Parasite antigens on the infected red cell surface are targets for naturally acquired immunity to malaria.

The feasibility of a malaria vaccine is supported by the fact that children in endemic areas develop naturally acquired immunity to disease. Development of disease immunity is characterized by a decrease in the frequency and severity of disease episodes over several years despite almost continuous infection, suggesting that immunity may develop through the acquisition of a repertoire of specific, protective antibodies directed against polymorphic target antigens. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a potentially important family of target antigens, because these proteins are inserted into the red cell surface and are prominently exposed and because they are highly polymorphic and undergo clonal antigenic variation, a mechanism of immune evasion maintained by a large family of var genes. In a large prospective study of Kenyan children, we have used the fact that anti-PfEMP1 antibodies agglutinate infected erythrocytes in a variant-specific manner, to show that the PfEMP1 variants expressed during episodes of clinical malaria were less likely to be recognized by the corresponding child's own preexisting antibody response than by that of children of the same age from the same community. In contrast, a heterologous parasite isolate was just as likely to be recognized. The apparent selective pressure exerted by established anti-PfEMP1 antibodies on infecting parasites supports the idea that such responses provide variant-specific protection against disease.

Polymorphism of a high molecular weight schizont antigen of the human malaria parasite Plasmodium falciparum.

Intraspecies antigenic diversity in the blood stages of the human malaria parasite Plasmodium falciparum was investigated using a collection of murine monoclonal antibodies and clones of the parasite. The results were as follows: (a) The schizont and merozoite stages of the parasite express on their surface clonally restricted antigens detectable by strain-specific antibodies in indirect immunofluorescence tests. (b) These restricted antigens are phenotypically stable characteristics of clones grown in vitro. (c) The molecules carrying the specific antigens were isolated by immunoprecipitation and were found to be parasite proteins ranging in size from Mr 190,000 to 200,000 between clones. (d) Comparative immunoprecipitation and peptide mapping of these molecules showed that each parasite clone expresses a protein that is antigenically and structurally distinct from the equivalent products of several other clones. (e) The different clonal products are, however, immunologically interrelated, since they possess determinants in common with all tested isolates of the parasite. (f) These polymorphic molecules are closely related to a previously described schizont protein of P. falciparum that is posttranslationally cleaved into fragments located on the merozoite surface. These findings show the existence of a family of related polymorphic schizont antigens (PSA) of P. falciparum, whose expression is clonally restricted, and indicate that these proteins have regions of constant and variable antigenicity. We propose that a system of immunological classification of the parasite can be developed based on the polymorphism of these proteins.

Possible pitfalls in the identification of glycophorin-binding proteins of Plasmodium falciparum.

Plasmodium falciparum proteins that bind to the putative erythrocyte receptor (glycophorin) have been identified in several laboratories by their ability to bind to glycophorin immobilized on aminoethyl-BioGel (AE-BioGel). We here report that several parasite proteins bind to AE-BioGel in the absence of coupled glycophorin. Binding is apparently due to the strong ion-exchange properties of the matrix, and is sensitive to ionic conditions such as the degree of equilibration of the matrix and the pH. The parasite proteins that bind to the blank column under appropriate conditions include proteins with the serological activities of S-antigen and Ag 23, which also bind to glycophorin-coupled AE-BioGel. In the light of these results, the glycophorin-binding specificity of these and other proteins reported to bind to glycophorin-coupled AE-BioGel will have to be reevaluated, preferably using a different support matrix.

Surface antigen expression on Plasmodium falciparum-infected erythrocytes is modified in alpha- and beta-thalassemia.

In an attempt to determine the mechanism whereby thalassemia in its milder forms may protect against malaria, we have examined the expression of neoantigen at the surface of Plasmodium falciparum-parasitized thalassemic red cells. Neoantigen expression was estimated by measurement of antibody bound after incubation in serum from adults living in a malaria-endemic area, using a quantitative radiometric antiglobulin assay. We found that P. falciparum-parasitized alpha- and beta-thalassemic red cells bind greater levels of antibody from endemic serum than controls: mean binding ratios (+/- SE), respectively, for alpha- and beta-thalassemia compared with controls were 1.69 +/- 0.12 and 1.23 +/- 0.06 on a cell for cell basis, and 1.97 +/- 0.11 and 1.47 +/- 0.08 after a correction for surface area differences. Binding of antibody increased exponentially during parasite maturation. In addition, we found a small but significant degree of binding of naturally occurring antibody to parasitized red cells, the extent of which was also greater in thalassemia. The apparent protective effect of thalassemia against malaria may be related to enhanced immune recognition and hence clearance of parasitized erythrocytes.

