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Plasmodium parasites, the eukaryotic pathogens that cause malaria, feature three distinct invasive forms tailored to the host environment they must navigate and invade for life cycle progression. One conserved feature of these invasive forms is the micronemes, apically oriented secretory organelles involved in egress, motility, adhesion, and invasion. Here we investigate the role of GPI-anchored micronemal antigen (GAMA), which shows a micronemal localization in all zoite forms of the rodent-infecting species Plasmodium berghei. ∆GAMA parasites are severely defective for invasion of the mosquito midgut. Once formed, oocysts develop normally, however, sporozoites are unable to egress and exhibit defective motility. Epitope-tagging of GAMA revealed tight temporal expression late during sporogony and showed that GAMA is shed during sporozoite gliding motility in a similar manner to circumsporozoite protein. Complementation of P. berghei knockout parasites with full-length P. falciparum GAMA partially restored infectivity to mosquitoes, indicating conservation of function across Plasmodium species. A suite of parasites with GAMA expressed under the promoters of CTRP, CAP380, and TRAP, further confirmed the involvement of GAMA in midgut infection, motility, and vertebrate infection. These data show GAMA's involvement in sporozoite motility, egress, and invasion, implicating GAMA as a regulator of microneme function., (© 2023 John Wiley & Sons Ltd.)

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 P36 mCherry 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.

Gliding motility and cell traversal by the Plasmodium ookinete and sporozoite invasive stages allow penetration of cellular barriers to establish infection of the mosquito vector and mammalian host, respectively. Motility and traversal are not observed in red cell infectious merozoites, and we have previously classified genes that are expressed in sporozoites but not merozoites (S genes) in order to identify proteins involved in these processes. The S4 gene has been described as criticaly involved in Cell Traversal for Ookinetes and Sporozoites (CelTOS), yet knockout parasites (s4/celtos¯) do not generate robust salivary gland sporozoite numbers, precluding a thorough analysis of S4/CelTOS function during host infection. We show here that a failure of oocysts to develop or survive in the midgut contributes to the poor mosquito infection by Plasmodium yoelii (Py) s4/celtos¯ rodent malaria parasites. We rescued this phenotype by expressing S4/CelTOS under the ookinete-specific circumsporozoite protein and thrombospondin-related anonymous protein-related protein (CTRP) promoter (S4/CelTOS CTRP ), generating robust numbers of salivary gland sporozoites lacking S4/CelTOS that were suitable for phenotypic analysis. Py S4/CelTOS CTRP sporozoites showed reduced infectivity in BALB/c mice when compared to wild-type sporozoites, although they appeared more infectious than sporozoites deficient in the related traversal protein PLP1/SPECT2 (Py plp1/spect2¯). Using in vitro assays, we substantiate the role of S4/CelTOS in sporozoite cell traversal, but also uncover a previously unappreciated role for this protein for sporozoite gliding motility., (© 2017 John Wiley & Sons Ltd.)

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., (Copyright © 2017, American Association for the Advancement of Science.)

Plasmodium 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(-) transition from liver stage to blood stage infection in mice. Our results show for the first time the importance of a Plasmodium RBP in the coordinated progression of late liver stage schizogony and maturation of new invasive forms., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Malaria, caused by Plasmodium parasites, imposes a significant health burden and live-attenuated parasites are being pursued as vaccines. Here, we report on the creation of a genetically attenuated parasite by the deletion of Plasmodium LINUP, encoding a liver stage nuclear protein. In the rodent parasite Plasmodium yoelii, LINUP expression was restricted to liver stage nuclei after the onset of liver stage schizogony. Compared to wildtype P. yoelii, P. yoelii LINUP gene deletion parasites (linup—) exhibited no phenotype in blood stages and mosquito stages but suffered developmental arrest late in liver stage schizogony with a pronounced defect in exo-erythrocytic merozoite formation. This defect caused severe attenuation of the liver stage-to-blood stage transition and immunization of mice with linup— parasites conferred robust protection against infectious sporozoite challenge. LINUP gene deletion in the human parasite Plasmodium falciparum also caused a severe defect in late liver stage differentiation. Importantly, P. falciparum linup— liver stages completely failed to transition from the liver stage to a viable blood stage infection in a humanized mouse model. These results suggest that P. falciparum LINUP is an ideal target for late liver stage attenuation that can be incorporated into a late liver stage-arresting replication competent whole parasite vaccine. A conserved Plasmodium protein, specific to the liver stage and localized in the nucleus of liver stage schizonts, plays a critical role in liver stage development and differentiation. [ABSTRACT FROM AUTHOR]

