Characterization of monoclonal antibodies directed against erythrocytic stage antigens of Plasmodium berghei.

Monoclonal antibodies recognizing various facets of the malaria parasite Plasmodium berghei and of the infected erythrocyte were obtained after generation of hybridomas between spleen cells from immunized mice and myeloma cells. The monoclonal antibodies were characterized by enzyme-linked immunosorbent assay, indirect immunofluorescence, immunoprecipitation of [35S]methionine-labeled proteins and immunoblotting. The most readily identified antigen was a parasite surface-associated protein of 230 kDa which is similar to the polymorphic schizont antigen described in a number of malarial species. In addition, three distinct antigens of 13, 31 and 120 kDa, which are external to the parasite, but within the infected erythrocyte were identified.

Evidence for major differences in ribosomal subunit proteins from Plasmodium berghei and rat liver.

Purified polysomes were isolated in high yield from the erythrocytic stages of the rodent malaria parasite, Plasmodium berghei, and from rat liver. Proteins extracted from the ribosomal subunits derived from these polysomes were fractionated and their number and molecular weights were estimated by two-dimensional polyacrylamide gel electrophoresis. Plasmodial small ribosomal subunits contained 30 proteins ranging in apparent molecular size from 11.7 to 40.7 kDa, while large subunits contained 35-36 proteins ranging from 12.1 to 42.6 kDa. None of these parasite proteins was shared by the two subunits nor altered in electrophoretic mobility by radioiodination. Rat liver 40 S ribosomal subunit proteins numbered 30 and ranged from 9.2 to 37.5 kDa, while liver 60 S subunits contained 41-43 proteins with apparent molecular sizes of 10.3-45.2 kDa. Coelectrophoresis of trace amounts of radioiodinated P. berghei ribosomal subunit proteins and stainable quantities of liver proteins demonstrated that most of these 139 parasite and host ribosomal proteins possessed different two-dimensional electrophoretic mobilities under the conditions of this study. Based upon a comparative analysis of P. berghei and rodent ribosomal RNA and these data, it was concluded that parasite and host ribosomes contain distinct ribosomal RNAs and ribosomal proteins.

Isolation and characterization of membrane proteins of Plasmodium berghei sporozoites.

Two immunologically significant proteins, Sp53 and Sp110, have been isolated from the sporozoites of Plasmodium berghei ANKA strain using different extraction procedures. In gel filtration studies the physicochemical characteristics of Sp53 and Sp110 appeared to be somewhat different. Both polypeptides could be purified using Sephacryl S300 column chromatography. The possible relationship of both Sp53 and Sp110 with sporozoite proteins described by other investigators is discussed.

Expression of the precursor of the major merozoite surface antigens during the hepatic stage of malaria.

The precursor of major merozoite surface antigens (PMMSA) and its proteolytic products are candidates for an asexual blood stage vaccine. Previous authors have shown that PMMSA epitopes are expressed in the liver or exoerythrocytic (EE) stage of malaria. Using Plasmodium berghei, we show that the molecular weight of the liver stage PMMSA is similar to that of the blood stage and that both EE and blood stage proteins are similarly processed. In the EE stage, it was synthesized toward the end of schizogony and appeared first to localize to the rough endoplasmic reticulum and then, as the cytomeres began to form, to the parasite plasmalemma. The EE and blood stage merozoites expressed similar amounts of this antigen as determined by indirect immunofluorescence.

Characteristics of multidrug resistance in Plasmodium and Leishmania: detection of P-glycoprotein-like components.

Multidrug-resistance (MDR) in neoplastic cells is frequently characterized by the overexpression of P-glycoprotein (PGP), a 170 kDa transmembrane glycoprotein that binds multiple cytotoxic drugs as well as calcium channel antagonists. Chloroquine resistance in Plasmodium falciparum appears to be analogous to MDR in neoplastic cells, where the induction of resistance with one drug confers resistance to other structurally and functionally unrelated drugs. To test the hypothesis that chloroquine resistance in P. falciparum and antimony resistance in Leishmania is mediated by a similar mechanism of MDR in mammalian neoplastic cells, a PGP-specific monoclonal antibody (C219) was used to determine the presence of PGP genes in resistant and sensitive Plasmodium and Leishmania parasites by indirect immunofluorescence assays and Western blotting procedures. These PGP-like components were detected in both drug-sensitive and -resistant Plasmodium and Leishmania cells. A 40-42 kDa component was observed to be greater in a chloroquine-resistant P. berghei (C line) than in a chloroquine-susceptible P line. Differences observed between Pentostam-resistant and -sensitive Leishmania promastigote clones and isolates included the increased expression of 96-106 and 23-25 kDa peptides in drug-resistant L. enrietti, and increased amounts of two different peptides in two drug-resistant L. panamensis clones (i.e., 96-106 and 43-45 kDa in WR-746-CL4, and 53 and 23-25 kDa in kDa) in amastigotes as in MDR KB carcinoma cells (KB-V1). Comparative indirect immunofluorescent studies suggested that a correlation existed between the degree of antimony susceptibility and the concentration of the moiety recognized by C219 in two L. panamensis clones. Binding of the C219 monoclonal antibody to the PGP-like component of Leishmania was blocked by Pentostam, while the binding of C219 to multiple-drug resistant KB-V1 PGP was not inhibited by Pentostam, regardless of the PGP concentration. This suggests some degree of specificity in the binding of Pentostam to the Leishmania PGP-like components. In addition, these studies have demonstrated that drug-sensitive Leishmania accumulate two to five times more 125Sb-Pentostam than resistant clones.

