Plasmodium falciparum antigens synthesized by schizonts and stabilized at the merozoite surface when schizonts mature in the presence of protease inhibitors

Schizonts of Plasmodium falciparum were cultured in medium containing a mixture of 10 micrograms/ml each of leupeptin, chymostatin, pepstatin, and antipain. The protease inhibitors did not inhibit macromolecular synthesis but were associated with decreased reinvasion of red cells and the accumulation of well preserved merozoites clustered around pigment granules (PCM, protease inhibitor clusters of merozoites). The parasite pellet from PCM cultures contained increased amounts of merozoite antigens, particularly at Mr 83, 73, 66, 45, and 17 kDa. The increases of the Mr 83, 73, and 45 kDa surface antigens observed in PCM had been observed also in similar merozoite clusters obtained by culturing schizonts in the presence of inhibitory antibodies. These three antigens are processed products of the abundant Mr 195 kDa schizont surface antigen. Liquid-phase double immunofluorescence of PCM demonstrated a residual red cell membrane through which monoclonal antibodies passed and reacted with the Mr 83, 73, and 45 kDa merozoite surface antigens or their precursors. The processes associated with normal reinvasion apparently involve protease(s), which plays a role(s) in the breakdown of the red cell membrane and the shedding of merozoite surface antigens. Interference with these processes by protease inhibitors is useful in increasing recoveries of merozoite antigens, as well as in elucidating mechanisms of reinvasion.     

Monoclonal antibody characterization of Plasmodium falciparum antigens in immune complexes formed when schizonts rupture in the presence of immune serum

When Plasmodium falciparum parasites are cultured with some immune sera, merozoites are agglutinated by antibodies to form immune clusters of merozoites and prevent their invasion into erythrocytes. Within these immune clusters of merozoites, several antigens that are normally found in the soluble fraction after detergent extraction accumulate in relatively insoluble immune complexes. From mice immunized with these immune complexes, we obtained hybridomas secreting monoclonal antibodies (mAb) that react with various immune clusters of merozoites antigens, including mAb 3D5, which recognizes a 101-kDa antigen (p101) and mAb, 5E3, which recognizes a 113-kDa antigen (p113). Both mAb reacted with antigens at the surface of schizonts, in the vacuolar space, and at the surface of merozoites before their release from schizont-infected cells. Both p101 and p113 were synthesized by mature trophozoites and young schizonts. In pulse-chase experiments, p113 was processed to 100-, 70-, 55-, and 50-kDa products. Both p101 and p113 appeared in the culture medium when schizont rupture occurred in normal culture medium but were found in immune complexes when schizont rupture occurred in the presence of immune serum. Antibodies in immune complexes, when dissociated with acid and used to probe immunoblots, reacted with affinity-purified p101 and p113. Antigens such as these, which are accessible at the parasite surface and react with antibodies present in immune serum that inhibits parasite invasion, are logical candidates to study in the search for a vaccine against the erythrocytic stages of malaria.     

Immunization of monkeys with a 140 kilodalton merozoite surface protein of Plasmodium knowlesi malaria: appearance of alternate forms of this protein

The merozoite is the invasive stage of the malaria parasite which is released by rupture of the schizont-infected erythrocyte. A monoclonal antibody against a 140 kilodalton (kDa) merozoite surface antigen of Plasmodium knowlesi was used to characterize and to purify this antigen. It was shown by pulse-chase metabolic labeling of mature schizonts that the 140 kDa merozoite antigen was the processed product of a 143 kDa schizont component, and that processing occurred at the time of erythrocyte rupture. Antiserum, prepared by immunizing a rabbit with the 143/140 kDa antigen purified by immunoaffinity chromatography with the monoclonal antibody, strongly inhibited invasion of erythrocytes in vitro; Fab fragments prepared from purified rabbit IgG were inactive at blocking invasion, suggesting that agglutination of merozoites was the mechanism of invasion inhibition. The purified 143/140 kDa antigen was used in Freund's adjuvant to immunize four rhesus monkeys. Two of the immunized animals developed fulminating infections on challenge with 10(4) schizonts, as did the three control animals. The remaining two immunized animals controlled their infections and developed chronic low-grade parasitemias. The animals which were partially protected were those that had developed anti-143/140 kDa antibodies capable of blocking invasion in vitro. Parasites were isolated from the chronic stage of infection (V5 population) and were compared with the original parasite population used for challenge (P population). Inhibition of invasion, immunofluorescence, and immunoprecipitation with anti-143/140 kDa monoclonal antibody, with immune rabbit, and with monkey sera showed that the 143/140 kDa surface antigen had been replaced by multiple cross-reacting alternate antigenic forms of the molecule in the V population. Thus, specific immune response directed against a purified merozoite surface antigen resulted in the replacement of this antigen by variant or mutant forms.     

