Correlative light-electron microscopy methods to characterize the ultrastructural features of the replicative and dormant liver stages of Plasmodium parasites.

BACKGROUND: The infection of the liver by Plasmodium parasites is an obligatory step leading to malaria disease. Following hepatocyte invasion, parasites differentiate into replicative liver stage schizonts and, in the case of Plasmodium species causing relapsing malaria, into hypnozoites that can lie dormant for extended periods of time before activating. The liver stages of Plasmodium remain elusive because of technical challenges, including low infection rate. This has been hindering experimentations with well-established technologies, such as electron microscopy. A deeper understanding of hypnozoite biology could prove essential in the development of radical cure therapeutics against malaria. RESULTS: The liver stages of the rodent parasite Plasmodium berghei, causing non-relapsing malaria, and the simian parasite Plasmodium cynomolgi, causing relapsing malaria, were characterized in human Huh7 cells or primary non-human primate hepatocytes using Correlative Light-Electron Microscopy (CLEM). Specifically, CLEM approaches that rely on GFP-expressing parasites (GFP-CLEM) or on an immunofluorescence assay (IFA-CLEM) were used for imaging liver stages. The results from P. berghei showed that host and parasite organelles can be identified and imaged at high resolution using both CLEM approaches. While IFA-CLEM was associated with more pronounced extraction of cellular content, samples' features were generally well preserved. Using IFA-CLEM, a collection of micrographs was acquired for P. cynomolgi liver stage schizonts and hypnozoites, demonstrating the potential of this approach for characterizing the liver stages of Plasmodium species causing relapsing malaria. CONCLUSIONS: A CLEM approach that does not rely on parasites expressing genetically encoded tags was developed, therefore suitable for imaging the liver stages of Plasmodium species that lack established protocols to perform genetic engineering. This study also provides a dataset that characterizes the ultrastructural features of liver stage schizonts and hypnozoites from the simian parasite species P. cynomolgi.

Drug discovery for parasitic diseases: powered by technology, enabled by pharmacology, informed by clinical science.

While prevention is a bedrock of public health, innovative therapeutics are needed to complement the armamentarium of interventions required to achieve disease control and elimination targets for neglected diseases. Extraordinary advances in drug discovery technologies have occurred over the past decades, along with accumulation of scientific knowledge and experience in pharmacological and clinical sciences that are transforming many aspects of drug R&D across disciplines. We reflect on how these advances have propelled drug discovery for parasitic infections, focusing on malaria, kinetoplastid diseases, and cryptosporidiosis. We also discuss challenges and research priorities to accelerate discovery and development of urgently needed novel antiparasitic drugs.

Global gene expression of human malaria parasite liver stages throughout intrahepatocytic development.

Plasmodium falciparum (Pf) is causing the greatest malaria burden, yet the liver stages (LS) of this most important parasite species have remained poorly studied. Here, we used a human liver-chimeric mouse model in combination with a novel fluorescent PfNF54 parasite line (PfNF54cspGFP) to isolate PfLS-infected hepatocytes and generate transcriptomes that cover the major LS developmental phases in human hepatocytes. RNA-seq analysis of early Pf LS trophozoites two days after infection, revealed a central role of translational regulation in the transformation of the extracellular invasive sporozoite into intracellular LS. The developmental time course gene expression analysis indicated that fatty acid biosynthesis, isoprenoid biosynthesis and iron metabolism are sustaining LS development along with amino acid metabolism and biosynthesis. Countering oxidative stress appears to play an important role during intrahepatic LS development. Furthermore, we observed expression of the variant PfEMP1 antigen-encoding var genes, and we confirmed expression of PfEMP1 protein during LS development. Transcriptome comparison of the late Pf liver stage schizonts with P. vivax (Pv) late liver stages revealed highly conserved gene expression profiles among orthologous genes. A notable difference however was the expression of genes regulating sexual stage commitment. While Pv schizonts expressed markers of sexual commitment, the Pf LS parasites were not sexually committed and showed expression of gametocytogenesis repression factors. Our results provide the first comprehensive gene expression profile of the human malaria parasite Pf LS isolated during in vivo intrahepatocytic development. This data will inform biological studies and the search for effective intervention strategies that can prevent infection.

Transcriptional profiling of hepatocytes infected with the replicative form of the malaria parasite Plasmodium cynomolgi.