Free-flow electrophoretic separation of Plasmodium berghei sporozoites.

Sporozoites of the rodent malaria, Plasmodium berghei, were obtained from infected Anopheles stephensi by grinding mosquitoes, prepurifying the material in a discontinuous Hypaque gradient and further purifying by means of continuous free-flow electrophoresis. Bacteria, debris, mitochondria, mitoplasts, and other contaminants were removed in the electric field. The isolated sporozoites were morphologically intact and were positive in indirect immunofluorescence assay. They were infective to mice prior to and following free-flow electrophoretic separation. The surface of the sporozoites exhibited a polysaccharide-rich layer. The predominant surface protein labelled after surface iodination had a molecular weight between 42,000 and 46,000 daltons.

Experimental studies of schistosomal pigment from Schistosoma japonicum.

The schistosomal and malarial pigments were distinguishable before and after extraction from the host liver. Presence of iron in both pigments was ascertained by the elemental X-ray analysis. Histochemically, however, schistosomal pigment was similar to that of malarial pigment.

The ribosomes of Plasmodium berghei: isolation and ribosomal ribonucleic acid analysis.

Ribosomes and high molecular weight ribosomal ribonucleic acid (rRNA) from the blood stages of Plasmodium berghei parasites were studied in preparations free from host ribosome contamination. Purified malarial ribosomes were isolated in high yield from a population of ultrastructurally intact, viable parasites by hypertonic lysis with Triton X-100 and differential centrifugation. These ribosomes were shown to be derived from active polysomes and could be dissociated into subunits by puromycin-0.5 M KCl treatment. Malarial rRNA extracted from purified 40S and 60S ribosomal subunits was characterized by electrophoretic, sedimentation and base ratio analyses. Like certain other protozoa, the P. berghei 40S ribosomal subunit possessed an exceptionally large RNA species (mol. wt 0.9 X 10(6), while RNA isolated from the parasite's 60S subunit (mol. wt 1.5 X 10(6)) was specifically 'nicked' to produce one large component (mol.wt 1.2 X 10(6)) and one small component (mol.wt 0.3 X 10(6)) in equimolar quantities. These rRNA's migrate identically on polyacrylamide gels after heating to 63 degrees C for 5 min or under denaturing conditions in the presence of formamide, indicating an absence of aggregation and non-specific degradation of the rRNA species. Base composition studies showed P. berghei rRNA to be low in guanosine and cytosine content, as is the case for protozoa generally.

Surface properties of extracellular malaria parasites: morphological and cytochemical study.

Morphological and cytochemical surface characteristics of isolated malaria parasites (Plasmodium berghei) and host erythrocytes were compared by electron microscopy by using thin section and carbon replica techniques. Erythrocytes were uniform in shape and had fine, granular surfaces. In contrast, free parasites exhibited a variety of sizes, shapes, and surface textures. Fine surface stippling was a common topographical feature of isolated parasites. Small, infective forms often had patterned surfaces resulting from the protuberance of an underlying thick intermediate layer. Results of cytochemical analysis using a sialophilic colloidal iron stain indicated that the malaria parasite's surface lacked exposed sialic acid groups which would normally give rise to a net negative surface charge common to erythrocytes. Biochemical assay demonstrated that malaria parasites contained about one-half the amount of sialic acid per unit weight as did control red cell extracts. Similarly, external acidic mucopolysaccharide coats of free parasites, as revealed by ruthenium red staining were extremely thin as compared with the thick glycocalyx layer of red cells. Lipid plaques at the surface of parasites and red cells were localized by lipophilic iron colloid staining. Although the gross patchwork distribution of plaques was somewhat similar for the two cell types, the parasites were stained more intensely and had a closer-knit patchwork pattern than those exhibited by the erythrocytes. Such findings indicate that there are slight differences in the arrangement of phospholipids at the surfaces of limiting membranes of host cells and parasites. The significance of the above cytochemical surface properties of the malaria parasite (which are seemingly akin to those of intracellular organelles is discussed in relation to certain host-parasite interactions, such as parasite adhesion to target cells and enhanced clearance of extracellular parasites.

