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The protozoan parasite Plasmodium, the causative agent of malaria, undergoes an obligatory stage of intra‐hepatic development before initiating a blood‐stage infection. Productive invasion of hepatocytes involves the formation of a parasitophorous vacuole (PV) generated by the invagination of the host cell plasma membrane. Surrounded by the PV membrane (PVM), the parasite undergoes extensive replication. During intracellular development in the hepatocyte, the parasites provoke the Plasmodium‐associated autophagy‐related (PAAR) response. This is characterized by a long‐lasting association of the autophagy marker protein, and ATG8 family member, LC3B with the PVM. LC3B localization at the PVM does not follow the canonical autophagy pathway since upstream events specific to canonical autophagy are dispensable. Here, we describe that LC3B localization at the PVM of Plasmodium parasites requires the V‐ATPase and its interaction with ATG16L1. The WD40 domain of ATG16L1 is crucial for its recruitment to the PVM. Thus, we provide new mechanistic insight into the previously described PAAR response targeting Plasmodium liver stage parasites. [ABSTRACT FROM AUTHOR]

Abstract 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.

Plasmodium is an obligate intracellular parasite that requires intense lipid synthesis for membrane biogenesis and survival. One of the principal membrane components is oleic acid, which is needed to maintain the membrane's biophysical properties and fluidity. The malaria parasite can modify fatty acids, and stearoyl‐CoA Δ9‐desaturase (Scd) is an enzyme that catalyzes the synthesis of oleic acid by desaturation of stearic acid. Scd is dispensable in P. falciparum blood stages; however, its role in mosquito and liver stages remains unknown. We show that P. berghei Scd localizes to the ER in the blood and liver stages. Disruption of Scd in the rodent malaria parasite P. berghei did not affect parasite blood stage propagation, mosquito stage development, or early liver‐stage development. However, when Scd KO sporozoites were inoculated intravenously or by mosquito bite into mice, they failed to initiate blood‐stage infection. Immunofluorescence analysis revealed that organelle biogenesis was impaired and merozoite formation was abolished, which initiates blood‐stage infections. Genetic complementation of the KO parasites restored merozoite formation to a level similar to that of WT parasites. Mice immunized with Scd KO sporozoites confer long‐lasting sterile protection against infectious sporozoite challenge. Thus, the Scd KO parasite is an appealing candidate for inducing protective pre‐erythrocytic immunity and hence its utility as a GAP. [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]

An essential step in the life cycle of malaria parasites is their egress from hepatocytes, which enables the transition from the asymptomatic liver stage to the pathogenic blood stage of infection. To exit the liver, Plasmodium parasites first disrupt the parasitophorous vacuole membrane that surrounds them during their intracellular replication. Subsequently, parasite‐filled structures called merosomes emerge from the infected cell. Shrouded by host plasma membrane, like in a Trojan horse, parasites enter the vasculature undetected by the host immune system and travel to the lung where merosomes rupture, parasites are released, and the blood infection stage begins. This complex, multi‐step process must be carefully orchestrated by the parasite and requires extensive manipulation of the infected host cell. This review aims to outline the known signaling pathways that trigger exit, highlight Plasmodium proteins that contribute to the release of liver‐stage merozoites, and summarize the accompanying changes to the hepatic host cell. [ABSTRACT FROM AUTHOR]

ABSTRACTPlasmodium parasite resistance to existing antimalarial drugs poses a devastating threat to the lives of many who depend on their efficacy. New antimalarial drugs and novel drug targets are in critical need, along with novel assays to accelerate their identification. Given the essentiality of protein synthesis throughout the complex parasite lifecycle, translation inhibitors are a promising drug class, capable of targeting the disease-causing blood stage of infection, as well as the asymptomatic liver stage, a crucial target for prophylaxis. To identify compounds capable of inhibiting liver stage parasite translation, we developed an assay to visualize and quantify translation in the Plasmodium berghei-HepG2 infection model. After labeling infected monolayers with o-propargyl puromycin (OPP), a functionalized analog of puromycin permitting subsequent bioorthogonal addition of a fluorophore to each OPP-terminated nascent polypeptide, we use automated confocal feedback microscopy followed by batch image segmentation and feature extraction to visualize and quantify the nascent proteome in individual P. berghei liver stage parasites and host cells simultaneously. After validation, we demonstrate specific, concentration-dependent liver stage translation inhibition by both parasite-selective and pan-eukaryotic active compounds and further show that acute pre-treatment and competition modes of the OPP assay can distinguish between known direct translation inhibitors and other compounds which we identify as indirect translation inhibitors. Using this framework, we identify a Malaria Box compound, MMV019266, as a direct translation inhibitor in P. berghei liver stages and confirm this potential mode of action in Plasmodium falciparum asexual blood stages.IMPORTANCEPlasmodium parasites cause malaria in humans. New multistage active antimalarial drugs are needed, and a promising class of drugs targets the core cellular process of translation, which has many potential molecular targets. During the obligate liver stage, Plasmodium parasites grow in metabolically active hepatocytes, making it challenging to study core cellular processes common to both host cells and parasites, as the signal from the host typically overwhelms that of the parasite. Here, we present and validate a flexible assay to quantify Plasmodium liver stage translation using a technique to fluorescently label the newly synthesized proteins of both host and parasite followed by computational separation of their respective nascent proteomes in confocal image sets. We use the assay to determine whether a test set of known compounds are direct or indirect liver stage translation inhibitors and show that the assay can also predict the mode of action for novel antimalarial compounds.

Plasmodium vivax causes the second highest number of malaria morbidity and mortality cases in humans. Several biological traits of this parasite species, including the formation of dormant stages (hypnozoites) that persist inside the liver for prolonged periods of time, present an obstacle for intervention measures and create a barrier for the elimination of malaria. Research into the biology of hypnozoites requires efficient systems for parasite transmission, liver stage cultivation and genetic modification. However, P. vivax research is hampered by the lack of an in vitro blood stage culture system, rendering it reliant on in vivo-derived, mainly patient, material for transmission and liver stage culture. This has also resulted in limited capability for genetic modification, creating a bottleneck in investigations into the mechanisms underlying the persistence of the parasite inside the liver. This bottleneck can be overcome through optimal use of the closely related and experimentally more amenable nonhuman primate (NHP) parasite, Plasmodium cynomolgi, as a model system. In this review, we discuss the genetic modification tools and liver stage cultivation platforms available for studying P. vivax persistent stages and highlight how their combined use may advance our understanding of hypnozoite biology. [ABSTRACT FROM AUTHOR]

Viral infection often induce the expression of murine fibrinogen‐like protein 2 (mFGL2) triggering immune coagulation, which causes severe liver pathogenesis via increased fibrin deposition and thrombosis in the microvasculature. We aimed to investigate the role of mFGL2 in the liver stage of malaria infections. We reveal that infection with malaria sporozoites also induces increased expression of mFGL2 and that this expression is primarily located within the liver Kupffer and endothelial cells. In addition, we report that inhibition of FGL2 has no significant effect on immune coagulation but increases the expression of inflammatory cytokines in the livers of infected mice. Interestingly, FGL2 deficiency had no significant impact on the development of liver stage malaria parasites or the pathogenesis of the infected liver. In contrast to viral infections, we conclude that mFGL2 does not contribute to either parasite development or liver pathology during these infections, revealing the unique features of this protein in liver‐stage malaria infections. [ABSTRACT FROM AUTHOR]