Intercellular adhesion molecule-1 and CD36 synergize to mediate adherence of Plasmodium falciparum-infected erythrocytes to cultured human microvascular endothelial cells.

We have compared the adhesion of Plasmodium falciparum-infected erythrocytes to human dermal microvascular endothelial cells (HDMEC) and human umbilical vein endothelial cells (HUVEC) and have assessed the relative roles of the receptors CD36 and intercellular adhesion molecule-1 (ICAM-1). HUVEC (a cell line that expresses high levels of ICAM-1 but no CD36) mediate low levels of adhesion, whereas HDMEC (which constitutively express CD36) mediate high levels of adhesion even before ICAM-1 induction ICAM-1 expression leads to yet greater levels of adhesion, which are inhibited both by anti-ICAM-1 and CD36 mAbs, despite no increase in the expression of CD36. The results indicate the presence of a substantial population of infected cells that require the presence of both receptors to establish adhesion. Synergy between these receptors could be demonstrated using a number of parasite lines, but it could not be predicted from the binding of these same parasite lines to purified ICAM-1 and CD36. This phenomenon could not be reproduced using either purified receptors presented on plastic, or formalin-fixed HDMEC, suggesting that receptor mobility is important. This is the first study to demonstrate receptor synergy in malaria cytoadherence to human endothelial cells, a phenomenon necessary for parasite survival and associated with disease severity.

Antigenic variation in Plasmodium falciparum: mechanisms and consequences.

In the past year, the major advances in malaria antigenic variation have been concerned with the transcription and switching of variant antigen genes, and the functional expression of regions of the major variant antigen. Also, new variant gene families have been discovered as a result of the Malaria Genome Project.

Identification of a 25 kDa polypeptide associated with the L antigen in low potassium-type sheep red cells.

Antisera to the L blood group antigen have been used, following radioiodination of low potassium-type sheep red cells and subsequent immunoprecipitation, to identify a polypeptide of the L antigen. Only LK, and not HK, cells express this 25 kDa component which is present in very low copy number.

Antigenic cross reactivity between p195 and a distinct protein of 100 kDa in Plasmodium falciparum.

A monoclonal antibody raised against merozoites of Plasmodium falciparum clone T9/96 was shown to react with an extremely strain specific epitope on a 195 kDa protein synthesized only by late trophozoites and schizonts. This protein was shown to exhibit all of the characteristics attributed to the molecule known variously as merozoite surface protein precursor, polymorphic schizont antigen and p195. The monoclonal antibody also identified a cross-reactive epitope on a distinct protein of 100 kDa in ring stage parasites which was shown to be synthesized throughout the asexual cycle and was not a processing product of p195. One-dimensional peptide mapping studies suggested that these two proteins share a degree of common sequence or structure.

Intraerythrocytic polyubiquitin expression in Plasmodium falciparum is subjected to developmental and heat-shock control.

The polyubiquitin gene of the human protozoan parasite Plasmodium falciparum (PfpUB) was cloned and shown to be comprised of five tandem repeats of the ubiquitin open reading frame, present as a single copy on chromosome 12. The 1672 bp of PfpUB is interrupted at the 5' end by a single intron of 526 bp. PfpUB expression is developmentally regulated in intraerythrocytic stages with a marked increase in both steady-state transcript and polyubiquitin protein levels in late trophozoite stages. On response to heat shock, late stage parasites (late trophozoites and schizonts) have a slightly elevated PfpUB transcript level as well as readily observable increases in the amount of polyubiquitin and ubiquitin-conjugated proteins.

Wild isolates of Plasmodium falciparum show extensive polymorphism in T cell epitopes of the circumsporozoite protein.

