Plasmodium Falciparum Erythrocyte Binding Antigen Research Articles

Immune responses to P falciparum antibodies in symptomatic malaria patients with variant hemoglobin genotypes in Ghana

BackgroundHaemoglobin (Hb) variants such as sickle cell trait (SCT/HbAS) play a role in protecting against clinical malaria, but little is known about the development of immune responses against malaria parasite (Plasmodium falciparum surface protein 230 (Pfs230) and Plasmodium falciparum erythrocyte binding antigen 175 region-3 (PfEBA175-3R)) and vector (on the An. gambiae Salivary Gland Protein-6 peptide 1 (gSG6-P1)) antigens in individuals with variants Hb genotypes. This study assessed antibody (IgG) responses against malaria parasite, Pfs230 and PfEBA175-3R and vector, gSG6-P1 in febrile individuals with variant Hb genotypes.MethodsThe study was conducted on symptomatic malaria patients attending various healthcare facilities throughout Ghana. Microscopy and ELISA were used to determine the natural IgG antibody levels of gSG6-P1, PfEBA175-3R & Pfs230, and Capillarys 2 Flex Piercing was used for Hb variants determination.ResultsOf the 600 symptomatic malaria patients, 50.0% of the participants had malaria parasites by microscopy. The majority 79.0% (398/504) of the participants had Hb AA, followed by HbAS variant at 11.3% (57/504) and HbAC 6.7% (34/504). There were significantly (p < 0.0001) reduced levels of gSG6-P1 IgG in individuals with both HbAC and HbAS genotypes compared to the HbAA genotype. The levels of gSG6-P1 IgG were significantly (p < 0.0001) higher in HbAS compared to HbAC. Similarly, Pfs230 IgG and PfEBA-175-3R IgG distributions observed across the haemoglobin variants were significantly higher in HbAC relative to HbAS.ConclusionThe study has shown that haemoglobin variants significantly influence the pattern of anti-gSG6-P1, Pfs230, and PfEBA-175 IgG levels in malaria-endemic population. The HbAS genotype is suggested to confer protection against malaria infection. Reduced exposure to infection ultimately reduces the induction of antibodies targeted against P. falciparum antigens.

Genetic polymorphism and natural selection of the erythrocyte binding antigen 175 region II in Plasmodium falciparum populations from Myanmar and Vietnam

Plasmodium falciparum erythrocyte binding antigen 175 (PfEBA-175) plays essential role in erythrocyte invasion by the parasite and is a leading vaccine candidate. However, its genetic diversity in global isolates is a concern in developing an universal vaccine incorporating this protein. This study aimed to investigate genetic polymorphisms and natural selection of pfeba-175 region II (RII) in Myanmar and Vietnam P. falciparum isolates. Vietnam pfeba-175 RII displayed a low genetic polymorphism, while Myanmar pfeba-175 RII showed high levels of genetic diversity across the region. Point mutations, deletion, and recombinations were main factors contributing to genetic diversities in P. falciparum populations. Global pfeba-175 RII revealed similar, but not identical, genetic polymorphisms and natural selection profiles. Despite profiles of amino acid substitutions differed among populations, five major amino acid changes (K279E, E403K, K481I, Q584K, and R664) were commonly detected in global pfeba-175 RII populations. Haplotype network and genetic differentiation analyses of global pfeba-175 RII populations demonstrated no geographical relationships. Non-neglectable level of genetic diversity was observed in global pfeba-175 RII populations, emphasizing the need to consider this when designing an effective vaccine based on this protein. This study underscores the importance of the continuous monitoring of genetic diversity of pfeba-175 RII in the global P. falciparum populations.

Moderately Neutralizing Epitopes in Nonfunctional Regions Dominate the Antibody Response to Plasmodium falciparum EBA-140.