ABSTRACT During vertebrate infection, obligate intracellular malaria parasites develop within a parasitophorous vacuole, which constitutes the interface between the parasite and its hepatocyte or erythrocyte host cells. To traverse this barrier, Plasmodium spp. utilize a dual-function pore formed by EXP2 for nutrient transport and, in the context of the PTEX translocon, effector protein export across the vacuole membrane. While critical to blood-stage survival, less is known about EXP2/PTEX function in the liver stage, although major differences in the export mechanism are suggested by absence of the PTEX unfoldase HSP101 in the intrahepatic vacuole. Here, we employed the glucosamine-activated glmS ribozyme to study the role of EXP2 during Plasmodium berghei liver-stage development in hepatoma cells. Insertion of the glmS sequence into the exp2 3′ untranslated region (UTR) enabled glucosamine-dependent depletion of EXP2 after hepatocyte invasion, allowing separation of EXP2 function during intrahepatic development from a recently reported role in hepatocyte invasion. Postinvasion EXP2 knockdown reduced parasite size and largely abolished expression of the mid- to late-liver-stage marker LISP2. As an orthogonal approach to monitor development, EXP2-glmS parasites and controls were engineered to express nanoluciferase. Activation of glmS after invasion substantially decreased luminescence in hepatoma monolayers and in culture supernatants at later time points corresponding to merosome detachment, which marks the culmination of liver-stage development. Collectively, our findings extend the utility of the glmS ribozyme to study protein function in the liver stage and reveal that EXP2 is important for intrahepatic parasite development, indicating that PTEX components also function at the hepatocyte-parasite interface. IMPORTANCE After the mosquito bite that initiates a Plasmodium infection, parasites first travel to the liver and develop in hepatocytes. This liver stage is asymptomatic but necessary for the parasite to transition to the merozoite form, which infects red blood cells and causes malaria. To take over their host cells, avoid immune defenses, and fuel their growth, these obligately intracellular parasites must import nutrients and export effector proteins across a vacuole membrane in which they reside. In the blood stage, these processes depend on a translocon called PTEX, but it is unclear if PTEX also functions during the liver stage. Here, we adapted the glmS ribozyme to control expression of EXP2, the membrane pore component of PTEX, during the liver stage of the rodent malaria parasite Plasmodium berghei. Our results show that EXP2 is important for intracellular development in the hepatocyte, revealing that PTEX components are also functionally important during liver-stage infection.

The Plasmodium liver stage represents a vulnerable therapeutic target to prevent disease progression as the parasite resides in the liver before clinical representation caused by intraerythrocytic development. However, most antimalarial drugs target the blood stage of the parasite's life cycle, and the few drugs that target the liver stage are lethal to patients with a glucose-6-phosphate dehydrogenase deficiency. Furthermore, implementation of in vitro liver models to study and develop novel therapeutics against the liver stage of human Plasmodium species remains challenging. In this review, we focus on the progression of in vitro liver models developed for human Plasmodium spp. parasites, provide a brief review on important assay requirements, and lastly present recommendations to improve models to enhance the discovery process of novel preclinical therapeutics. Over the decades, significant advances have been made to improve liver models that are able to support Plasmodium falciparum and Plasmodium vivax liver-stage parasites. The use of primary human hepatocytes (PHHs) has evolved to be commercially accessible, reproducible, and a biologically relevant system for Plasmodium liver studies. With optimal conditions, high-throughput screenings against Plasmodium liver stages can be performed using PHHs in 2D culture systems and high-content imaging. Liver models using PHH-derived spheroids better recapitulate in vivo physiology than commonly used cell lines and 2D culture systems. While models for studying the malaria parasite liver stages currently exist, further research contributions, improvements, and resource sharing are essential to achieve the mutual goal of malaria eradication. [ABSTRACT FROM AUTHOR]

Schistosoma is a genus of parasitic trematodes that undergoes complex migration in final hosts, finally developing into adult worms, which are responsible for egg production and disease dissemination. Recent studies documented the importance of extracellular vesicles (EVs) in the regulation of host-parasite interactions. Herein, we investigated the microRNA (miRNA) profiles of EVs isolated from host plasma at different stages of Schistosoma japonicum infection (lung stage: 3 days post-infection (dpi), and liver stages: 14 and 21 dpi) to identify miRNA cargo potentially involved in the pathogenesis and immune regulation of schistosomiasis. Characterization of the isolated plasma EVs revealed their diameter to be approximately 100 nm, containing typical EV markers such as Hsp70 and Tsg101. Deep sequencing analysis indicated the presence of 811 known and 15 novel miRNAs with an increasing number of differential miRNAs from the lung stage (27 miRNAs) to the liver stages (58 and 96 miRNAs at 14 and 21 dpi, respectively) in the plasma EVs of infected mice compared to EVs isolated from the uninfected control. In total, 324 plasma EV miRNAs were shown to be co-detected among different stages of infection and the validation of selected miRNAs showed trends of abundance similar to deep sequencing analysis. For example, miR-1a-3p and miR-122-5p showed higher abundance, whereas miR-150-3p and miR-126a showed lower abundance in the plasma EVs of infected mice at 3, 14, and 21 dpi as compared to those of uninfected mice. In addition, bioinformatic analysis combined with PCR validation of the miRNA targets, particularly those associated with the immune system and parasitic infectious disease, indicated a significant increase in the expression of Gbp7 and Ccr5 in contrast to the decreased expression of Fermt3, Akt1 , and IL-12a. Our results suggested that the abundance of miRNA cargo of the host plasma EVs was related to the stages of Schistosoma japonicum infection. Further studies on the roles of these miRNAs may reveal the regulatory mechanism of the host-parasite interaction. Moreover, the differentially abundant miRNA cargo in host EVs associated with S. japonicum infection may also provide valuable clues for identifying novel biomarkers for schistosomiasis diagnosis. [ABSTRACT FROM AUTHOR]

Host Genes and Drug Targets As Maria initiated her independent research career in Portugal, her focus on the study of the liver stage of I Plasmodium i infection intensified and expanded, and she soon reported the first transcriptome profile of the hepatic host cell throughout infection by malaria parasites ([1]). Keywords: malaria; liver stage; sporozoites; Plasmodium; hepatocyte EN malaria liver stage sporozoites Plasmodium hepatocyte 1 6 6 02/10/22 20220208 NES 220208 Introduction Mammalian infection by malaria parasites is initiated by the injection of I Plasmodium i sporozoites into the host's skin in preparation for a blood meal by an infected female I Anopheles i mosquito. A few years later, work from Maria's lab showed that I Plasmodium i liver stages induce a potent type I interferon (IFN) response that prompts host cell sensors to activate an immune cell-mediated response ([18]), the magnitude of which effectively inhibit re-infection in an IFN- -dependent way ([17]). Host-Mediated Modulation of Infection Maria's conviction that I Plasmodium i infection can be modulated by the host prompted her and her team to investigate whether the host's nutritional status influences the course of infection and pathology. [Extracted from the article]

