Irradiated Sporozoites Research Articles

Plasmodium berghei berghei: Irradiated sporozoites of the ANKA strain as Immunizing Antigens in mice

Mice were protected against challenge with infective Sporozoites following immunization with X-ray irradiated Sporozoites. The immunity lasted at least 8 weeks. Mice immune against sporozoite challenge remained fully susceptible to challenge with erythrocytic stages. Immunization of mice with extracts of mosquito thorax failed to protect them, indicating that mosquito antigens were not directly responsible for the immunity observed in the basic experiments.

Antibody-Induced Ultrastructural Changes of Malarial Sporozoites

Immunization with irradiated sporozoites produces a considerable degree protection against rodent, simian, and human malaria. This protection is in part antibody mediated. Antibodies neutralize sporozoites (SNA), i.e. abolish their infectivity, and cause, in vitro, the formation of a thread-like precipitate on the parasites (CSP reaction). The present study was undertaken to characterize the ultrastructural aspects of antibody-sporozoite interaction.

Intramuscular immunization of mice with irradiated Plasmodium berghei sporozoites. Enhancement of protection with albumin.

Abstract These studies have confirmed a previous report which demonstrated that intramuscularly injected irradiated sporozoites of Plasmodium berghei are far less effective than those injected intravenously in protectively immunizing mice against sporozoite-induced malaria. Because albumin has been shown to induce motility in Plasmodium sporozoites, attempts were made to determine whether the use of albumin in immunizing inocula given intramuscularly might enhance the protection thus obtained. The results showed that albumin did indeed significantly enhance the development of protective immunity, though this protection was still considerably less than that obtained by intravenous immunization. This effect of albumin may be due to its ability to induce motility in the injected sporozoites, thereby permitting them to reach and enter blood vessels more readily.

Plasmodium cynomolgi: X-irradiation and development of exo-erythrocytic schizonts in Macaca mulatta

Sporozoites of Plasmodium cynomolgi cynomolgi and the langur strain of P. cynomolgi were exposed to various dosages of X-irradiation while still in the salivary glands of living Anopheles atroparvus. The impact of this irradiation on the development of exo-erythrocytic stages in the liver of Macaca mulatta receiving the sporozoites was studied. Ten-krad doses apparently inactivated all sporozoites while lesser doses produced graded responses as reflected in morphologic changes in developing exo-erythrocytic schizonts. Five krad doses allowed the development of exo-erythrocytic schizonts generally normal in appearance and with only slightly reduced mean dimensions. In all of the sublethal radiation dosages used, reduced size and the appearance of a limiting membrane were the most prominent changes noted. Small but apparently normal schizonts as well as grossly modified forms were sometimes seen in the same biopsy. Some monkeys receiving irradiated sporozoites showed reduced parasitemias over those seen in controls.

Protective Immunity Produced by the Injection of X-Irradiated Sporozoites of Plasmodium berghei. II. Effects of Radiation on Sporozoites

The effects of X-radiation on the infectivity and development of sporozoites of Plasmodium berghei were determined. When susceptible mice were injected with X-irradiated sporozoites, results showed that the higher the dose of radiation, the smaller the percentage of mice that developed patent blood infections. Sporozoites irradiated with more than 10,000 rads failed to produce any blood infections. Small, but apparently mature, exoerythrocytic forms were found in the livers of rats that had been injected with X-irradiated sporozoites. Such forms were more numerous after a low dose of radiation, i.e., 2,000 rads, and were found to persist in the liver for at least 16 days after sporozoite injection. There may be a relationship between the presence of these forms and the protective immunity found in mice inoculated with irradiated sporozoites. Studies on the effects of ionizing radiations on parasitic protozoa have repeatedly shown that while an extremely high radiation dose is necessary to cause immediate death of the parasite, much lower doses can interfere with its infectivity (review by Kimball, 1955). Thus, doses of more than 100,000 roentgens (r) of X-rays were necessary to produce changes in motility in Trypanosoma gambiense in vitro, whereas a dose of 12,000 r abolished the infectivity of the trypanosome to mice (Halberstaedter, 1938). Although the immunogenicity of irradiated parasites, ranging from viruses (review by Luria, 1955) to helminths (I.A.E.A., 1964), has been investigated only a few such studies have been done with protozoa, e.g., Eimeria (Hein, in Urquhart, 1964), and Trypanosoma lewisi (Sanders and Wallace, 1966). These studies have shown that parasites irradiated with a dose which abolishes their reproductive potential and ability to produce patent infections may still retain their capacity to produce an immune response in a susceptible host. Much of the work on the effects of X-radiation on the malaria parasite has related primarily to the doses required to abolish the Received for publication 25 June 1968. * This work, contribution no. 430 from the Army Research Program on Malaria, was sponsored by the Commission on Malaria, Armed Forces Epidemiological Board, and was supported by the United States Army Medical Research and Development Command. t Department of Preventive Medicine. $ Department of Radiology. infectivity of the erythrocytic stages (Bennison and Coatney, 1945; Rigdon and Rudison, 1945; Ward, Bell, and Schneider, 1960; and Targett and Fulton, 1965), as well as the sporozoite stage (Bennison and Coatney, 1945). Ceithaml and Evans (1946) showed that erythrocytes parasitized by P. gallinaceum were capable of apparently normal oxygen uptake and glucose consumption after an X-ray dose sufficient to destroy reproductive capacity of the parasite (10,000 r). Only when the dose was increased to 30,000 r were these metabolic activities of the parasitized erythrocytes affected. The immunizing capability of X-irradiated erythrocytic stages of malaria parasites has been shown with P. gallinaceum (Ceithaml and Evans, 1946), and with P. berghei (Corradetti, Verolini, and Bucci, 1966; and Wellde and Sadun, 1967). P. gallinaceum sporozoites inactivated either with ultraviolet light or by drying were shown by Russell, Mulligan, and Mohan (1942) to be effective immunizing agents when injected into fowls. That such sporozoites might be more active agents of immunization than are inactivated erythrocytic parasites was suggested by the work of Richards (1966). Our own preliminary results (Nussenzweig et al., 1967) indicated the considerable protective effect induced by the injection of X-irradiated sporozoites of P. berghei into mice. The normal course of sporozoite-induced parasitemia in susceptible rodents, as well as quantitative aspects of the development of sporozoites into exoerythrocytic (E.E.) forms has been previously described (Vanderberg,

