Parasite polypeptides lost during schizogony and erythrocyte invasion by the malaria parasites, Plasmodium chabaudi and Plasmodium knowlesi

By biosynthetically labelling Plasmodium chabaudi and P. knowlesi stage-specific polypeptides and allowing continued development, schizogony and reinvasion in vivo or in vitro, we have identified parasite polypeptides not taken into the erythrocyte by the invading mezozoite. Three major and two minor parasite polypeptides synthesized by rings or mid-stage trophozoites of P. chabaudi were either degraded preferentially during further development, or lost during schizogony and reinvasion. For both P. chabaudi and P. knowlesi, a 250 000 mol. wt. polypeptide synthesized during maturation of trophozoites to schizonts and merozoites was not taken into the erythrocyte by the invading merozoite. The late stage synthesis of this polypeptide by P. chabaudi and its loss at schizogony and reinvasion was confirmed by immunofluorescence staining with a monoclonal antibody to this antigen. The importance of these antigens in the erythrocyte invasion process and in the induction and expression of immunity to malaria is discussed.     

Stage-specific protein synthesis by asexual blood stage parasites of Plasmodium knowlesi

P. knowlesi parasites with a maximum age distribution of 3 h were metabolically labelled with [³⁵S]methionine during 9 sequential non-overlapping intervals, from young rings to mature segmented schizonts. The proteins synthesised at the different stages were compared using sodium dodecyl sulphate-polyacrylamide gel electrophoresis; more than 40 polypeptides (M_r 20 000 to over 200 000) were identified in the different parasite preparations. The major polypeptides synthesised by rings and trophozoites of different ages were similar, but differences in minor polypeptides could always be recognised. At the onset of schizogony ring and trophozoite specific proteins ceased to be synthesised and proteins specific to schizogony emerged. In general, schizont-specific proteins were of higher molecular weight than ring stage proteins. Details of the morphological changes which occurred during the metabolic labelling episode permits correlation between parasite structure and synthesis of particular polypeptides. Comparison of parasite components metabolically labelled with [³H]glucosamine during defined periods of development also revealed stage-specific synthesis of glycoproteins. The extent to which proteins are altered after synthesis has been investigated by pulse-chase experiments.     

Stage-specific protein synthesis by asexual blood stage parasites of Plasmodium falciparum

Highly synchronised cultures of cloned Plasmodium falciparum (clone T9-94) were metabolically radiolabelled with [35S]methionine during eight consecutive non-overlapping intervals, while parasites developed from young rings to mature schizonts. Analysis of equal amounts of trichloroacetic acid precipitable radioactivity from each interval by sodium dodecyl sulphate-polyacrylamide gel electrophoresis fluorography allowed the stage specificity of protein synthesis to be investigated. More than forty polypeptides with molecular weights of 20 000 to 200 000 can be distinguished. While some proteins are synthesised throughout erythrocytic schizogony many are shown to be stage-specific. Among these are a range of high molecular weight proteins synthesised only during nuclear division. Detailed morphological information permits correlations to be made between synthesis of particular polypeptides and parasite structure.     

Monoclonal antibodies that protect in vivo against Plasmodium chabaudi recognize a 250,000-dalton parasite polypeptide.

Twenty monoclonal antibodies have been prepared to the erythrocytes from CBA/Ca mice infected with the rodent malaria Plasmodium chabaudi. By immunofluorescence, 15 of these antibodies recognized parasite antigens expressed only during the development of mature trophozoites to schizonts and merozoites, 2 recognized parasite antigens that were expressed throughout most of the intraerythrocytic cycle, and 3 recognized the membranes of all infected and uninfected erythrocytes. By immunoprecipitation of [35S]methionine-labeled, parasitized erythrocytes, parasite antigens recognized by all of the antiparasite antibodies were characterized. Eleven precipitated a 250,000-dalton parasite polypeptide which was synthesized and expressed late in the intraerythrocytic cell cycle and which appeared to be the major coat protein of the merozoites. In passive protection experiments, transfer of hyperimmune serum before infection with the parasite resulted in a delay in the rise of parasitemia, reduction in peak parasitemias, and a delay in the clearance of the parasitemia. Two monoclonal antibodies to the 250,000-dalton polypeptide had a similar but not as marked effect on parasitemia when given as a single dose before infection. When mixed and administered throughout the course of infection, their effects were greater. They had no influence on the course of Plasmodium berghei KSP11 parasitemia. Monoclonal antibodies to other parasite antigens and normal erythrocyte antigens failed to have a significant and reproducible effect on P. chabaudi parasitemia. The results suggest that this 250,000-dalton malaria parasite antigen may be important in the induction and expression of antibody-mediated immunity to malaria.     

