Preclinical characterization and target validation of the antimalarial pantothenamide MMV693183.

Drug resistance and a dire lack of transmission-blocking antimalarials hamper malaria elimination. Here, we present the pantothenamide MMV693183 as a first-in-class acetyl-CoA synthetase (AcAS) inhibitor to enter preclinical development. Our studies demonstrate attractive drug-like properties and in vivo efficacy in a humanized mouse model of Plasmodium falciparum infection. The compound shows single digit nanomolar in vitro activity against P. falciparum and P. vivax clinical isolates, and potently blocks P. falciparum transmission to Anopheles mosquitoes. Genetic and biochemical studies identify AcAS as the target of the MMV693183-derived antimetabolite, CoA-MMV693183. Pharmacokinetic-pharmacodynamic modelling predict that a single 30 mg oral dose is sufficient to cure a malaria infection in humans. Toxicology studies in rats indicate a \u3e 30-fold safety margin in relation to the predicted human efficacious exposure. In conclusion, MMV693183 represents a promising candidate for further (pre)clinical development with a novel mode of action for treatment of malaria and blocking transmission

Rôle de la protéine Histamine Releasing Factor (HRF) dans la transmission du parasite Plasmodium et dans le développement de la pathogénèse de la maladie

Recent findings have raised the hypothesis that clinical susceptibility to malaria may be related to allergy-type response. In human infection with P. falciparum, as well as in murine models of malaria, increased levels of histamine have been shown to be associated with disease severity. Histamine releasing factor (HRF), shown to be implicated in the release of pro-inflammatory histamine during late-phase allergy, was demonstrated to be produced by the parasite during mild and severe malaria infections suggesting that Plasmodium HRF may affect host immune responses and contributes to the pathogenesis. The objectives of this work were to evaluate the role of Plasmodium HRF in the development of the immune response and to determine whether its expression is associated with the severity of malaria disease by studying two HRF-deficient (hrfΔ) murine parasites (PbANKA and PbNK65). Infection with PbANKA-hrfΔ sporozoites showed a decrease in the frequency of ECM due to the impairment of the development of the mutant parasites in liver stages as a consequence of the up-regulation of IL-6. Infection with PbNK65-hrfΔ parasites confirmed the importance of HRF in enhancing the virulence of the parasite. Indeed, PbNK65-hrfΔ infection results in parasite clearance leading to a long-lasting protection and immune memory as reflected by an up-regulation of IL-6, a down-regulation of PD-1 expression on T cells and in the enhancement of Ab-mediated phagocytosis. HRF is the first parasite gene which directly modulates the host immune response.De études récentes suggèrent une forte relation entre susceptibilité au paludisme et réponse allergique. Des niveaux élevés d’histamine plasmatique et tissulaire ont été associés à la sévérité de la maladie chez l’homme infecté par P. falciparum et dans de nombreux modèles animaux. Histamine releasing factor (HRF), une protéine pro-inflammatoire libérant l'histamine au cours des processus allergiques, est produite par le parasite au cours des infections palustres modérées et sévères, suggérant l’implication du HRF dans l’altération des réponses immunitaires et dans la pathogenèse. Les objectifs de ce travail consistaient à évaluer le rôle de la protéine parasitaire HRF dans le développement de la réponse immunitaire et à déterminer si son expression est associée à la sévérité de la maladie en étudiant deux parasites murins, PbANKA et PbNK65, déficients pour la protéine HRF (hrfΔ). Les souris infectées avec des sporozoïtes PbANKA-hrfΔ ont montré une diminution de la fréquence du neuropaludisme associée à un déficit du développement des parasites mutants au cours du stade hépatique et à une augmentation précoce systémique d’IL-6. En outre, l'infection par les parasites PbNK65-hrfΔ est caractérisée par l’élimination du parasite conduisant à une protection durable et au développement d’une mémoire immunitaire caractérisée par une augmentation d’IL-6, une diminution de l’expression de PD-1 sur les cellules T et une amélioration de la phagocytose dépendante des anticorps, confirmant l'importance de la protéine HRF dans la virulence du parasite. HRF est le premier gène de parasite Plasmodium dont l’effet direct sur la réponse immunitaire de l’hôte est démontré

