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.

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.

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.

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.

Host-based Prophylaxis Successfully Targets Liver Stage Malaria Parasites.

Eliminating malaria parasites during the asymptomatic but obligate liver stages (LSs) of infection would stop disease and subsequent transmission. Unfortunately, only a single licensed drug that targets all LSs, Primaquine, is available. Targeting host proteins might significantly expand the repertoire of prophylactic drugs against malaria. Here, we demonstrate that both Bcl-2 inhibitors and P53 agonists dramatically reduce LS burden in a mouse malaria model in vitro and in vivo by altering the activity of key hepatocyte factors on which the parasite relies. Bcl-2 inhibitors act primarily by inducing apoptosis in infected hepatocytes, whereas P53 agonists eliminate parasites in an apoptosis-independent fashion. In combination, Bcl-2 inhibitors and P53 agonists act synergistically to delay, and in some cases completely prevent, the onset of blood stage disease. Both families of drugs are highly effective at doses that do not cause substantial hepatocyte cell death in vitro or liver damage in vivo. P53 agonists and Bcl-2 inhibitors were also effective when administered to humanized mice infected with Plasmodium falciparum. Our data demonstrate that host-based prophylaxis could be developed into an effective intervention strategy that eliminates LS parasites before the onset of clinical disease and thus opens a new avenue to prevent malaria.

Susceptibility to Plasmodium yoelii preerythrocytic infection in BALB/c substrains is determined at the point of hepatocyte invasion.

After transmission by Anopheles mosquitoes, Plasmodium sporozoites travel to the liver, infect hepatocytes, and rapidly develop as intrahepatocytic liver stages (LS). Rodent models of malaria exhibit large differences in the magnitude of liver infection, both between parasite species and between strains of mice. This has been mainly attributed to differences in innate immune responses and parasite infectivity. Here, we report that BALB/cByJ mice are more susceptible to Plasmodium yoelii preerythrocytic infection than BALB/cJ mice. This difference occurs at the level of early hepatocyte infection, but expression levels of reported host factors that are involved in infection do not correlate with susceptibility. Interestingly, BALB/cByJ hepatocytes are more frequently polyploid; thus, their susceptibility converges on the previously observed preference of sporozoites to infect polyploid hepatocytes. Gene expression analysis demonstrates hepatocyte-specific differences in mRNA abundance for numerous genes between BALB/cByJ and BALB/cJ mice, some of which encode hepatocyte surface molecules. These data suggest that a yet-unknown receptor for sporozoite infection, present at elevated levels on BALB/cByJ hepatocytes and also polyploid hepatocytes, might facilitate Plasmodium liver infection.

Of men in mice: the success and promise of humanized mouse models for human malaria parasite infections.

Forty percent of people worldwide are at risk of malaria infection, and despite control efforts it remains the most deadly parasitic disease. Unfortunately, rapid discovery and development of new interventions for malaria are hindered by the lack of small animal models that support the complex life cycles of the main parasite species infecting humans. Such tools must accommodate human parasite tropism for human tissue. Mouse models with human tissue developed to date have already enhanced our knowledge of human parasites, and are useful tools for assessing anti-parasitic interventions. Although these systems are imperfect, their continued refinement will likely broaden their utility. Some of the malaria parasite's interactions with human hepatocytes and human erythrocytes can already be modelled with available humanized mouse systems. However, interactions with other relevant human tissues such as the skin and immune system, as well as most transitions between life cycle stages in vivo will require refinement of existing humanized mouse models. Here, we review the recent successes achieved in modelling human malaria parasite biology in humanized mice, and discuss how these models have potential to become a valuable part of the toolbox used for understanding the biology of, and development of interventions to, malaria.

A next-generation genetically attenuated Plasmodium falciparum parasite created by triple gene deletion.

