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

The immunogenic Plasmodium knowlesi (H strain) Mr 74,000 protein in membranes of schizont-infected rhesus erythrocytes was purified on a large scale, free of other polypeptides as monitored by dodecyl sulfate polyacrylamide gel electrophoresis and isoelectric focusing. In a limited vaccination trial, four rhesus monkeys were immunized four consecutive times with the Mr 74,000 protein and Freund's complete and incomplete adjuvants. Two monkeys were injected with adjuvant only. Upon challenge with 10(4) viable P. knowlesi schizonts of the heterologous W strain, the control monkeys developed fatal parasitemias after 7 d. In contrast, the vaccinated monkeys exhibited a delayed onset of patent parasitemias and underwent self-cure on days 14 to 16 after peak parasitemias of between 7 and 11%. The protective immunity that was induced crossed different strains of P. knowlesi. Blood smears at the time of cure demonstrated limited reinfection, as indicated by the presence of normally appearing ring and trophozoite stages. The absence of schizont stages in the peripheral blood suggested a specific interruption of the erythrocytic schizogony at that stage. Immunochemical analyses of the rhesus sera revealed antibody only against the Mr 74,000 protein after the first two immunizations. Upon repeated antigen injection, antibodies reacted with components of Mr of approximately 102,000, 140,000, and 230,000 in addition to the Mr 74,000 protein. Besides immunological cross-reactivity, relatedness between all four immune-precipitated proteins was indicated by a greater than 50% tryptic peptide homology, suggesting that the Mr 230,000 component represents a precursor protein that is cleaved within the infected erythrocyte into proteins with Mr of approximately 140,000, 102,000, and 74,000.

Immunogenic antigens common to Plasmodium knowlesi and Plasmodium falciparum are expressed on the surface of infected erythrocytes.

Sera of Gambian individuals and rhesus monkeys immune against infections with Plasmodium falciparum and plasmodium knowlesi, respectively, were reacted with triton X-100-solubilized membranes of infected erythrocytes. Indirect immune precipitation with Staphylococcus aureus, Cowan strain A, followed by dodecylsulfate-polyacrylamide gel electrophoresis, were used to identify interspecies plasmodial antigens that were immunogenic in vivo. Both types of sera specifically precipitated Plasmodium-specific antigens with Mrs of 125,000, 90,000, and 65,000 to 50,000 from membranes of P. knowlesi-infected erythrocytes that had been labeled with 125I using the lactoperoxidase-catalyzed radioiodination or metabolically with 14C-amino acids. In addition, P. falciparum inhibited the precipitation of P. knowlesi antigens by the Gambian immune sera. Our results indicate, that during erythrocytic schizogony, interspecies Plasmodium antigens are exposed on the surfaces of infected erythrocytes.

Rhesus monkeys protected against Plasmodium knowlesi malaria produce antibodies against a 65,000-MrP. knowlesi glycoprotein at the surface of infected erythrocytes.

Sera from 27 rhesus monkeys immunized in various ways against the H strain of Plasmodium knowlesi were analyzed by quantitative crossed immunoelectrophoresis. The reaction of the sera was compared with a reference immune serum only reactive with P. knowlesi-specific 65,000-Mr glycoprotein-immune component 13 (gp65/ic13) in membranes of infected rhesus monkey erythrocytes. Triton X-100-solubilized, 125I-labeled membranes of schizont-infected erythrocytes were used as an antigen. Sera from 9 or 10 monkeys immunized by repeated infections with P. knowlesi reacted with gp65/ic13. In 6 of 10 sera, anti-gp65/ic13 was the only antibody reacting with host cell membrane proteins. In contrast, vaccination of 15 monkeys with predominantly sexual stages or trophozoites of P. knowlesi in Freund complete adjuvant resulted in protection against blood challenges in 7 monkeys, only 2 of which contained precipitating antibody against gp65/ic13. None of the sera from monkeys not protected by infections or vaccinations contained detectable levels of precipitating antibodies against gp65/ic13. Our data indicate that gp65/ic13 acts as a prominent immunogen in vivo during natural p. knowlesi infections of rhesus monkeys. There is a positive correlation suggested between anti-gp65/ic13 antibody and protection in the monkeys analyzed. This correlation does not apply to monkeys protected against P. knowlesi malaria by vaccination, pointing to other effective immune defense mechanisms.

