Polymorphism in the Plasmodium falciparum erythrocyte-binding ligand JESEBL/EBA-181 alters its receptor specificity

The malaria parasite lives within erythrocytes and depends on the binding of parasite ligands to host cell surface receptors for invasion. The most virulent human malaria parasite, Plasmodium falciparum, uses multiple ligands, including EBA-175, BAEBL, and JESEBL of the Duffy-binding-like (DBL) family of erythrocyte-binding proteins, for invasion of human erythrocytes. Region II of these parasite ligands is the erythrocyte-binding domain. Previously, we had shown that polymorphism in region II of BAEBL leads to different erythrocyte-binding specificities. We have now identified and characterized the binding specificity of six JESEBL variants. We sequenced region II of JESEBL from 20 P. falciparum clones collected from various parts of the world where malaria is endemic. We observed eight JESEBL variants that contained amino acid polymorphisms at five positions among all clones. Seven of the eight variants could be connected by a single base change that led to an amino acid change. We investigated the functional significance of these polymorphisms by transiently expressing region II from six of JESEBL variants on the surface of Chinese hamster ovary cells. We observed four erythrocyte-binding patterns to enzyme-treated erythrocytes. Thus, P. falciparum DBL ligands JESEBL and BAEBL can recognize multiple receptors on the erythrocyte surface. In contrast to Plasmodium vivax, which has disappeared from West Africa because of the Duffy-negative blood group, P. falciparum may have been successful in endemic areas because it has mutated the ligands of the DBL family to create multiple pathways of invasion, thus making selection of refractory erythrocytes unlikely.     

Evidence for erythrocyte-binding antigen 175 as a component of a ligand-blocking blood-stage malaria vaccine

The ligands that pathogens use to invade their target cells have often proven to be good targets for vaccine development. However, Plasmodium falciparum has redundant ligands that mediate invasion of erythrocytes. The first requirement for the development of a successful ligand-blocking malaria vaccine is the demonstration that antibodies induced to each ligand can block the erythrocyte invasion of parasites with polymorphic sequences. Because of P. falciparum's redundancy in erythrocyte invasion, each ligand needs to be studied under artificial conditions in which parasite invasion is restricted in its use of alternative pathways. Here we investigate the role of erythrocyte-binding antigen 175 (EBA-175), a parasite ligand that binds to sialic acid on glycophorin A, in the invasion of erythrocytes by 10 P. falciparum clones under conditions in which invasion is partially limited to the EBA-175-glycophorin A pathway, using chymotrypsin-treated erythrocytes. We show that the ability to invade erythrocytes for both sialic acid-independent and sialic acid-dependent pathways requires the EBA-175-glycophorin A pathway for erythrocyte invasion. Importantly, antibodies against region II of EBA-175 from the 3D7 clone blocked invasion of chymotrypsin-treated erythrocytes by >50% by all parasite clones studied, including those with multiple different mutations described in the literature. The one exception was FCR3, which had a similar sequence to 3D7 but only 30% inhibition of invasion of chymotrypsin-treated erythrocytes, indicating alternative pathways for invasion of chymotrypsin-treated erythrocytes. Our findings suggest that antibodies to region II of EBA-175, as one component of a ligand-blocking malaria vaccine, are largely unaffected by polymorphism in EBA-175.     

Receptor-binding residues lie in central regions of Duffy-binding-like domains involved in red cell invasion and cytoadherence by malaria parasites

Erythrocyte invasion by malaria parasites and cytoadherence of Plasmodium falciparum-infected erythrocytes to host capillaries are 2 key pathogenic mechanisms in malaria. The receptor-binding domains of erythrocyte-binding proteins (EBPs) such as Plasmodium falciparum EBA-175, which mediate invasion, and P falciparum erythrocyte membrane protein 1 (PfEMP-1) family members, which are encoded by var genes and mediate cytoadherence, have been mapped to conserved cysteine-rich domains referred to as Duffy-binding-like (DBL) domains. Here, we have mapped regions within DBL domains from EBPs and PfEMP-1 that contain receptor-binding residues. Using biochemical and molecular methods we demonstrate that the receptor-binding residues of parasite ligands that bind sialic acid on glycophorin A for invasion as well as complement receptor-1 and chondroitin sulfate A for cytoadherence map to central regions of DBL domains. In contrast, binding to intercellular adhesion molecule 1 (ICAM-1) requires both the central and terminal regions of DBLbetaC2 domains. Determination of functional regions within DBL domains is the first step toward understanding the structure-function bases for their interaction with diverse host receptors.     

