Application of gamma irradiation knowledge in tissue sterilisation: inactivation of malaria parasite.

Malaria is one of the exclusion criteria used in selecting tissue donors and the absence of this information can lead to rejection of tissues for transplant. The studies on the malaria parasite have been confined to low dose attenuation of parasites in blood for transfusion purposes. There is no published information relating to the inactivation of malaria parasites with irradiation for the sterilisation of tissues. A dose-surviving parasite population following radiation was replotted using D0 value from a published paper whereby D10 value of 41 Gy was obtained. Calculation of sterilisation dose for achieving SAL 10-6 of malaria parasites demonstrated the effectiveness of the sterilisation dose of 25 kGy being used in tissue banking.

Varying Immunizations With Plasmodium Radiation-Attenuated Sporozoites Alter Tissue-Specific CD8+ T Cell Dynamics.

Whole sporozoite vaccines represent one of the most promising strategies to induce protection against malaria. However, the development of efficient vaccination protocols still remains a major challenge. To understand how the generation of immunity is affected by variations in vaccination dosage and frequency, we systematically analyzed intrasplenic and intrahepatic CD8+ T cell responses following varied immunizations of mice with radiation-attenuated sporozoites. By combining experimental data and mathematical modeling, our analysis indicates a reversing role of spleen and liver in the generation of protective liver-resident CD8+ T cells during priming and booster injections: While the spleen acts as a critical source compartment during priming, the increase in vaccine-induced hepatic T cell levels is likely due to local reactivation in the liver in response to subsequent booster injections. Higher dosing accelerates the efficient generation of liver-resident CD8+ T cells by especially affecting their local reactivation. In addition, we determine the differentiation and migration pathway from splenic precursors toward hepatic memory cells thereby presenting a mechanistic framework for the impact of various vaccination protocols on these dynamics. Thus, our work provides important insights into organ-specific CD8+ T cell dynamics and their role and interplay in the formation of protective immunity against malaria.

Physical characterization of histidine-rich protein from Plasmodium lophurae.

The size and shape of Plasmodium lophurae histidine-rich protein have been determined by analytical centrifugation and electron microscopy. From the partial specific volume of 0.72 cc/g, the molecular weight was determined to be 43,000. The sedimentation velocity studies indicated a coefficient of 1.32 S in 0.9 M acetic acid (pH 3.5), monodispersity and significant asymmetry. Darkfield electron microscopy revealed the major species to be compact oblate spheroids 12 nm in width and extended filamentous particles of average length 35 nm by 1.5 nm. Analysis of the sequence of the protein by the method of Garnier et al. (J. Mol. Biol. (1978) 120, 97-120) predicted that 82% of its residues would be found in three long alpha-helices. The protein's CD spectrum has a strong resemblance to that of poly(L-histidine) at pH 4-5, where the homopolymer is thought to be in a right-handed alpha-helical form. A single helix containing 300 residues would be 45 nm long, the largest length found by electron microscopy. From the electron-microscopic data, sedimentation coefficients of 1.6 and 1.95 S, respectively, were calculated for flexible-coil and extended-rod models, in closer agreement with the measured value of 1.3 S than the value calculated for a spherical model. Thus, the major species in acetic acid is probably an incompletely extended rod which, as the pH is increased to neutrality, condenses to form spherical molecular aggregates seen in the malaria parasite.

Hepatic stages of malaria: specific and non-specific factors inhibiting the development.

Protection against pre-erythrocytic stages of malaria is possible, as demonstrated by the resistance obtained by immunizing with irradiated sporozoites. However, the involved mechanisms are more numerous and intricate than previously believed. Recently, the hepatic stage, rather than the sporozoite stage, has been seen as the target of immune attack.

Vaccination using radiation- or genetically attenuated live sporozoites.

The attenuation of Plasmodium parasites by either radiation or targeted gene deletion can result in viable sporozoites that invade the liver and subsequently arrest. The death of the growth-arrested liver stage parasite and the ensuing recognition by the immune system of parasite antigens promotes protective immunity in immunized mice and humans. The methods described below will enable researchers to determine the efficacy of radiation-attenuated and genetically attenuated rodent malaria sporozoite immunizations against infectious sporozoite challenge, and study protective immunity in immunized mice. In addition, by determining the time of arrest of genetically attenuated parasite liver stages and the mechanisms of clearance, researchers will be able to correlate biological features of the growth-arrested parasites with their ability to promote protective immunity.

Targeting Plasmodium liver stages: better late than never.

The worldwide burden of malaria can be substantially reduced using existing public health interventions. However, elimination of Plasmodium will require fundamentally different approaches. Novel experimental attenuation strategies for active immunization using knockout strains have recently stimulated renewed interest in whole-parasite vaccine approaches. Preventive drug administration during transmission of wild-type sporozoites is a complementary strategy for eliciting protective immune responses. These whole-cell immunization strategies are based on one fundamental principle: inducing protection by blocking parasite conversion from the clinically silent liver phase to the pathogenic intra-erythrocytic replication cycle. Here, we review the basis, evidence and targets for whole-cell-based vaccination strategies against the liver stage bottleneck in Plasmodium infections and discuss preclinical and clinical research opportunities.

Conserved protective mechanisms in radiation and genetically attenuated uis3(-) and uis4(-) Plasmodium sporozoites.

Immunization with radiation attenuated Plasmodium sporozoites (RAS) elicits sterile protective immunity against sporozoite challenge in murine models and in humans. Similarly to RAS, the genetically attenuated sporozoites (GAPs) named uis3(-), uis4(-) and P36p(-) have arrested growth during the liver stage development, and generate a powerful protective immune response in mice. We compared the protective mechanisms in P. yoelii RAS, uis3(-) and uis4(-) in BALB/c mice. In RAS and GAPs, sterile immunity is only achieved after one or more booster injections. There were no differences in the immune responses to the circumsporozoite protein (CSP) generated by RAS and GAPs. To evaluate the role of non-CSP T-cell antigens we immunized antibody deficient, CSP-transgenic BALB/c mice, that are T cell tolerant to CSP, with P. yoelii RAS or with uis3(-) or uis4(-) GAPs, and challenged them with wild type sporozoites. In every instance the parasite liver stage burden was approximately 3 logs higher in antibody deficient CSP transgenic mice as compared to antibody deficient mice alone. We conclude that CSP is a powerful protective antigen in both RAS and GAPs viz., uis3(-) and uis4(-) and that the protective mechanisms are similar independently of the method of sporozoite attenuation.

Hitting malaria before it hurts: attenuated Plasmodium liver stages.

Continuous natural exposure to Plasmodium transmission by infectious Anopheles mosquitoes leads to a gradual acquisition of immunological competence against malaria. The partial immunity, observed in adolescents and adults living in endemic areas, reduces morbidity and mortality without preventing parasite infection. In experimental animal models, long-lasting sterilizing immunity can be achieved with genetically attenuated Plasmodium liver stages. Can these findings be translated to accomplish sterile protection against natural malaria transmission in the high-risk group, young infants in sub-Saharan Africa?

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.