Variation in the immunodominant T cell epitopes Th2R and Th3R of the Plasmodium falciparum circumsporozoite protein has been analysed from Gambian clinical isolates using the polymerase chain reaction. The degree of polymorphism in these epitopes is more extensive than that found in several geographically diverse laboratory isolates. These findings strongly suggest that it will not be feasible to include all variants in a polyvalent subunit sporozoite vaccine.

Serological cross-reaction between high molecular weight proteins synthesized in blood schizonts of Plasmodium yoelii, Plasmodium chabaudi and Plasmodium falciparum.

A 230 000 molecular weight (MW) Plasmodium yoelii protein, a 250 000 MW P. chabaudi protein and a 195 000 MW P. falciparum protein, identified using monoclonal antibodies, have similar characteristics, and have been implicated as protective antigens. In this study the serological relationship between these proteins was investigated by Western transfer analysis. The monoclonal antibodies specific for each of the high molecular weight proteins did not cross-react with antigens of the other two parasites, but a polyvalent mouse serum raised against the purified 230 000 MW P. yoelii protein cross-reacted with the high molecular weight proteins of P. chabaudi and P. falciparum and also with the fragments derived from these proteins. This result indicates that these proteins belong to the same class of malaria parasite antigen.

Intraerythrocytic development and antigenicity of Plasmodium falciparum and comparison with simian and rodent malaria parasites.

Using sorbitol-synchronised cultures and metabolic labelling with [35S]methionine, the stage specificity of polypeptides synthesised by the intraerythrocytic stages of Plasmodium falciparum was studied. We confirmed that the synthesis of many polypeptides is restricted to defined morphological stages of parasite development, while other polypeptides are synthesised more or less throughout the cycle. The synthesis of at least 6 polypeptides was confined to the period of differentiation of mature trophozoites to schizonts and merozoites. Polypeptides synthesised by a cloned long-term passage isolate were very similar to those of a recently cultured uncloned isolate. Comparison of polypeptides synthesized during differentiation of mature trophozoites to schizonts and merozoites by P. falciparum with those of P. chabaudi and P. knowlesi showed that while P. chabaudi and P. knowlesi synthesised a 250 000 molecular weight polypeptide at this stage the apparently equivalent polypeptide of P. falciparum was of significantly lower molecular weight being 200 000. Using a surface immunoprecipitation technique, it was shown that this 200 000 mol. wt. polypeptide was accessible to antibodies on the surface of erythrocytes infected with mature trophozoites and schizonts. A 150 000 mol. wt. polypeptide was also accessible to antibodies. By comparing polypeptides synthesised during the differentiation of mature trophozoites to schizonts and merozoites with those recovered in the ring stage parasites after schizogony and erythrocyte invasion, it was shown that this 200 000 mol. wt. polypeptide and 140 000 and 120 000 mol. wt. polypeptides were not taken into the erythrocyte by the invading merozoite. The importance of these polypeptides in terms of the parasite biology and in the induction and expression of immunity to malaria is discussed.

A study of var gene transcription in vitro using universal var gene primers.

The polymorphic multigene family, var, encodes the variant antigen, Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), present on the surface of erythrocytes infected with the human malaria parasite, P. falciparum. PfEMP1 has been implicated in the pathology of malaria through its ability to bind to host endothelial receptors and uninfected erythrocytes. Understanding the relationship between host pathology, immune response and parasite variation is crucial, but requires a method of reliably detecting and differentiating all possible var genes. Several primer pairs used to date are biased and limited in their detection capacity. Here we describe a set of PCR primers that amplify the majority of var genes in the laboratory isolates 3D7 and A4, and appear to work equally well on all isolates tested. We use these universal primers to examine the relationship between var gene transcription as assessed by reverse transcriptase-PCR (RT-PCR) with that measured by Northern analysis of parasite RNA. Phenotypically selected young parasites have multiple transcripts detected by RT-PCR, but the full-length transcript appears to be homogeneous. In addition, we demonstrate that the choice of primers used for RT-PCR is crucial in data interpretation.

Identification of a schizont- and species-specific surface glycoprotein on erythrocytes infected with rodent malarias.