Liver-stage genetically attenuated malaria parasites (GAPs) are powerful immunogens that provide protection against sporozoite challenge. We previously generated two late liver-stage-arresting GAPs by deleting the stearoyl-CoA desaturase (Scd) or sporozoite conserved orthologous transcript 1 (Scot1) genes in Plasmodium berghei. Immunization with Scd or Scot1 GAP conferred complete protection against a sporozoite challenge. In a safety study, we observed rare breakthrough blood-stage infections in mice inoculated with high doses of sporozoites, indicating that both GAPs were incompletely attenuated. In this study, we generated a Scd/Scot1 GAP by dual gene deletion. This resulted in complete attenuation of the parasites in the liver and did not transition to blood-stage infection despite a high-dose sporozoite challenge. The Scd/Scot1 KO and WT GFP parasites were indistinguishable during blood, mosquito and early liver stage development. Moreover, Scd/Scot1 KO liver-stage schizonts exhibited an abnormal apicoplast biogenesis and nuclear division phenotype, failed to form hepatic merozoites, and exhibited late liver-stage arrest. Compared with early-arresting Speld KO immunization, late-stage liver-arresting Scd/Scot1 KO induces greater and broader CD8+ T-cell responses and elicits stage-transcending immunity that provides superior protection in C57BL/6 mice. These data prove that multiple gene deletions lead to complete attenuation of the parasite and support the development of late liver stage-arresting P. falciparum GAP. [ABSTRACT FROM AUTHOR]

The malaria vaccination landscape has seen significant advancements with the recent endorsement of RTS,S/AS01 and R21/Matrix-M vaccines, which target the pre-erythrocytic stages of Plasmodium falciparum (Pf) infection. However, several challenges remain to be addressed, including the incomplete protection afforded by these vaccines, their dependence on a single Pf antigen, and the fact that they were not designed to protect against P. vivax (Pv) malaria. Injectable formulations of whole-sporozoite (WSpz) malaria vaccines offer a promising alternative to existing subunit vaccines, with recent developments including genetically engineered parasites and optimized administration regimens. Clinical evaluations demonstrate varying efficacy, influenced by factors, such as immune status, prior exposure to malaria, and age. Despite significant progress, a few hurdles persist in vaccine production, deployment, and efficacy in malaria-endemic regions, particularly in children. Concurrently, transgenic parasites expressing Pv antigens emerge as potential solutions for PvWSpz vaccine development. Ongoing clinical studies and advancements in vaccine technology, including the recently described PfSPZ-LARC2 candidate, signify a hopeful future for WSpz malaria vaccines, which hold great promise in the global fight against malaria. This Review discusses challenges in developing malaria vaccines and promising recent developments in whole-sporozoite vaccines using genetically engineered parasites with a perspective on future directions of these vaccines for the prevention of malaria. [ABSTRACT FROM AUTHOR]