[Display omitted] • The carboxy-terminal domain is important for Plasmodium berghei UIS4 function. • Plasmodium falciparum ETRAMP8 and ETRAMP10.3 failed to substitute Pb UIS4. • Plasmodium chabaudi UIS4 successfully complemented Pb UIS4 function. • The mammalian host range of Plasmodium parasites is reflected by distinct ETRAMPs. Early transcribed membrane proteins form a unique protein family in malaria parasites. These molecules are expressed during Plasmodium intracellular phases and inserted at the parasite parasitophorus vacuole membrane, which constitutes the host–parasite interface. Upregulated in infectious sporozoites 4 (UIS4) is an essential early transcribed membrane protein of liver stages of the murine malaria model parasite Plasmodium berghei. Despite its relevance for liver stage maturation, the molecular functions of UIS4 remain elusive, and UIS4 orthologs in human malaria parasites have not yet been identified. In order to characterise functional domains of UIS4, we generated P. berghei parasites carrying a carboxy-terminally truncated version of UIS4. We observed that uis4Δc parasites are severely impaired in liver stage development, similar to uis4(−) parasites, indicating an important role of the C-terminal domain for UIS4 function. To test whether members of the P. falciparum early transcribed membrane protein family are potential UIS4 orthologs, we selected candidates based on structural homology and parasitophorous vacuole membrane localization. We generated transgenic P. berghei parasites where UIS4 was replaced by Plasmodium falciparum ETRAMP8 or ETRAMP10.3. Both early transcribed membrane proteins were expressed in transgenic parasite lines, but liver stage maturation was impaired, indicating that the selected early transcribed membrane proteins failed to substitute the function of UIS4. As a control, we included the UIS4 ortholog from the murine parasite Plasmodium chaubaudi. We observed that PcUIS4 successfully restores UIS4 function in P. berghei. Together, these results suggest that Plasmodium parasites express tailor-made parasitophorous vacuole membrane proteins that might at least partially explain the narrow host range of malaria parasites. [ABSTRACT FROM AUTHOR]

Upon transmission to the human host, Plasmodium sporozoites exit the skin, are taken up by the blood stream, and then travel to the liver where they infect and significantly modify a single hepatocyte. Low infection rates within the liver have made proteomic studies of infected hepatocytes challenging, particularly in vivo , and existing studies have been largely unable to consider how protein and phosphoprotein differences are altered at different spatial locations within the heterogeneous liver. Using digital spatial profiling, we characterized changes in host signaling during Plasmodium yoelii infection in vivo without disrupting the liver tissue. Moreover, we measured alterations in protein expression around infected hepatocytes and identified a subset of CD163+ Kupffer cells that migrate towards infected cells during infection. These data offer the first insight into the heterogeneous microenvironment that surrounds the infected hepatocyte and provide insights into how the parasite may alter its milieu to influence its survival and modulate immunity. [ABSTRACT FROM AUTHOR]

Abstract Background Immunization with attenuated malaria sporozoites protects humans from experimental malaria challenge by mosquito bite. Protection in humans is strongly correlated with the production of T cells targeting a heterogeneous population of pre-erythrocyte antigen proteoforms, including liver stage antigens. Currently, few T cell epitopes derived from Plasmodium falciparum, the major aetiologic agent of malaria in humans are known. Methods In this study both in vitro and in vivo malaria liver stage models were used to sequence host and pathogen proteoforms. Proteoforms from these diverse models were subjected to mild acid elution (of soluble forms), multi-dimensional fractionation, tandem mass spectrometry, and top-down bioinformatics analysis to identify proteoforms in their intact state. Results These results identify a group of host and malaria liver stage proteoforms that meet a 5% false discovery rate threshold. Conclusions This work provides proof-of-concept for the validity of this mass spectrometry/bioinformatic approach for future studies seeking to reveal malaria liver stage antigens towards vaccine development.

Upon transmission to the human host, Plasmodium sporozoites exit the skin, are taken up by the blood stream, and then travel to the liver where they infect and significantly modify a single hepatocyte. Low infection rates within the liver have made proteomic studies of infected hepatocytes challenging, particularly in vivo, and existing studies have been largely unable to consider how protein and phosphoprotein differences are altered at different spatial locations within the heterogeneous liver. Using digital spatial profiling, we characterized changes in host signaling during Plasmodium yoelii infection in vivo without disrupting the liver tissue. Moreover, we measured alterations in protein expression around infected hepatocytes and identified a subset of CD163+ Kupffer cells that migrate towards infected cells during infection. These data offer the first insight into the heterogeneous microenvironment that surrounds the infected hepatocyte and provide insights into how the parasite may alter its milieu to influence its survival and modulate immunity.

Plasmodium sporozoites invade hepatocytes, transform into liver stages, and replicate into thousands of merozoites that infect erythrocytes and cause malaria. Proteins secreted from micronemes play an essential role in hepatocyte invasion, and unneeded micronemes are subsequently discarded for replication. The liver-stage parasites are potent immunogens that prevent malarial infection. Late liver stage-arresting genetically attenuated parasites (GAPs) exhibit greater protective efficacy than early GAP. However, the number of late liver-stage GAPs for generating GAPs with multiple gene deletions is limited. Here, we identified Scot1 (Sporozoite Conserved Orthologous Transcript 1), which was previously shown to be upregulated in sporozoites, and by endogenous tagging with mCherry, we demonstrated that it is expressed in the sporozoite and liver stages in micronemes. Using targeted gene deletion in Plasmodium berghei , we showed that Scot1 is essential for late liver-stage development. Scot1 KO sporozoites grew normally into liver stages but failed to initiate blood-stage infection in mice due to impaired apicoplast biogenesis and merozoite formation. Bioinformatic studies suggested that Scot1 is a metal-small-molecule carrier protein. Remarkably, supplementation with metals in the culture of infected Scot1 KO cells did not rescue their phenotype. Immunization with Scot1 KO sporozoites in C57BL/6 mice confers protection against malaria via infection. These proof-of-concept studies will enable the generation of P. falciparum Scot1 mutants that could be exploited to generate GAP malaria vaccines.

Ivermectin, a broad-spectrum anti-parasitic drug, has been proposed as a novel vector control tool to reduce malaria transmission by mass drug administration. Ivermectin and some metabolites have mosquito-lethal effect, reducing Anopheles mosquito survival. Ivermectin inhibits liver stage development in a rodent malaria model, but no inhibition was observed in a primate malaria model or in a human malaria challenge trial. In the liver, cytochrome P450 3A4 and 3A5 enzymes metabolize ivermectin, which may impact drug efficacy. Thus, understanding ivermectin metabolism and assessing this impact on Plasmodium liver stage development is critical. Using primary human hepatocytes (PHHs), we characterized ivermectin metabolism and evaluated the efficacy of ivermectin and its primary metabolites M1 (3″- O -demethyl ivermectin) and M3 (4-hydroxymethyl ivermectin) against Plasmodium falciparum liver stages. Two different modes of ivermectin exposure were evaluated: prophylactic mode (days 0-3 post-infection) and curative mode (days 3-5 post-infection). We used two different PHH donors and modes to determine the inhibitory concentration (IC 50 ) of ivermectin, M1, M3, and the known anti-malarial drug pyrimethamine, with IC 50 values ranging from 1.391 to 14.44, 9.95-23.71, 4.767-8.384, and 0.9073-5.416 µM, respectively. In our PHH model, ivermectin and metabolites M1 and M3 demonstrated inhibitory activity against P. falciparum liver stages in curative treatment mode (days 3-5) and marginal activity in prophylactic treatment mode (days 0-3). Ivermectin had improved efficacy when co-administered with ketoconazole, a specific inhibitor of cytochrome P450 3A4 enzyme. Further studies should be performed to examine ivermectin liver stage efficacy when co-administered with CYP3A4 inhibitors and anti-malarial drugs to understand the pharmacokinetic and pharmacodynamic drug-drug interactions that enhance efficacy against human malaria parasites in vitro ., Competing Interests: The authors declare no conflict of interest.