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.     

Antibody responses to Plasmodium falciparum vaccine candidate antigens in three areas distinct with respect to altitude

Antibody levels against malaria antigens were measured among patients presenting with uncomplicated malaria at health centers from three locations in Zimbabwe (Bindura, Chiredzi and Kariba) that are distinct with regard to altitude and climatic conditions. Antibody levels were determined by ELISA using the antigens, apical membrane antigen 1 (AMA-1), erythrocyte binding antigen 175 (EBA-175), circumsporozoite surface protein (CSP), merozoite surface protein 1 (MSP-1) and Pfg27. For all the antigens tested, IgG and IgM levels were higher for Bindura (altitude 1100 m) compared to Kariba (<600 m, altitude) and Chiredzi (approximately 600 m, altitude) with the exception of IgG and IgM to AMA-1 and EBA-175 which were similar between Chiredzi and Bindura. Plasma samples were further analyzed for their functional activity by testing their ability to inhibit the growth of Plasmodium falciparum in culture. Our results, determined by microscopy and verified by the LDH assay revealed that plasma from the three locations had similar inhibitory activity against the growth of P. falciparum in vitro. Our data revealed that highest growth inhibition correlated with the highest levels of MSP-1 antibody values.     

Polyethylene glycol-coated red blood cells fail to bind glycophorin A-specific antibodies and are impervious to invasion by the Plasmodium falciparum malaria parasite

This study was designed to assess the binding of glycophorin A-specific antibodies to polyethylene glycol (PEG)-modified red blood cells (RBCs) and evaluate their resistance to invasion by Plasmodium falciparum malaria parasites. RBCs were conjugated with a range of concentrations (0.05 to 7.5 mM) of activated PEG derivatives of either 3.35 or 18.5 kd molecular mass. The binding of glycophorin A-specific antibodies was assessed by hemagglutination and flow cytometry. PEG-modified RBCs were assessed for their ability to form rosettes around Chinese hamster ovary (CHO) cells transiently expressing the glycophorin A binding domain of EBA-175, a P falciparum ligand crucial to RBC invasion. PEG-RBCs were also tested for their ability to be invaded by the malaria parasite. RBCs coated with 3.35 and 18.5 kd PEG demonstrated a dose-dependent inhibition of glycophorin A-specific antibody binding, CHO cell rosetting, and P falciparum invasion. These results indicate that glycophorin A epitopes responsible for antibody and parasite binding are concealed by PEG coating, rendering these cells resistant to P falciparum invasion. These studies confirm the effectiveness of PEG modification for masking RBC-surface glycoproteins. This may provide a means to prevent alloimmunization in the setting of RBC transfusion and suggests a novel method to enhance the effectiveness of exchange transfusion for the treatment of cerebral malaria.     

Characterization of naturally acquired antibodies to the non-repetitive flanking regions of the circumsporozoite protein of Plasmodium falciparum.

Antibody responses to the circumsporozoite (CS) protein of Plasmodium falciparum have previously been reported against the central repeating tetrapeptides of this protein. Segments of the protein flanking the repeat region also contain B-cell epitopes, but specific antibody responses have not been previously characterized. Longitudinal serum sets from 16 Thai adults who developed acute falciparum malaria were selected to represent a spectrum of antibody response to the repeat region (R32). These sera were assayed by enzyme-linked immunosorbent assay using as capture antigen a recombinant fusion protein, NS1(81)RLF, which contains both flanking regions, but lacks the NANP and NVDP repeats of the P. falciparum CS protein. Antibody responses to the repeatless flanking (RLF) regions were observed in all subjects, including five individuals who lacked detectable anti-R32 antibody responses. Anti-RLF antibody responses induced by natural infection appear to be short-lived and of low-to-moderate magnitude. Thus, if anti-RLF antibodies prove to be protective, derived vaccine candidates may require presentation of these epitopes with adjuvants or delivery systems that enhance immunogenicity.     