Plasmodium falciparum erythrocyte-binding antigen 140 (EBA-140) plays a role in tight junction formation during parasite invasion of red blood cells and is a potential vaccine candidate for malaria. Individuals in areas where malaria is endemic possess EBA-140-specific antibodies, and individuals with high antibody titers to this protein have a lower rate of reinfection by parasites. The red blood cell binding segment of EBA-140 is comprised of two Duffy-binding-like domains, called F1 and F2, that together create region II. The sialic acid-binding pocket of F1 is essential for binding, whereas the sialic acid-binding pocket in F2 appears dispensable. Here, we show that immunization of mice with the complete region II results in poorly neutralizing antibodies. In contrast, immunization of mice with the functionally relevant F1 domain of region II results in antibodies that confer a 2-fold increase in parasite neutralization compared to that of the F2 domain. Epitope mapping of diverse F1 and F2 monoclonal antibodies revealed that the functionally relevant F1 sialic acid-binding pocket is a privileged site inaccessible to antibodies, that the F2 sialic acid-binding pocket contains a nonneutralizing epitope, and that two additional epitopes reside in F1 on the opposite face from the sialic acid-binding pocket. These studies indicate that focusing the immune response to the functionally important F1 sialic acid binding pocket improves the protective immune response of EBA-140. These results have implications for improving future vaccine designs and emphasize the importance of structural vaccinology for malaria.

Plasmodium falciparum erythrocyte-binding antigen 175 triggers a biophysical change in the red blood cell that facilitates invasion

Invasion of the red blood cell (RBC) by the Plasmodium parasite defines the start of malaria disease pathogenesis. To date, experimental investigations into invasion have focused predominantly on the role of parasite adhesins or signaling pathways and the identity of binding receptors on the red cell surface. A potential role for signaling pathways within the erythrocyte, which might alter red cell biophysical properties to facilitate invasion, has largely been ignored. The parasite erythrocyte-binding antigen 175 (EBA175), a protein required for entry in most parasite strains, plays a key role by binding to glycophorin A (GPA) on the red cell surface, although the function of this binding interaction is unknown. Here, using real-time deformability cytometry and flicker spectroscopy to define biophysical properties of the erythrocyte, we show that EBA175 binding to GPA leads to an increase in the cytoskeletal tension of the red cell and a reduction in the bending modulus of the cell's membrane. We isolate the changes in the cytoskeleton and membrane and show that reduction in the bending modulus is directly correlated with parasite invasion efficiency. These data strongly imply that the malaria parasite primes the erythrocyte surface through its binding antigens, altering the biophysical nature of the target cell and thus reducing a critical energy barrier to invasion. This finding would constitute a major change in our concept of malaria parasite invasion, suggesting it is, in fact, a balance between parasite and host cell physical forces working together to facilitate entry.

Novel and cost-effective refolding of a recombinant receptor binding domain of Plasmodium falciparum EBA-175

Plasmodium falciparum erythrocyte binding antigen (EBA-175), which is a 175kDa protein, binds with sialic acid residues on glycophorin A during invasion of human erythrocytes. Receptor-binding residues of EBA-175 lie in a conserved, amino-terminal, cysteine-rich region, region F2 of EBA-175 (PfF2), that is homologous to the binding domains of other erythrocyte binding proteins. Recombinant PfF2 was expressed in Escherichia coli, purified from inclusion bodies, renatured by rapid dilution in presence of proline and purified to homogeneity by ion-exchange chromatography. Recombinant PfF2 was refolded at higher concentrations with improved refolding efficiency and yield. The refolded protein was characterized using biochemical methods and shown to be pure, homogenous and functional as it binds human erythrocytes with specificity. Immunization of mice and rabbits with recombinant PfF2 formulated with Freund’s and Montanide ISA720 adjuvants, elicited high and potent antibody responses. These observations support the development of a novel and cost-effective process for production of recombinant PfF2, a blood stage vaccine candidate for P. falciparum malaria.

A quantitative assay for binding and inhibition of Plasmodium falciparum Erythrocyte Binding Antigen 175 reveals high affinity binding depends on both DBL domains