Purification and some properties of malarial pigment.

Malarial pigment from erythrocytes infected with Plasmodium berghei was purified by treatment with sodium dodecyl sulphate solution, followed by incubation with Pancreatin. The purified pigment retained the apparently crystalline form of pigment within the parasite, rotated polarised light and had the same solubility characterisation as crude malarial pigment. It contained about 1% iron, all of which could be accounted for in terms of haemin. The iron of the pigment molecule is oxidised by the parasite to the ferric state.

Lipid composition and activity of a lytic factor isolated from Plasmodium berghei.

A fraction was obtained from Plasmodium berghei which induced hemolysis of the erythrocytes of mice and hamsters. This fraction, called lytic factor (LF), was found to be composed of a large amount of lipid material. An examination of the lipids showed the major lipids to be monoglycerides, diglycerides, triglycerides, fatty acids, long-chain alcohol, sterol, sterol ester, sterol glycoside, and two cerebrosides. The most abundant component found in the LF was sterol ester, followed in order by cerebrosides, sterol, and sterol glycoside. Lytic activity was found to be lost when samples were boiled for 5 min. An examination of the lipid composition of LF before and after boiling showed changes which may be useful in studies on the mechanism of activity of this factor. The fatty acid composition of the total lipid fraction of LF was examined by gas-liquid chromatography. The major fractions were 18:1 and 16:0 in unheated LF and 16:0 in the heated LF.

Relations between resistance to chloroquine and acidification of endocytic vesicle of Plasmodium berghei.

In order to visualize low-pH compartments of Plasmodium berghei strains we have used a basic congener of dinitrophenol, 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP) which concentrates in acidic compartments, and can be detected by immunocytochemistry with anti-dinitrophenol antibodies. We have demonstrated that in a P. berghei chloroquine-sensitive strain (N strain), DAMP accumulates in the endocytic vacuoles where haemoglobin degradation is occurring. These compartments which have recently been shown to concentrate 4-aminoquinoline drugs (Moreau, Prensier, Maalla & Fortier, 1986) have an acidic pH. Conversely DAMP was found scattered all over the cytoplasm in a P. berghei chloroquine-resistant strain; the same phenomenon was previously observed (Moreau et al. 1986) in the localization of a 4-aminoquinoline on this same strain. Monensin-induced swelling of acidic compartments (Boss & Morre, 1984) was used as a complementary method for the determination of low-pH compartments on P. berghei strains. All the data reported here suggest that chloroquine resistance in P. berghei RC may be related to an impairment in the acidification of endocytic vesicles.

Plasmodium berghei: cloning of the circumsporozoite protein gene.

A DNA fragment encoding the carboxy terminal 80% of the Plasmodium berghei circumsporozoite protein was selected from a genomic DNA expression library. Sequencing revealed that the P. berghei circumsporozoite protein was similar in overall structure to circumsporozoite proteins from other malaria species, although the central repeat region was unique in comprising two different blocks of tandem peptide repeats: 11 eight amino acid repeats with predominant sequence DPAPPNAN were followed by 16 two amino repeats, predominantly PQ. The P. berghei circumsporozoite protein exhibited limited, but about equal amino acid homology to circumsporozoite proteins from P. knowlesi, P. vivax, and P. falciparum, indicating that P. berghei is not closely related to any of these other malaria species. Cloning of the P. berghei circumsporozoite protein gene will allow direct testing of sporozoite vaccines in mice.

Cultivation of the exoerythrocytic stage of Plasmodium berghei in primary cultures of mouse hepatocytes and continuous mouse cell lines.

Plasmodium berghei exoerythrocytic (EE) stages have been cultured in vitro in human continuous cell lines and primary cultures of both human and rat hepatocytes. Although the predominant experimental model of irradiated sporozoite-induced protective immunity is the mouse, P. berghei has not been cultivated in primary mouse hepatocytes or in continuous mouse lines. Because of this, target cells are not available for determining if these immunized mice produce cytotoxic T lymphocytes (CTLs) that recognize P. berghei antigens expressed on hepatocytes in the context of class I major histocompatability (MHC) antigens. We report the development of methods for cultivating the (EE) stage of P. berghei in murine hepatocytes and in two cell lines derived from the livers of BALB/c mice; one line produced from a primary hepatocyte culture and the other produced by fusion of mouse hepatocytes with a continuous rat liver line. Mature parasites were detected by microscopy and by DNA probe in both cell lines, each of which supported complete development of P. berghei liver stages and production of infectious merozoites. Since class I MHC antigens are present on the surface of primary hepatocytes and the mouse X rat hybrid line, these cells can be used to detect cytotoxic T cells against liver stage parasites.