Antibodies against circumsporozoite proteins of Plasmodium falciparum induced by natural infection

Sera from 10 individuals who lived in a malaria endemic area, 10 patients with acute uncomplicated falciparum malaria and 10 patients with cerebral malaria and hyperimmune mouse serum were tested for their reactivities against Plasmodium falciparum sporozoite antigens by Western blot analysis using 125I-labeled staphylococcal protein A as the detecting reagent. These sera were shown by indirect immunofluorescence and/or circumsporozoite precipitation test to have antibodies reacting against the parasites. It was found that all serum antibodies from the three groups of individuals and the mouse serum reacted in a similar pattern with circumsporozoite (CS) proteins of P. falciparum. Ten sera from normal individuals were negative in all reactions. Monoclonal antibody (MAB) specific against CS proteins of the parasites showed that the proteins exhibited as four different molecular weight (MW) polypeptides, i.e., 67,000, 65,000, 60,000, and 58,000 daltons. These CS proteins of P. falciparum were found to be species and stage specific. Radioimmunoprecipitation using 35S-methionine-labeled parasites and sera of individuals from the various categories or MABs gave a similar result. Another protein antigen of P. falciparum sporozoites had a MW of 80,000 daltons. This antigen was not species specific, probably not membrane associated and was present in a minute quantity in the parasite's extract.     

A longitudinal study of naturally acquired cellular and humoral immune responses to a merozoite surface protein (MSP1) of Plasmodium falciparum in an area of seasonal malaria transmission

A longitudinal study of cellular and serological responses to the major merozoite surface protein of Plasmodium falciparum (PfMSP1) has been conducted in a malaria immune population living in The Gambia, where malaria transmission is seasonally endemic. Recombinant or native proteins representing the sequence of PfMSP1 from the Wellcome strain of P. falciparum were used in in vitro lymphocyte proliferation, cytokine and antibody assays. Cellular responses of individual donors fluctuated over time, independent of seasonal changes in malaria transmission whereas anti-PfMSP1 antibody levels were remarkably stable. At a population level, IFNγ responses were both more prevalent and of greater magnitude at the end of the rainy (malaria transmission) season than during the dry season. Responses of individuals lving in a rural village were compared with those of individuals living in an urban area with much lower levels of malaria transmission. Malaria infections were more likely to be symptomatic in urban dwellers than in inhabitants of rural villages but no significant differences in the level or prevalence of cellular or serological responses were seen between the two groups. However, urban dwellers with current symptomatic malaria infections had somewhat lower anti-PfMSP1 antibody levels than their healthy, non-parasitaemic neighbours.     

Diversity in the immunodominant determinants of the circumsporozoite protein of Plasmodium falciparum parasites from malaria-endemic regions of Papua New Guinea and Brazil.

To determine the nature and extent of variation in the T cell sites of the Plasmodium falciparum circumsporozoite (CS) protein, a candidate antigen in the development of a malaria vaccine, we cloned and sequenced 69 recombinant clones of the CS protein gene representing 18 and 17 P. falciparum isolates from infected individuals from Madang, Papua New Guinea (PNG), a holoendemic malaria region, and Paragaminos and Jacunda, Brazil, relatively low endemic regions, respectively. As previously known, the amino acid sequence polymorphism was restricted to the three immunodominant regions of the protein, Th1R-N1, Th2R, and Th3R. While some of the observed nonsilent mutations in the T cell determinants of the CS protein were similar to those previously identified, we have found new amino acid changes in each of the polymorphic sequences in parasites from PNG and Brazil. A comparison of the CS epitope sequences of parasites from PNG and Brazil with the previously known CS epitope sequences of parasites from Brazil and The Gambia showed the following: 1) polymorphism was found in the Th1R-N1, Th2R, and Th3R region; however, while amino acid substitutions in the Th1R-N1 and Th2R region tended to be conservative, the substitutions found in the Th3R region were not, suggesting that the Th3R epitope may be rapidly evolving to allow parasites to escape host antiparasite cytotoxic T cell-enforced immune responses, and 2) the CS proteins of P. falciparum from high malaria-transmission regions (PNG and The Gambia) appear more polymorphic than the CS proteins of parasites from relatively low malaria-endemic regions in Brazil, where P. falciparum infection has been recently established.     

