Seiji Waki

A Critical Role of Fc Receptor-Mediated Antibody-Dependent Phagocytosis in the Host Resistance to Blood-Stage Plasmodium berghei XAT Infection

Plasmodium berghei XAT is an irradiation-induced attenuated variant derived from the lethal strain P. berghei NK65, and its blood-stage parasites are spontaneously cleared in immune competent mice. In the present study, we studied the mechanism of host resistance to blood-stage malaria infection using P. berghei XAT. Infection enhanced Ab-dependent phagocytosis of PRBC by splenic macrophages in wild-type C57BL/6 mice. In contrast, FcR γ-chain knockout (FcRγ−/−) mice, which lack the ability to mediate Ab-dependent phagocytosis and Ab-dependent cell-mediated cytotoxicity through FcγRI, FcγRII, and FcγRIII, could not induce Ab-dependent phagocytic activity. These FcRγ−/− mice showed increased susceptibility to the P. berghei XAT infection, with eventually fatal results, although they produced comparable amounts of IFN-γ by spleen cells and anti-XAT Abs in serum. In addition, passive transfer of anti-XAT IgG obtained from wild-type mice that had recovered from infection into FcRγ−/− mice could not suppress the increase in parasitemia, and almost all of these mice died after marked parasitemia. In contrast, passive transfer of anti-XAT IgG into control wild-type mice inhibited the increase in parasitemia. IFN-γ−/− mice, which were highly susceptible to the P. berghei XAT infection, failed to induce Ab-dependent phagocytic activity and also showed reduced production of serum anti-XAT IgG2a isotype compared with control wild-type mice. These results suggest that FcR-mediated Ab-dependent phagocytosis, which is located downstream of IFN-γ production, is important as an effector mechanism to eliminate PRBC in blood-stage P. berghei XAT infection.

Plasmodium berghei: isolation and maintenance of an irradiation attenuated strain in the nude mouse.

Abstract An attenuated strain of malaria causing limited parasitemia in mice was derived from a highly virulent strain of Plasmodium berghei (NK65) which produced 100% lethality in mice. A pool of mouse blood infected with the original highly virulent P. berghei was exposed to 40 Krad irradiation and parasites were inoculated into nude mice as well as into thymus competent normal littermates. Thymus competent mice showed no parasitemia, while one out of the five nude mice inoculated with the irradiated parasites developed a slow and progressive parasitemia. These parasites induced a self-limiting parasitemia in thymus competent mice, even when a large inoculum was administered. Maintenance of the low virulence strain required passage through nude mice. After 50 passages at two weekly intervals, reversion to virulence did not occur. A single vaccination with the attenuated strain induced immunity in mice against a challenge inoculation with the original virulent strain. Specific IgG persisted at high titer for more than 9 weeks in mice receiving a single inoculation of the attenuated strain.

Interleukin-12-Dependent Mechanisms in the Clearance of Blood-Stage Murine Malaria Parasite Plasmodium berghei XAT, an Attenuated Variant of P. berghei NK65

The mechanism of development of host resistance to blood-stage malarial infection was studied by use of an irradiation-induced attenuated variant, Plasmodium berghei XAT, obtained from a lethal strain, P. berghei NK65. The infection enhanced mRNA expression of interleukin (IL)-12 p40 and also of interferon (IFN)-gamma, IL-4, IL-10, and cytokine-inducible nitric oxide synthase (iNOS) in spleen. Treatment of these mice with anti-IL-12 or anti-IFN-gamma led to the progression of parasitemia and fatal outcome. Anti-IL-12 treatment significantly reduced the secretion and mRNA expression of IFN-gamma and greatly diminished the augmentation of iNOS mRNA expression. In addition, recombinant IL-12 administration delayed the onset of parasitemia because of the enhanced IFN-gamma production. These results suggest that blood-stage P. berghei XAT infection induces IL-12 production, which is important for the development of host resistance via IFN-gamma production.

Plasmodium falciparum: attenuation by irradiation

The effect of irradiation on the in vitro growth of Plasmodium falciparum was investigated. The cultured malarial parasites at selected stages of development were exposed to gamma rays and the sensitivity of each stage was determined. The stages most sensitive to irradiation were the ring forms and the early trophozoites; late trophozoites were relatively insensitive. The greatest resistance was shown when parasites were irradiated at a time of transition from the late trophozoite and schizont stages to young ring forms. The characteristics of radiosensitive variation in the parasite cycle resembled that of mammalian cells. Growth curves of parasites exposed to doses of irradiation upto 150 gray had the same slope as nonirradiated controls but parasites which were exposed to 200 gray exhibited a growth curve which was less steep than that for parasites in other groups. Less than 10 organisms survived from the 10(6) parasites exposed to this high dose of irradiation; the possibility exists of obtaining radiation-attenuated P. falciparum.