Immunization with live-attenuated Plasmodium sporozoites completely protects against malaria infection. Genetic engineering offers a versatile platform to create live-attenuated sporozoite vaccine candidates. We previously generated a genetically attenuated parasite (GAP) by deleting the P52 and P36 genes in the NF54 wild-type (WT) strain of Plasmodium falciparum (Pf p52(-)/p36(-) GAP). Preclinical assessment of p52(-)/p36(-) GAP in a humanized mouse model indicated an early and severe liver stage growth defect. However, human exposure to >200 Pf p52(-)/p36(-) GAP-infected mosquito bites in a safety trial resulted in peripheral parasitemia in one of six volunteers, revealing that this GAP was incompletely attenuated. We have now created a triple gene deleted GAP by additionally removing the SAP1 gene (Pf p52(-)/p36(-)/sap1(-) GAP) and employed flippase (FLP)/flippase recognition target (FRT) recombination for drug selectable marker cassette removal. This next-generation GAP was indistinguishable from WT parasites in blood stage and mosquito stage development. Using an improved humanized mouse model transplanted with human hepatocytes and human red blood cells, we show that despite a high-dose sporozoite challenge, Pf p52(-)/p36(-)/sap1(-) GAP did not transition to blood stage infection and appeared to be completely attenuated. Thus, clinical testing of Pf p52(-)/p36(-)/sap1(-) GAP assessing safety, immunogenicity, and efficacy against sporozoite challenge is warranted.

Neuroimmunological blood brain barrier opening in experimental cerebral malaria.

Plasmodium falciparum malaria is responsible for nearly one million annual deaths worldwide. Because of the difficulty in monitoring the pathogenesis of cerebral malaria in humans, we conducted a study in various mouse models to better understand disease progression in experimental cerebral malaria (ECM). We compared the effect on the integrity of the blood brain barrier (BBB) and the histopathology of the brain of P. berghei ANKA, a known ECM model, P. berghei NK65, generally thought not to induce ECM, P. yoelii 17XL, originally reported to induce human cerebral malaria-like histopathology, and P. yoelii YM. As expected, P. berghei ANKA infection caused neurological signs, cerebral hemorrhages, and BBB dysfunction in CBA/CaJ and Swiss Webster mice, while Balb/c and A/J mice were resistant. Surprisingly, PbNK induced ECM in CBA/CaJ mice, while all other mice were resistant. P. yoelii 17XL and P. yoelii YM caused lethal hyperparasitemia in all mouse strains; histopathological alterations, BBB dysfunction, or neurological signs were not observed. Intravital imaging revealed that infected erythrocytes containing mature parasites passed slowly through capillaries making intimate contact with the endothelium, but did not arrest. Except for relatively rare microhemorrhages, mice with ECM presented no obvious histopathological alterations that would explain the widespread disruption of the BBB. Intravital imaging did reveal, however, that postcapillary venules, but not capillaries or arterioles, from mice with ECM, but not hyperparasitemia, exhibit platelet marginalization, extravascular fibrin deposition, CD14 expression, and extensive vascular leakage. Blockage of LFA-1 mediated cellular interactions prevented leukocyte adhesion, vascular leakage, neurological signs, and death from ECM. The endothelial barrier-stabilizing mediators imatinib and FTY720 inhibited vascular leakage and neurological signs and prolonged survival to ECM. Thus, it appears that neurological signs and coma in ECM are due to regulated opening of paracellular-junctional and transcellular-vesicular fluid transport pathways at the neuroimmunological BBB.

Perturbations of Plasmodium Puf2 expression and RNA-seq of Puf2-deficient sporozoites reveal a critical role in maintaining RNA homeostasis and parasite transmissibility.