Antibodies to the conserved C-terminal domain of the Plasmodium falciparum merozoite surface protein 1 and to the merozoite extract and their relationship with in vitro inhibitory antibodies and protection against clinical malaria in a Senegalese village

Antibodies to Plasmodium falciparum C-terminal merozoite surface protein 1 (PfMSP-1p19) have been correlated with protection against malaria, but this association may apply to many merozoite antigens. To address this question, we conducted a prospective serological study of 205 individuals in an active 5-month clinical survey in a Senegalese village where malaria is mesoendemic. Before the 2000 rainy season, antibody responses specific for recombinant baculovirus PfMSP-1p19 or merozoite extracts were compared with 2 in vitro functional antibody activities (inhibition of parasite grown and erythrocyte invasion) and with the number of clinical episodes during 5 months of follow-up. Antibody levels to PfMSP-1p19 and merozoite extract correlated, respectively, with erythrocyte invasion and parasite growth inhibition. Although antibody levels to both antigen preparations were associated with age, functional parameters were not. High levels of anti-PfMSP-1p19 immunoglobulin G were associated with reduced malaria in an age-adjusted multivariate analysis. These results support baculovirus PfMSP-1p19-based vaccine development.     

A longitudinal study of sero-reactivity to Plasmodium falciparum antigen in children and adult living in an endemic area of U.P.

Antibody levels of Pf RESA derived peptide R1 (EENVEHDA-C) from individuals living in malaria endemic areas correlated well with levels of endemicity. Serological and parasitological investigations were done in 32 adults (> 20 yrs) and 35 children (2-5 yrs) for three years; i.e. from 1992-95 periodically in village Piyawoli, U.P. Antibody levels against R1 peptide was estimated by ELISA, and blood smear for P. falciparum and P. vivax were screened using Jaswant Singh-Bhattacharya (JSB) staining. It appeared from our investigations that anti-R1 antibodies had a short span of life, i.e. 6-9 months. The longevity of these antibodies do not differ much in adults and children. The studies do not indicate any protective role for these antibodies. However, the levels of anti-R1 antibodies in a population living under malariogenic condition are related to Pf malaria endemicity.     

Targeted disruption of an erythrocyte binding antigen in Plasmodium falciparum is associated with a switch toward a sialic acid-independent pathway of invasion

Erythrocyte invasion by Plasmodium requires molecules present both on the merozoite surface and within the specialized organelles of the apical complex. The Plasmodium erythrocyte binding protein family includes the Plasmodium falciparum sialic acid-binding protein, EBA-175 (erythrocyte binding antigen-175), which binds sialic acid present on glycophorin A of human erythrocytes. We address the role of the conserved 3'-cysteine rich region, the transmembrane, and cytoplasmic domains through targeted gene disruption. Truncation of EBA-175 had no measurable effect on either the level of EBA-175 protein expression or its subcellular localization. Similarly, there appears to be no impairment in the ability of soluble EBA-175 to be released into the culture supernatant after schizont rupture. Additionally, the 3'-cys rich region, transmembrane, and cytoplasmic domains of EBA-175 are apparently non-essential for merozoite invasion. In contrast, erythrocyte invasion via the EBA-175/glycophorin A route appears to have been disrupted to such a degree that the mutant lines have undergone a stable switch in invasion phenotype. As such, EBA-175 appears to have been functionally inactivated within the truncation mutants. The sialic acid-independent invasion pathway within the mutant parasites accounts for approximately 85% of invasion into normal erythrocytes. These data demonstrate the ability of P. falciparum to utilize alternate pathways for invasion of red blood cells, a property that most likely provides a substantial survival advantage in terms of overcoming host receptor heterogeneity and/or immune pressure.     