Plasmodium berghei XAT: protective 155/160 kDa antigens are located in parasitophorous vacuoles of schizont-stage parasite

Effective blood-stage malaria vaccine candidates have been mainly developed from the proteins in exposed locations on the parasite such as the surface of free merozoites or infected red blood cells. In the present study, we identified and localized novel protective antigens derived from the blood-stage of Plasmodium berghei XAT after establishment of hybridomas producing protective monoclonal antibodies (mAbs) against the parasites. The protective antigens were expressed in schizonts but not in trophozoites, and located in the parasitophorous vacuoles in the infected erythrocyte cytoplasm. The antigens, with molecular weight of 155/160 kDa, were not identical to any merozoite/schizont antigens that have been reported as target molecules recognized by mAbs developed to rodent malaria parasites. The characterization of new malarial antigenic targets of potentially protective antibody responses following infection would give us new insights for the selection of candidate antigens for malaria vaccine.     

Plasmodium vivax: exoerythrocytic schizonts recognized by monoclonal antibodies against blood-stage schizonts

Exoerythrocytic parasites of Plasmodium vivax grown in human hepatoma cells in vitro were probed with monoclonal antibodies raised against other stages of P. vivax. Monoclonal antibodies specific for four independent antigens on blood-stage merozoites all reacted with exoerythrocytic schizonts and merozoites by immunostaining. The characteristic staining pattern of each monoclonal antibody was similar on both blood- and exoerythrocytic-stage parasites and appeared only in mature schizont segmenters. In contrast, a monoclonal antibody specific for the caveolar-vesicle complex of the infected host cell membrane and a second monoclonal antibody reacting with an unknown internal antigen did not appear to react with exoerythrocytic parasites. We confirm prior reports that monoclonal antibodies against the sporozoite immunodominant repeat antigen react with all exoerythrocytic-stage parasites, but note that as the exoerythrocytic parasite matures the immunostaining is concentrated in plaques reminiscent of germinal centers and apparently distinct from mature merozoites. These results indicate that mature merozoites from either exoerythrocytic or blood-stage parasites are antigenically very similar, but that stage-specific antigens may be found in specialized structures present only in a specific host cell type.     

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts.  Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies.       

Protective antigens of bloodstage Plasmodium knowlesi parasites

The simian malaria Plasmodium knowlesi provides many favourable features as an experimental model; it can be grown in vivo or in vitro. Parasites of defined variant specificity and stage of development are readily obtained and both the natural host and a highly susceptible host are available for experimental infection and vaccination trials. Proteins synthesized by erythrocytic P. knowlesi parasites are characteristic of the developmental stage, as are the alterations that the parasite induces in the red cell surface. Erythrocytic merozoites are anatomically and biochemically complex, their surface alone is covered by at least eight distinct polypeptides. Immune serum from merozoite-immunized rhesus recognizes many parasite components, especially those synthesized by schizonts. All of the merozoite surface components and some of the schizont-infected red cell surface antigens are recognized by such immune sera. Rhesus monkeys rendered immune by repeated infection may by contrast recognize comparatively few antigens; a positive correlation was established for these 'naturally' immunized monkeys between protection and antibody directed against a 74 000 molecular mass antigen. Immunization with this purified antigen confers partial protection. Other putative protective antigens have been identified by monoclonal antibodies that inhibit merozoite invasion of red cells in vitro. The antigens recognized by inhibitory monoclonal antibodies are synthesized exclusively by schizonts and are processed, at the time of schizont rupture and merozoite release, to smaller molecules that are present on the merozoite surface. The multiplicity of protective antigens is clearly demonstrated by the fact that seven distinct merozoite surface antigens are recognized by three different inhibitory monoclonals. None of the protective antigens identified are variant or strain specific.     