BACKGROUND: The zoonotic simian parasite Plasmodium cynomolgi develops into replicating schizonts and dormant hypnozoites during the infection of hepatocytes and is used as a model organism to study relapsing malaria. The transcriptional profiling of P. cynomolgi liver stages was previously reported and revealed many important biological features of the parasite but left out the host response to malaria infection. METHODS: Previously published RNA sequencing data were used to quantify the expression of host genes in rhesus macaque hepatocytes infected with P. cynomolgi in comparison to either cells from uninfected samples or uninfected bystander cells. RESULTS: Although the dataset could not be used to resolve the transcriptional profile of hypnozoite-infected hepatocytes, it provided a snapshot of the host response to liver stage schizonts at 9-10 day post-infection and identified specific host pathways that are modulated during the exo-erythrocytic stage of P. cynomolgi. CONCLUSIONS: This study constitutes a valuable resource characterizing the hepatocyte response to P. cynomolgi infection and provides a framework to build on future research that aims at understanding hepatocyte-parasite interactions during relapsing malaria infection.

Plasmodium vivax latent liver infection is characterized by persistent hypnozoites, hypnozoite-derived schizonts, and time-dependent efficacy of primaquine.

Plasmodium vivax is a malaria-causing pathogen that establishes a dormant form in the liver (the hypnozoite), which can activate weeks, months, or years after the primary infection to cause a relapse, characterized by secondary blood-stage infection. These asymptomatic and undetectable latent liver infections present a significant obstacle to the goal of global malaria eradication. We use a human liver-chimeric mouse model (FRG huHep) to study P. vivax hypnozoite latency and activation in an in vivo model system. Functional activation of hypnozoites and formation of secondary schizonts is demonstrated by first eliminating primary liver schizonts using a schizont-specific antimalarial tool compound, and then measuring recurrence of secondary liver schizonts in the tissue and an increase in parasite RNA within the liver. We also reveal that, while primaquine does not immediately eliminate hypnozoites from the liver, it arrests developing schizonts and prevents activation of hypnozoites, consistent with its clinical activity in humans. Our findings demonstrate that the FRG huHep model can be used to study the biology of P. vivax infection and latency and assess the activity of anti-relapse drugs.

Mass Spectrometry Identification of Biomarkers in Extracellular Vesicles From Plasmodium vivax Liver Hypnozoite Infections.

Latent liver stages termed hypnozoites cause relapsing Plasmodium vivax malaria infection and represent a major obstacle in the goal of malaria elimination. Hypnozoites are clinically undetectable, and presently, there are no biomarkers of this persistent parasite reservoir in the human liver. Here, we have identified parasite and human proteins associated with extracellular vesicles (EVs) secreted from in vivo infections exclusively containing hypnozoites. We used P. vivax-infected human liver-chimeric (huHEP) FRG KO mice treated with the schizonticidal experimental drug MMV048 as hypnozoite infection model. Immunofluorescence-based quantification of P. vivax liver forms showed that MMV048 removed schizonts from chimeric mice livers. Proteomic analysis of EVs derived from FRG huHEP mice showed that human EV cargo from infected FRG huHEP mice contain inflammation markers associated with active schizont replication and identified 66 P. vivax proteins. To identify hypnozoite-specific proteins associated with EVs, we mined the proteome data from MMV048-treated mice and performed an analysis involving intragroup and intergroup comparisons across all experimental conditions followed by a peptide compatibility analysis with predicted spectra to warrant robust identification. Only one protein fulfilled this stringent top-down selection, a putative filamin domain-containing protein. This study sets the stage to unveil biological features of human liver infections and identify biomarkers of hypnozoite infection associated with EVs.

Partial protection against P. vivax infection diminishes hypnozoite burden and blood-stage relapses.

Latent forms of Plasmodium vivax, called hypnozoites, cause malaria relapses from the liver into the bloodstream and are a major obstacle to malaria eradication. To experimentally assess the impact of a partially protective pre-erythrocytic vaccine on reducing Plasmodium vivax relapses, we developed a liver-humanized mouse model that allows monitoring of relapses directly in the blood. We passively infused these mice with a suboptimal dose of an antibody that targets the circumsporozoite protein prior to challenge with P. vivax sporozoites. Although this regimen did not completely prevent primary infection, antibody-treated mice experienced 62% fewer relapses. The data constitute unprecedented direct experimental evidence that suboptimal efficacy of infection-blocking antibodies, while not completely preventing primary infection, has a pronounced benefit in reducing the number of relapses. These findings suggest that a partially efficacious pre-erythrocytic Plasmodium vivax vaccine can have a disproportionately high impact in positive public health outcomes.