Arthropod Saliva and Its Role in Pathogen Transmission: Insect Saliva: Mosquito saliva, skin, allergy, and the outcome of Malaria infection - from mice to men

International audienceSkin provides the first line of defense against exoantigens including pathogens and allergens and is composed of a complex immune network that subsequently influences the systemic immune response. Arthropod saliva has profound effects on pathogen transmission, and the immunological response to saliva leads to both Type 1 and Type 4 hypersensitivity reactions, classically associated with allergic diseases. Such reactions result in immune deviation toward a Th2 response and a dysfunctional Th1 response, with significant consequences for the immune response to malaria and leishmaniasis parasites. In malaria, mast cells situated in the skin play a key role in linking these two responses producing histamine and thymic stromal lymphopoietin, master regulators of allergic inflammation. Increasing evidence from mouse models to natural infections in human populations provides support for considering the immune response to malaria within an allergic context and that saliva and its allergenic nature through direct response by immune effectors in the skin have significant immediate and long-term effects for the outcome of infection by malaria parasites and the development of clinical immunity. Together with tick saliva, sand fly saliva has been thoroughly investigated in numerous studies. It targets host pharmacology as well as its immune system. Sand fly salivary proteins act on different types of cells, including monocytes, neutrophils, dendritic cells, and lymphocytes. Therefore, these proteins modulate the innate immune response, leading to the production of mediators that favor the establishment of infection by Leishmania parasites. Besides that, it has been demonstrated that sand fly saliva induces apoptosis of neutrophils, increasing the parasite load in in vitro experiments. On the other hand, salivary proteins are immunogenic and induce humoral and cellular responses in mammalian hosts. These responses protect the host against an infection by Leishmania sp. because the delayed-type hypersensitivity response induced by after frequent exposure to bites, or by immunization with whole saliva or their proteins, creates an inhospitable environment that inhibits parasite growth. The robust protection against Leishmania conferred by immunity to several distinct salivary candidates suggests that their inclusion as part of a vaccine formulation may enhance its efficacy

Using Cryopreserved <i>Plasmodium falciparum</i> Sporozoites in a Humanized Mouse Model to Study Early Malaria Infection Processes and Test Prophylactic Treatments

In addition to vector control, long-lasting insecticidal nets and case management, the prevention of infection through vaccination and/or chemoprevention are playing an increasing role in the drive to eradicate malaria. These preventative approaches represent opportunities for improvement: new drugs may be discovered that target the early infectious stages of the Plasmodium parasite in the liver (rather than the symptomatic, abundant blood stage), and new, exciting vaccination technologies have recently been validated (using mRNA or novel adjuvants). Exploiting these possibilities requires the availability of humanized mouse models that support P. falciparum infection yet avoid the hazardous use of infectious mosquitoes. Here, we show that commercially available P. falciparum sporozoites and FRG mice carrying human hepatocytes and red blood cells faithfully recapitulate the early human malaria disease process, presenting an opportunity to use this model for the evaluation of prophylactic treatments with a novel mode of action

Malaria Parasite-Infected Erythrocytes Secrete PfCK1, the Plasmodium Homologue of the Pleiotropic Protein Kinase Casein Kinase 1

Casein kinase 1 (CK1) is a pleiotropic protein kinase implicated in several fundamental processes of eukaryotic cell biology. Plasmodium falciparum encodes a single CK1 isoform, PfCK1, that is expressed at all stages of the parasite's life cycle. We have previously shown that the pfck1 gene cannot be disrupted, but that the locus can be modified if no loss-of-function is incurred, suggesting an important role for this kinase in intra-erythrocytic asexual proliferation. Here, we report on the use of parasite lines expressing GFP- or His-tagged PfCK1 from the endogenous locus to investigate (i) the dynamics of PfCK1 localisation during the asexual cycle in red blood cells, and (ii) potential interactors of PfCK1, so as to gain insight into the involvement of the enzyme in specific cellular processes. Immunofluorescence analysis reveals a dynamic localisation of PfCK1, with evidence for a pool of the enzyme being directed to the membrane of the host erythrocyte in the early stages of infection, followed by a predominantly intra-parasite localisation in trophozoites and schizonts and association with micronemes in merozoites. Furthermore, we present strong evidence that a pool of enzymatically active PfCK1 is secreted into the culture supernatant, demonstrating that PfCK1 is an ectokinase. Our interactome experiments and ensuing kinase assays using recombinant PfCK1 to phosphorylate putative interactors in vitro suggest an involvement of PfCK1 in many cellular processes such as mRNA splicing, protein trafficking, ribosomal, and host cell invasion