Development ofEimeria dispersa Tyzzer, 1929, from bobwhite quail (Colinis virginianus), in bovine kidney cell cultures

Summary The development ofEimeria dispersa Tyzzer, a parasite of bobwhite quail, in Madin-Darby bovine kidney cell cultures was investigated. Excysted sporozoites were inoculated into Leighton tubes containing cell monolayers on glass coverglasses and maintained in minimum essential medium supplemented with heat-inactivated fetal calf serum. Sporozoites became intracellular within 2 h. Sporozoite-shaped schizonts, schizonts with developing merozoites, and mature first-generation schizonts were seen 24 h postinoculation. Intracellular first-generation merozoites, second-generation trophozoites, and early second-generation schizonts containing two nuclei were first observed 72 h postinoculation. Second-generation schizonts containing developing merozoites as well as mature second-generation schizonts were first seen 96h postinoculation. Gametogony was not observed.DM developing merozoite - HN host nucleus - IM intracellular merozoite - M merozoite - N nucleus - R refractile body - RB residual body - V parasitophorous vacuole     

Identification of a Plasmodium chabaudi antigen present in the membrane of ring stage infected erythrocytes

A novel antigen of asexual blood stages of the rodent malaria parasite Plasmodium chabaudi, was detected by means of a modified indirect immunofluorescence assay (IFA), using glutaraldehyde fixed and air dried monolayers of P. chabaudi infected erythrocytes. P. chabaudi hyperimmune sera gave a distinct surface immunofluorescence of erythrocytes infected with early stages of the parasite. Fixation and drying of the erythrocytes was necessary for the antigenic activity to be exposed. The antigens were species specific as P. chabaudi hyperimmune serum only stained P. chabaudi but not P. yoelii or P. falciparum infected erythrocytes. The antigenic activity involved in the IFA was resistant to trypsin, phospholipases and neuraminidase but not to pronase, suggesting that the antigens were polypeptides. The surface immunofluorescence was inhibited by an extract of parasitized erythrocytes, but not by similar extracts of normal erythrocytes. The inhibitory antigens were soluble and heat stable (100 degrees C, 5 min). For identification and characterization of the antigens, antibodies were isolated by acid elution from monolayers of infected erythrocytes and monoclonal antibodies were produced. Probing in immunoblotting of extracts of parasitized erythrocytes separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis with the eluted antibodies, showed that they reacted consistently with a polypeptide of Mr 105 000 (Pch105). The Pch105 antigen shares many characteristics with Pf155, a P. falciparum antigen considered as a candidate for a vaccine against that parasite.     

Fragments of the polymorphic Mr 185,000 glycoprotein from the surface of isolated Plasmodium falciparum merozoites form an antigenic complex