Further Studies on the Plasmodium berghei-Anopheles stephensi: Rodent System of Mammalian Malaria

Techniques for laboratory maintenance of the Plasmodium berghei-Anopheles stephensi system for rodent malaria are described. The system permits the regular production of sporozoite-induced blood infections in susceptible rodents, as well as the regular attainment of preerythrocytic schizonts in the livers of young white rats. The most sensitive measures of the infectivity of a pool of sporozoites are the time to patency of infected animals, and the percentage of sporozoites which become preerythrocytic schizonts. Three kinds of variability in this infectivity were noted: 1) a variability between different pools of sporozoites, 2) an interspecific variability with tree rats, young white rats, and mice being susceptible in that order, 3) an intraspecific variability, with some animals of a given species being less susceptible than others. Repetitive sporozoite induction of rodent malaria in the laboratory by means of the Plasmodium berghei-Anopheles stephensi system was reported by us in a previous paper (Nussenzweig et al., 1966). Hamsters, tree rats (Thamnomys surdaster), and young white rats were shown to be suitable laboratory hosts, whereas white mice (CF#1 strain) were found to be less readily infected. This difficulty in infecting mice was partially circumvented with the use of an inbred strain of mouse (A/J from Jackson Memorial Laboratories) that was more susceptible to sporozoite-induced infection (Most et al., 1966). Since then, improvements in our technique have enabled us to produce and harvest a greater number of more infective sporozoites. Our experiences with this new system have demonstrated its value in both basic and applied investigations. The sporozoites have been used in chemotherapeutic studies (Most, Herman, and Schoenfeld, 1967), as well as in studies on specific immunity (Nussenzweig et al., 1967) and nonspecific resistance (Nussenzweig, 1967) to sporozoiteinduced infection. The relative ease with which this model may be set up leads us to report our techniques and the characteristics of the sporozoite-induced infections. This paper forms a background for other studies: modification of sporozoites by X-radiation Received for publication 14 May 1968. * This work, contribution no. 384 from the Army Research Program on Malaria, was sponsored by the Commission on Malaria, Armed Forces Epidemiological Board, and was supported by the United States Army Medical Research and Development Command. (Vanderberg et al., 1968), and studies on the use of these irradiated sporozoites as agents for immunization (Nussenzweig et al., 1967). MATERIALS AND METHODS Rearing of mosquitoes Anopheles stephensi were reared in an insectary maintained at 28 C and about 85% relative humidity. Rearing pans containing third and fourth instar larvae were moved to an insectary maintained at 21 C and about 85% relative humidity. The slowing down of development resulting from this drop in temperature allowed us to harvest pupae every other day, instead of daily. Pupae were harvested by means of the cold water technique of Weathersby (1963). Emerged adults were fed 10% glucose solution prior to their infective feeding on hamsters. All mosquitoes were kept on a daily cycle of 16 hr of light and 8 hr of darkness. Infection of hamsters to be used as sources of gametocytes Hamsters used for mosquito feeding were 3 to 5 weeks old. They were infected with an intraperitoneal inoculation of heart blood from a hamster infected with the NK strain of P. berghei berghei. Blood from a donor animal was found to be most useful when the animal was in the rapidly rising phase of an infection in which 20 to 35% of its erythrocytes were parasitized. To guard against senescence of the P. berghei strain only a few hamster to hamster passages of the parasite were used before interposition of a sexual cycle by mosquito passage. More recently we have been fee ng mosquitoes only on hamsters which had been infected with blood from a sporozoite-induced infection. Infection of mosquitoes P ior to their infective blood meal,. mosquitoes were kept at 21 C. The hamsters were anesthetized with an intraperitoneal injection of thialbarbitone sodium (Kemithal?), and placed on top of a cage containing about 250 mosquitoes in the 21 C insectary. The mosquitoes were allowed to feed through