Malaria's cycle of infection requires parasite transmission between a mosquito vector and a mammalian host. We here demonstrate that the Plasmodium yoelii Pumilio-FBF family member Puf2 allows the sporozoite to remain infectious in the mosquito salivary glands while awaiting transmission. Puf2 mediates this solely through its RNA-binding domain (RBD) likely by stabilizing or hastening the degradation of specific mRNAs. Puf2 traffics to sporozoite cytosolic granules, which are negative for several markers of stress granules and P-bodies, and disappear rapidly after infection of hepatocytes. In contrast to previously described Plasmodium berghei pbpuf2(-) parasites, pypuf2(-) sporozoites have no apparent defect in host infection when tested early in salivary gland residence, but become progressively non-infectious and prematurely transform into EEFs during prolonged salivary gland residence. The premature overexpression of Puf2 in oocysts causes striking deregulation of sporozoite maturation and infectivity while extension of Puf2 expression in liverstages causes no defect, suggesting that the presence of Puf2 alone is not sufficient for its functions. Finally, by conducting the first comparative RNA-seq analysis of Plasmodium sporozoites, we find that Puf2 may play a role in directly or indirectly maintaining the homeostasis of specific transcripts. These findings uncover requirements for maintaining a window of opportunity for the malaria parasite to accommodate the unpredictable moment of transmission from mosquito to mammalian host.

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.

Plasmodium yoelii macrophage migration inhibitory factor is necessary for efficient liver-stage development.

Mammalian macrophage migration inhibitory factor (MIF) is a multifaceted cytokine involved in both extracellular and intracellular functions. Malaria parasites express a MIF homologue that might modulate host immune responses against blood-stage parasites, but the potential importance of MIF against other life cycle stages remains unstudied. In this study, we characterized the MIF homologue of Plasmodium yoelii throughout the life cycle, with emphasis on preerythrocytic stages. P. yoelii MIF (Py-MIF) was expressed in blood-stage parasites and detected at low levels in mosquito salivary gland sporozoites. MIF expression was strong throughout liver-stage development and localized to the cytoplasm of the parasite, with no evidence of release into the host hepatocyte. To examine the importance of Py-MIF for liver-stage development, we generated a Py-mif knockout parasite (P. yoelii Δmif). P. yoelii Δmif parasites grew normally as asexual erythrocytic-stage parasites and showed normal infection of mosquitoes. In contrast, the P. yoelii Δmif strain was attenuated during the liver stage. Mice infected with P. yoelii Δmif sporozoites either did not develop blood-stage parasitemia or exhibited a delay in the onset of blood-stage patency. Furthermore, P. yoelii Δmif parasites exhibited growth retardation in vivo. Combined, the data indicate that Plasmodium MIF is important for liver-stage development of P. yoelii, during which it is likely to play an intrinsic role in parasite development rather than modulating host immune responses to infection.

Disruption of the Plasmodium falciparum liver-stage antigen-1 locus causes a differentiation defect in late liver-stage parasites.

The malaria parasite Plasmodium falciparum infects humans and first targets the liver where liver-stage parasites undergo pre-erythrocytic replication. Liver-stage antigen-1 (LSA-1) is currently the only identified P. falciparum protein for which expression is restricted to liver stages. Yet, the importance of LSA-1 for liver-stage parasite development remains unknown. Here we deleted LSA-1 in the NF54 strain of P. falciparum and analysed the lsa-1(-) parasites throughout their life cycle. lsa-1(-) sporozoites had normal gliding motility and invasion into hepatocytes. Six days after infection of a hepatocytic cell line, lsa-1(-) parasites exhibited a moderate phenotype with an ~50% reduction of late liver-stage forms when compared with wild type. Strikingly, lsa-1(-) parasites growing in SCID/Alb-uPA mice with humanized livers showed a severe defect in late liver-stage differentiation and exo-erythrocytic merozoite formation 7 days after infection, a time point when wild-type parasites develop into mature merozoites. The lsa-1(-) parasites also showed aberrant liver-stage expression of key parasite proteins apical membrane antigen-1 and circumsporozoite protein. Our data show that LSA-1 plays a critical role during late liver-stage schizogony and is thus important in the parasite transition from the liver to blood. LSA-1 is the first P. falciparum protein identified to be required for this transitional stage of the parasite life cycle.

