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.

Tryptophan C-mannosylation is critical for Plasmodium falciparum transmission.

Tryptophan C-mannosylation stabilizes proteins bearing a thrombospondin repeat (TSR) domain in metazoans. Here we show that Plasmodium falciparum expresses a DPY19 tryptophan C-mannosyltransferase in the endoplasmic reticulum and that DPY19-deficiency abolishes C-glycosylation, destabilizes members of the TRAP adhesin family and inhibits transmission to mosquitoes. Imaging P. falciparum gametogenesis in its entirety in four dimensions using lattice light-sheet microscopy reveals defects in ΔDPY19 gametocyte egress and exflagellation. While egress is diminished, ΔDPY19 microgametes still fertilize macrogametes, forming ookinetes, but these are abrogated for mosquito infection. The gametogenesis defects correspond with destabilization of MTRAP, which we show is C-mannosylated in P. falciparum, and the ookinete defect is concordant with defective CTRP secretion on the ΔDPY19 background. Genetic complementation of DPY19 restores ookinete infectivity, sporozoite production and C-mannosylation activity. Therefore, tryptophan C-mannosylation by DPY19 ensures TSR protein quality control at two lifecycle stages for successful transmission of the human malaria parasite.

Epitope-coated polymer particles elicit neutralising antibodies against Plasmodium falciparum sporozoites.

The current Malaria RTS,S vaccine is based on virus-like particles (VLPs) comprising the NANP repetitive epitopes from the cicumsporozoite protein (CSP) of Plasmodium falciparum. This vaccine has limited efficacy, only preventing severe disease in about 30% of vaccinated individuals. A more efficacious vaccine is urgently needed to combat malaria. Here we developed a particulate malaria vaccine based on the same CSP epitopes but using biopolymer particles (BPs) as an antigen carrier system. Specific B- and T-cell epitope-coated BPs were assembled in vivo inside an engineered endotoxin-free mutant of Escherichia coli. A high-yield production process leading to ~27% BP vaccine weight over biomass was established. The epitope-coated BPs were purified and their composition, i.e., the polymer core and epitope identity, was confirmed. Epitope-coated BPs were used alongside soluble peptide epitopes and empty BPs to vaccinate sheep. Epitope-coated BPs showed enhanced immunogenicity by inducing anti-NANP antibody titre of EC50 > 150,000 that were at least 20 times higher than induced by the soluble peptides. We concluded that the additional T-cell epitope was not required as it did not enhance immunogenicity when compared with the B-cell epitope-coated BPs. Antibodies specifically bound to the surface of Plasmodium falciparum sporozoites and efficiently inhibited sporozoite motility and traversal of human hepatocytes. This study demonstrated the utility of biologically self-assembled epitope-coated BPs as an epitope carrier for inclusion in next-generation malaria vaccines.

Property activity refinement of 2-anilino 4-amino substituted quinazolines as antimalarials with fast acting asexual parasite activity.

Malaria is a devastating disease caused by Plasmodium parasites. Emerging resistance against current antimalarial therapeutics has engendered the need to develop antimalarials with novel structural classes. We recently described the identification and initial optimization of the 2-anilino quinazoline antimalarial class. Here, we refine the physicochemical properties of this antimalarial class with the aim to improve aqueous solubility and metabolism and to reduce adverse promiscuity. We show the physicochemical properties of this class are intricately balanced with asexual parasite activity and human cell cytotoxicity. Structural modifications we have implemented improved LipE, aqueous solubility and in vitro metabolism while preserving fast acting P. falciparum asexual stage activity. The lead compounds demonstrated equipotent activity against P. knowlesi parasites and were not predisposed to resistance mechanisms of clinically used antimalarials. The optimized compounds exhibited modest activity against early-stage gametocytes, but no activity against pre-erythrocytic liver parasites. Confoundingly, the refined physicochemical properties installed in the compounds did not engender improved oral efficacy in a P. berghei mouse model of malaria compared to earlier studies on the 2-anilino quinazoline class. This study provides the framework for further development of this antimalarial class.

Platelet derived growth factor receptor ß (PDGFRß) is a host receptor for the human malaria parasite adhesin TRAP.

Following their inoculation by the bite of an infected Anopheles mosquito, the malaria parasite sporozoite forms travel from the bite site in the skin into the bloodstream, which transports them to the liver. The thrombospondin-related anonymous protein (TRAP) is a type 1 transmembrane protein that is released from secretory organelles and relocalized on the sporozoite plasma membrane. TRAP is required for sporozoite motility and host infection, and its extracellular portion contains adhesive domains that are predicted to engage host receptors. Here, we identified the human platelet-derived growth factor receptor ß (hPDGFRß) as one such protein receptor. Deletion constructs showed that the von Willebrand factor type A and thrombospondin repeat domains of TRAP are both required for optimal binding to hPDGFRß-expressing cells. We also demonstrate that this interaction is conserved in the human-infective parasite Plasmodium vivax, but not the rodent-infective parasite Plasmodium yoelii. We observed expression of hPDGFRß mainly in cells associated with the vasculature suggesting that TRAP:hPDGFRß interaction may play a role in the recognition of blood vessels by invading sporozoites.

