Antibodies against the Plasmodium falciparum receptor binding domain of EBA-175 block invasion pathways that do not involve sialic acids

The 175-kDa Plasmodium falciparum erythrocyte binding protein (EBA-175) binds to its receptor, sialic acids on glycophorin A. The binding region within EBA-175 is a cysteine-rich region identified as region II. Antibodies against region II block the binding of native EBA-175 to erythrocytes. We identified a P. falciparum strain, FVO, that could not invade erythrocytes devoid of sialic acids due to prior neuraminidase treatment, and in addition, we used a strain, 3D7, that could invade such sialic acid-depleted erythrocytes. We used these two strains to study the capacity of anti-region II antibodies to inhibit FVO and 3D7 parasite development in vitro. Analysis of growth-inhibitory effects of purified FVO anti-region II immunoglobulin G (IgG) with the FVO and 3D7 strains resulted in similar levels of growth inhibition. FVO and 3D7 strains were inhibited between 28 and 56% compared to control IgG. There appeared to be no intracellular growth retardation or killing of either isolate, suggesting that invasion was indeed inhibited. Incubation of recombinant region II with anti-region II IgG reversed the growth inhibition. These results suggest that antibodies against region II can also interfere with merozoite invasion pathways that do not involve sialic acids. The fact that EBA-175 has such a universal and yet susceptible role in erythrocyte invasion clearly supports its inclusion in a multivalent malaria vaccine.     

Sialic acid-dependent binding of baculovirus-expressed recombinant antigens from Plasmodium falciparum EBA-175 to Glycophorin A

The Plasmodium falciparum Erythrocyte Binding Antigen-175, EBA-175, is a soluble merozoite stage parasite protein which binds to glycophorin A surface receptors on human erythrocytes. We have expressed two conserved cysteine-rich regions, region II and region VI, of this protein as soluble His-tagged polypeptides in insect cell culture, and have tested their function in erythrocyte and glycophorin A binding assays. Recombinant region II polypeptides comprised of the F2 sub-domain or the entire region II (F1 and F2 sub-domains together) bound to erythrocytes and to purified glycophorin A in a manner similar to the binding of native P. falciparum EBA-175 to human red cells. Removal of sialic acid residues from the red cell surface totally abolished recombinant region II binding, while trypsin treatment of the erythrocyte surface reduced but did not eliminate recombinant region II binding. Synthetic peptides from three discontinuous regions of the F2 sub-domain of region II inhibited human erythrocyte cell binding and glycophorin A receptor recognition. Immune sera raised against EBA-175 recombinant proteins recognized native P. falciparum-derived EBA-175, and sera from malaria-immune adults recognized recombinant antigens attesting to both the antigenicity and immunogenicity of proteins. These results suggest that the functionally-active recombinant region II domain of EBA-175 may be an attractive candidate for inclusion in multi-component asexual blood stage vaccines.     

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.     

Antibodies raised against receptor-binding domain of Plasmodium knowlesi Duffy binding protein inhibit erythrocyte invasion

Erythrocyte invasion by malaria parasites requires specific receptor-ligand interactions. Plasmodium vivax and Plasmodium knowlesi are completely dependent on binding the Duffy blood group antigen to invade human erythrocytes. P. knowlesi invades rhesus erythrocytes by multiple pathways using the Duffy antigen as well as alternative receptors. Plasmodium falciparum binds sialic acid residues on glycophorin A as well as other sialic acid-independent receptors to invade human erythrocytes. Parasite proteins that mediate these interactions belong to a family of erythrocyte binding proteins, which includes the P. vivax Duffy binding protein, 175 kDa P. falciparum erythrocyte binding antigen (EBA-175), P. knowlesi alpha protein, which binds human and rhesus Duffy antigens, and P. knowlesi beta and gamma proteins, which bind Duffy-independent receptors on rhesus erythrocytes. The receptor-binding domains of these proteins lie in conserved, N-terminal, cysteine-rich regions that are referred to as region II. Here, we have examined the feasibility of inhibiting erythrocyte invasion with antibodies directed against receptor-binding domains of erythrocyte binding proteins. Region II of P. knowelsi alpha protein (Pk(alpha)RII), which binds the Duffy antigen, was expressed as a secreted protein in insect cells and purified from culture supernatants. Rabbit antibodies raised against recombinant Pk(alpha)RII were tested for inhibition of erythrocyte binding and invasion. Antibodies raised against Pk(alpha)RII inhibit P. knowlesi invasion of both human and rhesus erythrocytes. These data provide support for the development of recombinant vaccines based on the homologous binding domains of P. vivax Duffy binding protein and P. falciparum EBA-175.     