Plasmodium falciparum: two antigens of similar size are located in different compartments of the rhoptry

Two previously described antigens, AMA-1 and QF3, which are located in the rhoptries of Plasmodium falciparum merozoites have polypeptides of similar relative molecular masses. On immunoblots, antibodies to both antigens recognized polypeptides of relative molecular mass 80,000 and 62,000 in all isolates tested. Two-dimensional electrophoresis showed that the isoelectric points of the two antigens were different. QF3 being more basic than AMA-1. AMA-1 was soluble in Triton X-114 whereas QF3 partitioned into the aqueous phase after temperature-dependent phase separation. In immunoelectron microscopic studies. QF3 was found in the body of the rhoptry whereas AMA-1 was consistently found in the neck of the rhoptry. Both antigens gave a punctate double-dot pattern in mature schizonts and merozoites when visualized by fluorescence microscopy, but AMA-1 antibodies also appeared to label the merozoite surface. QF3 was also detected in ring-infected erythrocytes whereas AMA-1 was not. Synthesis of both antigens was first observed in mature trophozoites and immature schizonts. Pulse-chase experiments showed that the Mr 80,000 polypeptide of the AMA-1 gene was subject to immediate processing to the Mr 62,000 product. This cleavage pattern was not stage specific. The Mr 80,000 polypeptide of QF3 was derived from a short-lived Mr 84,000 precursor polypeptide. Processing of the Mr 80,000 polypeptide to an Mr 62,000 polypeptide was restricted to the period of merozoite maturation and reinvasion. Hence AMA-1 and QF3 are different antigens with polypeptides of similar size but located in different compartments of the merozoite rhoptries.     

Proteins and antigens of merozoites and sporozoites of Eimeria bovis (Apicomplexa)

Proteins and antigens of first-generation merozoites and sporozoites of Eimeria bovis were examined using standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and lactoperoxidase iodination procedures. SDS-PAGE gels revealed both common and unique protein bands in merozoite and sporozoite extracts, ranging in molecular weight (Mr) from 15,000 to 215,000. Nitrocellulose immunoblots of separated proteins, when probed with sera obtained from immunized calves, revealed numerous IgG-binding antigens of Mr 18,000 to 180,000 in merozoites and Mr 28,000 to approximately 118,000 in sporozoites. Although merozoite and sporozoite preparations each contained antigens of different molecular weights, 4 antigens had the same migratory distance in both preparations (Mr 58,000, 70,000, 83,000, 98,000). Of 3 types of immune sera used to probe immunoblots, serum taken from a calf that had been inoculated with oocysts of E. bovis and boosted 10 wk later by subcutaneous injection with 2 X 10(7) live merozoites emulsified in Freund's complete adjuvant consistently identified and reacted more intensely with more antigens of merozoites and sporozoites than the other immune sera tested. Autoradiographic analysis of radioiodinated parasites revealed major surface proteins on merozoites of between 15,000 and 18,000 Mr and 3 surface proteins on sporozoites of Mr 28,000, 77,000, and 183,000. All but the 183,000 protein elicited an IgG antibody response in the host.     

Lipidic vacuoles in Plasmodium knowlesi erythrocytic schizonts

Electron microscopy of schizont development in erythrocytic Plasmodium knowlesi has revealed that spheroidal vacuoles 250 nm in diameter with semi-dense contents appear at the periphery of the parasite prior to the budding of merozoites. When treated with non-polar solvents, their contents are completely extracted, and after fixation in tannic-glutaraldehyde they contain regular lamellae with a periodicity of 5.5 nm. Both of these reactions are typical of lipids. Some of these structures are associated with phagosomal vacuoles which may contribute to their lamellae. They disappear at the onset of merozoite formation, but membranous whorls of various sizes continue to be associated with the schizont surface during budding of merozoites. It is suggested that the lipidic vacuoles are a source of preformed lipid which can be utilized rapidly during the generation of merozoites.     

Localization of protective 143/140 kDa antigens of Plasmodium knowlesi by the use of antibodies and ultracryomicrotomy

Immune sera from mice immunized with the 143/140 kDa protein have been shown to partially block erythrocyte invasion by P. knowlesi merozoites. Therefore, immunoelectron microscopy utilizing ultracryomicrotomy, antibody to 143/140 kDa protein, and protein A gold particles were used to determine the precise localization of this protein in malarial parasites. Gold particles were not seen associated with young trophozoites but appeared in the parasite cytoplasm as the parasites grew to multi-nucleate schizonts. In presegmenter-schizonts, gold particles were associated with the well-developed endoplasmic reticulum, the parasite plasma membrane, and the parasitophorous vacuole membrane. The surface of merozoites was covered with gold particles. Maurer's clefts, which appeared in Plasmodium infected erythrocytes, were also associated with gold particles. These observations suggest that 143/140 kDa protective malarial proteins may be synthesized in the endoplasmic reticulum of P. knowlesi schizonts before being transported to the surface of the schizonts and merozoites. Shedding of the merozoite surface coat may be responsible for the presence of the 143/140 kDa proteins in the parasitophorous vacuole and Maurer's clefts.     