Malaria parasites have a complex life cycle and undergo replication and population expansion within vertebrate hosts and mosquito vectors. These developmental transitions rely on changes in gene expression and chromatin reorganization that result in the activation and silencing of stage-specific genes. The ApiAp2 family of DNA-binding proteins plays an important role in regulating gene expression in malaria parasites. Here, we characterized the ApiAp2 protein in Plasmodium berghei , which we termed Ap2-D. In silico analysis revealed that Ap2-D has three beta-sheets followed by a helix at the C-terminus for DNA binding. Using gene tagging with 3XHA-mCherry, we found that Ap2-D is expressed in Plasmodium blood stages and is present in the parasite cytoplasm and nucleus. Surprisingly, our gene deletion study revealed a completely dispensable role for Ap2-D in the entirety of the P. berghei life cycle. Ap2-D KO parasites were found to grow in the blood successfully and progress through the mosquito midgut and salivary glands. Sporozoites isolated from mosquito salivary glands were infective for hepatocytes and achieved similar patency as WT in mice. We emphasize the importance of genetic validation of antimalarial drug targets before progressing them to drug discovery.

Hepatocyte invasion by Plasmodium sporozoites represents a promising target for innovative antimalarial therapy, but the molecular events mediating this process are still largely uncharacterized. We previously showed that Plasmodium falciparum sporozoite entry into hepatocytes strictly requires CD81. However, CD81-overexpressing human hepatoma cells remain refractory to P. falciparum infection, suggesting the existence of additional host factors necessary for sporozoite entry. Here, through differential transcriptomic analysis of human hepatocytes and hepatoma HepG2-CD81 cells, the transmembrane protein Aquaporin-9 (AQP9) was found to be among the most downregulated genes in hepatoma cells. RNA silencing showed that sporozoite invasion of hepatocytes requires AQP9 expression. AQP9 overexpression in hepatocytes increased their permissiveness to P. falciparum. Moreover, chemical disruption with the AQP9 inhibitor phloretin markedly inhibited hepatocyte infection. Our findings identify AQP9 as a novel host factor required for P. falciparum sporozoite hepatocyte-entry and indicate that AQP9 could be a potential therapeutic target.

Hepatocyte invasion by Plasmodium sporozoites represents a promising target for innovative antimalarial therapy, but the molecular events mediating this process are still largely uncharacterized. We previously showed that Plasmodium falciparum sporozoite entry into hepatocytes strictly requires CD81. However, CD81-overexpressing human hepatoma cells remain refractory to P. falciparum infection, suggesting the existence of additional host factors necessary for sporozoite entry. Here, through differential transcriptomic analysis of human hepatocytes and hepatoma HepG2-CD81 cells, the transmembrane protein Aquaporin-9 (AQP9) was found to be among the most downregulated genes in hepatoma cells. RNA silencing showed that sporozoite invasion of hepatocytes requires AQP9 expression. AQP9 overexpression in hepatocytes increased their permissiveness to P. falciparum. Moreover, chemical disruption with the AQP9 inhibitor phloretin markedly inhibited hepatocyte infection. Our findings identify AQP9 as a novel host factor required for P. falciparum sporozoite hepatocyte-entry and indicate that AQP9 could be a potential therapeutic target. [ABSTRACT FROM AUTHOR]

Major challenges have been encountered regarding the development of highly efficient subunit malaria vaccines, and so whole-parasite vaccines have regained attention in recent years. The whole-killed blood-stage vaccine (WKV) is advantageous as it can be easily manufactured and efficiently induced protective immunity against a blood-stage challenge, as well as inducing cross-stage protection against both the liver and sexual-stages. However, it necessitates a high dose of parasitized red blood cell (pRBC) lysate for immunization, and this raises concerns regarding its safety and low immunogenicity. Knowledge of the major components of WKV that can induce or evade the host immune response, and the development of appropriate human-compatible adjuvants will greatly help to optimize the WKV. Therefore, we argue that the further development of the WKV is worthwhile to control and potentially eradicate malaria worldwide.

Major challenges have been encountered regarding the development of highly efficient subunit malaria vaccines, and so whole-parasite vaccines have regained attention in recent years. The whole-killed blood-stage vaccine (WKV) is advantageous as it can be easily manufactured and efficiently induced protective immunity against a blood-stage challenge, as well as inducing cross-stage protection against both the liver and sexual-stages. However, it necessitates a high dose of parasitized red blood cell (pRBC) lysate for immunization, and this raises concerns regarding its safety and low immunogenicity. Knowledge of the major components of WKV that can induce or evade the host immune response, and the development of appropriate human-compatible adjuvants will greatly help to optimize the WKV. Therefore, we argue that the further development of the WKV is worthwhile to control and potentially eradicate malaria worldwide. [ABSTRACT FROM AUTHOR]

Malaria remains a devastating global health problem, resulting in many annual deaths due to the complications of severe malaria. However, in endemic regions, individuals can acquire 'clinical immunity' to malaria, characterized by a decrease in severe malaria episodes and an increase of asymptomatic Plasmodium falciparum infections. Recently, it has been reported that tolerance to 'clinical malaria' and reduced disease severity correlates with a decrease in the numbers of circulating Vγ9Vδ2 T cells, the major subset of γδ T cells in the human peripheral blood. This is particularly interesting as this population typically undergoes dramatic expansions during acute Plasmodium infections and was previously shown to play antiparasitic functions. Thus, regulated γδ T‐cell responses may be critical to balance immune protection with severe pathology, particularly as both seem to rely on the same pro‐inflammatory cytokines, most notably TNF and IFN‐γ. This has been clearly demonstrated in mouse models of experimental cerebral malaria (ECM) based on Plasmodium berghei ANKA infection. Furthermore, our recent studies suggest that the natural course of Plasmodium infection, mimicked in mice through mosquito bite or sporozoite inoculation, includes a major pathogenic component in ECM that depends on γδ T cells and IFN‐γ production in the asymptomatic liver stage, where parasite virulence is seemingly set and determines pathology in the subsequent blood stage. Here, we discuss these and other recent advances in our understanding of the complex—protective versus pathogenic—functions of γδ T cells in malaria. [ABSTRACT FROM AUTHOR]