Merozoites of the human malaria parasite Plasmodium falciparum express on their surface several antigens derived from a polymorphic glycoprotein precursor of Mr 185,000 synthesised earlier on by trophozoites and schizonts. A panel of 18 monoclonal antibodies against a range of different specificities of the precursor was used to characterise its mature products in spontaneously released merozoites. Merozoites released by [35S]methionine or [14C]glucosamine-labelled schizonts, or surface 125I-labelled purified merozoites, were extracted in detergents, and the antigens were detected by immunoprecipitation or Western blotting. We show that a nonglycosylated peptide of Mr 80,000 and two glycosylated fragments of Mr 40,000 and Mr 16,000, all derived from the precursor, are exposed on the surface of the mature merozoite. Precipitations from extracts in different detergents indicate that the 80 and 40 kDa fragments can form a non-covalent complex with each other and two additional major surface antigens of 36 and 22 kDa. Several antibodies react strongly with the complex but not with its dissociated subunits, thus indicating presence of conformational epitopes. Other epitopes are positively mapped on different dissociated subunits by immunoprecipitation and Western blotting. The 80 and 40 kDa antigens each carry a different polymorphic marker epitope, and both of these markers are absent on the 16 kDa fragment. The 40 and 16 kDa glycoproteins share common epitopes, and the latter may be derived from the former fragments. Only epitopes present on the 16 kDa antigen, but not those specific for the larger fragments, are detectable by immunofluorescence in the ring-stage. This indicates that the whole or a part of the 16 kDa antigen remains on the parasite through the invasion process.     

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.     

Serum antibody immunoglobulin G of mice convalescent from Plasmodium yoelii infection inhibits growth of Plasmodium falciparum in vitro: blood stage antigens of P. falciparum involved in interspecies cross-reactive inhibition of parasite growth.

We demonstrated that antibodies in the serum of BALB/c mice convalescent from Plasmodium yoelii infection inhibit the in vitro growth of Plasmodium falciparum. Blood stage P. falciparum antigens that cross-react with the convalescent-phase mouse serum antibodies were identified and partially characterized. Convalescent-phase mouse serum immunoglobulin G (IgG) reacted with P. falciparum lysates at up to a 1:15,000 dilution of the immune sera and bound to P. falciparum-parasitized erythrocytes at up to a 1:5,000 dilution of the sera. The cross-reactive moieties of antigens in parasite lysates were resistant to oxidation by periodate but sensitive to trypsinization. About 15 polypeptides (M(r)s of 15,000 to 110,000) of P. falciparum blood stages were recognized by the convalescent-phase mouse anti-P. yoelii sera; many of these antigens were metabolically 35S labeled and specifically immunoprecipitated. Also, virtually all of the cross-reactive antigens were recognized by human malaria-immune sera. The anti-P. yoelii serum antibodies bound, with high affinity, to at least five of the cross-reactive antigens (M(r)s of 107,000, 84,000, 53,000, 36,000, and 30,000). By phase separation in Triton X-114, eight interspecies cross-reactive antigens (M(r)s of 84,000, 76,000, 51,000, 31,000, 29,000, 28,000, 23,000, and 22,000) were found to be integral membrane proteins. Convalescent-phase mouse serum IgG strongly inhibited the differentiation of P. falciparum from schizonts to rings; 75 micrograms of IgG per ml caused 80% inhibition of release of merozoites and their invasion into erythrocytes. On the other hand, the anti-P. yoelii serum antibodies also inhibited intracellular development of P. falciparum from rings to schizonts; 25 micrograms of IgG per ml caused 50% inhibition. Inhibition of P. falciparum growth by anti-P. yoelii serum IgG suggests that some of the interspecies cross-reactive antigens contain important conserved epitopes and induce protective antibodies against P. falciparum.     

Identification and characterization of a conserved,stage-specific gene product of Plasmodium falciparum recognized by parasite growth inhibitory antibodies

