Immunization of humans with whole sporozoites confers complete, sterilizing immunity against malaria infection. However, achieving consistent safety while maintaining immunogenicity of whole parasite vaccines remains a formidable challenge. We generated a genetically attenuated Plasmodium falciparum (Pf) malaria parasite by deleting three genes expressed in the pre-erythrocytic stage (Pf p52/p36/sap1). We then tested the safety and immunogenicity of the genetically engineered (Pf GAP3KO) sporozoites in human volunteers. Pf GAP3KO sporozoites were delivered to 10 volunteers using infected mosquito bites with a single exposure consisting of 150 to 200 bites per subject. All subjects remained blood stage-negative and developed inhibitory antibodies to sporozoites. GAP3KO rodent malaria parasites engendered complete, protracted immunity against infectious sporozoite challenge in mice. The results warrant further clinical testing of Pf GAP3KO and its potential development into a vaccine strain.
Within the liver, Plasmodium sporozoites traverse cells searching for a “suitable” hepatocyte, invading these cells through a process that results in the formation of a parasitophorous vacuole (PV), within which the parasite undergoes intracellular replication as a liver stage. It was previously established that two members of the Plasmodium s48/45 protein family, P36 and P52, are essential for productive invasion of host hepatocytes by sporozoites as their simultaneous deletion results in growth-arrested parasites that lack a PV. Recent studies point toward a pathway of entry possibly involving the interaction of P36 with hepatocyte receptors EphA2, CD81, and SR-B1. However, the relationship between P36 and P52 during sporozoite invasion remains unknown. Here we show that parasites with a single P52 or P36 gene deletion each lack a PV after hepatocyte invasion, thereby pheno-copying the lack of a PV observed for the P52/P36 dual gene deletion parasite line. This indicates that both proteins are equally important in the establishment of a PV and act in the same pathway. We created a Plasmodium yoelii P36mCherry tagged parasite line that allowed us to visualize the subcellular localization of P36 and found that it partially co-localizes with P52 in the sporozoite secretory microneme organelles. Furthermore, through co-immunoprecipitation studies in vivo, we determined that P36 and P52 form a protein complex in sporozoites, indicating a concerted function for both proteins within the PV formation pathway. However, upon sporozoite stimulation, only P36 was released as a secreted protein while P52 was not. Our results support a model in which the putatively glycosylphosphatidylinositol (GPI)-anchored P52 may serve as a scaffold to facilitate the interaction of secreted P36 with the host cell during sporozoite invasion of hepatocytes.
Genetically attenuated malaria parasites (GAP) that arrest during liver stage development are powerful immunogens and afford complete and durable protection against sporozoite infection. Late liver stage-arresting GAP provide superior protection against sporozoite challenge in mice compared to early live stage-arresting attenuated parasites. However, very few late liver stage-arresting GAP have been generated to date. Therefore, identification of additional loci that are critical for late liver stage development and can be used to generate novel late liver stage-arresting GAPs is of importance. We further explored genetic attenuation in by combining two gene deletions, and (), that each cause late liver stage arrest with various degrees of infrequent breakthrough to blood stage infection. The dual gene deletion resulted in a synthetic lethal phenotype that caused complete attenuation in a highly susceptible mouse strain. arrested late in liver stage development and did not persist in livers beyond 3 days after infection. Immunization with this GAP elicited robust protective antibody responses in outbred and inbred mice against sporozoites, liver stages, and blood stages as well as eliciting protective liver-resident T cells. The immunization afforded protection against both sporozoite challenge and blood stage challenge. These findings provide evidence that completely attenuated late liver stage-arresting GAP are achievable via the synthetic lethal approach and might enable a path forward for the creation of a completely attenuated late liver stage-arresting GAP.
bPlasmodium parasites employ posttranscriptional regulatory mechanisms as their life cycle transitions between host cell invasion and replication within both the mosquito vector and mammalian host. RNA binding proteins (RBPs) provide one mechanism for modulation of RNA function. To explore the role of Plasmodium RBPs during parasite replication, we searched for RBPs that might play a role during liver stage development, the parasite stage that exhibits the most extensive growth and replication. We identified a parasite ortholog of the Mei2 (Meiosis inhibited 2) RBP that is conserved among Plasmodium species (PlasMei2) and exclusively transcribed in liver stage parasites. Epitope-tagged Plasmodium yoelii PlasMei2 was expressed only during liver stage schizogony and showed an apparent granular cytoplasmic location. Knockout of PlasMei2 (plasmei2 ؊ ) in P. yoelii only affected late liver stage development. The P. yoelii plasmei2 ؊ liver stage size increased progressively until late in development, similar to wild-type parasite development. However, P. yoelii plasmei2 ؊ liver stage schizonts exhibited an abnormal DNA segregation phenotype and failed to form exoerythrocytic merozoites. Consequently the cellular integrity of P. yoelii plasmei2 ؊ liver stages became increasingly compromised late in development and the majority of P. yoelii plasmei2 ؊ underwent cell death by the time wild-type liver stages mature and release merozoites. This resulted in a complete block of P. yoelii plasmei2 Malaria is caused by unicellular eukaryotic parasites of the genus Plasmodium. The life cycles of the Plasmodium parasites that cause human disease (including the major human parasite Plasmodium falciparum) and other mammalian parasites such as rodent malaria parasites (including P. berghei and P. yoelii) are similar. The parasites progress through an elaborate life cycle, part within an anopheline mosquito vector and part within the mammalian host. Within the host, the sporozoite stage first infects hepatocytes, within which the parasite then develops as a liver stage (also known as an exoerythrocytic form), an acyclic process that occurs once during a malaria infection. Intrahepatocytic parasite development progresses in a cellular process known as schizogony, an endomitotic process characterized by an enormous increase in cell mass with concomitant DNA replication and organelle replication but without cytokinesis. It is only in the final hours of liver stage schizogony that organelles and DNA completely segregate and are apportioned into tens of thousands of rapidly differentiating exoerythrocytic invasive merozoites. These are released and initiate the infection of erythrocytes. Erythrocytic schizogony is cyclic and generates approximately 20 to 30 new merozoites in each cycle. Parasite schizogony and merozoite formation in both hepatocytes and erythrocytes likely employ shared gene regulatory networks and cell regulatory mechanisms, but it is possible that because exoerythrocytic schizogony exceeds erythrocytic schizogony by m...
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