The protozoan parasite Plasmodium, causative agent of malaria, invades hepatocytes by invaginating the host cell plasma membrane and forming a parasitophorous vacuole membrane (PVM). Surrounded by this PVM, the parasite undergoes extensive replication. Parasites inside a PVM provoke the Plasmodium‐associated autophagy‐related (PAAR) response. This is characterised by a long‐lasting association of the autophagy marker protein LC3 with the PVM, which is not preceded by phosphatidylinositol 3‐phosphate (PI3P)‐labelling. Prior to productive invasion, sporozoites transmigrate several cells and here we describe that a proportion of traversing sporozoites become trapped in a transient traversal vacuole, provoking a host cell response that clearly differs from the PAAR response. These trapped sporozoites provoke PI3P‐labelling of the surrounding vacuolar membrane immediately after cell entry, followed by transient LC3‐labelling and elimination of the parasite by lysosomal acidification. Our data suggest that this PI3P response is not only restricted to sporozoites trapped during transmigration but also affects invaded parasites residing in a compromised vacuole. Thus, host cells can employ a pathway distinct from the previously described PAAR response to efficiently recognise and eliminate Plasmodium parasites. [ABSTRACT FROM AUTHOR]

The Plasmodium parasites that cause malaria undergo an obligate, asymptomatic developmental stage in the host liver before initiating the symptomatic blood-stage infection. The parasite liver stage is a key intervention point for antimalarial chemoprophylaxis: successful targeting of liver-stage parasites prevents disease development in individuals and can help to reduce parasite transmission in populations, as the gametocyte forms that transmit infection to mosquitos are exclusively found in the blood stage. Antimalarial drugs that can target multiple parasite stages are thus highly desirable, and one emerging cellular target for such multistage active compounds is the process of protein synthesis or translation. Quantitative study of liver stage translation, and thus mechanistic evaluation of translation inhibitors against liver stage parasites, is not amenable to the methods allowing quantification of asexual blood stage translation, such as radiolabeled amino acid incorporation or lysate-based translation of reporter transcripts. Here, we present a method using o-propargyl puromycin (OPP) labeling of host and parasite nascent proteomes in the P. berghei -HepG2 infection model, followed by automated confocal image acquisition and computational separation of P. berghei vs. H. sapiens nascent proteome signals to allow simultaneous readout of the effects of translation inhibitors on both host and parasite. This protocol details our HepG2 cell culture and infected monolayer handling optimized for microscopy, our OPP labeling workflow, and our approach to automated confocal imaging, image processing, and data analysis. Key features • Uses the o-propargyl puromycin labeling technique developed by Liu et al. to quantitatively analyze protein synthesis in Plasmodium berghei liver-stage parasites in actively translating hepatoma cells. • This quantitative approach should be adaptable for other puromycin-sensitive intracellular pathogens residing in actively translating host cells. • The P. berghei -infected HepG2 recovery and reseeding protocol presented here is of use in applications beyond nascent proteome labeling and quantification., Competing Interests: Competing interestsThe authors declare no competing interests., (©Copyright : © 2024 The Authors; This is an open access article under the CC BY-NC license.)

Transgenic reporter lines of malaria parasites that express fluorescent or luminescent proteins are valuable tools for drug and vaccine screening assays as well as to interrogate parasite gene function. Different Plasmodium falciparum (Pf ) reporter lines exist, however nearly all have been created in the African NF54/3D7 laboratory strain. Here we describe the generation of novel reporter lines, using CRISPR/Cas9 gene modification, both in the standard Pf NF54 background and in a recently described Cambodian P. falciparum NF135.C10 line. Sporozoites of this line show more effective hepatocyte invasion and enhanced liver merozoite development compared to Pf NF54. We first generated Pf NF54 reporter parasites to analyze two novel promoters for constitutive and high expression of mCherry-luciferase and GFP in blood and mosquito stages. The promoter sequences were selected based on available transcriptome data and are derived from two housekeeping genes, i.e., translation initiation factor SUI1, putative (sui1, PF3D7_1243600) and 40S ribosomal protein S30 (40s, PF3D7_0219200). We then generated and characterized reporter lines in the Pf NF135.C10 line which express GFP driven by the sui1 and 40s promoters as well as by the previously used ef1α promoter (GFP@ef1α, GFP@sui1, GFP@40s). The GFP@40s reporter line showed strongest GFP expression in liver stages as compared to the other two lines. The strength of reporter expression by the 40s promoter throughout the complete life cycle, including liver stages, makes transgenic lines expressing reporters by the 40s promoter valuable novel tools for analyses of P. falciparum.

ABSTRACT The apicomplexan parasites Plasmodium spp. are the causative agents of malaria, a disease that poses a significant global health burden. Plasmodium spp. initiate infection of the human host by transforming and replicating within hepatocytes. This liver stage (LS) is poorly understood compared to other Plasmodium life stages, which has hindered our ability to target these parasites for disease prevention. We conducted an extensive transcriptome sequencing (RNA-Seq) analysis throughout the Plasmodium berghei LS, covering as early as 2 h postinfection (hpi) and extending to 48 hpi. Our data revealed that hundreds of genes are differentially expressed at 2 hpi and that multiple genes shown to be important for later infection are upregulated as early as 12 hpi. Using hierarchical clustering along with coexpression analysis, we identified clusters functionally enriched for important liver-stage processes such as interactions with the host cell and redox homeostasis. Furthermore, some of these clusters were highly correlated to the expression of ApiAP2 transcription factors, while showing enrichment of mostly uncharacterized DNA binding motifs. This finding indicates potential LS targets for these transcription factors, while also hinting at alternative uncharacterized DNA binding motifs and transcription factors during this stage. Our work presents a window into the previously undescribed transcriptome of Plasmodium upon host hepatocyte infection to enable a comprehensive view of the parasite’s LS. These findings also provide a blueprint for future studies that extend hypotheses concerning LS gene function in P. berghei to human-infective Plasmodium parasites. IMPORTANCE The LS of Plasmodium infection is an asymptomatic yet necessary stage for producing blood-infective parasites, the causative agents of malaria. Blocking the liver stage of the life cycle can prevent clinical malaria, but relatively less is known about the parasite’s biology at this stage. Using the rodent model P. berghei, we investigated whole-transcriptome changes occurring as early as 2 hpi of hepatocytes. The transcriptional profiles of early time points (2, 4, 12, and 18 hpi) have not been accessible before due to the technical challenges associated with liver-stage infections. Our data now provide insights into these early parasite fluxes that may facilitate establishment of infection, transformation, and replication in the liver.

Here, we present the construction of an attenuated herpes simplex virus type-1 (HSV-1)-vectored vaccine, expressing three liver-stage (LS) malaria parasite exported proteins (EXP1, UIS3 and TMP21) as fusion proteins with the VP26 viral capsid protein. Intramuscular and subcutaneous immunizations of mice with a pooled vaccine, composed of the three attenuated virus strains expressing each LS antigen, induced sterile protection against the intravenous challenge of Plasmodium yoelii 17X-NL salivary gland sporozoites. Our data suggest that this malaria vaccine may be effective in preventing malaria parasite infection using practical routes of immunization in humans.