We have identified a novel conserved protein of Plasmodium falciparum, designated D13, that is stage-specifically expressed in asexual blood stages of the parasite. The predicted open reading frame (ORF) D13 contains 863 amino acids with a calculated molecular mass of 99.7 kDa and displays a repeat region composed of pentapeptide motives. Northern blot analysis with lysates of synchronized blood stage parasites showed that D13 is highly expressed at the mRNA level during schizogony. The first N'-terminal 138 amino acids of D13 were expressed in Escherichia coli and the purified protein was used to generate anti-D13 monoclonal antibodies (MAbs). Using total lysates of blood stage parasites and Western blot analysis, these MAbs stained one single band of approximately 100 kDa, corresponding to the predicted molecular mass of ORF D13. Western blot analysis demonstrated further that D13 is expressed during schizogony, declines rapidly in early ring stages and is undetectable in trophozoites. D13 protein is localized in individual merozoites in a distinct area, as demonstrated by indirect immunofluorescence analysis. After subcellular fractionation, D13 was confined to the pelleted fraction of the parasite lysate and its extraction by alkaline carbonate buffer treatment indicated that D13 is not a membrane-integral protein. Inclusion of certain anti-D13 MAbs into in vitro cultures of blood stage parasites resulted in considerable reduction in parasite growth. The N'-terminal domain encompassing 158 amino acids is 94 and 95%, respectively, identical at the amino acid level between Plasmodium knowlesi, Plasmodium yoelii, and P. falciparum. In summary, we describe a novel stage-specifically expressed, highly conserved gene product of P. falciparum that is recognized by parasite growth inhibitory antibodies.     

Identification of the Plasmodium chabaudi homologue of merozoite surface proteins 4 and 5 of Plasmodium falciparum

Previous studies of Plasmodium falciparum have identified a region of chromosome 2 in which are clustered three genes for glycosylphosphatidylinositol (GPI)-anchored merozoite surface proteins, MSP2, MSP5, and MSP4, arranged in tandem. MSP4 and MSP5 both encode proteins 272 residues long that contain hydrophobic signal sequences, GPI attachment signals, and a single epidermal growth factor (EGF)-like domain at their carboxyl termini. Nevertheless, the remainder of their protein coding regions are quite dissimilar. The locations and similar structural features of these genes suggest that they have arisen from a gene duplication event. Here we describe the identification of the syntenic region of the genome in the murine malaria parasite, Plasmodium chabaudi adami DS. Only one open reading frame is present in this region, and it encodes a protein with structural features reminiscent of both MSP4 and MSP5, including a single EGF-like domain. Accordingly, the gene has been designated PcMSP4/5. The homologue of the P. falciparum MSP2 gene could not be found in P. chabaudi; however, the amino terminus of the PcMSP4/5 protein shows similarity to that of MSP2. The PcMSP4/5 gene encodes a protein with an apparent molecular mass of 36 kDa, and this protein is detected in mature stages of the parasite. The protein partitions in the detergent-enriched phase after Triton X-114 fractionation and is localized to the surfaces of trophozoites and developing and free merozoites. The PcMSP4/5 gene is transcribed in both ring and trophozoite stages but appears to be spliced in a stage-specific manner such that the central intron is spliced from the mRNA in the parasitic stage in which the protein is expressed.     

Apical location of a novel EGF-like domain-containing protein of Plasmodium falciparum

Using bioinformatics analyses of the unfinished malaria genome sequence, we have identified a novel protein of Plasmodium falciparum that contains two epidermal growth factor (EGF)-like domains near the C-terminus of the protein. The sequence contains a single open reading frame of 1572bp with the potential to encode a protein of 524 residues containing hydrophobic regions at the extreme N- and C-termini which appear to represent signal peptide and glycosylphosphatidylinositol (GPI)-attachment sites, respectively. RT-PCR analysis has confirmed that the novel gene is transcribed in asexual stages of P. falciparum. Antibodies to the EGF-like domains of the novel protein are highly specific and do not cross-react with the EGF-like domains of MSP1, MSP4, MSP5 or MSP8 expressed as GST fusion proteins. Antisera to the C-terminal fragments react with two bands of 80 and 36kDa in P. falciparum parasite lysates whereas antisera to the most N-terminal fusion protein only recognises the 80kDa band, suggesting that the novel protein may undergo processing in a similar way to MSP1 and MSP8, but with fewer cleavage events. Immunoblot analysis of stage-specific parasite samples reveals that the protein is present in trophozoites, schizonts and in isolated merozoites. The protein partitions in the detergent-enriched phase after Triton X-114 fractionation and is localised to the surfaces of trophozoites, schizonts and free merozoites in an apical distribution. Based on the accepted nomenclature in the field we now designate this protein MSP10. We have shown that the MSP10 fusion proteins are in a conformation that can be recognised by human immune sera and that there is very limited sequence diversity in an approximately lkb region of MSP10, encompassing the two EGF-like domains. A sequence similar to MSP10 can be identified in the available P. yoelii genomic sequence, offering the possibility of ascertaining whether this novel protein can induce host protective responses in an in vivo model.     