Metabolic labelling of P. knowlesi-specific glycoproteins in membranes of parasitized rhesus monkey erythrocytes.

Rhesus monkey erythrocytes infected with P. knowlesi at high degrees of synchrony were metabolically labeled in vitro during the early schizont stage, using 14C-amino acids and 14C-glucosamine as precursors. Parasite-specific proteins in purified schizonts and host-cell membranes were characterized by their isoelectric points and molecular masses, using isoelectric focusing in polyacrylamide and dodecyl sulfate polyacrylamide gel electrophoresis, respectively. Both 14C-amino acids and 14C-glucosamine caused labelling of 60,000-90,000D proteins/glycoproteins, focusing between pH 4.5 and pH 5.2. The plasmodial parasites synthesized gangliosides and/or other glycolipids, leading to their appearance in the host cell membrane.

Two Plasmodium knowlesi-specific antigens on the surface of schizont-infected Rhesus monkey erythrocytes induce antibody production in immune hosts.

Purified schizonts (6--10 nuclei) and membranes of schizont-infected erythrocytes from the Malaysian and Philippine strain of Plasmodium knowlesi are analyzed immunochemically using immunoglobulin of rhesus monkey hyperimmune sera against schizonts and of sera from naturally immune monkeys. The anti-schizont Ig identifies less than 20 immune components in Triton X-100-solubilized schizonts and membranes of infected cells. Of these antigens, 9 (component 1, 3, 4, 5, 6, 10, 11, 18, and 20) are common to parasites and membranes of infected erythrocytes, and 12 (2A,B, 6, 8, 9, 12, 13p, 14, 16A,B, 19 A,Bp, 21, 22p, and 23) are predominantly found in the parasite; 4 components (13i, 19A,Bi, 22A, B, and 24) are unique to the membrane of infected erythrocytes. Only three parasite-specific components (1, 13, and 19) are exposed on the surface of parasitized erythrocytes as revealed by both lactoperoxidase-catalyzed radioiodination and extensive absorption of anti-schizont Ig using intact infected erythrocytes. Two plasmodium-specific antigens (1 and 13) on the surface of infected erythrocytes are recognized by sera of rhesus monkeys rendered naturally immune against P. knowlesi infections and, therefore, represent antigens in vivo. Analyses of schizonts and membranes of parasitized erythrocytes of the two different strains of P. knowlesi yields only some minor quantitative, but no qualitative differences when analyzed with both types of antisera. Importantly, components 1 and 13 appear identical in both strains.

Fractionation of Plasmodium knowlesi-induced antigens of rhesus monkey erythrocyte membranes.

In order to characterize parasite-induced host cell membrane antigens, the plasma membranes of Plasmodium knowlesi-infected rhesus erythrocytes have been compared with those of normal red cells and purified schizonts by immunochemical and biochemical techniques. Host cell membranes and schizonts were separated by differential centrifugation following nitrogen decompression. Isolated schizonts were further fractionated into several subcellular compartments. Crossed-immune electrophoresis, against monkey anti-schizont serum, of Triton X-100-solubilized material identified 7 P. knowlesi-specific antigens, of which 4 could be detected only in the host cell membranes. These membranes also contained 3 proteins, with relative molecular masses of 55 000, 65 000 and 90 000 and isoelectric points at pH 4.5, 4.5 and 5.2, respectively, which are lacking in normal membranes. Pulse-chase experiments with ((14)C)-glucosamine showed that these parasite-induced host cell membrane components are glycoproteins.