Targeting the Extrinsic Pathway of Hepatocyte Apoptosis Promotes Clearance of Plasmodium Liver Infection.

Plasmodium sporozoites infect the liver and develop into exoerythrocytic merozoites that initiate blood-stage disease. The hepatocyte molecular pathways that permit or abrogate parasite replication and merozoite formation have not been thoroughly explored, and a deeper understanding may identify therapeutic strategies to mitigate malaria. Cellular inhibitor of apoptosis (cIAP) proteins regulate cell survival and are co-opted by intracellular pathogens to support development. Here, we show that cIAP1 levels are upregulated during Plasmodium liver infection and that genetic or pharmacological targeting of cIAPs using clinical-stage antagonists preferentially kills infected hepatocytes and promotes immunity. Using gene-targeted mice, the mechanism was defined as TNF-TNFR1-mediated apoptosis via caspases 3 and 8 to clear parasites. This study reveals the importance of cIAPs to Plasmodium infection and demonstrates that host-directed antimalarial drugs can eliminate liver parasites and induce immunity while likely providing a high barrier to resistance in the parasite.

Dual Plasmepsin-Targeting Antimalarial Agents Disrupt Multiple Stages of the Malaria Parasite Life Cycle.

Artemisin combination therapy (ACT) is the main treatment option for malaria, which is caused by the intracellular parasite Plasmodium. However, increased resistance to ACT highlights the importance of finding new drugs. Recently, the aspartic proteases Plasmepsin IX and X (PMIX and PMX) were identified as promising drug targets. In this study, we describe dual inhibitors of PMIX and PMX, including WM382, that block multiple stages of the Plasmodium life cycle. We demonstrate that PMX is a master modulator of merozoite invasion and direct maturation of proteins required for invasion, parasite development, and egress. Oral administration of WM382 cured mice of P. berghei and prevented blood infection from the liver. In addition, WM382 was efficacious against P. falciparum asexual infection in humanized mice and prevented transmission to mosquitoes. Selection of resistant P. falciparum in vitro was not achievable. Together, these show that dual PMIX and PMX inhibitors are promising candidates for malaria treatment and prevention.

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.

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.

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.

Plasmodium yoelii S4/CelTOS is important for sporozoite gliding motility and cell traversal.

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

Humoral protection against mosquito bite-transmitted Plasmodium falciparum infection in humanized mice.

A malaria vaccine that prevents infection will be an important new tool in continued efforts of malaria elimination, and such vaccines are under intense development for the major human malaria parasite Plasmodium falciparum (Pf). Antibodies elicited by vaccines can block the initial phases of parasite infection when sporozoites are deposited into the skin by mosquito bite and then target the liver for further development. However, there are currently no standardized in vivo preclinical models that can measure the inhibitory activity of antibody specificities against Pf sporozoite infection via mosquito bite. Here, we use human liver-chimeric mice as a challenge model to assess prevention of natural Pf sporozoite infection by antibodies. We demonstrate that these mice are consistently infected with Pf by mosquito bite and that this challenge can be combined with passive transfer of either monoclonal antibodies or polyclonal human IgG from immune serum to measure antibody-mediated blocking of parasite infection using bioluminescent imaging. This methodology is useful to down-select functional antibodies and to investigate mechanisms or immune correlates of protection in clinical trials, thereby informing rational vaccine optimization.

Complete attenuation of genetically engineered Plasmodium falciparum sporozoites in human subjects.

Immunization of humans with whole sporozoites confers complete, sterilizing immunity against malaria infection. However, achieving consistent safety while maintaining immunogenicity of whole parasite vaccines remains a formidable challenge. We generated a genetically attenuated Plasmodium falciparum (Pf) malaria parasite by deleting three genes expressed in the pre-erythrocytic stage (Pf p52-/p36-/sap1-). We then tested the safety and immunogenicity of the genetically engineered (Pf GAP3KO) sporozoites in human volunteers. Pf GAP3KO sporozoites were delivered to 10 volunteers using infected mosquito bites with a single exposure consisting of 150 to 200 bites per subject. All subjects remained blood stage-negative and developed inhibitory antibodies to sporozoites. GAP3KO rodent malaria parasites engendered complete, protracted immunity against infectious sporozoite challenge in mice. The results warrant further clinical testing of Pf GAP3KO and its potential development into a vaccine strain.

An Opsonic Phagocytosis Assay for Plasmodium falciparum Sporozoites.