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.     

Effect of codon optimization on expression levels of a functionally folded malaria vaccine candidate in prokaryotic and eukaryotic expression systems

We have produced two synthetic genes that code for the F2 domain located within region II of the 175-kDa Plasmodium falciparum erythrocyte binding antigen (EBA-175) to determine the effects of codon alteration on protein expression in homologous and heterologous host systems. EBA-175 plays a key role in the process of merozoite invasion into erythrocytes through a specific receptor-ligand interaction. The F2 domain of EBA-175 is the ligand that binds to the glycophorin A receptor on human erythrocytes and is therefore a target of vaccine development efforts. We designed synthetic genes based on P. falciparum, Escherichia coli, and Pichia codon usage and expressed recombinant F2 in E. coli and Pichia pastoris. Compared to the expression of the native F2 sequence, conversion to prokaryote (E. coli)- or eukaryote (Pichia)-based codon usage dramatically improved the levels of recombinant protein expression in both E. coli and P. pastoris. The majority of the protein expressed in E. coli, however, was produced as inclusion bodies. The protein expressed in P. pastoris, on the other hand, was expressed as a secreted, soluble protein. The P. pastoris-produced protein was superior to that produced in E. coli based on its ability to bind to red blood cells. Consistent with these observations, the antibodies generated against the Pichia-produced protein prevented the binding of recombinant EBA to red blood cells. These antibodies recognize EBA-175 present on merozoites as well as in sporozoites by immunofluorescence. Our results suggest that the Pichia-based EBA-F2 vaccine construct has further potential to be developed for clinical use.     

A novel Plasmodium falciparum erythrocyte binding protein-2 (EBP2/BAEBL) involved in erythrocyte receptor binding

The 175-kDa erythrocyte binding protein (EBA-175) of Plasmodium falciparum and Duffy antigen binding proteins of P. vivax and P. knowlesi are members of a protein family. The features of this protein family include a cysteine-rich motif present in the erythrocyte receptor-binding domain. We identify here a novel 140-kDa P. falciparum erythrocyte binding protein (EBP2/BAEBL) containing the signature cysteine-rich motif by comparative analysis of gene sequence information. Polyclonal antibodies generated by immunization with an EBP2/BAEBL DNA vaccine immunoprecipitated a 140-kDa protein from P. falciparum schizont-infected erythrocyte lysates. Similar to EBA-175, the binding of EBP2/BAEBL to human erythrocytes was dependent on sialic acids because neuraminidase treatment of those erythrocytes rendered them incapable of binding, but differed from EBA-175 in that trypsin treatment decreased EBP2/BAEBL binding by only twofold compared to a 10-fold reduction in EBA-175 binding. Antibodies raised against the putative erythrocyte-binding domain of EBP2/BAEBL effectively blocked the binding of native EBP2/BAEBL to erythrocytes. These functional antibodies localize EBP2/BAEBL to the invasive apical end of the merozoite. We identify EBP2/BAEBL as a paralogue of EBA-175 and as a novel P. falciparum vaccine candidate.     

Inhibitory Humoral Responses to the Plasmodium falciparum Vaccine Candidate EBA-175 Are Independent of the Erythrocyte Invasion Pathway

Plasmodium falciparum utilizes multiple ligand-receptor interactions for invasion. The invasion ligand EBA-175 is being developed as a major blood-stage vaccine candidate. EBA-175 mediates parasite invasion of host erythrocytes in a sialic acid-dependent manner through its binding to the erythrocyte receptor glycophorin A. In this study, we addressed the ability of naturally acquired human antibodies against the EBA-175 RII erythrocyte-binding domain to inhibit parasite invasion of ex vivo isolates, in relationship to the sialic acid dependence of these parasites. We have determined the presence of antibodies to the EBA-175 RII domain by enzyme-linked immunosorbent assay (ELISA) in individuals from areas of Senegal where malaria is endemic with high and low transmission. Using affinity-purified human antibodies to the EBA-175 RII domain from pooled patient plasma, we have measured the invasion pathway as well as the invasion inhibition of clinical isolates from Senegalese patients in ex vivo assays. Our results suggest that naturally acquired anti-EBA-175 RII antibodies significantly inhibit invasion of Senegalese parasites and that these responses can be significantly enhanced through limiting other ligand-receptor interactions. However, the extent of this functional inhibition by EBA-175 antibodies is not associated with the sialic acid dependence of the parasite strain, suggesting that erythrocyte invasion pathway usage by parasite strains is not driven by antibodies targeting the EBA-175/glycophorin A interaction. This work has implications for vaccine design based on the RII domain of EBA-175 in the context of alternative invasion pathways.     