Even though malaria is preventable and curable, it has become a serious threat to mankind. In 2016, there were an estimated 216 million cases of malaria across the world. The biology of its causative agent, i.e. Plasmodium parasite is full of complex mechanisms. There are five Plasmodium species responsible for malaria in humans, viz. Plasmodium falciparum, P. vivax, P. malariae, P. ovale and recently identified P. knowlesi that normally infect apes. In humans, malaria is spread by the injection of Plasmodium sporozoites through the bite of infectious Anopheles’ female mosquito during their blood meal. From the time of entry into human skin till the development into the asexual forms, the parasite undergoes several transformations. This review attempts to understand the science behind the pre-erythrocytic liver stage of Plasmodium. Research articles explaining parasite biology, cell-traversal, transformation stages, cell-egress process, etc. were retrieved from PubMed and google scholar database. Various known and unknown mechanisms and strategies used by the malaria parasite P. berghei in rodent models have been discussed in this review. Limited or no information was available for humans, due to technical feasibility and complexity of parasite’s life cycle. Hence, it was concluded that there is an urgent need to investigate the hepatic invasion, traversal and egress mechanism of P. falciparum and P. vivax for developing novel therapeutics to fight against malaria.

Abstract Background Eradication of malaria is difficult because of the ability of hypnozoite, the dormant liver-stage form of Plasmodium vivax, to cause relapse in patients. Research efforts to better understand the biology of P. vivax hypnozoite and design relapse prevention strategies have been hampered by the lack of a robust and reliable model for in vitro culture of liver-stage parasites. Although the HC-04 hepatoma cell line is used for culturing liver-stage forms of Plasmodium, these cells proliferate unrestrictedly and detach from the culture dish after several days, which limits their usefulness in a long-term hypnozoite assay. Methods A novel immortalized hepatocyte-like cell line (imHC) was evaluated for the capability to support P. vivax sporozoite infection. First, expression of basic hepatocyte markers and all major malaria sporozoite-associated host receptors in imHC was investigated. Next, in vitro hepatocyte infectivity and intracellular development of sporozoites in imHC were determined using an indirect immunofluorescence assay. Cytochrome P450 isotype activity was also measured to determine the ability of imHC to metabolize drugs. Finally, the anti-liver-stage agent primaquine was used to test this model for a drug sensitivity assay. Results imHCs maintained major hepatic functions and expressed the essential factors CD81, SR-BI and EphA2, which are required for host entry and development of the parasite in the liver. imHCs could be maintained long-term in a monolayer without overgrowth and thus served as a good, supportive substrate for the invasion and growth of P. vivax liver stages, including hypnozoites. The observed high drug metabolism activity and potent responses in liver-stage parasites to primaquine highlight the potential use of this imHC model for antimalarial drug screening. Conclusions imHCs, which maintain a hepatocyte phenotype and drug-metabolizing enzyme expression, constitute an alternative host for in vitro Plasmodium liver-stage studies, particularly those addressing the biology of P. vivax hypnozoite. They potentially offer a novel, robust model for screening drugs against liver-stage parasites.

Transgenic reporter lines of malaria parasites that express fluorescent or luminescent proteins are valuable tools for drug and vaccine screening assays as well as to interrogate parasite gene function. Different Plasmodium falciparum (Pf) reporter lines exist, however nearly all have been created in the African NF54/3D7 laboratory strain. Here we describe the generation of novel reporter lines, using CRISPR/Cas9 gene modification, both in the standard Pf NF54 background and in a recently described Cambodian P. falciparum NF135.C10 line. Sporozoites of this line show more effective hepatocyte invasion and enhanced liver merozoite development compared to Pf NF54. We first generated Pf NF54 reporter parasites to analyze two novel promoters for constitutive and high expression of mCherry-luciferase and GFP in blood and mosquito stages. The promoter sequences were selected based on available transcriptome data and are derived from two housekeeping genes, i.e., translation initiation factor SUI1, putative (sui1 , PF3D7_1243600) and 40S ribosomal protein S30 (40s , PF3D7_0219200). We then generated and characterized reporter lines in the Pf NF135.C10 line which express GFP driven by the sui1 and 40s promoters as well as by the previously used ef1 α promoter (GFP@ef1 α, GFP@sui1, GFP@40s). The GFP@40s reporter line showed strongest GFP expression in liver stages as compared to the other two lines. The strength of reporter expression by the 40s promoter throughout the complete life cycle, including liver stages, makes transgenic lines expressing reporters by the 40s promoter valuable novel tools for analyses of P. falciparum. [ABSTRACT FROM AUTHOR]

Background: Immunization with attenuated malaria sporozoites protects humans from experimental malaria challenge by mosquito bite. Protection in humans is strongly correlated with the production of T cells targeting a heterogeneous population of pre-erythrocyte antigen proteoforms, including liver stage antigens. Currently, few T cell epitopes derived from Plasmodium falciparum, the major aetiologic agent of malaria in humans are known. Methods: In this study both in vitro and in vivo malaria liver stage models were used to sequence host and pathogen proteoforms. Proteoforms from these diverse models were subjected to mild acid elution (of soluble forms), multi-dimensional fractionation, tandem mass spectrometry, and top-down bioinformatics analysis to identify proteoforms in their intact state. Results: These results identify a group of host and malaria liver stage proteoforms that meet a 5% false discovery rate threshold. Conclusions: This work provides proof-of-concept for the validity of this mass spectrometry/bioinformatic approach for future studies seeking to reveal malaria liver stage antigens towards vaccine development. [ABSTRACT FROM AUTHOR]

Plasmodium vivax hypnozoites persist in the liver, cause malaria relapse and represent a major challenge to malaria elimination. Our previous transcriptomic study provided a novel molecular framework to enhance our understanding of the hypnozoite biology (Voorberg-van der Wel A, et al., 2017). In this dataset, we identified and characterized the Liver-Specific Protein 2 (LISP2) protein as an early molecular marker of liver stage development. Immunofluorescence analysis of hepatocytes infected with relapsing malaria parasites, in vitro (P. cynomolgi) and in vivo (P. vivax), reveals that LISP2 expression discriminates between dormant hypnozoites and early developing parasites. We further demonstrate that prophylactic drugs selectively kill all LISP2-positive parasites, while LISP2-negative hypnozoites are only sensitive to anti-relapse drug tafenoquine. Our results provide novel biological insights in the initiation of liver stage schizogony and an early marker suitable for the development of drug discovery assays predictive of anti-relapse activity.

In vitro studies of liver stage (LS) development of the human malaria parasite Plasmodium falciparum are technically challenging; therefore, fundamental questions about hepatocyte receptors for invasion that can be targeted to prevent infection remain unanswered. To identify novel receptors and to further understand human hepatocyte susceptibility to P. falciparum sporozoite invasion, we created an optimized in vitro system by mimicking in vivo liver conditions and using the subcloned HC-04.J7 cell line that supports mean infection rates of 3–5% and early development of P. falciparum exoerythrocytic forms—a 3- to 5-fold improvement on current in vitro hepatocarcinoma models for P. falciparum invasion. We juxtaposed this invasion-susceptible cell line with an invasion-resistant cell line (HepG2) and performed comparative proteomics and RNA-seq analyses to identify host cell surface molecules and pathways important for sporozoite invasion of host cells. We identified and investigated a hepatocyte cell surface heparan sulfate proteoglycan, glypican-3, as a putative mediator of sporozoite invasion. We also noted the involvement of pathways that implicate the importance of the metabolic state of the hepatocyte in supporting LS development. Our study highlights important features of hepatocyte biology, and specifically the potential role of glypican-3, in mediating P. falciparum sporozoite invasion. Additionally, it establishes a simple in vitro system to study the LS with improved invasion efficiency. This work paves the way for the greater malaria and liver biology communities to explore fundamental questions of hepatocyte-pathogen interactions and extend the system to other human malaria parasite species, like P. vivax.