A Humanized Mouse Model for Plasmodium vivax to Test Interventions that Block Liver Stage to Blood Stage Transition and Blood Stage Infection.

The human malaria parasite Plasmodium vivax remains vastly understudied, mainly due to the lack of suitable laboratory models. Here, we report a humanized mouse model to test interventions that block P. vivax parasite transition from liver stage infection to blood stage infection. Human liver-chimeric FRGN huHep mice infected with P. vivax sporozoites were infused with human reticulocytes, allowing transition of exo-erythrocytic merozoites to reticulocyte infection and development into all erythrocytic forms, including gametocytes, in vivo. In order to test the utility of this model for preclinical assessment of interventions, the invasion blocking potential of a monoclonal antibody targeting the essential interaction of the P. vivax Duffy Binding Protein with the Duffy antigen receptor was tested by passive immunization. This antibody inhibited invasion by over 95%, providing unprecedented in vivo evidence that PvDBP constitutes a promising blood stage vaccine candidate and proving our model highly suitable to test blood stage interventions.

The Plasmodium liver-specific protein 2 (LISP2) is an early marker of liver stage development.

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.

Beyond Blood Smears: Qualification of Plasmodium 18S rRNA as a Biomarker for Controlled Human Malaria Infections.

18S rRNA is a biomarker that provides an alternative to thick blood smears in controlled human malaria infection (CHMI) trials. We reviewed data from CHMI trials at non-endemic sites that used blood smears and Plasmodium 18S rRNA/rDNA biomarker nucleic acid tests (NATs) for time to positivity. We validated a multiplex quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for Plasmodium 18S rRNA, prospectively compared blood smears and qRT-PCR for three trials, and modeled treatment effects at different biomarker-defined parasite densities to assess the impact on infection detection, symptom reduction, and measured intervention efficacy. Literature review demonstrated accelerated NAT-based infection detection compared with blood smears (mean acceleration: 3.2-3.6 days). For prospectively tested trials, the validated Plasmodium 18S rRNA qRT-PCR positivity was earlier (7.6 days; 95% CI: 7.1-8.1 days) than blood smears (11.0 days; 95% CI: 10.3-11.8 days) and significantly preceded the onset of grade 2 malaria-related symptoms (12.2 days; 95% CI: 10.6-13.3 days). Discrepant analysis showed that the risk of a blood smear-positive, biomarker-negative result was negligible. Data modeling predicted that treatment triggered by specific biomarker-defined thresholds can differentiate complete, partial, and non-protective outcomes and eliminate many grade 2 and most grade 3 malaria-related symptoms post-CHMI. Plasmodium 18S rRNA is a sensitive and specific biomarker that can justifiably replace blood smears for infection detection in CHMI trials in non-endemic settings. This study led to biomarker qualification through the U.S. Food and Drug Administration for use in CHMI studies at non-endemic sites, which will facilitate biomarker use for the qualified context of use in drug and vaccine trials.

The Micronemal Plasmodium Proteins P36 and P52 Act in Concert to Establish the Replication-Permissive Compartment Within Infected Hepatocytes.

Within the liver, Plasmodium sporozoites traverse cells searching for a "suitable" hepatocyte, invading these cells through a process that results in the formation of a parasitophorous vacuole (PV), within which the parasite undergoes intracellular replication as a liver stage. It was previously established that two members of the Plasmodium s48/45 protein family, P36 and P52, are essential for productive invasion of host hepatocytes by sporozoites as their simultaneous deletion results in growth-arrested parasites that lack a PV. Recent studies point toward a pathway of entry possibly involving the interaction of P36 with hepatocyte receptors EphA2, CD81, and SR-B1. However, the relationship between P36 and P52 during sporozoite invasion remains unknown. Here we show that parasites with a single P52 or P36 gene deletion each lack a PV after hepatocyte invasion, thereby pheno-copying the lack of a PV observed for the P52/P36 dual gene deletion parasite line. This indicates that both proteins are equally important in the establishment of a PV and act in the same pathway. We created a Plasmodium yoelii P36mCherry tagged parasite line that allowed us to visualize the subcellular localization of P36 and found that it partially co-localizes with P52 in the sporozoite secretory microneme organelles. Furthermore, through co-immunoprecipitation studies in vivo, we determined that P36 and P52 form a protein complex in sporozoites, indicating a concerted function for both proteins within the PV formation pathway. However, upon sporozoite stimulation, only P36 was released as a secreted protein while P52 was not. Our results support a model in which the putatively glycosylphosphatidylinositol (GPI)-anchored P52 may serve as a scaffold to facilitate the interaction of secreted P36 with the host cell during sporozoite invasion of hepatocytes.