Measurement of antibody levels against region II of the erythrocyte-binding antigen 175 of Plasmodium falciparum in an area of malaria holoendemicity in western Kenya

Region II of the 175-kDa erythrocyte-binding antigen (EBA-175RII) of Plasmodium falciparum is functionally important in sialic acid-dependent erythrocyte invasion and is considered a prime target for an invasion-blocking vaccine. The objectives of this study were to (i) determine the prevalence of anti-EBA-175RII antibodies in a naturally exposed population, (ii) determine whether naturally acquired antibodies have a functional role by inhibiting binding of EBA-175RII to erythrocytes, and (iii) determine whether antibodies against EBA-175RII correlate with immunity to clinical malaria. We treated 301 lifelong residents of an area of malaria holoendemicity in western Kenya for malaria, monitored them during a high-transmission season, and identified 33 individuals who were asymptomatic despite parasitemia (clinically immune). We also identified 50 clinically susceptible individuals to serve as controls. These 83 individuals were treated and monitored again during the subsequent low-transmission season. Anti-EBA-175RII antibodies were present in 98.7% of the individuals studied. The antibody levels were relatively stable between the beginning and end of the high-transmission season and correlated with the plasma EBA-175RII erythrocyte-binding-inhibitory activity. There was no difference in anti-EBA-175RII levels or plasma EBA-175RII erythrocyte-binding-inhibitory activity between clinically immune and clinically susceptible groups. However, these parameters were higher in nonparasitemic than in parasitemic individuals at enrollment. These results suggest that although antibodies against EBA-175RII may be effective in suppressing some of the wild parasite strains, EBA-175RII is unlikely to be effective as a monovalent vaccine against malaria, perhaps due to allelic heterogeneity and/or presence of sialic acid-independent strains.     

Characterization of the 175-kilodalton erythrocyte binding antigen of Plasmodium falciparum

EBA-175 is a soluble 175-kDa Plasmodium falciparum antigen that is released into culture supernatants during rupture of schizont-infected erythrocytes. EBA-175 binds to erythrocytes and binding is sialic acid-dependent. A clone expressing the gene encoding EBA-175 was obtained previously by screening a genomic DNA expression library with antibodies that had been affinity-purified from EBA-175. Antibodies were raised against a 43-amino-acid peptide (EBA-peptide 4) synthesized according to the deduced amino acid sequence. Antibodies to peptide 4 and affinity-purified antibodies specific for EBA-175 were used to characterize further EBA-175 giving the following results: (1) EBA-175 differs biochemically and immunologically from other reported malarial antigens; (2) the EBA-175s from six geographical isolates of P. falciparum are antigenically conserved; (3) EBA-175 is expressed during schizogony as a 190-kDa protein which is larger than the culture supernatant form of the antigen. The 190-kDa form of the protein is recovered from the cell pellet in schizont-infected erythrocytes and partitions to the soluble fraction when extracted with detergent; (4) release of soluble EBA-175 into the culture supernatant coincides with schizont rupture; (5) there was no observable change in pI (pI=6.86) by isoelectric focusing between the cellular and supernatant species of the protein; and (6) release of EBA-175 into the culture supernatant is inhibited by the addition of chymostatin and leupeptin. The continued research into the role of EBA-175 during erythrocyte invasion may aid in vaccine development for malaria.     

Low prevalence of antibodies to preerythrocytic but not blood-stage Plasmodium falciparum antigens in an area of unstable malaria transmission compared to prevalence in an area of stable malaria transmission