Erythrocytes infected with mature trophozoites of Plasmodium chabaudi and reticulocytes infected with P. berghei were labelled metabolically in vitro with [35S]methionine. The labelled cells were incubated with normal and immune serum and washed to remove unbound antibody. Solubilisation of the antibody-coated cells in detergent was followed by co-precipitation of antibody/antigen complexes and analysis of the immunoprecipitates by SDS-PAGE and fluorography. One major parasite polypeptide of 250 000 daltons was found to be exposed to antibody in both species. A labelled band of the same molecular weight could be identified by immunoprecipitation and SDS-PAGE analysis of P. chabaudi-infected cells that had been surface-labelled with periodate/NaB3H4. This molecule also incorporated [3H]glucosamine in short term cultures of mature parasitised erythrocytes. The results suggest that a 250 000 dalton glycoprotein which is synthesised only by late trophozoites or schizonts is exposed either on the surface of the infected erythrocyte, the surface of the merozoite, or both. Furthermore, the exposed portion of the molecule was not immunologically cross-reactive in the two Plasmodium species, but some cross reaction was detectable in total parasite lysates. The significance of these findings to protective immunity is discussed.

Stage specific protein and nucleic acid synthesis during the asexual cycle of the rodent malaria Plasmodium chabaudi.

The asexual, intraerythrocytic stage of Plasmodium chabaudi develops synchronously in CBA mice. This in vivo synchrony has been exploited in in vitro pulse-labelling experiments to investigate the stage specificity of macromolecular synthesis by malaria parasites. Groups of mice were infected on day 0 with P. chabaudi and on day 3, individual mice were killed at three hour intervals, and the parasitised blood labelled in vitro for 2 h with radioactive precursors of protein or nucleic acid synthesis. By taking 11 samples covering one and one-third parasite division cycles, it was shown that the synthesis of many parasite polypeptides was restricted to defined morphological stages of parasite development. Other polypeptides were synthesised more or less continuously during the growth cycle. The synthesis of at least 6 polypeptides was confined to schizont or merozoite differentiation. RNA synthesis was shown to increase in rate steadily during parasite growth and to fall sharply during merozoite invasion. Approximately 40% of DNA synthesis was shown to occur during trophozoite growth, but the majority (60%) was confined to a short 4-6 h period at or just before schizogony.

Analysis of adhesive domains from the A4VAR Plasmodium falciparum erythrocyte membrane protein-1 identifies a CD36 binding domain.

The A4VAR is a variant antigen expressed by a clonal line that binds CD36 and intercellular adhesion molecule-1, ICAM-1. We have cloned and sequenced the extracellular domain coded by the A4var gene. To probe the relationship between A4var expression and parasite adhesion to ICAM-1, var mRNA and protein expression were analyzed in an enriched population of A4 parasites that displayed higher ICAM-1 binding. By Northern analyses, A4var was the predominant var message and antisera raised against a recombinant A4VAR protein reacted with the majority of infected erythrocytes, reinforcing previous conclusions that A4VAR binds ICAM-1. A4VAR contains five Duffy-binding like (DBL) domains, and two cysteine-rich interdomain regions (CIDR) domains. DBL and CIDR domains from A4VAR were expressed in mammalian cells to determine which regions mediate binding to CD36 and ICAM-1. Using several different binding assays, the A4VAR CIDR1 was the only domain found to bind CD36. In contrast, the same assays were unable to identify the ICAM-1 binding domain in A4VAR. This is the first time that each of the DBL and CIDR domains from a Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) have been systematically expressed and tested for binding. These results confirm that CIDR1 is sufficient to bind CD36 without any apparent contribution from other domains.

A possible molecular basis for strain specific immunity to malaria.

A 250 kDa antigen implicated in the induction of protective immunity to Plasmodium chabaudi was examined with a panel of 11 monoclonal antibodies in cloned parasite lines. 2 antibodies cross-reacted with the different parasite lines while 9 were specific for one line. This antigenic diversity was correlated with major differences in one dimensional peptide maps between the purified antigen from different lines of parasites. The peptide maps also revealed some apparently conserved structure which may have been responsible for the antigenic cross reactivity. Using cloned lines of P. falciparum and a second series of monoclonal antibodies, similar antigenic and structural diversity was evident in the equivalent antigen from the important human pathogen. These findings are discussed with relationship to the induction of protective immunity to malaria.