Transcriptomic analysis reveals reduced transcriptional activity in the malaria parasite Plasmodium cynomolgi during progression into dormancy.

Relapses of Plasmodium dormant liver hypnozoites compromise malaria eradication efforts. New radical cure drugs are urgently needed, yet the vast gap in knowledge of hypnozoite biology impedes drug discovery. We previously unraveled the transcriptome of 6 to 7 day-old P. cynomolgi liver stages, highlighting pathways associated with hypnozoite dormancy (Voorberg-van der Wel et al., 2017). We now extend these findings by transcriptome profiling of 9 to 10 day-old liver stage parasites, thus revealing for the first time the maturation of the dormant stage over time. Although progression of dormancy leads to a 10-fold decrease in transcription and expression of only 840 genes, including genes associated with housekeeping functions, we show that pathways involved in quiescence, energy metabolism and maintenance of genome integrity remain the prevalent pathways active in mature hypnozoites.

A recombinant antibody against Plasmodium vivax UIS4 for distinguishing replicating from dormant liver stages.

BACKGROUND: Plasmodium vivax is the most geographically widespread of the human malaria parasites, causing 50,000 to 100,000 deaths annually. Plasmodium vivax parasites have the unique feature of forming dormant liver stages (hypnozoites) that can reactivate weeks or months after a parasite-infected mosquito bite, leading to new symptomatic blood stage infections. Efforts to eliminate P. vivax malaria likely will need to target the persistent hypnozoites in the liver. Therefore, research on P. vivax liver stages necessitates a marker for clearly distinguishing between actively replicating parasites and dormant hypnozoites. Hypnozoites possess a densely fluorescent prominence in the parasitophorous vacuole membrane (PVM) when stained with antibodies against the PVM-resident protein Upregulated in Infectious Sporozoites 4 (PvUIS4), resulting in a key feature recognizable for quantification of hypnozoites. Thus, PvUIS4 staining, in combination with the characteristic small size of the parasite, is currently the only hypnozoite-specific morphological marker available. RESULTS: Here, the generation and validation of a recombinant monoclonal antibody against PvUIS4 (α-rUIS4 mAb) is described. The variable heavy and light chain domains of an α-PvUIS4 hybridoma were cloned into murine IgG1 and IgK expression vectors. These expression plasmids were co-transfected into HEK293 cells and mature IgG was purified from culture supernatants. It is shown that the α-rUIS4 mAb binds to its target with high affinity. It reliably stains the schizont PVM and the hypnozoite-specific PVM prominence, enabling the visual differentiation of hypnozoites from replicating liver stages by immunofluorescence assays in different in vitro settings, as well as in liver sections from P. vivax infected liver-chimeric mice. The antibody functions reliably against all four parasite isolates tested and will be an important tool in the identification of the elusive hypnozoite. CONCLUSIONS: The α-rUIS4 mAb is a versatile tool for distinguishing replicating P. vivax liver stages from dormant hypnozoites, making it a valuable resource that can be deployed throughout laboratories worldwide.

Characterization of Plasmodium vivax Proteins in Plasma-Derived Exosomes From Malaria-Infected Liver-Chimeric Humanized Mice.

Exosomes are extracellular vesicles of endocytic origin containing molecular signatures implying the cell of origin; thus, they offer a unique opportunity to discover biomarkers of disease. Plasmodium vivax, responsible for more than half of all malaria cases outside Africa, is a major obstacle in the goal of malaria elimination due to the presence of dormant liver stages (hypnozoites), which after the initial infection may reactivate to cause disease. Hypnozoite infection is asymptomatic and there are currently no diagnostic tools to detect their presence. The human liver-chimeric (FRG huHep) mouse is a robust P. vivax infection model for exo-erythrocytic development of liver stages, including hypnozoites. We studied the proteome of plasma-derived exosomes isolated from P. vivax infected FRG huHep mice with the objective of identifying liver-stage expressed parasite proteins indicative of infection. Proteomic analysis of these exosomes showed the presence of 290 and 234 proteins from mouse and human origin, respectively, including canonical exosomal markers. Human proteins include proteins previously detected in liver-derived exosomes, highlighting the potential of this chimeric mouse model to study plasma exosomes derived unequivocally from human hepatocytes. Noticeably, we identified 17 parasite proteins including enzymes, surface proteins, components of the endocytic pathway and translation machinery, as well as uncharacterized proteins. Western blot analysis validated the presence of human arginase-I and an uncharacterized P. vivax protein in plasma-derived exosomes. This study represents a proof-of-principle that plasma-derived exosomes from P. vivax infected FRG-huHep mice contain human hepatocyte and P. vivax proteins with the potential to unveil biological features of liver infection and identify biomarkers of hypnozoite infection.

