Some of the earliest malaria vaccine studies in experimental models were based on the use of irradiation-attenuated whole malaria parasites 
. However, due to the perceived difficulties related to their safety and large scale production, the whole parasite approach was not considered to be a practical vaccine strategy. In recent years, the limited success of sub-unit based vaccines in clinical studies has reignited the interest in whole parasite based malaria vaccines 
. Notwithstanding their potential value as candidate vaccines, studies with radiation-attenuated parasites offer an excellent opportunity to explore host pathogenesis and to examine the immune mechanisms induced by protective vaccines against malaria.
In the current study, a single, non-adjuvanted immunization with 107
irradiated blood-stage parasites protected CD1 mice from parasitemia and severe disease, while up to two immunizations with 103
irradiated parasites were not protective. This anti-parasite immunity is consistent with previous studies of irradiated blood-stage malaria parasites that showed protection from parasitemia and severe malaria anemia in different model systems 
. The protection in the previous studies was achieved without adjuvants, and it also required the intravenous delivery of large numbers of blood-stage parasites.
To our knowledge, the current study is the first to also demonstrate the effectiveness of irradiated blood-stage parasites for protection against ECM, the most pathogenic consequence of malaria infection. A single immunization with 107
irradiated parasites protected against ECM, while two immunizations of 103
irradiated parasites did not protect. However, in contrast to immunized CD1 mice, the immunized C57BL/6 mice that had acquired anti-disease immunity against ECM did not acquire anti-parasite immunity. Similar observations have been noted in other experimental studies 
, suggesting that anti-disease immunity and anti-parasite immunity act through distinct mechanisms. This notion is further supported by the observations of distinct immune mechanisms that confer immunity against severe malaria and parasitemia in adults living in endemic areas 
The mechanism of protection in this study is not known, and thus it is not clear what relative roles humoral and cellular responses may have played for this protection. The protective immunization against ECM in this study was associated with a parasite-specific antibody response that increased following a boost during a challenge infection. However, whether these antibodies played any role against protection from ECM (C57Bl/6 mice) or parasitemia (CD1 mice) is not clear. In previous studies, antibody responses have been shown to be important to some whole parasite vaccines 
, and it has been proposed that part of their protective effects may come from an increased clearance of opsonized parasites 
. In the current study, the immunized ECM-protected mice did not have significantly lower blood parasitemia levels compared to non-protected mice, suggesting that bulk parasite clearance is unlikely to fully explain protection. However, the spleen sizes of protected mice increased 2-fold during a virulent infection, and since this can be indicative of increased parasite clearance and/or immune cell recruitment, further experiments will be necessary to address this question in detail. The ECM-protective immunization was also associated with a reduced parasite-specific IFN-γ response in the spleen during a virulent infection. In mice that have a Th1 bias such as C57BL/6, splenic IFN-γ production during a blood-stage Pb-A
infection is thought to promote inflammatory responses that contribute to ECM pathology 
. In contrast, the non-protective immunization in this study did not stimulate anti-parasite antibodies and it only modestly reduced parasite-specific IFN-γ expression during an infection, indicating that it was less effective at stimulating both antibody and cellular responses.
While elevated levels of splenic IFN-γ during the fulminant phase of Pb-A infection in mice are associated with susceptibility to ECM, a very early IFN-γ response in the spleen during infection has been associated with resistance to ECM 
. In a previous study, it was proposed that CD 8+ T cells were a source of the early splenic IFN-γ responses during Pb-A
infection, and the authors provided evidence that NK cells, NKT cells, and γδ T cells did not significantly contribute to this IFN-γ signal. In the current study, the unchallenged, IrrPb immunized mice produced detectable levels of splenic IFN-γ when stimulated in vitro with Pb-A
antigen. While the source of this splenic IFN-γ produced upon immunization with IrrPb was not studied, we cannot discount the possibility that antigenically primed CD 4+ T cells or some other splenic cells were responsible for the IFN-γ production through an immunological feedback mechanism, since ECM-protected IrrPb immunized mice had lower splenic IFN-γ levels after Pb-A
Recently, it has been proposed that immunization with subpatent doses of viable blood-stage parasites that have been genetically attenuated or curtailed by drug cure can provide protection from a virulent challenge 
. These are intriguing findings, and may in part be linked to the degree to which the attenuated parasites are able to replicate and persist to stimulate the immune system without causing severe disease symptoms. In the current study, even the largest inoculums of irradiated parasites fell below the limit of blood-smear detection within a few days, suggesting that these parasites were capable of little or no replication. This level of attenuation was necessary because it appeared that replication competent blood-stage Pb-A
also remained virulent to produce fulminant infections and severe disease. In another study, genetically attenuated, blood-stage Pb-A
parasites that retained an ability to replicate carried a very high risk of severe disease themselves, although mice that survived the initial vaccination developed robust immunity against both parasites and disease during subsequent challenge infections 
. In the current study, low numbers of the non-replicating parasites failed to induce anti-parasite or anti-disease immunity.
Although immunization with a high dose of irradiated Pb-A
protected mice against ECM in this study, the protected mice still accumulated CD8 T cells in their brain vasculature similar to ECM-susceptible mice. Previous studies have shown that brain sequestered CD8 T cells are necessary but not sufficient for ECM in the Pb-A
. Similar to this study, perforin-deficient mice that were resistant to ECM still accumulated activated effector CD8 T cells (CD44+, CD62L−, CD69+) in their brains 
. Further experimentation will be needed to explore the mechanism of ECM resistance induced by irradiated blood stage parasites.
In summary, inline with our results in the P. berghei model, it is possible that a single inoculation with a high dose of replication-deficient P. falciparum parasites might protect children in endemic areas from parasite burden and/or cerebral malaria. In either scenario, such a vaccine might lower the prospect of death from severe malaria during their vulnerable early years and thus provide them with the opportunity to develop clinical immunity after continued parasite exposure.