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.

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Both IFN-gamma and IL-12 play critical roles in defence against malaria. In a previous study, using Plasmodium yoelii model, C57BL/6 IFN-gamma receptor deficient mice (IFN-gammaR-/-) failed to develop protective immunity after a single immunization with irradiated sporozoites, but were protected after multiple immunizations. In contrast, in another study, BALB/c IFN-gamma gene knockout mice (IFN-gamma-/-) and BALB/c IL-12-deficient mice (IL-12p40-/- and IL-12p35-/-) were unable to mount protective immune response even after multiple immunizations with the same irradiated parasites. To better define the role of IFN-gamma and IL-12p40 in sterile protection, we selected the C57BL/6 model. Wild-type and IL-12p40-/- mice were immunized with a single or multiple doses of P. berghei irradiated sporozoites. While the wild-type mice were able to rapidly produce IFN-gamma and mount a protective immune response after a single immunization with irradiated sporozoites, IL-12p40-/- mice were neither able to produce IFN-gamma nor were protected. However, both strains of mice were able to produce IFN-gamma and were protected after three doses of irradiated sporozoites. Protection was partially and largely mediated by CD4+ T cells and CD8+ T cells, respectively. Thus, IL-12p40 plays an important role in mediating early protection by irradiated sporozoite immunization but is dispensable for protective immunity induced by several immunizations with irradiated sporozoites. Moreover, treatment of hyperimmune IL-12p40-/- mice with rhIL-18 bp-Fc, an inhibitor of IL-18 activity, did not abrogate protection indicating that IL-18 is not required for the effector phase of the immune response; it remains possible, however, that IL-18 may compensate for the lack of IL-12p40 in the induction phase of the immune response.