Malaria is a mosquito-borne disease that is transmitted by inoculation of the Plasmodium parasite sporozoite stage. Sporozoites invade hepatocytes, transform into liver stages, and subsequent liver-stage development ultimately results in release of pathogenic merozoites. Liver stages of the parasite are a prime target for malaria vaccines because they can be completely eliminated by sterilizing immune responses, thereby preventing malarial infection. Using expression profiling, we previously identified genes that are only expressed in the pre-erythrocytic stages of the parasite. Here, we show by reverse genetics that one identified gene, UIS3 (upregulated in infective sporozoites gene 3), is essential for early liver-stage development. uis3-deficient sporozoites infect hepatocytes but are unable to establish blood-stage infections in vivo, and thus do not lead to disease. Immunization with uis3-deficient sporozoites confers complete protection against infectious sporozoite challenge in a rodent malaria model. This protection is sustained and stage specific. Our findings demonstrate that a safe and effective, genetically attenuated whole-organism malaria vaccine is possible.

Malaria infection starts when sporozoites are transmitted to the mammalian host during a mosquito bite. Sporozoites enter the blood circulation, reach the liver, and infect hepatocytes. The formation of a parasitophorous vacuole (PV) establishes their intracellular niche. Recently, two members of the 6-Cys domain protein family, P52 and P36, were each shown to play an important albeit nonessential role in Plasmodium berghei sporozoite infectivity for the rodent host. Here, we generated p52/p36-deficient Plasmodium yoelii parasites by the simultaneous deletion of both genes using a single genetic manipulation. p52/p36-deficient parasites exhibited normal progression through the life cycle during blood-stage infection, transmission to mosquitoes, mosquito-stage development, and sporozoite infection of the salivary glands. p52/p36-deficient sporozoites also showed normal motility and cell traversal activity. However, immunofluorescence analysis and electron microscopic observations revealed that p52/p36-deficient parasites did not form a PV within hepatocytes in vitro and in vivo. The p52/p36-deficient parasites localized as free entities in the host cell cytoplasm or the host cell nucleoplasm and did not develop as liver stages. Consequently, they did not cause blood-stage infections even at high sporozoite inoculation doses. Mice immunized with p52/p36-deficient sporozoites were completely protected against infectious sporozoite challenge. Our results demonstrate for the first time the generation of two-locus gene deletion-attenuated parasites that infect the liver but do not progress to blood-stage infection. The study will critically guide the design of Plasmodium falciparum live attenuated malaria vaccines.

Irradiation-attenuated sporozoite vaccinations confer sterile protection against malaria infection in animal models and humans. Persistent, nonreplicating parasite forms in the liver are presumably necessary for the maintenance of sterile immunity. A novel vaccine approach uses genetically attenuated parasites (GAPs) that undergo arrested development during liver infection. The fate of GAPs after immunization, their persistence in vaccinated animals, and the immune mechanisms that mediate protection are unknown. To examine the developmental defects of genetically attenuated liver stages in vivo, we created deletions of the UIS3 and UIS4 loci in the Plasmodium yoelii rodent malaria model (Pyuis3[-] and Pyuis4[-]). The low 50% infectious dose of P. yoelii in BALB/c mice provides the most sensitive infectivity model. We show that P. yoelii GAPs reach the liver, invade hepatocytes, and develop a parasitophorous vacuole but do not significantly persist 40 h after infection. A single dose of Pyuis4(-) sporozoites conferred complete protection, but full protection by Pyuis3(-) sporozoites required at least 2 immunizations. CD8(+) T cells were essential for protection, but CD4(+) T cells were not. Our results show that genetically distinct GAPs confer different degrees of protective efficacy and that live vaccine persistence in the liver is not necessary to sustain long-lasting protection. These findings have important implications for the development of a P. falciparum GAP malaria vaccine.