Epidemiology of malaria in a hypoendemic Brazilian Amazon migrant population: a cohort study

The present study describes aspects of the epidemiology of malaria in a migrant population living in a hypoendemic area in Brazil using an open cohort study design. Rural settlement residents in Leonislandia, Peixoto de Azevedo, Mato Grosso, Brazil were followed from September 1996 to April 1997. At baseline, an interview and malaria diagnoses were carried out and spleen size was measured. Incident cases were detected through follow-up visits and laboratory records. Cox regression was used to assess risk factors for time to malaria onset. Eighty percent (n = 414) of the study population (n = 521) contributed follow-up data. Overall, malaria prevalence during any study visit ranged from 0.3% to 5.4% and the malaria incidence rate (IR) was 4.49 (95% confidence interval = 3.66, 5.46) per 100 person-months. The IR of Plasmodium vivax malaria was approximately four times higher than the IR for P. falciparum malaria during follow-up. Among individuals who had had malaria during his or her lifetime, 14.03% reported hospitalization (median duration = 3 days) and 70.1% reported days of work lost (median duration = 4 days for P. falciparum malaria and 3 days for P. vivax malaria) related to the last malaria episode. No important risk factor was associated with the malaria IR. The fact that neither work-related factors nor age was associated with the risk of malaria indicates that indoor/peri-domiciliary transmission by the local vector is more important or as important as workplace-related transmission.     

pfcrt Polymorphism and the spread of chloroquine resistance in Plasmodium falciparum populations across the Amazon Basin

The widespread occurrence of drug-resistant malaria parasites in South America presents a formidable obstacle to disease control in this region. To characterize parasite populations and the chloroquine-resistance profile of Plasmodium falciparum in the Amazon Basin, we analyzed a DNA segment of the pfcrt gene, spanning codons 72-76, and genotyped 15 microsatellite (MS) markers in 98 isolates from 6 areas of Brazil, Peru, and Colombia where malaria is endemic. The K76T mutation, which is critical for chloroquine resistance, was found in all isolates. Five pfcrt haplotypes (S[tct]MNT, S[agt]MNT, CMNT, CMET, and CIET) were observed, including 1 previously found in Asian/African isolates. MS genotyping showed relatively homogeneous genetic backgrounds among the isolates, with an average of 3.8 alleles per marker. Isolates with identical 15-loci MS haplotypes were found in different locations, suggesting relatively free gene flow across the Amazon Basin. Allopatric isolates carrying SMNT and CMNT haplotypes have similar genetic backgrounds, although parasites carrying the CIET haplotype have some exclusive MS alleles, suggesting that parasites with CIET alleles were likely to have been introduced into Brazil from Asia or Africa. This study provides the first evidence of the Asian pfcrt allele in Brazil and a detailed analysis of P. falciparum populations, with respect to pfcrt haplotypes, in the Amazon Basin.     

Characterization of a Plasmodium falciparum erythrocyte-binding protein paralogous to EBA-175

A member of a Plasmodium receptor family for erythrocyte invasion was identified on chromosome 13 from the Plasmodium falciparum genome sequence of the Sanger Centre (Cambridge, U.K.). The protein (named BAEBL) has homology to EBA-175, a P. falciparum receptor that binds specifically to sialic acid and the peptide backbone of glycophorin A on erythrocytes. Both EBA-175 and BAEBL localize to the micronemes, organelles at the invasive ends of the parasites that contain other members of the family. Like EBA-175, the erythrocyte receptor for BAEBL is destroyed by neuraminidase and trypsin, indicating that the erythrocyte receptor is a sialoglycoprotein. Its specificity, however, differs from that of EBA-175 in that BAEBL can bind to erythrocytes that lack glycophorin A, the receptor for EBA-175. It has reduced binding to erythrocytes with the Gerbich mutation found in another erythrocyte, sialoglycoprotein (glycophorin C/D). The interest in BAEBL's reduced binding to Gerbich erythrocytes derives from the high frequency of the Gerbich phenotype in some regions of Papua New Guinea where P. falciparum is hyperendemic.     