In areas where levels of transmission of Plasmodium falciparum are high and stable, the age-related acquisition of high-level immunoglobulin G (IgG) antibodies to preerythrocytic circumsporozoite protein (CSP) and liver-stage antigen 1 (LSA-1) has been associated with protection from clinical malaria. In contrast, age-related protection from malaria develops slowly or not at all in residents of epidemic-prone areas with unstable low levels of malaria transmission. We hypothesized that this suboptimal clinical and parasitological immunity may in part be due to reduced antibodies to CSP or LSA-1 and/or vaccine candidate blood-stage antigens. Frequencies and levels of IgG antibodies to CSP, LSA-1, thrombospondin-related adhesive protein (TRAP), apical membrane antigen 1 (AMA-1), erythrocyte binding antigen 175 (EBA-175), and merozoite surface protein 1 (MSP-1) were compared in 243 Kenyans living in a highland area of unstable transmission and 210 residents of a nearby lowland area of stable transmission. Levels of antibodies to CSP, LSA-1, TRAP, and AMA-1 in the oldest age group (>40 years) in the unstable transmission area were lower than or similar to those of children 2 to 6 years old in the stable transmission area. Only 3.3% of individuals in the unstable transmission area had high levels of IgG (>2 arbitrary units) to both CSP and LSA-1, compared to 43.3% of individuals in the stable transmission area. In contrast, antibody levels to and frequencies of MSP-1 and EBA-175 were similar in adults in areas of stable and unstable malaria transmission. Suboptimal immunity to malaria in areas of unstable malaria transmission may relate in part to infrequent high-level antibodies to preerythrocytic antigens and AMA-1.     

Erythrocyte invasion phenotypes of Plasmodium falciparum in The Gambia

In vitro experimentation with Plasmodium falciparum has determined that a number of different receptor-ligand interactions are involved in the invasion of erythrocytes. Most culture-adapted parasite isolates use a mechanism of invasion that depends primarily on the erythrocyte sialoglycoprotein glycophorin A (GYPA) and erythrocyte-binding antigen 175 (EBA-175) of the parasite blood-stage merozoite. However, a minority of culture-adapted parasites and a majority of Indian field isolates can apparently invade by other means. Here, erythrocyte invasion phenotypes of P. falciparum field isolates in Africa were studied. For 38 Gambian isolates, invasion of neuraminidase-treated and trypsin-treated erythrocytes was inhibited, on average, by more than 60 and 85%, respectively, indicating a high level of dependence on sialic acid and trypsin-sensitive proteins on the erythrocyte surface. These results support the hypothesis that African P. falciparum parasites use GYPA as a primary receptor for invasion. However, the considerable variation among isolates confirms the idea that alternative receptors are also used by many parasites. Three amino acid polymorphisms in the GYPA-binding region of EBA-175 (region II) were not significantly associated with invasion phenotype. There was variation among isolates in the selectivity index (i.e., a statistical tendency toward aggregation or multiple invasions of host erythrocytes), but this variation did not correlate with enzyme-determined invasion phenotype or with eba-175 alleles. Overall, these invasion phenotypes in Africa support a vaccine strategy of inhibiting EBA-175 binding to GYPA but suggest that parasites with alternative phenotypes would be selected for if this strategy were used alone.     

A recombinant baculovirus-expressed Plasmodium falciparum receptor-binding domain of erythrocyte binding protein EBA-175 biologically mimics native protein

EBA-175 of Plasmodium falciparum is a merozoite ligand that binds its receptor glycophorin A on erythrocytes during invasion. The ligand-receptor interaction is dependent on sialic acids as well as the protein backbone of glycophorin A. Region II (RII) of EBA-175 has been defined as the receptor-binding domain. RII is divided into regions F1 and F2, which contain duplicated cysteine motifs. We expressed RII in a baculovirus and show that RII binds erythrocytes with a specificity identical to that of the native protein. We found that, consistent with the binding of erythrocytes to COS cells expressing F2, recombinant baculovirus-expressed F2 bound erythrocytes. About 20% of all baculovirus-expressed RII is N-glycosylated, unlike native P. falciparum proteins that remain essentially unglycosylated. However, glycosylation of recombinant RII did not affect its immunogenicity. Antibodies raised against both glycosylated and unglycosylated baculovirus-expressed RII recognized P. falciparum schizonts in immunofluorescence assays and also gave similar enzyme-linked immunosorbent assay titers. Furthermore, these antibodies have similar abilities to block native EBA-175 binding to erythrocytes. These results allow the development of RII as a vaccine candidate for preclinical assessment.     