Author Correction: A comprehensive model for assessment of liver stage therapies targeting Plasmodium vivax and Plasmodium falciparum.

The original version of this Article contained an error in the spelling of Richard Thomson-Luque, which was incorrectly given as Richard Thomson Luque. This error has now been corrected in both the PDF and HTML versions of the Article.

A comprehensive model for assessment of liver stage therapies targeting Plasmodium vivax and Plasmodium falciparum.

Malaria liver stages represent an ideal therapeutic target with a bottleneck in parasite load and reduced clinical symptoms; however, current in vitro pre-erythrocytic (PE) models for Plasmodium vivax and P. falciparum lack the efficiency necessary for rapid identification and effective evaluation of new vaccines and drugs, especially targeting late liver-stage development and hypnozoites. Herein we report the development of a 384-well plate culture system using commercially available materials, including cryopreserved primary human hepatocytes. Hepatocyte physiology is maintained for at least 30 days and supports development of P. vivax hypnozoites and complete maturation of P. vivax and P. falciparum schizonts. Our multimodal analysis in antimalarial therapeutic research identifies important PE inhibition mechanisms: immune antibodies against sporozoite surface proteins functionally inhibit liver stage development and ion homeostasis is essential for schizont and hypnozoite viability. This model can be implemented in laboratories in disease-endemic areas to accelerate vaccine and drug discovery research.

Plasmodium falciparum Liver Stage Infection and Transition to Stable Blood Stage Infection in Liver-Humanized and Blood-Humanized FRGN KO Mice Enables Testing of Blood Stage Inhibitory Antibodies (Reticulocyte-Binding Protein Homolog 5) In Vivo.

The invention of liver-humanized mouse models has made it possible to directly study the preerythrocytic stages of Plasmodium falciparum. In contrast, the current models to directly study blood stage infection in vivo are extremely limited. Humanization of the mouse blood stream is achievable by frequent injections of human red blood cells (hRBCs) and is currently the only system with which to study human malaria blood stage infections in a small animal model. Infections have been primarily achieved by direct injection of P. falciparum-infected RBCs but as such, this modality of infection does not model the natural route of infection by mosquito bite and lacks the transition of parasites from liver stage infection to blood stage infection. Including these life cycle transition points in a small animal model is of relevance for testing therapeutic interventions. To this end, we used FRGN KO mice that were engrafted with human hepatocytes and performed a blood exchange under immune modulation to engraft the animals with more than 50% hRBCs. These mice were infected by mosquito bite with sporozoite stages of a luciferase-expressing P. falciparum parasite, resulting in noninvasively measurable liver stage burden by in vivo bioluminescent imaging (IVIS) at days 5-7 postinfection. Transition to blood stage infection was observed by IVIS from day 8 onward and then blood stage parasitemia increased with a kinetic similar to that observed in controlled human malaria infection. To assess the utility of this model, we tested whether a monoclonal antibody targeting the erythrocyte invasion ligand reticulocyte-binding protein homolog 5 (with known growth inhibitory activity in vitro) was capable of blocking blood stage infection in vivo when parasites emerge from the liver and found it highly effective. Together, these results show that a combined liver-humanized and blood-humanized FRGN mouse model infected with luciferase-expressing P. falciparum will be a useful tool to study P. falciparum preerythrocytic and erythrocytic stages and enables the testing of interventions that target either one or both stages of parasite infection.

In Vitro Culture, Drug Sensitivity, and Transcriptome of Plasmodium Vivax Hypnozoites.