Plasmodium yoelii: Induction of attenuated mutants by irradiation

When erythrocytic forms of Plasmodium yoelii nigeriensis, which is invariably fatal in mice, were exposed to X rays, the dose to reduce surviving parasites to one millionth was 100 gray (10 Krad). A suspension of 5 X 10(6) per ml of parasitized erythrocyte was irradiated at 100 gray, and 0.2 ml aliquots were inoculated into 22 mice. Eleven mice showed patent parasitemia, and in these the growth curves were less steep than that found in nonirradiated parasites. The infections of 8 mice of the 11 were self-resolving, and the attenuated feature of the parasites maintained following a limited number of blood passages. The parasites were slowly growing even in nude mice and cause self-resolving infections in intact mice. BALB/c mice immunized with the attenuated parasites were protected against subsequent challenge infections with the original virulent erythrocytic and sporogonic forms. These findings indicate that attenuated mutants of malaria parasites can be readily induced by this method.

A new technique for drug susceptibility tests for Plasmodium falciparum by ethidium bromide fluoroassay

When Plasmodium falciparum parasites were stained with the fluorescent dye ethidium bromide, the fluorescence intensity of the solubilized parasites was correlated with the amount of nucleic acid present. It was possible to monitor development of the parasites from ring form to schizont by the fluoroassay. The assay system was applied to in vitro drug susceptibility tests on malaria parasites.

Immunity to an attenuated variant of Plasmodium berghei : role of some non-specific factors

Plasmodium berghei XAT, an attenuated variant of lethal P. berghei, causes a resolving infection in Balb/c mice from which they recover in about 3 weeks. The parasitaemia displays an early peak at about 5 days, followed by a steep drop in parasite number associated with the appearance of degenerating forms inside mature erythrocytes; the parasites remaining are inside reticulocytes. By contrast, no degenerating parasites were seen in infections caused by the virulent parent, which was mainly confined to mature erythrocytes. However, P. berghei XAT was no more sensitive to reactive O2 metabolites, generated by alloxan, or to tumour necrosis serum, than its virulent parent. Furthermore, its early drop in parasitaemia was unaffected by silica. The drop still occurred in the absence of T cells, although the infection was then ultimately lethal, and it was not mediated by NK cells since it occurred in nude mice treated with anti-asialo GM1 serum to abolish NK cell activity. However, it was absent in splenectomized mice, in which P. berghei XAT infection was lethal. Thus, the attenuation of P. berghei XAT infection is not due to increased susceptibility to some of the agents thought to cause parasite destruction, but to some other mechanism in which the spleen is involved.

Effect of recombinant human colony-stimulating factor on the course of parasitaemia in non-lethal rodent malaria.

The effect of repeated subcutaneous injections of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on the attenuatedPlasmodium berghei XAT infection in CBA mice was examined. When mice were injeected with rhG-CSF daily beginning 2 days before infection, the neutrophil count in the peripheral blood increased 5 times higher than that of control mice and the development of parasitaemia was suppressed significantly during the early phase of the infection. This suppressive effect of rhG-CSF was reduced by treatment of the mice with either anti-interferon (IFN)-γ or antitumour necrosis factor (TNF)-α immunoglobulins. These results suggest that neutrophils may have a role in immunity against the parasites and that IFN-γ and TNF-α are possibly involved.

X-ray sensitivity and DNA synthesis in synchronous culture ofPlasmodium falciparum

The relationship between parasite development and sensitivity to irradiation with X-rays was investigated during a single synchronous cycle ofPlasmodium falciparum in culture. The sensitivity of the parasites to irradiation was closely correlated with the phases of DNA synthesis. Their sensitivity was greatest at the ring stage in development, but decreased at the trophozoite stage when DNA synthesis begins. Lowest sensitivity was found when DNA synthesis was most rapid as the parasites were transforming from late trophozoite to schizont forms. These findings suggest that DNA is the target of the lethal radiation damage in the parasites.

An alternative approach to malaria vaccine with a permanent attenuated mutant from a high virulence Plasmodium berghei strain.

An alternative approach to malaria vaccine with the use of Plasmodium berghei NK65XAT (XAT) is reviewed. XAT is a permanent low virulence strain derived from high virulence P. berghei NK65 (NK65) by irradiation. Although one organism of parent NK65 could kill one mouse, as many as 10(7) XAT parasites caused modest self limiting parasitaemia in immuno-competent mice. In the mice recovered from XAT infection, long lasting immunity to challenge not only by parent NK65, but also by ANKA so far as different species of rodent Plasmodia was seen. The XAT parasites invaded selectively into immature erythrocytes. Because of this feature, the attenuated parasite might induce potent and long-lasting immunity presumably with the background of MHC antigen expression on infected cells. Immunopathologic reactions in mice infected with XAT were modest comparing to those seen in mice with parent NK65 infection. Attenuation was also tested using P. yoelii nigeriensis with which cyclical transmission with A. stephensi was established. Although similar attenuation occurred by X-ray irradiation, produced parasites eventually reverted to virulence after several animal passages. Irradiation was also attempted to induce attenuated P. falciparum mutant and a parasite of a slow multiplication feature was obtained in an experiment. We would propose an alternative approach in the study of malaria vaccine using attenuated live organisms which confers potent and long lasting immunity to the host.