Age-Dependent IgG Subclass Responses to Plasmodium falciparum EBA-175 Are Differentially Associated with Incidence of Malaria in Mozambican Children

Plasmodium falciparum blood-stage antigens such as merozoite surface protein 1 (MSP-1), apical membrane antigen 1 (AMA-1), and the 175-kDa erythrocyte binding antigen (EBA-175) are considered important targets of naturally acquired immunity to malaria. However, it is not clear whether antibodies to these antigens are effectors in protection against clinical disease or mere markers of exposure. In the context of a randomized, placebo-controlled trial of intermittent preventive treatment in infants conducted between 2002 and 2004, antibody responses to Plasmodium falciparum blood-stage antigens in a cohort of 302 Mozambican children were evaluated by immunofluorescence antibody test and enzyme-linked immunosorbent assay at 5, 9, 12, and 24 months of age. We found that IgG subclass responses to EBA-175 were differentially associated with the incidence of malaria in the follow-up period. A double amount of cytophilic IgG1 or IgG3 was associated with a significant decrease in the incidence of malaria (incidence rate ratio [IRR] = 0.49, 95% confidence interval [CI] = 0.25 to 0.97, and P = 0.026 and IRR = 0.44, CI = 0.19 to 0.98, and P = 0.037, respectively), while a double amount of noncytophilic IgG4 was significantly correlated with an increased incidence of malaria (IRR = 3.07, CI = 1.08 to 8.78, P = 0.020). No significant associations between antibodies to the 19-kDa fragment of MSP-1 (MSP-1₁₉) or AMA-1 and incidence of malaria were found. Age, previous episodes of malaria, present infection, and neighborhood of residence were the main factors influencing levels of antibodies to all merozoite antigens. Deeper understanding of the acquisition of antibodies against vaccine target antigens in early infancy is crucial for the rational development and deployment of malaria control tools in this vulnerable population.     

Binding of Plasmodium falciparum 175-kilodalton erythrocyte binding antigen and invasion of murine erythrocytes requires N-acetylneuraminic acid but not its O-acetylated form.

Sialic acid on human erythrocytes is involved in invasion by the human malaria parasite, Plasmodium falciparum. Mouse erythrocytes were used as a reagent to explore the question of whether erythrocyte sialic acid functions as a nonspecific negative charge or whether the sialic acid is a necessary structural part of the receptor for merozoites. Human erythrocytes contain N-acetylneuraminic acid (Neu5Ac), whereas mouse erythrocytes, which are also invaded by P. falciparum merozoites, contain 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2) and N-glycoloylneuraminic acid (Neu5Gc), in addition to Neu5Ac. We compared the effects of sialidase and influenza C virus esterase treatments of mouse erythrocytes on invasion and the binding of a 175-kDa P. falciparum protein (EBA-175), a sialic acid-dependent malaria ligand implicated in the invasion process. Sialidase-treated mouse erythrocytes were refractory to invasion by P. falciparum merozoites and failed to bind EBA-175. Influenza C virus esterase, which converts Neu5,9Ac2 to Neu5Ac, increased both invasion efficiency and EBA-175 binding to mouse erythrocytes. Thus, the parasite and EBA-175 discriminate between Neu5Ac and Neu5,9Ac2, that is, the C-9 acetyl group interferes with EBA-175 binding and invasion by P. falciparum merozoites. This indicates that sialic acid is part of a receptor for invasion.     

Invasion profiles of Brazilian field isolates of Plasmodium falciparum: phenotypic and genotypic analyses

The invasion of red blood cells (RBCs) by Plasmodium falciparum is dependent on multiple molecular interactions between erythrocyte receptors and parasite ligands. Invasion studies using culture-adapted parasite strains have indicated significant receptor heterogeneity. It is not known whether this heterogeneity reflects the parasite invasion arsenal in the field. We have studied the invasion phenotypes of 14 distinct field isolates from the Legal Amazon areas of Brazil by using erythrocyte invasion assays to investigate invasion into normal, enzyme-treated, and clinical-mutant RBCs. Analysis of these isolates revealed four distinct invasion profiles. Using En(a-) cells to get an unequivocal estimate of the use of glycophorin A (GPA) as a receptor, we found that the 175-kDa erythrocyte-binding antigen (EBA-175)/GPA pathway was used by a minority of the parasite isolates studied. Although polymorphism of region II domains at specific amino acid positions in both EBA-140 and EBA-181 was found in these field isolates, this did not correlate with invasion profiles and thus receptor selectivity. These studies have further confirmed the existence of a significant diversity of invasion pathways in nature and suggest that additional parasite ligands will have to be targeted to devise global vaccines that will work in the field.     

