Plasmodium parasites have a complex life cycle alternating between a mosquito and a vertebrate host. Following the bite of an Anopheles female mosquito, Plasmodium sporozoites are transmitted from the skin to the liver; their first place of replication within the host. Successfully invaded sporozoites undergo a massive replication and growth involving asynchronous DNA replication and division that results in the generation of tens of thousands or even hundreds of thousands of merozoites depending on the Plasmodium species. The generation of a high number of daughter parasites requires biogenesis and segregation of organelles to finally reach a relatively synchronous cytokinesis event. At the end of liver stage (LS) development, merozoites are packed into merosomes and released into the bloodstream. They are then liberated and infect red blood cells to again produce merozoites by schizogony for the erythrocytic stage of the life cycle. Although parasite LS and asexual blood stage (ABS) differ in many respects, important similarities exist between the two. This review focuses on the cell division of Plasmodium parasite LS in comparison with other life cycle stages especially the parasite blood stage.

RTS,S and R21 are the only vaccines recommended by the WHO to protect children from Plasmodium falciparum (Pf) clinical malaria. Both vaccines target the Pf sporozoite surface protein circumsporozoite protein (CSP). Recent studies showed that human antibodies neutralize Pf sporozoites most efficiently when simultaneously binding to the PfCSP NANP repeat and the NPDP junction domain. However, neither RTS,S nor R21 targets this junction domain. To test the potential of the NPDP junction domain and other sites of PfCSP as innovative vaccine targets, we developed multiple vaccine candidates based on cucumber mosaic virus-like particles (CuMV_TT-VLPs). These candidates vary in several aspects: the number of targeted NANP repeats, the presence or absence of the junction domain, the cleavage site, and up to three NVDP repeats within the target sequence. Immunogenicity and efficacy studies were conducted in BALB/c mice, utilizing chimeric Plasmodium berghei (Pb) sporozoites, in which the endogenous CSP has been replaced by PfCSP (Pb/PfCSP). We observed a positive association between the number of targeted NANP repeats and the induction of specific IgM/IgG antibodies. Elevated humoral responses led to enhanced protection against parasitemia after Pb/PfCSP sporozoite challenge. Especially high-avidity/affinity antibody formation and vaccine protection were NANP repeat-dependent. Intriguingly, vaccine efficacy was not enhanced by targeting sites on PfCSP other than the NANP repeats. Our data emphasize the dominant role of the NANP repeat region for induction of protective antibodies. Furthermore, we present here novel malaria vaccine candidates with an excellent immunogenic profile that confer sterile protection in mice, even in absence of adjuvants.

Summary: Faithful chromosome segregation relies on precise kinetochore–microtubule interactions and checkpoint surveillance, yet the molecular basis of these processes varies widely across eukaryotes and is only beginning to be defined in apicomplexan parasites. In the malaria parasite Plasmodium berghei , chromosome segregation is especially critical during transmission from host to mosquito: rapid mitoses generate male gametes, and subsequent meiosis in the zygote seeds the next generation of infection. Here, we identify Aurora-related kinase 1 (ARK1) as a central regulator of chromosome segregation in both mitotic and meiotic contexts. ARK1 localises to spindle poles, spindles, and kinetochores, and its depletion results in short and multipolar spindles, kinetochore misalignment, and failed chromosome partitioning. ARK1 forms a minimal Chromosomal Passenger Complex (CPC) with INCENP1 during male gametogenesis, but a more elaborate CPC with INCENP2, kinetochores, centromeric histones, and spindle assembly checkpoint proteins during meiosis. This stage-specific modularity allows Plasmodium to prioritise male gamete formation whilst safeguarding faithful chromosome inheritance during zygote development, ensuring parasite transmission to the mosquito. Our findings demonstrate that conserved CPC principles are rewired in Plasmodium , highlighting both the plasticity of eukaryotic checkpoint control and a potential vulnerability for blocking malaria transmission.

Malaria caused by Plasmodium parasites remains a large health burden. One approach to combat this disease involves vaccinating individuals with whole sporozoites that have been genetically modified to arrest their development at a specific stage in the liver by targeted gene deletion, resulting in a genetically attenuated parasite (GAP). Through a comprehensive phenotyping screen, we identified the hscb gene, encoding a putative iron-sulfur protein assembly chaperone, as crucial for liver stage development, making it a suitable candidate gene for GAP generation. Parasites lacking Plasmodium berghei HscB (PbHscB) exhibited normal sporozoite production in mosquitoes, but their liver stage development was severely impaired, characterized by slow growth and delayed expression of merozoite surface protein 1 (MSP1). In vivo experiments demonstrated that PbHscB-deficient parasites exhibited a delay in prepatency of 2–4 days, emphasizing the significance of PbHscB for exo-erythrocytic development. Although knockout of PbHscB alone allowed breakthrough infections, it is a potent candidate for a dual gene deletion strategy. PlasMei2, an RNA-binding protein, was previously found to be crucial for the completion of liver stage development. We generated a PbHscB-PbMei2-double attenuated parasite line, serving as a late liver stage-arresting replication-competent (LARC) GAP, providing a solid block of liver-to-blood stage transition.

Malaria remains a major global health burden, and the emergence of resistance to blood stage antimalarials underscores the need for new interventions targeting earlier stages of the parasite’s life cycle. The pre-erythrocytic liver stage represents a critical bottleneck and an attractive target for chemotherapeutic and prophylactic interventions. In this study, we functionally characterized the putative E3 ubiquitin ligase Trophozoite Exported Protein 1 (TEX1; PBANKA_0102200) in Plasmodium berghei using gene knockout, tagging, and imaging approaches across the mosquito and liver stages. TEX1 knockout parasites (PbTEX1-KO) showed impaired development during mosquito-stage transitions, with significant reductions in ookinete formation, oocyst numbers, and sporozoites reaching the salivary glands. In hepatic stages, TEX1-KO parasites displayed reduced growth, abnormal nuclear division, and impaired liver stage maturation, ultimately leading to a dramatic decline in detached cell formation and blood stage infectivity. Endogenous C-terminal tagging of TEX1 with GFP and 3×HA revealed a discrete subnuclear localization pattern, indicating a critical role in DNA synthesis and/or mitotic regulation. Our findings reveal that TEX1 is required for nuclear replication and division and successful development in both the mosquito and liver stages of Plasmodium. Given its pivotal role and nuclear localization during hepatic schizogony, TEX1 represents a promising target for the development of liver stage antimalarial interventions.