Plasmodium falciparum malaria remains the deadliest parasitic disease worldwide. Vaccines targeting the preerythrocytic sporozoite and liver stages have the potential to entirely prevent blood-stage infection and disease, as well as onward transmission. Sporozoite surface and secreted proteins are leading candidates for inclusion in a preerythrocytic stage-specific, antibody-based vaccine. Preclinical functional assays to identify humoral correlates of protection in vitro and to validate novel sporozoite protein targets for inclusion in multisubunit vaccines currently do not consider the interaction of sporozoite-targeting antibodies with other components of the immune system. Here, we describe the development of a simple flow cytometric assay to quantitatively assess the ability of antibodies directed against P. falciparum sporozoites to facilitate their phagocytosis. We demonstrate that this sporozoite opsonic phagocytosis assay (SOPA) is compatible with both monoclonal antibodies and human immune serum and can be performed using cryopreserved P. falciparum sporozoites. This simple, accessible assay will aid with the assessment of antibody responses to vaccination with Plasmodium antigens and their interaction with phagocytic cells of the immune system.

Delta-9-tetrahydrocannabinol disrupts hippocampal neuroplasticity and neurogenesis in trained, but not untrained adolescent Sprague-Dawley rats.

Cannabis is the most widely used illicit drug, and disruption of learning and memory are commonly reported consequences of cannabis use. We have previously demonstrated a spatial learning impairment by ∆(9)-tetrahydrocannabinol (THC) in adolescent Sprague-Dawley rats (Steel et al., 2011). The molecular mechanisms underlying behavioural impairment by cannabis remain poorly understood, although the importance of adaptive changes in neuroplasticity (synaptic number and strength) and neurogenesis during learning are accepted. Here we aimed to identify any effects of THC on the early induction of these adaptive processes supporting learning, so we conducted our analyses at the mid-training point of our previous study. Both untrained and trained (15 days of training) adolescent (P28-P42) Sprague-Dawley rats were treated daily with THC (6 mg/kg i.p.) or its vehicle, and changes in the levels of markers of hippocampal neuroplasticity (CB1R, PSD95, synapsin-I, synapsin-III) and neurogenesis (Ki67, DCX, PSA-NCAM, BrdU labelling) by training were measured. Training of control animals, but not THC-treated animals increased neuroplasticity marker levels. However training of THC-treated animals, but not control animals reduced immature neuronal marker levels. Levels of hippocampal proliferation, and survival of the BrdU-labelled progeny of these divisions were unaffected by THC in trained and untrained animals. These data show a smaller neuroplastic response, and a reduction of new-born neuronal levels not attributable to effects on proliferation or survival by THC-treatment during training. Importantly no effects of THC were seen in the absence of training, indicating that these effects represent specific impairments by THC on training-induced responses.

Learning impairment by Δ(9)-tetrahydrocannabinol in adolescence is attributable to deficits in chunking.

Cannabis is the most popular illicit drug used by adolescents. Yet, there are only a few studies that have examined the effects of cannabis use on learning and memory during this sensitive and important neurodevelopmental stage. Male adolescent Sprague-Dawley rats were treated with Δ(9)-tetrahydrocannabinol (THC, 6 mg/kg) daily for 27 days and concurrently trained in a spatial learning and memory task. The chronic effects of cannabis use were specifically examined by assessing animal behaviour during the 'postacute' period (17 h after drug exposure), when minimal acute drug burden is expected to be present. The postacute period is a good model for cannabis use patterns in human adolescents. In addition, we investigated whether the hierarchical organization of working memory (chunking) was impaired by THC-treatment. We show that THC exposure impairs adolescent learning when tested in the postacute period, and that THC impairs the ability of animals to use a chunking strategy.

Acute in utero morphine exposure slows G2/M phase transition in radial glial and basal progenitor cells in the dorsal telencephalon of the E15.5 embryonic mouse.

The antiproliferative effects of opiate exposure on neurogenesis in vitro have been well documented, but the effects of opiates on brain development in vivo are less well understood. We have recently shown that mu opioid receptors are expressed on radial glia of the lateral ventricle, the neuronal and glial progenitor cells of the developing cortex. In the present study we show that in vivo morphine treatment of the E15.5 mouse increases the length of the G(2)/M phase of the radial glial cell cycle in the dorsal telencephalon, as well as slows interkinetic nuclear migration of radial glial nuclei from the basal ventricular zone to the apical surface. A prolonged G(2)/M phase was also observed in basal progenitor cells. Although morphine exposure altered the duration of the cell cycle for progenitor cells in the embryonic telencephalon, it did not affect whether the progenitors remained proliferative and re-entered the S phase, or whether they exited the cell cycle and became quiescent. In addition, morphine treatment did not change the proportion of basal to apical mitoses. These findings indicate that opioid signalling plays a role in cell cycle progression of both radial glia and basal progenitor cells in vivo in the developing cerebral cortex.