Distinct Th1- and Th2-Type prenatal cytokine responses to Plasmodium falciparum erythrocyte invasion ligands

Prenatal immunity to Plasmodium falciparum merozoite proteins involved in erythrocyte invasion may contribute to the partial protection against malaria that is acquired during infancy in areas of stable malaria transmission. We examined newborn and maternal cytokine and antibody responses to merozoite surface protein-1 (MSP-1), ribosomal phosphoprotein P0 (PfP0), and region II of erythrocyte binding antigen-175 (EBA-175) in infant-mother pairs in Kenya. Overall, 82 of 167 (50%), 106 of 176 (60%), and 38 of 84 (45%) cord blood lymphocytes (CBL) from newborns produced one or more cytokines in response to MSP-1, PfP0, and EBA-175, respectively. Newborns of primigravid and/or malaria-infected women were more likely to have antigen-responsive CBL than were newborns of multigravid and/or uninfected women at delivery. Newborn cytokine responses did not match those of their mothers and fell into three distinct categories, Th1 (21 of 55 CBL donors produced only gamma interferon and/or interleukin 2 [IL-2]), Th2 (21 of 55 produced only IL-5 and/or IL-13), and mixed Th1/Th2 (13 of 55). Newborns produced more IL-10 than adults. High and low levels of cord blood IL-12 p70 production induced by anti-CD40 activation were associated with malaria-specific Th1 and Th2 responses, respectively. Antigen-responsive CBL in some newborns were detected only after depletion of IL-10-secreting CD8 cells with enrichment for CD4 cells. These data indicate that prenatal sensitization to blood-stage Plasmodium falciparum occurs frequently in areas where malaria is holoendemic. Modulation of this immunity, possibly by maternal parity and malaria, may affect the acquisition of protective immunity against malaria during infancy.     

Identification of a Potent Combination of Key Plasmodium falciparum Merozoite Antigens That Elicit Strain-Transcending Parasite-Neutralizing Antibodies

Blood-stage malaria vaccines that target single Plasmodium falciparum antigens involved in erythrocyte invasion have not induced optimal protection in field trials. Blood-stage malaria vaccine development has faced two major hurdles, antigenic polymorphisms and molecular redundancy, which have led to an inability to demonstrate potent, strain-transcending, invasion-inhibitory antibodies. Vaccines that target multiple invasion-related parasite proteins may inhibit erythrocyte invasion more efficiently. Our approach is to develop a receptor-blocking blood-stage vaccine against P. falciparum that targets the erythrocyte binding domains of multiple parasite adhesins, blocking their interaction with their receptors and thus inhibiting erythrocyte invasion. However, with numerous invasion ligands, the challenge is to identify combinations that elicit potent strain-transcending invasion inhibition. We evaluated the invasion-inhibitory activities of 20 different triple combinations of antibodies mixed in vitro against a diverse set of six key merozoite ligands, including the novel ligands P. falciparum apical asparagine-rich protein (PfAARP), EBA-175 (PfF2), P. falciparum reticulocyte binding-like homologous protein 1 (PfRH1), PfRH2, PfRH4, and Plasmodium thrombospondin apical merozoite protein (PTRAMP), which are localized in different apical organelles and are translocated to the merozoite surface at different time points during invasion. They bind erythrocytes with different specificities and are thus involved in distinct invasion pathways. The antibody combination of EBA-175 (PfF2), PfRH2, and PfAARP produced the most efficacious strain-transcending inhibition of erythrocyte invasion against diverse P. falciparum clones. This potent antigen combination was selected for coimmunization as a mixture that induced balanced antibody responses against each antigen and inhibited erythrocyte invasion efficiently. We have thus demonstrated a novel two-step screening approach to identify a potent antigen combination that elicits strong strain-transcending invasion inhibition, supporting its development as a receptor-blocking malaria vaccine.     

Disruption of the C-terminal region of EBA-175 in the Dd2/Nm clone of Plasmodium falciparum does not affect erythrocyte invasion

EBA-175 is a Plasmodium falciparum micronemal protein that binds to sialic acid in the context of the peptide backbone of glycophorin A and has been implicated in sialic acid-dependent invasion of erythrocytes. The existence of an alternative invasion pathway has been suggested by the finding that the P. falciparum clone Dd2/Nm can invade sialic acid-depleted erythrocytes. To study the role of EBA-175 in this alternative pathway, we have generated Dd2/Nm clones expressing a truncated form of EBA-175 that lacks region 6 and the cytoplasmic domain. The protein still appears to be localized to the apical end in the vicinity of the micronemes, suggesting that region 6 and the cytoplasmic domain are not involved in EBA-175 trafficking to the micronemes. In these genetically modified clones, the level of truncated EBA-175 protein expression was greatly reduced. EBA-175-disrupted clones displayed normal rates of invasion of untreated and enzyme-treated human and animal erythrocytes, suggesting a lack of involvement of EBA-175 in this alternative invasion pathway.