Harry Most

Immunization of man against sporozite-induced falciparum malaria

Based on successful work with nonhuman malarias, an attempt was made to immunize man against mosquito-borne stages of Plasmodium falciparum. Strict ethical guidelines were followed. Mosquitoes carrying sporozoites of P. falciparum were X-irradiated and then fed on volunteers who had not previously been exposed to malaria. The sporozoites were inactivated at a minimum dosage of 15,000 rads, and did not produce adverse reactions in the volunteers. Three volunteers were each exposed during 84 days to 379 infected irradiated mosquitoes, and on day 98 were fed on by nonirradiated mosquitoes heavily infected with homologous strain P. falciparum. One of these men did not develop malaria, and continued to be immunized during the ensuing 217 days with 819 infected mosquitoes. On day 327, when antisporozoite antibody was first demonstrated in his serum by the circumsporozoite precipitation test, he was fed on by nonirradiated mosquitoes carrying homologous strain P. falciparum, and did not develop malaria. Possible causes of his failure to become infected were investigated, and it was concluded that he had become immunized to falciparum sporozoites of that strain. His continued susceptibility to strain-specific falciparum malaria induced by direct blood transfer was demonstrated.

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

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.

Plasmodium berghei: accelerated clearance of sporozoites from blood as part of immune-mechanism in mice.

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,

Parasitic infections in a closed community. Results of a 10-year survey in Willowbrook State School.

Abstract The maximum circulation time, as well as the rate of clearance, of viable, infective, Plasmodium berghei sporozoites was determined after intravenous injection into immunized and normal mice. In animals actively immunized by the previous injection of X-irradiated sporozoites, the circulation time of infective sporozoites was considerably reduced as a consequence of an increased clearance rate of the parasites. The transfer of large volumes of immune serum into normal mice also resulted in a considerably increased disappearance rate of infective sporozoites from their peripheral blood. Exo-erythrocytic forms (EEF) of the parasite were either not detectable or greatly reduced in number in the livers of both groups of actively and passively immunized animals, as compared to controls. However, in spite of the apparent absence of EEF in some of these animals and the reduced number of EEF in all the others, protection was never complete in the passively immunized animals, all of them developing parasitemia after a prolonged prepatent period. This differed markedly from the results obtained in the actively immunized animals, the majority of which was totally protected. Together with other experimental evidence, this seems to indicate that in addition to the important role of immune serum, another, as yet undetermined mechanism might be necessary to bring about complete protection to malaria.

The biology of a newly isolated strain of Plasmodium berghei in a rodent host and in experimental mosquito vectors.

Abstract A study designed to elucidate the causes of mass morbidity and widespread intestinal disorders in Willowbrook State School has revealed the role of parasitic agents in the aetiology of the diseases. Strongyloides stercoralis, Hymenolepis nana and Giardia lamblia were the predominant pathogenic agents. Their spread was limited, however, to the childrens buildings and to the severely mentally retarded. 522 patients with S. stercoralis and 322 with H. nana were detected during the period 1962–1970. Annual stool surveys served to trace symptomless carriers, to determine levels of endemicity and to assess the effects of treatment and control. Factors which influence the spread of parasitic infections are discussed in the light of findings of the survey and preventive measures for custodial institutions are outlined.

PLASMODIUM BERGHEI: CYCLICAL TRANSMISSIONS BY EXPERIMENTALLY INFECTED ANOPHELES QUADRIMACULATUS.

Abstract 1. 1) A strain of Plasmodium berghei , Kasapa S.P. 7307, recently isolated from the Belgian Congo, has been cyclically transmitted through Anopheles quadrimaculatus, Anopheles aztecus , and young white rats three successive times. Sporozoites from crushed oocysts of heavily infected mosquitoes were used as inoculum. 2. 2) The newly isolated “passage” strains were designated as N.Y.U. 1 , N.Y.U. 2 , and N.Y.U. 3 Strains of P. berghei (New York University Strains). 3. 3) The pre-patent period in four sporozoite infected young rats were: 71 hours, 96 hours, 8 days, and 10 days. 4. 4) Parasites found in the peripheral blood at the early “flooding” period of 71 hours after sporozoite inoculation are described in detail. 5. 5) A description is given of the different developmental stages of the sporozonic cycle as observed in the newly isolated strains, under experimental conditions.