Eimeria tenella: use of a monoclonal antibody in determining the intracellular fate of the refractile body organelles and the effect on in vitro development

A monoclonal antibody, which recognizes the refractile body of Eimeria sporozoites, was used to study the developmental fate of this organelle during asexual development of E. tenella and to determine the effect of this monoclonal antibody on in vitro development of the parasite. Through use of immunofluorescent antibody and gold-labeling techniques at the light and electron microscopy level, the refractile body at 48 to 96 hr postinoculation was found to separate into 6 to 10 small globules, then diffuse throughout the schizont cytoplasm, and eventually reconcentrate as a small dot of material in each of the mature first-generation merozoites. The schizont did not develop to maturity if diffusion of the refractile body did not occur. The refractile body material was quickly lost as the merozoite left the schizont and invaded new cells and was not detected in any later developmental stages. The in vitro development of first- and second-generation schizonts of E. tenella was greatly inhibited (up to 100%) with exposure to the monoclonal antibody. There was an increase in the number of schizonts with nondispersed refractile body in the monoclonal antibody-treated cells when compared to the untreated controls, and the few mature schizonts seen had up to a 50-fold decrease in the number of merozoites. Immunofluorescent antibody labeling of the refractile body of intracellular sporozoites and schizonts treated in vitro with the monoclonal antibody for 24-96 hr postinoculation indicated that the antibody had crossed the host cell and parasite plasma membrane during incubation.     

Gametocyte formation by the progeny of single Plasmodium falciparum schizonts

Gametocytogenesis of the malaria parasite Plasmodium falciparum was studied in monolayers of erythrocytes attached to tissue culture dishes. Merozoites produced by single schizonts in erythrocytes overlaying the monolayer infected the attached erythrocytes and produced clusters of progeny. Parasites in these readily indentifiable clusters then underwent either asexual growth or sexual differentiation. The progeny of most schizonts yielded no gametocytes. However, the progeny of those schizonts that did yield gametocytes showed a marked tendency to produce multiple gametocytes. Gametocytogenesis, therefore, was not random. Instead, the progeny of certain schizonts were committed to produce gametes. However, even those clusters containing several gametocytes also contained asexual forms. Therefore, not all merozoites of a single schizont were committed to gametocytogenesis. In those cells infected with two or more merozoites the formation of a gametocyte was usually associated with a block in the further development of other parasites.     

Investigations into the fire structure of the asexual developmental stages of Frenkelia in the liver of the bank vole (author's transl)]

Bank voles (Clethrionomys glareolus) were infected by stomach tube with Frenkelia sporocysts from the faeces of buzzards (Buteo buteo). The voles were sacrificed at regular intervals and their livers examined electronmicroscopically. Seven days p.i. developmental stages of Frenkelia could be detected in liver parenchymal cells. The youngest schizonts detected are enveloped by a pellicle consisting of two membranes. This pellicle, which is in direct contact with the host cell mitochondria, shows marked invaginations which increase with the development of the schizont. A parasitophorous vacuole is not detectable. In developing schizonts numerous sections through nuclei with nucleic spindles and merozoite anlagen (dome-shaped) structures) are visible. It is not clear whether there are several nuclei or a section through one large and lobed nucleus. Within the merozoite anlagen the conoid and the subpellicular microtubules are formed first. By the prolongation of the dome-shaped structures towards the posterior pole, the nucleus and the other newly formed cell organelles are incorporated into the forming merozoite. The posterior pole of the merozoite still remains open at this stage of development. With increasing differentiation the merozoites become lancet-shaped, their apical poles bing always directed towards the periphery of the schizont. The outer membrane of the pellicle of the schizont forms the outer part of the pellicle of the merozoites by invaginating around them. At this stage of development the inner membrane of the pellicle of the schizont is no longer detectable. Thus the typical pellicle of the motile stages of sporozoaonsisting of three membranes is formed. In the centre of the merozoites which lie freely in the liver cell a residual body is present. The host cell reacts against the parasites by forming a thick border of mitochondria and distinct endoplasmic reticulum.     