The 140/130/105 kilodalton protein complex in the rhoptries of Plasmodium falciparum consists of discrete polypeptides

Four monoclonal antibodies (MAbs) recognise an antigen localised in the rhoptries of Plasmodium falciparum merozoites using both indirect immunofluorescence assay and immunoelectron microscopy with immunogold labeling. All MAbs immunoprecipitated bands at 140, 130 and 105 kDa from [35S]methionine-labeled parasites; however, one MAb immunoblotted only the 130 kDa protein and another MAb immunoblotted the 105 kDa protein. The affinity purified antigen complex consisted of proteins of 140, 130, 105 and 98 kDa. The individual proteins were subjected to peptide mapping with Staphylococcus aureus V8 protease; the 98 kDa protein was a degradation product of the 105 kDa protein and the 140, 130, and 105 kDa proteins were found to be unrelated. The antigen complex was synthesised at the mid trophozoite stage and was considered to be soluble as judged by release from mature schizonts by freeze/thaw lysis. One of the MAbs inhibited parasite growth and/or merozoite invasion of erythrocytes, in vitro, to a small but significant extent.     

Antibodies Reactive with the N-Terminal Domain of Plasmodium falciparum Serine Repeat Antigen Inhibit Cell Proliferation by Agglutinating Merozoites and Schizonts

The serine repeat antigen (SERA) is a vaccine candidate antigen of Plasmodium falciparum. Immunization of mice with Escherichia coli-produced recombinant protein of the SERA N-terminal domain (SE47') induced an antiserum that was inhibitory to parasite growth in vitro. Affinity-purified mouse antibodies specific to the recombinant protein inhibited parasite growth between the schizont and ring stages but not between the ring and schizont stages. When Percoll-purified schizonts were cultured with the affinity-purified SE47'-specific antibodies, schizonts and merozoites were agglutinated. Indirect-immunofluorescence assays with unfixed parasite cells showed that SE47'-specific immunoglobulin G (IgG) bound to SERA molecules on rupturing schizonts and merozoites but the IgG did not react with the schizont-infected erythrocytes (RBC). Furthermore, double-fluorescence staining against SE47'-specific IgG and anti-human RBC membrane IgG showed that the RBC membrane disappeared from SE47'-specific-IgG-bound schizonts after cultivation. These observations suggest that the SE47'-specific antibodies inhibit parasite growth by cross-linking SERA molecules that are associated with merozoites in rupturing schizonts with partly broken RBC and parasitophorous vacuole membranes, blocking merozoite release.     

Effect of jasplakinolide on the growth,invasion, and actin cytoskeleton of Plasmodium falciparum

The effect of jasplakinolide (JAS), an actin-polymerizing and filament-stabilizing drug, on the growth, invasion, and actin cytoskeleton of Plasmodium falciparum was examined. Jasplakinolide markedly decreased the parasitemia in a synchronized culture of P. falciparum strain FCR-3 in a time- and concentration-dependent manner. The decrease became evident at day 2 at concentrations of 0.3 micro M and above, and parasites finally disappeared at day 4. Giemsa-stained smears of P. falciparum-infected erythrocytes demonstrated that there was no effect on the development of schizonts from ring forms. Merozoites were released from the infected erythrocytes in a normal manner with and without JAS. However, there were no ring form-infected erythrocytes when JAS was administered, even after the release of merozoites. This indicates that the merozoites exposed to JAS failed to invade erythrocytes. The inhibitory effect of JAS on the parasitemia was reversed by the removal of the drug after exposure to 1 micro M of JAS for 1 day. Electron microscopy revealed that the merozoites treated with JAS showed a protrusion of the apical end which contained the microfilament structure. Immunoblot analysis indicated that the JAS treatment increased F-actin filaments of merozoites but had no effect on those of the trophozoites and schizonts. Therefore, this study demonstrated that JAS has an antimalarial activity.     