Vaccination with infectious Plasmodium falciparum (Pf) sporozoites (SPZ) administered with antimalarial drugs (PfSPZ-CVac), confers superior sterilizing protection against infection when compared to vaccination with replication-deficient, radiation-attenuated PfSPZ. However, the requirement for drug administration constitutes a major limitation for PfSPZ-CVac. To obviate this limitation, we generated late liver stage-arresting replication competent (LARC) parasites by deletion of the Mei2 and LINUP genes (mei2–/linup– or LARC2). We show that Plasmodium yoelii (Py) LARC2 sporozoites did not cause breakthrough blood stage infections and engendered durable sterilizing immunity against various infectious sporozoite challenges in diverse strains of mice. We next genetically engineered a PfLARC2 parasite strain that was devoid of extraneous DNA and produced cryopreserved PfSPZ-LARC2. PfSPZ-LARC2 liver stages replicated robustly in liver-humanized mice but displayed severe defects in late liver stage differentiation and did not form liver stage merozoites. This resulted in complete abrogation of parasite transition to viable blood stage infection. Therefore, PfSPZ-LARC2 is the next-generation vaccine strain expected to unite the safety profile of radiation-attenuated PfSPZ with the superior protective efficacy of PfSPZ-CVac. Synopsis: Malaria persists as a formidable global health threat, necessitating development of a vaccine that protects against malaria parasite infection. A late liver stage-arresting replication competent (LARC) parasite vaccine was engineered by deletion of the Mei2 and LINUP genes (mei2-/linup- or LARC2) in Plasmodium yoelii (PyLARC2) and Plasmodium falciparum (PfSPZ-LARC2). PyLARC2 did not show any breakthrough blood stage infections in mice. PyLARC2 elicited durable sterilizing immunity against diverse sporozoite challenges in a range of mouse strains. PfSPZ-LARC2 liver stages showed robust replication in liver-humanized mice but did not transition to viable blood stage infection. Cryopreserved PfSPZ-LARC2 vaccine was successfully produced. PfSPZ-LARC2 vaccine is the next-generation vaccine strain designed to unite the safety attributes of radiation-attenuated PfSPZ with the superior protective efficacy of PfSPZ-CVac. Malaria persists as a formidable global health threat, necessitating development of a vaccine that protects against malaria parasite infection. A late liver stage-arresting replication competent (LARC) parasite vaccine was engineered by deletion of the Mei2 and LINUP genes (mei2-/linup- or LARC2) in Plasmodium yoelii (PyLARC2) and Plasmodium falciparum (PfSPZ-LARC2). [ABSTRACT FROM AUTHOR]