Plasmodium falciparum Erythrocyte Binding Antigen 175 (PfEBA-175) engages Glycophorin A (GpA) on erythrocytes during malaria infection. The two Duffy binding like domains (F1 and F2) of PfEBA-175 that form region II (RII) are necessary for binding GpA, and are the target of neutralizing antibodies. Recombinant production of RII in Pichia pastoris and baculovirus has required mutations to prevent aberrant glycosylation or deglycosylation resulting in modifications to the protein surface that may affect antibody recognition and binding. In this study, we developed a recombinant system in Escherichia coli to obtain RII and F2 without mutations or glycosylation through oxidative refolding. The system produced refolded protein with high yields and purity, and without the need for mutations or deglycosylation. Biophysical characterization indicated both proteins are well behaved and correctly folded. We also demonstrate the recombinant proteins are functional, and develop a quantitative functional flow cytometry binding assay for erythrocyte binding ideally suited to measure inhibition by antibodies and inhibitors. This assay showed far greater binding of RII to erythrocytes over F2 and that binding of RII is inhibited by a neutralizing antibody and sialyllactose, while galactose had no effect on binding. These studies form the framework to measure inhibition by antibodies and small molecules that target PfEBA-175 in a rapid and quantitative manner using RII that is unmodified or mutated. This approach has significant advantages over current methods for examining receptor–ligand interactions and is applicable to other erythrocyte binding proteins used by the parasite.

The upstream sequence segment of the C-terminal cysteine-rich domain is required for microneme trafficking of Plasmodium falciparum erythrocyte binding antigen 175

Erythrocyte invasion is a critical step for survival of Plasmodium parasites, the causative agents of malaria, in their host and recognition of the host cell receptors by Plasmodium erythrocyte-binding-like (EBL) proteins plays an important role. Although EBL subcellular localization was shown to be closely linked to parasite virulence in the rodent model of malaria, the trafficking of EBL to micronemes, the secretory organelle in the invasive parasite is not fully understood. In this study, we assessed the impact of the deletion and amino acid replacement of Plasmodium falciparum EBL (EBA-175) using transgenic P. falciparum lines expressing modified EBA-175. We found that, in addition to a signal peptide and a cysteine rich region (region 6) to the cytoplasmic tail, a previously unrecognized sequence segment in region 5 was required for correct microneme trafficking of EBA-175. Replacement of Arg or Phe residues in this segment altered microneme trafficking, suggesting that the sequence itself contained critical information. Based on these findings, we propose that the sequence segment in region 5 is also required for the recognition of EBA-175 by the trafficking machinery to direct this protein to the microneme. Our results provide key information to clarify an as yet unidentified EBA-175 trafficking mechanism.

Crystal and Solution Structures of Plasmodium falciparum Erythrocyte-binding Antigen 140 Reveal Determinants of Receptor Specificity during Erythrocyte Invasion

Erythrocyte-binding antigen 140 (PfEBA-140) is a critical Plasmodium falciparum erythrocyte invasion ligand that engages glycophorin C on host erythrocytes during malaria infection. The minimal receptor-binding region of PfEBA-140 contains two conserved Duffy binding-like (DBL) domains, a fold unique to Plasmodium species. Here, we present the crystal structure of the receptor-binding region of PfEBA-140 at 2.4 Å resolution. The two-domain binding region is present as a monomer in the asymmetric unit, and the structure reveals novel features in PfEBA-140 that are likely determinants of receptor specificity. Analysis by small-angle x-ray scattering demonstrated that the minimal binding region is monomeric in solution, consistent with the crystal structure. Erythrocyte binding assays showed that the full-length binding region containing the tandem DBL domains is required for erythrocyte engagement, suggesting that both domains contain critical receptor contact sites. The electrostatic surface of PfEBA-140 elucidates a basic patch that constitutes a putative high-affinity binding interface spanning both DBL domains. Mutation of residues within this interface results in severely diminished erythrocyte binding. This study provides insight into the structural basis and mechanism of PfEBA-140 receptor engagement and forms a basis for future studies of this critical interaction. In addition, the solution and crystal structures allow the first identification of likely determinants of erythrocyte receptor specificity for P. falciparum invasion ligands. A complete understanding of the PfEBA-140 erythrocyte invasion pathway will aid in the design of invasion inhibitory therapeutics and vaccines.