Importance: Plasmodium parasites cause malaria in humans. New multistage active antimalarial drugs are needed, and a promising class of drugs targets the core cellular process of translation, which has many potential molecular targets. During the obligate liver stage, Plasmodium parasites grow in metabolically active hepatocytes, making it challenging to study core cellular processes common to both host cells and parasites, as the signal from the host typically overwhelms that of the parasite. Here, we present and validate a flexible assay to quantify Plasmodium liver stage translation using a technique to fluorescently label the newly synthesized proteins of both host and parasite followed by computational separation of their respective nascent proteomes in confocal image sets. We use the assay to determine whether a test set of known compounds are direct or indirect liver stage translation inhibitors and show that the assay can also predict the mode of action for novel antimalarial compounds., Competing Interests: The authors declare no conflict of interest.

Cerebral malaria (CM) is a major cause of death due to Plasmodium infection. Both parasite and host factors contribute to the onset of CM, but the precise cellular and molecular mechanisms that contribute to its pathogenesis remain poorly characterized. Unlike conventional αβ-T cells, previous studies on murine γδ-T cells failed to identify a nonredundant role for this T cell subset in experimental cerebral malaria (ECM). Here we show that mice lacking γδ-T cells are resistant to ECM when infected with Plasmodium berghei ANKA sporozoites, the liver-infective form of the parasite and the natural route of infection, in contrast with their susceptible phenotype if challenged with P. berghei ANKA-infected red blood cells that bypass the liver stage of infection. Strikingly, the presence of γδ-T cells enhanced the expression of Plasmodium immunogenic factors and exacerbated subsequent systemic and brain-infiltrating inflammatory αβ-T cell responses. These phenomena were dependent on the proinflammatory cytokine IFN-γ, which was required during liver stage for modulation of the parasite transcriptome, as well as for downstream immune-mediated pathology. Our work reveals an unanticipated critical role of γδ-T cells in the development of ECM upon Plasmodium liver-stage infection. [ABSTRACT FROM AUTHOR]

In vitro studies of liver stage (LS) development of the human malaria parasite Plasmodium falciparum are technically challenging; therefore, fundamental questions about hepatocyte receptors for invasion that can be targeted to prevent infection remain unanswered. To identify novel receptors and to further understand human hepatocyte susceptibility to P. falciparum sporozoite invasion, we created an optimized in vitro system by mimicking in vivo liver conditions and using the subcloned HC-04.J7 cell line that supports mean infection rates of 3–5% and early development of P. falciparum exoerythrocytic forms—a 3- to 5-fold improvement on current in vitro hepatocarcinoma models for P. falciparum invasion. We juxtaposed this invasion-susceptible cell line with an invasion-resistant cell line (HepG2) and performed comparative proteomics and RNA-seq analyses to identify host cell surface molecules and pathways important for sporozoite invasion of host cells. We identified and investigated a hepatocyte cell surface heparan sulfate proteoglycan, glypican-3, as a putative mediator of sporozoite invasion. We also noted the involvement of pathways that implicate the importance of the metabolic state of the hepatocyte in supporting LS development. Our study highlights important features of hepatocyte biology, and specifically the potential role of glypican-3, in mediating P. falciparum sporozoite invasion. Additionally, it establishes a simple in vitro system to study the LS with improved invasion efficiency. This work paves the way for the greater malaria and liver biology communities to explore fundamental questions of hepatocyte-pathogen interactions and extend the system to other human malaria parasite species, like P. vivax. [ABSTRACT FROM AUTHOR]

Malaria remains one of the most prevalent infectious diseases worldwide, primarily affecting some of the most vulnerable populations around the globe. Despite achievements in the treatment of this devastating disease, there is still an urgent need for the discovery of new drugs that tackle infection by Plasmodium parasites. However, de novo drug development is a costly and time-consuming process. An alternative strategy is to evaluate the anti-plasmodial activity of compounds that are already approved for other purposes, an approach known as drug repurposing. Here, we will review efforts to assess the anti-plasmodial activity of existing drugs, with an emphasis on the obligatory and clinically silent liver stage of infection. We will also review the current knowledge on the classes of compounds that might be therapeutically relevant against Plasmodium in the context of other communicable diseases that are prevalent in regions where malaria is endemic. Repositioning existing compounds may constitute a faster solution to the current gap of prophylactic and therapeutic drugs that act on Plasmodium parasites, overall contributing to the global effort of malaria eradication.

The need for a malaria vaccine is indisputable. A single vaccine for Plasmodium pre-erythrocytic stages targeting the major sporozoite antigen circumsporozoite protein (CSP) has had partial success. Additionally, CD8+ T cells targeting liver-stage (LS) antigens induced by live attenuated sporozoite vaccines were associated with protection in human challenge experiments. To further evaluate protection mediated by LS antigens, we focused on exported pre-erythrocytic proteins (exported protein 1 (EXP1), profilin (PFN), exported protein 2 (EXP2), inhibitor of cysteine proteases (ICP), transmembrane protein 21 (TMP21), and upregulated in infective sporozoites-3 (UIS3)) expressed in all Plasmodium species and designed optimized, synthetic DNA (synDNA) immunogens. SynDNA antigen cocktails were tested with and without the molecular adjuvant plasmid IL-33. Immunized animals developed robust T cell responses including induction of antigen-specific liver-localized CD8+ T cells, which were enhanced by the co-delivery of plasmid IL-33. In total, 100% of mice in adjuvanted groups and 71%−88% in non-adjuvanted groups were protected from blood-stage disease following Plasmodium yoelii sporozoite challenge. This study supports the potential of synDNA LS antigens as vaccine components for malaria parasite infection.

ABSTRACT During their development within the vertebrate host, Plasmodium parasites infect hepatocytes and red blood cells. Within these cells, parasites are surrounded by a parasitophorous vacuole membrane (PVM). The PVM plays an essential role for the interaction of parasites with their host cells; however, only a limited number of proteins of this membrane have been identified so far. This is partially because systematic proteomic analysis of the protein content of the PVM has been difficult in the past, due to difficulties encountered in attempts to separate the PVM from other membranes such as the parasite plasma membrane. In this study, we adapted the BioID technique to in vitro-cultivated Plasmodium berghei blood stage parasites and utilized the promiscuous biotin ligase BirA* fused to PVM-resident exported protein 1 to biotinylate proteins of the PVM. These we further processed by affinity purification, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and label-free quantitation, leading to a list of 61 known and candidate PVM proteins. Seven proteins were analyzed further during blood and liver stage development. This resulted in the identification of three novel PVM proteins, which were the serine/threonine protein phosphatase UIS2 (PlasmoDB accession no. PBANKA_1328000) and two conserved Plasmodium proteins with unknown functions (PBANKA_0519300 and PBANKA_0509000). In conclusion, our report expands the number of known PVM proteins and experimentally validates BioID as a powerful method to screen for novel constituents of specific cellular compartments in P. berghei. IMPORTANCE Intracellular pathogens are often surrounded by a host-cell derived membrane. This membrane is modified by the pathogens to their own needs and is crucial for their intracellular lifestyle. In Plasmodium parasites, this membrane is referred to as the PVM and only a limited number of its proteins are known so far. Here, we applied in rodent P. berghei parasites a method called BioID, which is based on biotinylation of proximal and interacting proteins by the promiscuous biotin ligase BirA*, and demonstrated its usefulness in identification of novel PVM proteins.