Immunization with Plasmodium sporozoites, either attenuated or administered under the cover of an antimalarial drug, can induce strong protection against malaria in pre-clinical murine models, as well as in human trials. Previous studies have suggested that whole-sporozoite (WSpz) formulations based on parasites with longer liver stage development induce higher protection, but a comparative analysis of four different WSpz formulations has not been reported. We employed a rodent model of malaria to analyze the effect of immunization dosage on the protective efficacy of WSpz formulations consisting of (i) early liver arresting genetically attenuated parasites (EA-GAP) or (ii) radiation-attenuated sporozoites (RAS), (iii) late arresting GAP (LA-GAP), and (iv) sporozoites administered under chemoprophylaxis, that are eliminated upon release into the bloodstream (CPS). Our results show that, unlike all other WSpz formulations, EA-GAP fails to confer complete protection against an infectious challenge at any immunization dosage employed, suggesting that a minimum threshold of liver development is required to elicit fully effective immune responses. Moreover, while immunization with RAS, LA-GAP and CPS WSpz yields comparable, dosage-dependent protection, protection by EA-GAP WSpz peaks at an intermediate dosage and markedly decreases thereafter. In-depth immunological analyses suggest that effector CD8+ T cells elicited by EA-GAP WSpz immunization have limited developmental plasticity, with a potential negative impact on the functional versatility of memory cells and, thus, on protective immunity. Our findings point towards dismissing EA-GAP from prioritization for WSpz malaria vaccination and enhance our understanding of the complexity of the protection elicited by these WSpz vaccine candidates, guiding their future optimization. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Invasion of host cells by apicomplexan parasites such as Toxoplasma and Plasmodium spp requires the sequential secretion of the parasite apical organelles, the micronemes and the rhoptries. The claudin-like apicomplexan microneme protein (CLAMP) is a conserved protein that plays an essential role during invasion by Toxoplasma gondii tachyzoites and in Plasmodium falciparum asexual blood stages. CLAMP is also expressed in Plasmodium sporozoites, the mosquito-transmitted forms of the malaria parasite, but its role in this stage is still unknown. CLAMP is essential for Plasmodium blood stage growth and is refractory to conventional gene deletion. To circumvent this obstacle and study the function of CLAMP in sporozoites, we used a conditional genome editing strategy based on the dimerisable Cre recombinase in the rodent malaria model parasite P. berghei. We successfully deleted clamp gene in P. berghei transmission stages and analyzed the functional consequences on sporozoite infectivity. In mosquitoes, sporozoite development and egress from oocysts was not affected in conditional mutants. However, invasion of the mosquito salivary glands was dramatically reduced upon deletion of clamp gene. In addition, CLAMP-deficient sporozoites were impaired in cell traversal and productive invasion of mammalian hepatocytes. This severe phenotype was associated with major defects in gliding motility and with reduced shedding of the sporozoite adhesin TRAP. Expansion microscopy revealed partial colocalization of CLAMP and TRAP in a subset of micronemes, and a distinct accumulation of CLAMP at the apical tip of sporozoites. Collectively, these results demonstrate that CLAMP is essential across invasive stages of the malaria parasite, and support a role of the protein upstream of host cell invasion, possibly by regulating the secretion or function of adhesins in Plasmodium sporozoites. Author summary: Plasmodium parasites, the causative agents of malaria, are transmitted during the bite of an infected mosquito. Infectious parasite stages known as sporozoites are released from the insect salivary glands and injected into the host skin. Sporozoites rapidly migrate to the host liver, invade hepatocytes and differentiate into the next invasive forms, the merozoites, which invade and replicate inside red blood cells. Sporozoite motility and host cell invasion rely on the secretion of apical organelles called micronemes and rhoptries. Here we characterize the function of a microneme protein expressed both in merozoites and sporozoites, the claudin-like protein CLAMP. We used a conditional genome editing strategy in a rodent malaria model to generate CLAMP-deficient sporozoites. In the absence of CLAMP, sporozoites failed to invade mosquito salivary glands and mammalian hepatocytes, and showed defects in gliding motility and microneme secretion. Our data establish that CLAMP plays an essential role across Plasmodium invasive stages, and might represent a potential target for transmission-blocking antimalarial strategies. [ABSTRACT FROM AUTHOR]

Malaria, a devastating febrile illness caused by protozoan parasites, sickened 247,000,000 people in 2021 and killed 619,000, mostly children and pregnant women in sub-Saharan Africa. A highly effective vaccine is urgently needed, especially for Plasmodium falciparum (Pf), the deadliest human malaria parasite. Sporozoites (SPZ), the parasite stage transmitted by Anopheles mosquitoes to humans, are the only vaccine immunogen achieving >90% efficacy against Pf infection. This review describes >30 clinical trials of PfSPZ vaccines in the U.S.A., Europe, Africa, and Asia, based on first-hand knowledge of the trials and PubMed searches of 'sporozoites,' 'malaria,' and 'vaccines.' First generation (radiation-attenuated) PfSPZ vaccines are safe, well tolerated, 80–100% efficacious against homologous controlled human malaria infection (CHMI) and provide 18–19 months protection without boosting in Africa. Second generation chemo-attenuated PfSPZ are more potent, 100% efficacious against stringent heterologous (variant strain) CHMI, but require a co-administered drug, raising safety concerns. Third generation, late liver stage-arresting, replication competent (LARC), genetically-attenuated PfSPZ are expected to be both safe and highly efficacious. Overall, PfSPZ vaccines meet safety, tolerability, and efficacy requirements for protecting pregnant women and travelers exposed to Pf in Africa, with licensure for these populations possible within 5 years. Protecting children and mass vaccination programs to block transmission and eliminate malaria are long-term objectives. [ABSTRACT FROM AUTHOR]