Drug-resistance transfer among rodent plasmodia. 1. Acquisition of resistance to pyrimethamine by a drug-sensitive strain of Plasmodium berghei in the course of its concomitant development with a pyrimethamine-resistant P. vinckei strain.

A number of strains of Plasmodium berghei were isolated from sporozoites of Anopheles dureni. Laboratory-bred Anopheles quadrimaculatus fed on carriers of the newly isolated strains showed overwhelming midgut infections and moderate or mild salivary gland infections. Successive cyclic transmissions by the bite of experimentally infected A. quadrimaculatus in laboratorybred tree rats (Thamnomys surdaster) were carried out.

Studies on filariasis. III. Partial growth of the mammalian stages of Dirofilaria immitis in vitro

A study on transfer of drug resistance between species of malaria parasites was carried out in mixed infections of P. berghei and P. vinckei . The biological characters which served as contrasting markers for separation of the two species were the inability of the ‘old’ blood-passaged strain of P. vinckei to develop in suitable mosquito vectors and its failure to produce parasitaemia in the adult golden hamster. Facility of sporogonic development and cyclical transmission and a pronounced virulence for the adult hamster marked the N.K. 65 strain of P. berghei . Development of a maximal and stable resistance to 200 mg/kg pyrimethamine in the P. vinckei strain was achieved by a gradual increase in drug level in successive blood transfers. Mixed, simultaneous infections of pyrimethamine-resistant P. vinckei and sensitive P. berghei were experimentally induced in white mice. Subinoculations of the mixed infection at the height of parasitaemia was carried out in adult hamsters. Suppression of the P. vinckei infection in the hamster by innate resistance and the reappearance of P. berghei in the blood was noted. Tests with the maximal concentration of pyrimethamine (200 mg/kg) showed the acquisition of drug resistance in some of the P. berghei infected hamsters. Persistence of the acquired resistance was demonstrated following fifteen successive blood transfers in the absence of drug pressure and after five cyclical transmissions. Parallel control experiments with pyrimethamine-sensitive P. vinckei and P. berghei in mixed infections failed to reveal any enhancement in drug resistance. No spontaneous drug-resistant mutant was found in the sensitive P. berghei after exposure to a single course of treatment with the drug, and persistence of parasites in some treated animals was considered as individual variations in host reactions to the drug. This resistance never persisted after blood transfer or cyclical transmission. No loss in drug resistance had been found following transfer of the resistant R strain from mouse to hamster. A marked difference in the course of the plasmodial infection and in the length of survival of mice inoculated with the drug-resistant P. berghei was noted following treatment with pyrimethamine in various concentrations. It is suggested that these differences represent a selective action of the drug on a heterogenic P. berghei population, consisting of drug-resistant and drug-sensitive parasites in the blood of the host. Injection of sonified material from pyrimethamine-resistant P. vinckei and P. berghei into mice inoculated with a drug-sensitive P. berghei failed to induce enhancement of resistance to the drug. The phenomenon of drug-resistance transfer observed under experimental conditions in rodent malaria is viewed in the light of similar phenomena in micro-organisms. We gratefully acknowledge the assistance rendered by Mr Jerold Sklarsh. This paper is Contribution no. N 408 from the Army Research Programme on Malaria. The work was carried out under the sponsorship of the Commission on Malaria, Armed Forces Epidemiological Board, and supported in part by the United States Office of the Surgeon General, Department of the Army.

Tree Rat, Thamnomys surdaster surdaster, in Laboratory Research

Abstract Invasive third-stage larvae of Dirofilaria immitis obtained from experimentally infected Anopheles quadrimaculatus were maintained in a culture medium in a perfusion apparatus for 30 days at 37 °C. Molting of the larvae was observed, and an increase in size from an initial length of 1059 μ and a 27.68 μ width to an average 1510 μ length and 34.70 μ width at 12 days, and a 1825 μ length and 38.70 μ width at 30 days, were recorded. Blunting and rounding of the anterior ends in the growing larvae were conspicuous, and the caudal papillae were prominent. These changes are comparable to those occurring in larvae of similar age found in experimentally infected dogs. The results of the experiments are discussed and compared in the light of previous attempts to grow D. immitis in vitro.