Neutral proteases involved in the reinvasion of erythrocytes by Plasmodium merozoites

Neutral proteases of Plasmodium sp erythrocytic stages were studied by means of a sensitive fluorogenic method and gelatin-SDS-PAGE. The substrates gluconoyl-Val-Leu-Gly-Lys(or Arg)-3-amido-9-ethylcarbazole were selectively hydrolyzed by an endopeptidase from rodent Plasmodium berghei (Pb) and Plasmodium chabaudi (Pc) and from human Plasmodium falciparum (Pf) parasites. These endopeptidases were purified from 100,000-g soluble schizont extract by high pressure liquid chromatography; they have a similar Mr of 68,000 in SDS-PAGE, and an optimal activity at pH 7.4. The Pb 68 and Pf 68 endopeptidases were localized in schizonts and also in merozoites as shown by indirect immunofluorescence on Pb merozoites and by the identification of the Pf 68 endopeptidase activity in free viable merozoites. The Pb 68 and Pf 68 endopeptidases belong to the class of cysteine proteases. Analysis by gelatin-SDS-PAGE of a Pb 68 endopeptidase-enriched fraction showed a reproducible 95,000 proteolytic band. The initial extracts showed a similar 95,000 proteolytic band, and also 2 other 90,000 and 85,000 major bands. During reinvasion experiments, it was possible to recover a 95,000 and a 40,000 protease band from supernates of cultures grown in a semidefined medium without serum. Hydrophilic peptide derivatives related to the substrate of Pf 68 endopeptidase are shown to be potential inhibitors of the Pf reinvasion process in vitro.     

Ultrastructure of shizonts and merozoites of Sarcocystis falcatula in the lungs of budgerigars (Melopsittacus undulatus).

Transmission electron microscopy was used to study the ultrastructure of schizogony of Sarcocystis falcatula in the lungs of budgerigars (Melopsittacus undulatus). Schizogony occurred exclusively by endopolygeny within endothelial cells of pulmonary capillaries, venules, and small veins. Early schizonts were elongate with a large nucleus and nucleolus, surrounded by a pellicle consisting of a plasmalemma and an inner single membrane, and contained most of the organelles and inclusion bodies found in merozoites of Sarcocystis species. As development proceeded, schizonts increased in size and conformed to the shapes of the pulmonary blood vessels. As micronemes, dense granules, the conoid, and subpellicular microtubules disappeared, there was an increase in the size and number of mitochondria, Golgi complexes, and Golgi adjuncts (apicoplasts). As the nucleus elongated, there was a progressive increase in the number of spindles located at various intervals along the nuclear envelope. Eventually, 2 merozoites formed internally immediately above each spindle. During endopolygeny, a portion of the nucleus was incorporated into each merozoite bud along with 1 or 2 Golgi adjuncts, a Golgi complex, mitochondria, endoplasmic reticulum, and ribosomes. During merozoite formation, micronemes appeared in close association with the Golgi complex and gradually increased in number. The pellicle invaginated around the merozoites so they budded at the schizont surface leaving behind a small, central residual body. Dense granules appeared after merozoites were completely formed. Schizonts were 24 x 6.8 microm and contained 24-96 merozoites. Merozoites were 5.1 x 1.8 microm and were found free in the pulmonary air passages and pulmonary capillaries and within nearly all cells of the lung except red blood cells.     

Ultrastructural Study of Schizogony in Eimeria callospermophili*

SYNOPSIS The development of 1st generation schizonts of Eimeria callospermophili was studied with cell cultures and with experimentally infected host animals, Spermophilus armatus. Sporozoite-shaped schizonts each had 5-10 nuclei and all of the organelles of the sporozoite; each nucleus had a nucleolus and an associated Golgi apparatus. In stages immediately preceding merozoite formation, an intranuclear spindle apparatus with conical polar areas were observed near the outer margin of each nucleus. Two centrioles, each having 9 single peripheral tubules and one central tubule, were observed near each pole in some specimens. Merozoite formation began internally, with anlagen of 2 merozoites developing near each nucleus. The inner membrane of the merozoites first appeared as 2 dense thickenings adjacent to the polar cones and centrioles; subpellicular microtubules appeared simultaneously. Two anterior annuli and the conoid formed between the 2 thickenings. Vesicles, possibly of Golgi origin, were located next to the forming inner membrane. As the forming merozoites underwent elongation, a rhoptries anlage, a Golgi apparatus, refractile bodies, and mitochondria were incorporated into each. Sporozoite-shaped schizonts with merozoite anlagen transformed into spheroid or ovoid schizonts; at this time the conoid, rhoptries, micronemes, and the inner membrane of the pellicle gradually disappeared; several small refractile bodies were formed from the larger one. When development was about 1/3 complete, the immature merozoites began to grow outward from the surface of the schizont. In this phase of development, the single surface membrane of the schizont became the outer membrane of the merozoite's pellicle, and additional organelles, including the nucleus, were incorporated. Finally, the merozoites became pinched off, leaving a residual body. Development in cell cultures and host tissues was similar. This type of schizogony, previously undescribed in Eimeria, is compared with corresponding stages of development in other species of Eimeria and Sporozoa.     

