The mechanisms responsible for malarial immunity induced by repetitive injections of X-irradiated sporozoites have not been fully established. We demonstrate here that a single injection of irradiated sporozoites induced, as soon as 24 h after, a non-permissive state to hepatocyte reinfection with sporozoites in vitro. The same effect was observed when malarial blood forms, irradiated promastigotes of Leishmania infantum, adjuvants (muramyl dipeptide, poly acidylic uridylic) or interferon-gamma was injected. Activation of the nitric oxide (NO) pathway in the hepatocyte by these factors was found to be responsible for hepatocyte refractory status. Additionally, this metabolic pathway is involved in protection given by repeated injections of irradiated sporozoites since protection could be reversed by treating mice at the time of sporozoite challenge with a competitive inhibitor (NG-monomethyl-L-arginine) of the NO pathway. These results suggest that, in view of an antisporozoite vaccine, further studies are needed to find out how to activate specifically a long-lasting nonspecific immune response.

Immunization of mammals with irradiated malaria sporozoites protects from a subsequent contact with the parasite. Protective immunity is directed against the pre‐erythrocytic stages of the parasite, sporozoites and liver stages. Specific antibodies neutralize part of the infectious sporozoites injected by the mosquito vector, while liver stages are the target of a cellular immune response which is mediated by T cells. In this study, we evaluated the T‐cell dependent protection induced by the injection of P. berghei irradiated sporozoites and the contribution of perforin and of the receptor/ligand system CD95/CD95L, two T cell‐dependent mechanisms known to mediate elimination of target cells. Wild type, perforin deficient, CD95 mutant, CD95L mutant and perforin deficient/CD95L mutant mice were immunized with P. berghei irradiated sporozoites and submitted to a challenge with infectious sporozoites. All mice immunized with P. berghei irradiated sporozoites were protected against a sporozoite challenge, including perforin deficient/CD95L mutant animals. These results indicate that T cells do not kill malaria‐infected hepatocytes via one of the known pathways, but rather that activated parasite‐specific T cells produce cytokines which activate in cascade other mechanisms responsible for the intracellular elimination of the parasite.

A polypeptide of 69 amino acids (PbCS 242–310) encompassing the C‐terminal region of the circumsporozoite protein of Plasmodium berghei (PbCS) was generated using solid‐phase peptide synthesis. The immunological and protective properties of peptide PbCS 242–310 were studied in BALB/c mice (H‐2d). Two subcutaneous injections, in the presence of IFA at the base of the tail, generated (i) high titers of anti‐peptide antibodies which also recognized the native P. berghei CS protein, (ii) cytolytic T cells specific for the Kd‐restricted peptide PbCS 245–253 and (iii) partial CD8+‐dependent protection against sporozoite‐induced malaria. The same frequencies of peptide PbCS 245–253 specific CD8+ T cells were found by IFN‐γ ELISPOT in the draining lymph nodes of animals immunized with the short optimal CTL peptide 245–253 or with the polypeptide 242–310, indicating that the longer polypeptide can be processed and presented in vivo in the context of MHC class I as efficiently as the short CTL peptide. Interestingly, higher levels of IFN‐γ producing CD8+ T cells and protection were observed when the four cysteine residues present in the C‐terminal peptide were fully oxidized. These findings underline the potential importance of the chemical nature of the C‐terminal fragment on the activation of the immune system and concomitant protection.

We previously reported that low doses of interleukin-1 strongly inhibited in vitro development of the hepatic stages of Plasmodiumfalciparum and P. yoelii. Among several hypotheses, we considered the role of C-reactive protein (CRP), a major acute-phase reactant whose concentration increases markedly in infectious disorders. We demonstrated that human hepatocytes cultured in the presence of interleukin-1 released, as early as 30 min after stimulation, an increased amount of CRP. We then established that CRP bound to the P. falciparum and P. yoelii sporozoite surface membranes, probably via a phosphorylcholine binding site. Experiments in which CRP was added to rat hepatocyte monolayers during or after inoculation confirmed that the target of the CRP-mediated inhibition was at the very early phase of infection. These in vitro functional activities were confirmed in an in vivo model; rats with increased levels of CRP in serum following an injection of turpentine oil were found to be largely protected against an inoculation of P. yoelii sporozoites. The same results were observed in animals inoculated with sporozoites previously incubated in purified CRP or in sera of rats pretreated with turpentine oil. The latter effect was inhibited after incubation of serum from turpentineinjected rats with anti-CRP serum.

Six tetraoxanes had 50% inhibitory concentrations in the range of 10 to 100 ng/ml against Plasmodium falciparum, whereas the corresponding hexaoxonanes had minimal antimalarial activity. The lack of ironmediated reactivity of the hexaoxonanes may explain their low activity compared to the tetraoxanes, the latter of which are able to undergo iron(II)-mediated activation.The antimalarial artemisinin (qinghaosu) contains a pharmacophoric peroxide bond within its 1,2,4-trioxane heterocycle. The complex structure of artemisinin is an incentive to identify more synthetically accessible antimalarial peroxides (10,13,15,18). One of the most structurally simple classes of antimalarial synthetic peroxides are 1,2,4,5-tetraoxanes (2,5,6,12,16), as exemplified by WR 148999 (Fig. 1). Tetraoxanes such as WR 148999 (20) differ considerably in structure from artemisinin, are readily prepared in one step from substituted cyclohexanones, and possess good antimalarial activity, although they are significantly less active than the semisynthetic artemisinins.In this study, we disclose the comparative antimalarial activities of six pairs of 1,2,4,5-tetraoxanes (peroxide dimers) 1a-1f and 1,2,4,5,7,8-hexaoxonanes (peroxide trimers) 2a-2f ( Fig. 2) to further elucidate the structure-activity-relationship of 1,2,4,5-tetraoxanes and, to our knowledge, record for the first time the antimalarial activity of 1,2,4,5,7,8-hexaoxonanes. The latter group of synthetic peroxides has been investigated primarily as synthetic precursors to macrocyclic lactones of perfumery interest (17). As previously described (7, 8), tetraoxanes 1a-1f and hexaoxonanes 2a-2f were prepared either by peroxidation of the corresponding ketone derivatives in H 2 SO 4 /CH 3 CN or by ozonolysis of the corresponding ketone O-methyl oximes. 1,3-Dioxolane 3, the nonperoxidic isostere of tetraoxane 1d, was prepared by ketalization of cyclohexanone with 1,1-dimethanolcyclohexane (TsOH, refluxing toluene) in 35% yield after Kugelrohr distillation (melting point 53 to 56°C) and characterized by 1 H and 13 C NMR and elemental analysis.As previously described (6), in vitro and in vivo antimalarial activities were measured using the chloroquine-resistant K1 and chloroquine-sensitive NF54 strains of Plasmodium falciparum-and P. berghei-infected mice, respectively. Groups of three P. berghei-infected MORO mice were treated 1 day postinfection with 100-mg/kg oral and subcutaneous doses of compounds dissolved or suspended in a solubilizing 3% ethanol and 7% Tween 80 vehicle. Antimalarial activity was measured by determining the percent reduction in parasitemia on day 3 postinfection compared to an untreated control group.The data in Table 1 show a clear demarcation in the antimalarial activities of tetraoxanes 1 and hexaoxonanes 2 against P. falciparum. Tetraoxanes 1 had 50% inhibitory concentrations (IC 50 s) in the range of 10 to 100 ng/ml, whereas all hexaoxonanes had minimal antimalarial activity; only three of these (2a, 2d, and 2f) had IC 50 s of Ͻ10,000 ng/ml. The previously rep...