Research note: HLA degenerate T-cell epitopes from Plasmodium falciparum liver stage-specific antigen 1 (LSA-1) are highly conserved in isolates from geographically distinct areas

Considerable effort is directed at the development of a malaria vaccine that elicits antigen-specific T-cell responses against pre-erythrocytic antigens of Plasmodium falciparum. Genetic restriction of host T-cell responses and polymorphism of target epitopes on parasite antigens pose obstacles to the development of such a vaccine. Liver stage-specific antigen-1 (LSA-1) is a prime candidate vaccine antigen and five T-cell epitopes that are degenerately restricted by HLA molecules common in most populations have been identified on LSA-1. To define the extent of polymorphism within these T-cell epitopes, the N-terminal non-repetitive region of the LSA-1 gene from Malaysian P. falciparum field isolates was sequenced and compared with data of isolates from Brazil, Kenya and Papua New Guinea. Three of the T-cell epitopes were completely conserved while the remaining two were highly conserved in the isolates examined. Our findings underscore the potential of including these HLA-degenerate T-cell epitopes of LSA-1 in a subunit vaccine.     

Cross-reactive cellular immune response to circumsporozoite proteins of Plasmodium vivax and P. falciparum in malaria-exposed individuals

Acquired immunity against the recombinant circumsporozoite protein of P. falciparum (rPfCS) or P. vivax (rPvCS) was studied in two malarious areas of the Brazilian Amazon. Cellular responsiveness, evaluated by proliferative assays, was detected in about 45% of individuals who had recovered from recent acute malaria infections. Peripheral blood mononuclear cells of individuals whose last malaria infection was by P. vivax responded more to the rCS proteins than those who had P. falciparum . Since in P. vivax infections hypnozoites in the liver retain CS antigen, this stage may have contributed to the increased cellular response. The unexpected result was that in primoinfections by P. falciparum or P. vivax the proliferative response did not correspond to the rPfCS and rPvCS, respectively. Furthermore, among the malaria-exposed individuals, there was a positive correlation between the intensity of the responses to the two rCS proteins. Our results suggest that cross-reactive epitopes exist in the CS protein of P. falciparum and P. vivax . In the areas studied, the frequency of antibodies against rPvCS and/or rPfCS ranged from 43% to 11%. Species-specific antibodies against the CS protein were detected in the primoinfected individuals. Some individuals living in the endemic area but with no clinical history of malaria were positive by serology (8%) or by in vitro proliferation (21%). However, antibodies and cellular responses against rCS were detected only in malaria-exposed individuals, since those living outside the endemic area were all negative.     

Seroepidemiologic studies of humoral immune response to the Plasmodium falciparum antigens in Thailand.

We have investigated seroreactivity against Plasmodium falciparum crude parasite antigens, the P. falciparum ring-infected erythrocyte surface antigen (Pf155/RESA), as well as against two synthetic peptides (EENV)6 and (EENVEHDA)3 that represent important epitopes of Pf155/RESA. The study population consisted of 421 children and adult Thais living in an area with moderate malaria transmission. We related these serologic findings to some important epidemiologic baseline data collected in the study area. The parasite rate in study subjects was 18.76%. Sixty-two percent were seropositive to crude P. falciparum antigens, 30.3% to the Pf155/RESA antigen, 23.05% to (EENV)6, and 20.17% to (EENVEHDA)3. Antibody responses to crude P. falciparum antigens and to Pf155/RESA were age dependent and increased with exposure. There was evidence that Pf155/RESA antibodies might play a role in protective immunity in this population. Since Pf155/RESA is a potential vaccine candidate antigen, the information obtained from these field studies will provide some seroepidemiologic baseline data for subsequent vaccine trials.