Plasmodium knowlesi-induced antigens in membranes of parasitized rhesus monkey erythrocytes.

Abstract

Highly purified Plasmodium knowlesi schizonts were used to produce a hyperimmune anti-parasite serum in a rhesus monkey. Proteins of membranes from normal and P. knowlesi-infected erythrocytes, as well as purified schizonts, were solubilized in 1% Triton X-100 and analyzed by bidimensional electrophoretic techniques. Of seven parasite-specific antigens identified in membranes of parasitized erythrocytes by crossed immune electrophoresis against monkey anti-parasite serum, only three could be detected in the purified schizonts. Bidimensional focusing-dodecyl sulfate/polyacrylamide gel electrophoresis of membranes from parasitized cells revealed three proteins, in the 55,000-90,000 molecular weight region, with isoelectric points between pH 4.5 and pH 5.2,-that could not be detected in normal membranes or purified schizonts. Membranes of normal erythrocytes and uninfected erythrocytes that had been incubated with sera from monkeys with 25-50% parasitemia did not react with the monkey anti-parasite serum.Inoculation of owl or rhesus monkeys with intraerythrocytic stages of Plasmodium falciparum (1) or P. knowlesi merozoites (2), with complete Freund's adjuvant, produces effective animalarial immunity. Moreover, Collins et al. (3) have isolated a heat-stable soluble antigen from sera of P. knowlesi-infected monkeys which when injected with complete adjuvant, will suppress parasitemia and reduce mortality of animals challenged with homologous parasites. These experiments show that antimalarial immunity can be achieved experimentally by vaccination with parasite antigens. However, they do not provide information about parasite-induced host-cell antigens. These were first recognized by Brown et al. (4), who showed that the sera of rhesus monkeys chronically infected with, or immune to infection by, a given strain of P. knowlesi contained antibodies that agglutinate erythrocytes containing schizonts of that strain of P. knowles . Possibly related antigens have also been detected by immune electron microscopy of erythrocytes infected with P. falciparum (5), but it is not known whether any such antigens can elicit a protective immune response.We have approached this area by biochemical and immunochemical techniques, first developing a reliable method for the isolation of schizonts and host-cell plasma membranes from P. I6mowlessinfected rhesus erythrocytes (6) and now analyzing these fractions, by two-dimensional isoelectric focusing-dodecyl sulfate (DodSO41)/polyacrylamidp gel electrophoresis and by immunoelectrophoretic methods using high-titer monkey antisera raised against purified schizonts.MATERIALS AND METHODS Chemicals. Reagents for electrophoretic separations were as in refs. 6-8; other chemicals were as in ref. 6. Monkeys. Rhesus monkeys (Macaca mulatta), weighing 4-8 kg, obtained from the Primate Import Company (Port Washington, NY), were first infected after about a 4-week quarantine period (Tb-negative). Cure was by an initial intramuscular injection of 20 mg of chloroquine per kg, followed by two...

Isolation and characterization of the plasma membrane of human erythrocytes infected with the malarial parasite Plasmodium falciparum.

“…However, our results do cast doubt on whether parasite proteins act as structural components of knobs and question the insertion of neoproteins into the erythrocyte membrane (23,24,26,27). Indeed, the mechanism whereby neoproteins would be transported from the parasite rough endoplasmic reticulum, across the double membrane complex surrounding the parasite, and then finally inserted into the host cell membrane remains both unknown and difficult to envision.…”

“…USA 80 (1983) 1091 erythrocyte membrane, possibly at the level of the spectrin-actin network. Indeed, alteration of erythrocyte membrane proteins, and particularly spectrin, has been reported (18)(19)(20)(21)(22)(23)(24). Such modifications could lead to a local deformation of the membrane, as is seen with the knobs, and could explain the specific immunolabeling of knobs (2, 3) by exposing host cell antigens that would otherwise remain buried in or below the membrane.…”

Protective Plasmodium knowlesi Mr 74,000 antigen in membranes of schizont-infected rhesus erythrocytes.

“…The cells were incubated at a [35S]methionine concentration of 0.2 mCi/ml for 3 h. The incubator was maintained at 37°C and equilibrated with humidified O2/C02/N2 (5.5%:5%:89.5%, voi/vol). After labeling, the parasitized erythrocytes were washed three times in RPMI 1640, 0.01 M, in unlabeled methionine and then disrupted by nitrogen decompression (22,28). Subcellular fractionation into parasites and host cell membranes was as described elsewhere (29).…”

“…The Mr 74,000 protein was isolated from purified membranes of P. knowlesi-infected rhesus erythrocytes (containing 4-6 nuclei schizonts). Of the 4 × 10 H cells used for subcellular fractionation (28,29,31), 4 × 10 l° were labeled metabolically with [35S]methionine as described above. One half of the host cell membrane proteins were solubilized by two sequential extractions in Triton X-100 and immune precipitated with rhesus immune Ig (monkey 153) (11); the other half was equilibrated in sample buffer.…”

“…Purified schizont-infected erythrocytes (22,28) were adjusted to a hematocrit of 10% using methionine-free RPMI 1640 fortified with 10% dialyzed, heat-inactivated FCS. The cells were incubated at a [35S]methionine concentration of 0.2 mCi/ml for 3 h. The incubator was maintained at 37°C and equilibrated with humidified O2/C02/N2 (5.5%:5%:89.5%, voi/vol).…”