Current status of the Plasmodium falciparum genome project.

The Plasmodium falciparum Genome Project is a collaborative effort by many laboratories that will provide detailed molecular information about the parasite, which may be used for developing practical control measures. Initial goals are to prepare an electronically indexed clone bank containing partially sequenced clones representing up to 80% of the parasite's genes and to prepare an ordered set of overlapping clones spanning each of the parasite's 14 chromosomes. Currently, clones of genomic DNA, prepared as yeast artificial chromosomes, are arranged into contigs covering approximately 70% of the genome of parasite clone 3D7, gene sequence tags are available from more than contigs covering approximately 70% of the genome of parasite clone 3D7, gene sequence tags are available from more than 20% of the parasite's genes, and approximately 5% of the parasite's genes are tentatively identified from similarity searches of entries in the international sequence databases. A total of > 0.5 Mb of P. falciparum sequence tag data is available. The gene sequence tags are presently being used to complete YAC contig assembly and localize the cloned genes to positions on the physical map in preparation for sequencing the genome. Routes of access to project information and services are described.

Protection by alpha-thalassaemia against Plasmodium falciparum malaria: modified surface antigen expression rather than impaired growth or cytoadherence.

We have attempted to determine the cellular mechanism by which alpha-thalassaemia may protect against Plasmodium falciparum malaria. Invasion and development of P. falciparum in the microcytic red cells of two-gene deletion forms of alpha-thalassaemia when measured morphologically or by [3H]hypoxanthine incorporation were normal compared to controls. Normal invasion rates were also observed following schizogony in thalassaemic red cells. Neither the addition of the oxidant menadione, 30% oxygen, nor modified medium, produced differential damage to parasites within thalassaemic cells. Furthermore, there were no significant differences in the binding of P. falciparum-parasitized alpha-thalassaemic and normal cells to C32 melanoma cells in vitro. However, when neoantigen expression on the surface of infected thalassaemic cells was estimated using a quantitative radiometric antiglobulin assay, clear differences were observed. It was found that alpha-thalassaemic cells bound higher levels of antibody from serum obtained from individuals living in a malaria endemic area than control normal red cells. The binding ratio for thalassaemic compared with controls was 1.69 on a cell-for-cell basis, and 1.97 when related to surface area. The binding of antibody from immune serum increased exponentially during parasite maturation. We also found increased binding of naturally occurring antibody present in non-immune serum to parasitized thalassaemic red cells which also increased during parasite maturation. We conclude that the protection afforded by thalassaemia against malaria may not reside in the ability of parasites to enter, grow or cytoadhere to endothelium in such cells, but may be related to immune recognition and subsequent clearance of parasitized red cells.

The interaction between Plasmodium falciparum and P. vivax in children on Espiritu Santo island, Vanuatu.

Studies of the prevalence and incidence of malaria were conducted in children < 10 years old living in 10 rural villages on the island of Espiritu Santo, Vanuatu, south-west Pacific. Malaria prevalence remained stable at 30% throughout the year but the relative contributions of the 2 major species were highly dependent on season. Plasmodium falciparum predominated in the long wet season (November-May) and P. vivax in the dry season (June-October). Case definitions for malaria, derived using a multiple logistic regression method, showed that parasite densities associated with clinical disease were low; case definitions for P. falciparum (> 1000 parasites/microL in children > 1 year old and > 500 microL in infants) and P. vivax (> 500 parasites/microL at all ages) were both associated with a specificity and sensitivity of > 90%. Like prevalence data, malaria morbidity was highly seasonal; 80% of clinical P. falciparum infections occurred in the wet season and 66% of clinical P. vivax in the dry season. Mixed infections were rare. Malaria was important cause of morbidity with children < 5 years old experiencing 1.3-3.0 episodes of clinical malaria per year and 23% of fevers being attributable to malaria in this age group. Children aged 5-9 years continued to suffer one episode of clinical malaria per year. The peak incidence of P. vivax malaria occurred earlier in life than the peak incidence of P. falciparum malaria. The possible interactions between these 2 parasite species are discussed.