Whole-sporozoite (WSp) malaria vaccines induce protective immune responses in animal malaria models and in humans. A recent clinical trial with a WSp vaccine comprising genetically attenuated parasites (GAP) which arrest growth early in the liver (PfSPZ-GA1), showed that GAPs can be safely administered to humans and immunogenicity is comparable to radiation-attenuated PfSPZ Vaccine. GAPs that arrest late in the liver stage (LA-GAP) have potential for increased potency as shown in rodent malaria models. Here we describe the generation of four putative P. falciparum LA-GAPs, generated by CRISPR/Cas9-mediated gene deletion. One out of four gene-deletion mutants produced sporozoites in sufficient numbers for further preclinical evaluation. This mutant, PfΔmei2, lacking the mei2-like RNA gene, showed late liver growth arrest in human liver-chimeric mice with human erythrocytes, absence of unwanted genetic alterations and sensitivity to antimalarial drugs. These features of PfΔmei2 make it a promising vaccine candidate, supporting further clinical evaluation. PfΔmei2 (GA2) has passed regulatory approval for safety and efficacy testing in humans based on the findings reported in this study. [ABSTRACT FROM AUTHOR]

Apicomplexa are obligatory intracellular parasites that sense and actively invade host cells. Invasion is a conserved process that relies on the timely and spatially controlled exocytosis of unique specialized secretory organelles termed micronemes and rhoptries. Microneme exocytosis starts first and likely controls the intricate mechanism of rhoptry secretion. To assemble the invasion machinery, micronemal proteins—associated with the surface of the parasite—interact and form complexes with rhoptry proteins, which in turn are targeted into the host cell. This review covers the molecular advances regarding microneme and rhoptry exocytosis and focuses on how the proteins discharged from these two compartments work in synergy to drive a successful invasion event. Particular emphasis is given to the structure and molecular components of the rhoptry secretion apparatus, and to the current conceptual framework of rhoptry exocytosis that may constitute an unconventional eukaryotic secretory machinery closely related to the one described in ciliates. [ABSTRACT FROM AUTHOR]

During transmission of malaria‐causing parasites from mosquitoes to mammals, Plasmodium sporozoites migrate rapidly in the skin to search for a blood vessel. The high migratory speed and narrow passages taken by the parasites suggest considerable strain on the sporozoites to maintain their shape. Here, we show that the membrane‐associated protein, concavin, is important for the maintenance of the Plasmodium sporozoite shape inside salivary glands of mosquitoes and during migration in the skin. Concavin‐GFP localizes at the cytoplasmic periphery and concavin(−) sporozoites progressively round up upon entry of salivary glands. Rounded concavin(−) sporozoites fail to pass through the narrow salivary ducts and are rarely ejected by mosquitoes, while normally shaped concavin(−) sporozoites are transmitted. Strikingly, motile concavin(−) sporozoites disintegrate while migrating through the skin leading to parasite arrest or death and decreased transmission efficiency. Collectively, we suggest that concavin contributes to cell shape maintenance by riveting the plasma membrane to the subtending inner membrane complex. Interfering with cell shape maintenance pathways might hence provide a new strategy to prevent a malaria infection. Synopsis: Concavin, a membrane‐associated Plasmodium protein, is essential for sporozoite shape maintenance. Parasites lacking concavin deform in the mosquito salivary gland and disintegrate while migrating in the skin, which limits transmission. Concavin, a membrane‐associated protein, is essential for Plasmodium shape maintenance.In the absence of concavin Plasmodium sporozoites deform and round up.Thickened and deformed Plasmodium sporozoites fail to be transmitted by mosquitoes.Parasites migrating in the skin disintegrate in the absence of concavin. [ABSTRACT FROM AUTHOR]