Distribution and Genetic Diversity of Plasmodium falciparum Erythrocyte Binding Antigen 175 and Clinical Outcome of Malaria in the Kassena-Nankana District

Distribution and Genetic Diversity of Plasmodium falciparum Erythrocyte Binding Antigen 175 and Clinical Outcome of Malaria in the Kassena-Nankana District

Immunogenicity of a recombinant malaria vaccine based on receptor binding domain of Plasmodium falciparum EBA-175

Malaria parasites require specific receptor–ligand interactions to invade host erythrocytes. The 175 kDa Plasmodium falciparum erythrocyte binding antigen (EBA-175) binds sialic acid residues on glycophorin A to mediate erythrocyte invasion. The amino-terminal, conserved, cysteine-rich region of EBA-175, referred to as F2, contains receptor-binding sites. We propose to develop a recombinant malaria vaccine based on region F2. Recombinant P. falciparum region F2 (PfF2) was expressed in Escherichia coli, purified from inclusion bodies under denaturing conditions by metal affinity chromatography, renatured by oxidative refolding and purified further by ion-exchange and gel filtration chromatography. Recombinant PfF2 was characterized and shown to be pure, homogeneous and functionally active in that it binds human erythrocytes with specificity. The immunogenicity of recombinant PfF2 formulated with three human compatible adjuvants, namely, Montanide ISA720, AS02A and alum was tested in mice. All the formulations tested elicited high titer antibodies that block erythrocyte invasion in vitro . The AS02 formulation yielded sera with the highest end-point ELISA titers followed by Montanide ISA720 and alum. Analysis of cellular immune responses indicated that all formulations resulted in significant splenocyte proliferation. Analysis of cytokines secreted by proliferating splenocytes indicated that all the adjuvant formulations tested induced Th1 type responses. These results suggest that recombinant PfF2 formulated with human compatible adjuvants is immunogenic and can elicit high titer invasion inhibitory antibodies providing support for further clinical development of this promising vaccine candidate.

Contrasting signatures of selection on the Plasmodium falciparum erythrocyte binding antigen gene family

Erythrocyte binding antigens of Plasmodium falciparum are involved in erythrocyte invasion, and may be targets of acquired immunity. Of the five eba genes, protein products have been detected for eba-175, eba-181 and eba-140, but not for ψeba-165 or ebl-1, providing opportunity for comparative analysis of genetic variation to identify selection. Region II of each of these genes was sequenced from a cross-sectional sample of parasites in an endemic Kenyan population, and the frequency distributions of polymorphisms analysed. A positive value of Tajima's D was observed for eba-175 ( D = 1.13) indicating an excess of intermediate frequency polymorphisms, while all other genes had negative values, the most negative being ebl-1 ( D = −2.35) followed by ψeba-165 ( D = −1.79). The eba-175 and ebl-1 genes were then studied in a sample of parasites from Thailand, for which a positive Tajima's D value was again observed for eba-175 ( D = 1.79), and a negative value for ebl-1 ( D = −1.85). This indicates that eba-175 is under balancing selection in each population, in strong contrast to the other members of the gene family, particularly ebl-1 and ψeba-165 that may have been under recent directional selection. Population expansion simulations were performed under a neutral model, further supporting the departures from neutrality of these genes.

Erythrocyte-binding antigen 175 mediates invasion in Plasmodium falciparum utilizing sialic acid-dependent and -independent pathways.

The Plasmodium falciparum erythrocyte-binding antigen 175 (EBA-175) is a ligand for merozoite invasion into human erythrocytes that binds to glycophorin A in a sialic acid-dependent manner. P. falciparum strain W2mef depends on sialic acid for invasion of erythrocytes, whereas 3D7 is sialic acid-independent. We generated parasites that lack expression or express truncated forms of EBA-175 in W2mef and 3D7. Lack of EBA-175 expression in W2mef parasites was associated with a switch to sialic acid-independent invasion. 3D7 parasites lacking expression of EBA-175 showed no alteration in their ability to utilize sialic acid-independent pathways. Strikingly, both W2mef and 3D7 parasites lacking EBA-175 expression invaded chymotrypsin-treated erythrocytes inefficiently compared with the parental lines. This loss of function suggests that the EBA-175/glycophorin A ligand-receptor interaction is the major chymotrypsin-resistant invasion pathway. Parasite lines with truncated EBA-175 had invasion phenotypes equivalent to parasites lacking expression of EBA-175. The EBA-175 ligand is functional in erythrocyte invasion by merozoites that utilize either sialic acid-dependent or -independent invasion pathways. This finding suggests a model where a minimal affinity supplied by multiple ligand-receptor interactions is required for successful invasion and has implications for EBA-175 as a malaria vaccine candidate.