Preerythrocytic, live-attenuated Plasmodium falciparum vaccine candidates by design.

Falciparum malaria is initiated when Anopheles mosquitoes transmit the Plasmodium sporozoite stage during a blood meal. Irradiated sporozoites confer sterile protection against subsequent malaria infection in animal models and humans. This level of protection is unmatched by current recombinant malaria vaccines. However, the live-attenuated vaccine approach faces formidable obstacles, including development of accurate, reproducible attenuation techniques. We tested whether Plasmodium falciparum could be attenuated at the early liver stage by genetic engineering. The P. falciparum genetically attenuated parasites (GAPs) harbor individual deletions or simultaneous deletions of the sporozoite-expressed genes P52 and P36. Gene deletions were done by double-cross-over recombination to avoid genetic reversion of the knockout parasites. The gene deletions did not affect parasite replication throughout the erythrocytic cycle, gametocyte production, mosquito infections, and sporozoite production rates. However, the deletions caused parasite developmental arrest during hepatocyte infection. The double-gene deletion line exhibited a more severe intrahepatocytic growth defect compared with the single-gene deletion lines, and it did not persist. This defect was assessed in an in vitro liver-stage growth assay and in a chimeric mouse model harboring human hepatocytes. The strong phenotype of the double knockout GAP justifies its human testing as a whole-organism vaccine candidate using the established sporozoite challenge model. GAPs might provide a safe and reproducible platform to develop an efficacious whole-cell malaria vaccine that prevents infection at the preerythrocytic stage.

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.

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.

The modulation of CD40 ligand signaling by transmembrane CD28 splice variant in human T cells.

The role of CD40 ligand (CD40L)/CD40 signaling in T cell-dependent B cell differentiation and maturation has been amply documented. The mechanism of CD40 signaling in B cells has been well established, whereas the signaling mechanism of CD40L in T cell costimulation remains unknown. In this study we show that CD28i, a transmembrane splice variant of CD28 costimulatory receptor, complexes with CD40L in human T cells. The cross-linking of CD40L resulted in the coendocytosis of CD28i with CD40L. The tyrosine phosphorylation of CD28i followed the cross-linking of CD40L, and the overexpression of CD28i augmented the c-Jun NH2-terminal kinase, p21-activated kinase 2, and nuclear factor kappaB activation. These data indicate that CD28i, by functioning as a signaling adaptor, transduces CD40L signaling as well as CD28 signaling in human T cells.

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.

Targeted deletion of SAP1 abolishes the expression of infectivity factors necessary for successful malaria parasite liver infection.

Malaria parasite sporozoites prepare for transmission to a mammalian host by upregulation of UIS (Upregulated in Infectious Sporozoites) genes. A number of UIS gene products are essential for the establishment of the intrahepatocytic niche. However, the factors that regulate the expression of genes involved in gain of infectivity for the liver are unknown. Herein, we show that a conserved Plasmodium sporozoite low-complexity asparagine-rich protein, SAP1 (Sporozoite Asparagine-rich Protein 1), has an essential role in malaria parasite liver infection. Targeted deletion of SAP1 in the rodent malaria parasite Plasmodium yoelii generated mutant parasites that traverse and invade hepatocytes normally but cannot initiate liver-stage development in vitro and in vivo. Moreover, immunizations with Pysap1(-) sporozoites confer long-lasting sterile protection against wild-type sporozoite infection. Strikingly, lack of SAP1 abolished expression of essential UIS genes including UIS3, UIS4 and P52 but not the constitutively expressed genes encoding, among others, sporozoite proteins CSP and TRAP. SAP1 localization to the cell interior but not the nucleus of sporozoites suggests its involvement in a post-transcriptional mechanism of gene expression control. These findings demonstrate that SAP1 is essential for liver infection possibly by functioning as a selective regulator controlling the expression of infectivity-associated parasite effector genes.

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.