Glycoprotein recognition mediates attachment of Plasmodium chabaudi to mouse erythrocytes

The interaction between Plasmodium falciparum merozoites and human erythrocytes is mediated by specific parasite proteins and sialoglycoproteins (SGPs) on the surface of the host cell. To investigate whether a similar mechanism functions in rodent malaria, a series of experiments was performed to identify the proteins involved in the interaction of Plasmodium chabaudi parasites and mouse erythrocytes. Labeled parasite proteins incubated with purified mouse SGP bound specifically to glycoprotein 2.1. Two parasite proteins (72 and 126 kilodaltons [kDa]) were coprecipitated with antibody directed to mouse erythrocyte membrane proteins. The lower band (72 kDa) as well as a band of 105 kDa were also observed to bind to N-acetyl-D-galactosamine affinity columns, suggesting a carbohydrate component in the binding of these parasites to erythrocytes. These experiments indicate that P. chabaudi possesses specific proteins which recognized SGP on the surface of murine erythrocytes in a manner similar to that of the merozoites of P. falciparum. Thus P. chabaudi in mice may provide an in vivo model of the human parasite for testing ways to inhibit merozoite recognition and invasion of host cells.     

Characterisation of the rhoph2 gene of Plasmodium falciparum and Plasmodium yoelii

The high molecular mass protein complex (RhopH) in the rhoptries of the malaria parasite consists of three distinct polypeptides with estimated sizes in Plasmodium falciparum of 155kDa (PfRhopH1), 140kDa (PfRhopH2) and 110kDa (PfRhopH3). Using a number of reagents, including a new mAb 4E10 that is specific for the PfRhopH complex, it was shown that the RhopH complex is synthesised during schizogony and transferred intact to the ring stage in newly invaded erythrocytes. The genes encoding RhopH1 and RhopH3 have already been identified and characterised in both P. falciparum and Plasmodium yoelii. In this report, we describe the identification of the gene for RhopH2 in both these parasite species. Peptide sequences were obtained from purified RhopH2 proteins and used to generate oligonucleotide primers and search malaria sequence databases. In a parallel approach, mAb 4E10 was used to identify a clone coding for RhopH2 from a P. falciparum cDNA library. The sequences of both P. falciparum and P. yoelii genes for RhopH2 were completed and compared. They both contain nine introns and there is a high degree of similarity between the deduced amino acid sequences of the two proteins. The P. falciparum gene is a single copy gene located on chromosome 9, and is transcribed in schizonts.     

Guanosine triphosphate cyclohydrolase in Plasmodium falciparum and other Plasmodium species

GTP cyclohydrolase (EC 3.5.4.16), the first enzyme in the pteridine pathway leading to the de novo formation of folic acid, has been identified and isolated from the human malaria parasite, Plasmodium falciparum. The enzyme was purified 200-fold by high performance size-exclusion chromatography on a TSK-G-3000 SW protein column. The molecular weight was estimated at 300 000. Optimal enzyme activity was observed at pH 8.0 and 42 degrees C. The Km for GTP was 54.6 microM. Products of the enzyme reaction were identified as the carbon-8 of GTP and D-erythro-dihydroneopterin triphosphate. ATP was a competitive inhibitor (Ki = 600 microM) of the enzyme. Activity of the enzyme was Mg2+-independent, whereas Mn2+, Cu2+ and Hg2+ (5 mM) were inhibitory. GTP cyclohydrolase activity was also identified in a murine parasite, Plasmodium berghei, and a simian parasite, Plasmodium knowlesi. Activity of the enzyme in P. knowlesi, an intrinsically synchronous quotidian parasite, was found to be dependent on the stage of parasite development.     

