protease-resistant properties of the erythrocyte receptor suggests that it is not glycophorin A or C. Additionally, analysis of mutant erythrocytes from humans has shown that EBA140 does not bind glycophorin B. Interestingly, we have identified a parasite line that lacks the eba140 gene, suggesting that this protein is not essential for in vitro invasion. These results suggest that EBA140 may be involved in merozoite invasion using a sialic acid-dependent receptor on human erythrocytes.     

Identifying Plasmodium falciparum EBA-175 homologue sequences that specifically bind to human erythrocytes

Erythrocyte binding antigen-160 (EBA-160) protein is a Plasmodium falciparum antigen homologue from the erythrocyte binding protein family (EBP). It has been shown that the EBP family plays a role in parasite binding to the erythrocyte surface. The EBA-160 sequence has been chemically synthesised in seventy 20-mer sequential peptides covering the entire 3D7 protein strain, each of which was tested in erythrocyte binding assays to identify possible EBA-160 functional regions. Five EBA-160 high activity binding peptides (HABPs) specifically binding to erythrocytes with high affinity were identified. Dissociation constants lay between 200 and 460 nM and Hill coefficients between 1.5 and 2.3. Erythrocyte membrane protein binding peptide cross-linking assays using SDS-PAGE showed that these peptides bound specifically to 12, 28, and 44 kDa erythrocyte membrane proteins. The nature of these receptor sites was studied in peptide binding assays using enzyme-treated erythrocytes. HABPs were able to block merozoite in vitro invasion of erythrocytes. HABPs’ potential as anti-malarial vaccine candidates is also discussed.     

Evidence for diversifying selection on erythrocyte-binding antigens of Plasmodium falciparum and P. vivax

Malaria parasite antigens involved in erythrocyte invasion are primary vaccine candidates. The erythrocyte-binding antigen 175K (EBA-175) of Plasmodium falciparum binds to glycophorin A on the human erythrocyte surface via an N-terminal cysteine-rich region (termed region II) and is a target of antibody responses. A survey of polymorphism in a malaria-endemic population shows that nucleotide alleles in eba-175 region II occur at more intermediate frequencies than expected under neutrality, but polymorphisms in the homologous domains of two closely related genes, eba-140 (encoding a second erythrocyte-binding protein) and psieba-165 (a putative pseudogene), show an opposite trend. McDonald-Kreitman tests employing interspecific comparison with the orthologous genes in P. reichenowi (a closely related parasite of chimpanzees) reveal a significant excess of nonsynonymous polymorphism in P. falciparum eba-175 but not in eba-140. An analysis of the Duffy-binding protein gene, encoding a major erythrocyte-binding antigen in the other common human malaria parasite P. vivax, also reveals a significant excess of nonsynonymous polymorphisms when compared with divergence from its ortholog in P. knowlesi (a closely related parasite of macaques). The results suggest that EBA-175 in P. falciparum and DBP in P. vivax are both under diversifying selection from acquired human immune responses.     

Antibodies targeting the PfRH1 binding domain inhibit invasion of Plasmodium falciparum merozoites

Invasion by the malaria merozoite depends on recognition of specific erythrocyte surface receptors by parasite ligands. Plasmodium falciparum uses multiple ligands, including at least two gene families, reticulocyte binding protein homologues (RBLs) and erythrocyte binding proteins/ligands (EBLs). The combination of different RBLs and EBLs expressed in a merozoite defines the invasion pathway utilized and could also play a role in parasite virulence. The binding regions of EBLs lie in a conserved cysteine-rich domain while the binding domain of RBL is still not well characterized. Here, we identify the erythrocyte binding region of the P. falciparum reticulocyte binding protein homologue 1 (PfRH1) and show that antibodies raised against the functional binding region efficiently inhibit invasion. In addition, we directly demonstrate that changes in the expression of RBLs can constitute an immune evasion mechanism of the malaria merozoite.     

Targeting sialic acid dependent and independent pathways of invasion in Plasmodium falciparum

The pathology of malaria is a consequence of the parasitaemia which develops through the cyclical asexual replication of parasites in a patient's red blood cells. Multiple parasite ligand-erythrocyte receptor interactions must occur for successful Plasmodium invasion of the human red cell. Two major malaria ligand families have been implicated in these variable ligand-receptor interactions used by Plasmodium falciparum to invade human red cells: the micronemal proteins from the Erythrocyte Binding Ligands (EBL) family and the rhoptry proteins from the Reticulocyte binding Homolog (PfRH) family. Ligands from the EBL family largely govern the sialic acid (SA) dependent pathways of invasion and the RH family ligands (except for RH1) mediate SA independent invasion. In an attempt to dissect out the invasion inhibitory effects of antibodies against ligands from both pathways, we have used EBA-175 and RH5 as model members of each pathway. Mice were immunized with either region II of EBA-175 produced in Pichia pastoris or full-length RH5 produced by the wheat germ cell-free system, or a combination of the two antigens to look for synergistic inhibitory effects of the induced antibodies. Sera obtained from these immunizations were tested for native antigen recognition and for efficacy in invasion inhibition assays. Results obtained show promise for the potential use of such hybrid vaccines to induce antibodies that can block multiple parasite ligand-red cell receptor interactions and thus inhibit parasite invasion.