The unique relapsing nature of Plasmodium vivax infection is a major barrier to malaria eradication. Upon infection, dormant liver-stage forms, hypnozoites, linger for weeks to months and then relapse to cause recurrent blood-stage infection. Very little is known about hypnozoite biology; definitive biomarkers are lacking and in vitro platforms that support phenotypic studies are needed. Here, we recapitulate the entire liver stage of P. vivax in vitro, using a multiwell format that incorporates micropatterned primary human hepatocyte co-cultures (MPCCs). MPCCs feature key aspects of P. vivax biology, including establishment of persistent small forms and growing schizonts, merosome release, and subsequent infection of reticulocytes. We find that the small forms exhibit previously described hallmarks of hypnozoites, and we pilot MPCCs as a tool for testing candidate anti-hypnozoite drugs. Finally, we employ a hybrid capture strategy and RNA sequencing to describe the hypnozoite transcriptome and gain insight into its biology.

Immunization of Malaria-Preexposed Volunteers With PfSPZ Vaccine Elicits Long-Lived IgM Invasion-Inhibitory and Complement-Fixing Antibodies.

Background: The assessment of antibody responses after immunization with radiation-attenuated, aseptic, purified, cryopreserved Plasmodium falciparum sporozoites (Sanaria PfSPZ Vaccine) has focused on IgG isotype antibodies. Here, we aimed to investigate if P. falciparum sporozoite binding and invasion-inhibitory IgM antibodies are induced following immunization of malaria-preexposed volunteers with PfSPZ Vaccine. Methods: Using serum from volunteers immunized with PfSPZ, we measured vaccine-induced IgG and IgM antibodies to P. falciparum circumsporozoite protein (PfCSP) via ELISA. Function of this serum as well as IgM antibody fractions was measured via in vitro in an inhibition of sporozoite invasion assay. These IgM antibody fractions were also measured for binding to sporozoites by immunofluorescence assay and complement fixation on whole sporozoites. Results: We found that in addition to anti-PfCSP IgG, malaria-preexposed volunteers developed anti-PfCSP IgM antibodies after immunization with PfSPZ Vaccine and that these IgM antibodies inhibited P. falciparum sporozoite invasion of hepatocytes in vitro. These IgM plasma fractions also fixed complement to whole P. falciparum sporozoites. Conclusions: This is the first finding that PfCSP and P. falciparum sporozoite-binding IgM antibodies are induced following immunization of PfSPZ Vaccine in malaria-preexposed individuals and that IgM antibodies can inhibit P. falciparum sporozoite invasion into hepatocytes in vitro and fix complement on sporozoites. These findings indicate that the immunological assessment of PfSPZ Vaccine-induced antibody responses could be more sensitive if they include parasite-specific IgM in addition to IgG antibodies. Clinical Trials Registration: NCT02132299.

Assessing drug efficacy against Plasmodium falciparum liver stages in vivo.

Malaria eradication necessitates new tools to fight the evolving and complex Plasmodium pathogens. These tools include prophylactic drugs that eliminate Plasmodium liver stages and consequently prevent clinical disease, decrease transmission, and reduce the propensity for resistance development. Currently, the identification of these drugs relies on in vitro P. falciparum liver stage assays or in vivo causal prophylaxis assays using rodent malaria parasites; there is no method to directly test in vivo liver stage activity of candidate antimalarials against the human malaria-causing parasite P. falciparum. Here, we use a liver-chimeric humanized mouse (FRG huHep) to demonstrate in vivo P. falciparum liver stage development and describe the efficacy of clinically used and candidate antimalarials with prophylactic activity. We show that daily administration of atovaquone-proguanil (ATQ-PG; ATQ, 30 mg/kg, and PG, 10 mg/kg) protects 5 of 5 mice from liver stage infection, consistent with the use in humans as a causal prophylactic drug. Single-dose primaquine (60 mg/kg) has similar activity to that observed in humans, demonstrating the activity of this drug (and its active metabolites) in FRG huHep mice. We also show that DSM265, a selective Plasmodial dihydroorotate dehydrogenase inhibitor with causal prophylactic activity in humans, reduces liver stage burden in FRG huHep mice. Finally, we measured liver stage-to-blood stage transition of the parasite, the ultimate readout of prophylactic activity and measurement of infective capacity of parasites in the liver, to show that ATQ-PG reduces blood stage patency to below the limit of quantitation by quantitative PCR (qPCR). The FRG huHep model, thus, provides a platform for preclinical evaluation of drug candidates for liver stage causal prophylactic activity, pharmacokinetic/pharmacodynamics studies, and biological studies to investigate the mechanism of action of liver stage active antimalarials.