Rat monoclonal antibodies which inhibit the in vitro multiplication of plasmodium knowlesi

A total of 28 double cloned monoclonal antibodies specific for Plasmodium knowlesi were raised by fusion of Y3 rat myeloma cells with spleen cells of A0 rats immunized with W1 variant isolated merozoites. Four of these antibodies reacted positively in a solid phase radioimmunoassay against glutaraldehyde-fixed schizonts but gave no detectable reaction on indirect immunofluorescence against methanol-fixed schizonts or merozoites. The remaining 24 antibodies could be divided into 13 distinctive immunofluorescent categories on the basis of their patterns of binding to schizonts and merozoites and reactivity with Plasmodium falciparum. Eight antibodies were studied for their ability to inhibit the in vitro multiplication of W1 P. knowlesi as assessed by parasite incorporation of ³H-amino acids and parasite counts. Partially purified antibody preparations from ascitic fluids were all inhibitory for parasite growth; however, when fully purified antibodies were tested, six of the eight proved to be non-inhibitory. Two of the purified antibodies, both IgG2a isotype, inhibited the in vitro multiplication of P. knowlesi in a dose-dependent manner. Inhibition was not associated with detectable damage to intracellular parasites, suggesting that the inhibitory monoclonal antibodies act by blocking the reinfection of red cells by newly released merozoites. On immunofluorescent analysis both inhibitory antibodies bound to methanol-fixed schizonts, with the intensity increasing for progressively more mature parasites; both reacted diffusely with isolated merozoites, and neither cross-reacted with P. falciparum. Both bound specifically to a single metabolically labelled polypeptide which appears to be a minor parasite component and has an approximate molecular weight of 66,000 when analysed by SDS-PAGE fluorography. The putative protective antigen of P. knowlesi has potential interest as a vaccine against P. knowlesi malaria.     

Merozoite surface protein 8 of Plasmodium falciparum contains two epidermal growth factor-like domains

By motif searching of the unfinished sequences in the Malaria Genome Sequencing Project databases we have identified a novel EGF-like domain-containing protein of Plasmodium falciparum. The sequence lies within a single open reading frame of 1791 bp and is predicted to encode a polypeptide of 597 amino acids. There are hydrophobic regions at the extreme N- and C-termini, which could represent secretory signal peptide and GPI attachment sites, respectively. Similar to MSP1, there are two EGF-like domains located near the C-terminus. RT-PCR analysis of the novel gene shows that it is transcribed in asexual stages of the malaria parasite. We have expressed portions of the protein as recombinant GST fusions in Escherichia coli and raised antisera in rabbits. Antibodies to the EGF-like domains of the novel protein are highly specific and do not cross-react with the EGF-like domains of MSP1, MSP4 or MSP5 expressed as GST fusion proteins. Antiserum raised to the most C-terminal region of the protein reacts with four bands of 98, 50, 25 and 19 kDa in P. falciparum parasite lysates whereas antisera to the N-terminal fusion proteins recognise the 98 and 50 kDa bands, suggesting that the novel protein may undergo processing in a similar way to MSP1. Immunoblot analysis of stage-specific parasite samples reveals that the protein is present throughout the parasite asexual life cycle and in isolated merozoites, with the smaller fragments present in ring stage parasites. The protein partitions in the detergent-enriched phase after Triton X-114 fractionation and is localized to the surfaces of trophozoites, schizonts and free merozoites by indirect immunofluorescence. Antisera to the C-terminus stain the surface of rings, whereas antisera to the N-terminus do not, suggesting that a fragment of the protein is carried into the developing ring stage parasite. Based on the accepted nomenclature in the field we designate this protein MSP8. We have shown that the MSP8 fusion proteins are in a conformation that can be recognised by human immune sera and that there is very limited diversity in the MSP8 gene sequences from various P. falciparum laboratory isolates. MSP8 shows significant similarity to the recently reported sequence of the protective P. yoelii merozoite surface protein pypAg-2 [Burns JM, Belk CC, Dunn PD. Infect Immun 2000;68:6189-95.] suggesting that the two proteins are homologues. Taken together, these findings suggest that MSP8/pypAg-2 may play an important role in the process of red cell invasion and is a potential malaria vaccine candidate.