Abstract Emergence of drug resistance and targeting all stages of the parasite life cycle are currently the major challenges in antimalarial chemotherapy. Molecular hybridization combining two scaffolds in a single molecule is an innovative strategy for achieving these goals. In this work, a series of novel quinoxaline 1,4-di- N -oxide hybrids containing either chloroquine or primaquine pharmacophores was designed, synthesized and tested against both chloroquine sensitive and multidrug resistant strains of Plasmodium falciparum. Only chloroquine-based compounds exhibited potent blood stage activity with compounds 4b and 4e being the most active and selective hybrids at this parasite stage. Based on their intraerythrocytic activity and selectivity or their chemical nature, seven hybrids were then evaluated against the liver stage of Plasmodium yoelii , Plasmodium berghei and Plasmodium falciparum infections. Compound 4b was the only chloroquine-quinoxaline 1,4-di- N -oxide hybrid with a moderate liver activity, whereas compound 6a and 6b were identified as the most active primaquine-based hybrids against exoerythrocytic stages, displaying enhanced liver activity against P. yoelii and P. berghei , respectively, and better SI values than primaquine. Although both primaquine-quinoxaline 1,4-di- N -oxide hybrids slightly reduced the infection of mosquitoes, they inhibited sporogony of P. berghei and compound 6a showed 92% blocking of transmission. In vivo liver efficacy assays revealed that compound 6a showed causal prophylactic activity affording parasitaemia reduction of up to 95% on day 4. Absence of genotoxicity and in vivo acute toxicity were also determined. These results suggest the approach of primaquine-quinoxaline 1,4-di- N -oxide hybrids as new potential dual-acting antimalarials for further investigation. Graphical abstract Image 1 Highlights - New chloroquine/primaquine–quinoxaline 1,4-di- N- oxide (QdNO) hybrids were synthesized. - Chloroquine-based 4b was the most active in blood stage with a moderate liver activity. - Primaquine-QdNO 6a / 6b displayed better liver Py/Pb activity and SI than primaquine. - Hybrid 6a showed causal prophylactic activity and high inhibition of sporogony (92%). - Absence of genotoxicity and in vivo acute toxicity for primaquine-QdNO 6a was found. [ABSTRACT FROM AUTHOR]

Hybrid compounds may play a critical role in the context of the malaria eradication agenda, which will benefit from therapeutic tools active against the symptomatic erythrocytic stage of Plasmodium infection, and also capable of eliminating liver stage parasites. To address the need for efficient multistage antiplasmodial compounds, a small library of 1,2,4,5-tetraoxane-8- aminoquinoline hybrids, with the metabolically labile C-5 position of the 8-aminoquinoline moiety blocked with aryl groups, was synthesized and screened for antiplasmodial activity and metabolic stability. The hybrid compounds inhibited development of intra-erythrocytic forms of the multidrug-resistant Plasmodium falciparum W2 strain, with EC 50 values in the nM range, and with low cytotoxicity against mammalian cells. The compounds also inhibited the development of P. berghei liver stage parasites, with the most potent compounds displaying EC 50 values in the low μM range. SAR analysis revealed that unbranched linkers between the endoperoxide and 8-aminoquinoline pharmacophores are most beneficial for dual antiplasmodial activity. Importantly, hybrids were significantly more potent than a 1:1 mixture of 8-aminoquinoline-tetraoxane, highlighting the superiority of the hybrid approach over the combination therapy. Furthermore, aryl substituents at C-5 of the 8-aminoquinoline moiety improve the compounds' metabolic stability when compared with their primaquine (i.e. C-5 unsubstituted) counterparts. Overall, this study reveals that blocking the quinoline C-5 position does not result in loss of dual-stage antimalarial activity, and that tetraoxane-8- aminoquinoline hybrids are an attractive approach to achieve elimination of exo- and intraerythrocytic parasites, thus with the potential to be used in malaria eradication campaigns. [ABSTRACT FROM AUTHOR]

Background: Eradication of malaria is difficult because of the ability of hypnozoite, the dormant liver-stage form of Plasmodium vivax, to cause relapse in patients. Research efforts to better understand the biology of P. vivax hypnozoite and design relapse prevention strategies have been hampered by the lack of a robust and reliable model for in vitro culture of liver-stage parasites. Although the HC-04 hepatoma cell line is used for culturing liver-stage forms of Plasmodium, these cells proliferate unrestrictedly and detach from the culture dish after several days, which limits their usefulness in a long-term hypnozoite assay. Methods: A novel immortalized hepatocyte-like cell line (imHC) was evaluated for the capability to support P. vivax sporozoite infection. First, expression of basic hepatocyte markers and all major malaria sporozoite-associated host receptors in imHC was investigated. Next, in vitro hepatocyte infectivity and intracellular development of sporozoites in imHC were determined using an indirect immunofluorescence assay. Cytochrome P450 isotype activity was also measured to determine the ability of imHC to metabolize drugs. Finally, the anti-liver-stage agent primaquine was used to test this model for a drug sensitivity assay. Results: imHCs maintained major hepatic functions and expressed the essential factors CD81, SR-BI and EphA2, which are required for host entry and development of the parasite in the liver. imHCs could be maintained long-term in a monolayer without overgrowth and thus served as a good, supportive substrate for the invasion and growth of P. vivax liver stages, including hypnozoites. The observed high drug metabolism activity and potent responses in liverstage parasites to primaquine highlight the potential use of this imHC model for antimalarial drug screening. Conclusions: imHCs, which maintain a hepatocyte phenotype and drug-metabolizing enzyme expression, constitute an alternative host for in vitro Plasmodium liver-stage studies, particularly those addressing the biology of P. vivax hypnozoite. They potentially offer a novel, robust model for screening drugs against liver-stage parasites. [ABSTRACT FROM AUTHOR]

The intracellular development and differentiation of the Plasmodium parasite in the host liver is a prerequisite for the actual onset of malaria disease pathology. Since liver-stage infection is clinically silent and can be completely eliminated by sterilizing immune responses, it is a promising target for urgently needed innovative antimalarial drugs and/or vaccines. Discovered more than 65 years ago, these stages remain poorly understood regarding their molecular repertoire and interaction with their host cells in comparison to the pathogenic erythrocytic stages. The differentiating and replicative intrahepatic parasite resides in a membranous compartment called the parasitophorous vacuole, separating it from the host-cell cytoplasm. Here we outline seminal work that contributed to our present understanding of the fundamental dynamic cellular processes of the intrahepatic malarial parasite with both specific host-cell factors and compartments. [ABSTRACT FROM AUTHOR]