Ultrastructural localization of protective and nonprotective Plasmodium falciparum proteins using serum samples from vaccinated Aotus monkeys

Postembedding immunoelectron microscopy, using pooled serum samples from a recent vaccination experiment involving Aotus monkeys, was used to localize immune targets in Plasmodium falciparum-infected erythrocytes and free merozoites. Serum samples from Aotus monkeys, protected completely by immunization with the P. falciparum merozoite surface coat precursor protein, identified immune targets on the surface of free and intracellular merozoites as well as the cytoplasm, plasma membrane, and parasitophorous vacuole membrane of immature schizonts. Serum samples from unprotected monkeys, which had been immunized with a complex of 143-kDa, 132-kDa, and 102-kDa polypeptides reacted specifically with the rhoptries of immature schizonts and mature merozoites.     

Fine Structure of the Neogregarine Farinocystis tribolii Weiser, 1953. Developmental Stages in Merogony.

SYNOPSIS. The fine structure of schizonts and free merozoites of the neogregarine Farinocystis tribolii Weiser, and their development in the fat body of larval Tribolium castaneum were studied.
The surface of a multinucleate schizont and that of a uninucleate merozoite is covered by a double-layered membrane. Rhoptries and micronemes are present. The cytoplasm is packed with ribosomes and also contains dark bodies. Mitochondria are of the vesicular type. The spherical nucleus of the schizont and merozoite contains a large nucleolus. The anterior end of the merozoite has a typical conoidal complex composed of a conoid and a polar ring with 22 subpellicular mirotubules projecting from it.
New findings are a membranous septum across the body of the merozoite at 2/3 of its length below the nucleus and a highly osmiophilic spiral structure in the perinuclear space close to the Golgi complex. In addition, we found some "developmental stages" of the latter structure.     

Plasmodium vivax, P. cynomolgi, and P. knowlesi: identification of homologue proteins associated with the surface of merozoites

We have identified a Plasmodium vivax merozoite surface protein (MSP) that migrates on SDS-polyacrylamide gels at a Mr of about 185 kDa. This protein was recognized by a P. vivax monoclonal antibody (mAb) that localizes the protein by immunofluorescence to the surface of merozoites and also immunoprecipitates this protein from NP-40 detergent extracts of [35S]methionine metabolically radiolabeled P. vivax schizonts. The P. vivax MSP does not become biosynthetically radiolabeled with [3H]glucoamine, [3H]myristate, [3H]palmitate, or [3H]mannose, indicating that this P. vivax MSP is not posttranslationally modified and bound to the merozoite membrane by a glycosylphosphatidylinositol (GPI) lipid anchor. Thus, in this respect, this protein is different from members of the MSP-1 protein family and from MSP-2 and MSP-4 of P. falciparum. The mAb cross-reacts with and outlines the surface of P. cynomolgi merozoites and immunoprecipitates a 150-kDa P. cynomolgi homologue. The mAb was used as an affinity reagent to purify the native homologous MSP from NP-40 extracts of P. cynomolgi mature schizonts in order to develop a specific polyclonal antiserum. The resulting anti-PcyMSP rabbit antiserum cross-reacts strongly with the P. vivax 185-kDa MSP and also recognizes an analogous 110-kDa protein from P. knowlesi. We have determined via an immunodepletion experiment that the 110-kDa P. knowlesi MSP corresponds to the PK 110 protein partially characterized earlier (Perler et al. 1987). The potential of P. vivax MSP as a vaccine candidate was addressed by conducting in vitro inhibition of erythrocyte invasion assays, and the IgG fraction of both the P. vivax MSP mAb and the P. cynomolgi MSP rabbit antiserum significantly inhibited entry of P. vivax merozoites. We denote, on a preliminary basis, these antigenically related merozite surface proteins PvMSP-185, PcyMSP-150, and PkMSP-110.