Plasmodium falciparum erythrocyte invasion through glycophorin C and selection for Gerbich negativity in human populations.

Geographic overlap between malaria and the occurrence of mutant hemoglobin and erythrocyte surface proteins has indicated that polymorphisms in human genes have been selected by severe malaria. Deletion of exon 3 in the glycophorin C gene (called GYPCDeltaex3 here) has been found in Melanesians; this alteration changes the serologic phenotype of the Gerbich (Ge) blood group system, resulting in Ge negativity. The GYPCDeltaex3 allele reaches a high frequency (46.5%) in coastal areas of Papua New Guinea where malaria is hyperendemic. The Plasmodium falciparum erythrocyte-binding antigen 140 (EBA140, also known as BAEBL) binds with high affinity to the surface of human erythrocytes. Here we show that the receptor for EBA140 is glycophorin C (GYPC) and that this interaction mediates a principal P. falciparum invasion pathway into human erythrocytes. EBA140 does not bind to GYPC in Ge-negative erythrocytes, nor can P. falciparum invade such cells using this invasion pathway. This provides compelling evidence that Ge negativity has arisen in Melanesian populations through natural selection by severe malaria.

Bacterially expressed and refolded receptor binding domain of Plasmodium falciparum EBA-175 elicits invasion inhibitory antibodies

Malaria parasites make specific receptor–ligand interactions to invade erythrocytes. A 175 kDa Plasmodium falciparum erythrocyte binding antigen (EBA-175) binds sialic acid residues on glycophorin A during invasion of human erythrocytes. The receptor-binding domain of EBA-175 lies in a conserved, amino-terminal, cysteine-rich region, region F2 of EBA-175 (PfF2), that is homologous to the binding domains of other erythrocyte binding proteins such as Plasmodium vivax Duffy binding protein. We have developed methods to produce recombinant PfF2 in its functional form. Recombinant PfF2 was expressed in Escherichia coli, purified from inclusion bodies, renatured by oxidative refolding and purified to homogeneity by ion-exchange and gel filtration chromatography. Refolded PfF2 has been characterized using biochemical and biophysical methods and shown to be pure, homogenous and functional in that it binds human erythrocytes with specificity. Immunization with refolded PfF2 yields high titre antibodies that efficiently inhibit P. falciparum invasion of erythrocytes in vitro. Importantly, antibodies raised against PfF2 block invasion by a P. falciparum field isolate that invades erythrocytes using multiple pathways. These observations support the development of recombinant PfF2 as a vaccine candidate for P. falciparum malaria.

Baculovirus-mediated expression of Plasmodium falciparum erythrocyte binding antigen 175 polypeptides and their recognition by human antibodies.

The erythrocyte binding antigen EBA-175 is a 175-kDa Plasmodium falciparum protein which mediates merozoite invasion of erythrocytes in a sialic acid-dependent manner. The purpose of this study was to produce recombinant EBA-175 polypeptide domains which have previously been identified as being involved in the interaction of EBA-175 with erythrocytes and to determine whether these polypeptides are recognized by malaria-specific antibodies. The eba-175 gene was cloned by PCR from genomic DNA isolated from the 3D7 strain of P. falciparum. The predicted protein sequence was highly conserved with that predicted from the published eba-175 gene sequences from the Camp and FCR-3 strains of P. falciparum and contained the F segment divergent region. Purified recombinant EBA-175 polypeptide fragments, expressed as glutathione S-transferase fusion proteins in insect cells by using the baculovirus system, were recognized by antibodies present in serum from a drug-cured, malaria-immune Aotus nancymai monkey. The fusion proteins were also recognized by antibodies present in sera from individuals residing in areas where malaria is endemic. In both cases the antibodies specifically recognized the EBA-175 polypeptide portion of the fusion proteins. Antibodies raised in rabbits immunized with the recombinant fusion proteins recognized parasite proteins present in schizont-infected erythrocytes. Our results suggest that these regions of the EBA-175 protein are targets for the immune response against malaria and support their further study as possible vaccine components.

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.