Hepatic Inflammation Confers Protective Immunity Against Liver Stages of Malaria Parasite.

Deciphering the mechanisms by which Plasmodium parasites develop inside hepatocytes is an important step toward the understanding of malaria pathogenesis. We propose that the nature and the magnitude of the inflammatory response in the liver are key for the establishment of the infection. Here, we used mice deficient in the multidrug resistance-2 gene (Mdr2-/-)-encoded phospholipid flippase leading to the development of liver inflammation. Infection of Mdr2-/- mice with Plasmodium berghei ANKA (PbANKA) sporozoites (SPZ) resulted in the blockade of hepatic exo-erythrocytic forms (EEFs) with no further development into blood stage parasites. Interestingly, cultured primary hepatocytes from mutant and wild-type mice are equally effective in supporting EEF development. The abortive infection resulted in a long-lasting immunity in Mdr2-/- mice against infectious SPZ where neutrophils and IL-6 appear as key effector components along with CD8+ and CD4+ effector and central memory T cells. Inflammation-induced breakdown of liver tolerance promotes anti-parasite immunity and provides new approaches for the design of effective vaccines against malaria disease

Protection against malaria in mice is induced by blood stage–arresting histamine-releasing factor ( HRF )–deficient parasites

International audienceAlthough most vaccines against blood stage malaria in development today use subunit preparations, live attenuated parasites confer significantly broader and more lasting protection. In recent years, Plasmodium genetically attenuated parasites (GAPs) have been generated in rodent models that cause self-resolving blood stage infections and induce strong protection. All such GAPs generated so far bear mutations in housekeeping genes important for parasite development in red blood cells. In this study, using a Plasmodium berghei model compatible with tracking anti-blood stage immune responses over time, we report a novel blood stage GAP that lacks a secreted factor related to histamine-releasing factor (HRF). Lack of HRF causes an IL-6 increase, which boosts T and B cell responses to resolve infection and leave a cross-stage, cross-species, and lasting immunity. Mutant-induced protection involves a combination of antiparasite IgG2c antibodies and Fc gamma R+ CD11b(+) cell phagocytes, especially neutrophils, which are sufficient to confer protection. This immune-boosting GAP highlights an important role of opsonized parasite-mediated phagocytosis, which may be central to protection induced by all self-resolving blood stage GAP infections

Interaction between PfCK1 and PfNapL, PfCK2a or PfRON3.

<p><b>(A) Interaction between native PfCK1 and PfNapL, PfCK2a or PfRON3.</b> Immunoprecipitation was performed on protein extracts from transgenic parasites expressing GFP-tagged PfCK1 and from wild-type 3D7 parasites using GFP-trap beads. Detection of GFP, PfCK2a, PfNapL and PfRON3 was then performed by western-blot on the immunoprecipitates using the <i>ad hoc</i> antibodies. Lane 1: total extracts from PfCK1-GFP parasites; lane 2: total extracts from 3D7 WT parasites; lane 3: immunoprecipitates from PfCK1-GFP parasites; lane 4: immunoprecipitates from 3D7 WT parasites. <b>(B) Recombinant PfCK1 and PfCK2α interact <i>in vitro</i>.</b> GST-PfCK1 was incubated with His-PfCK2α and complexes containing the CK1 GST-tagged protein were then purified using glutathione agarose beads. The His-tagged proteins were detected by Western blot analysis using an anti-His antibody and the corresponding Coomassie blue-stained gels are shown. Lane1: Bound material after incubation of GST agarose beads with soluble His-PfCK2α; lane 2: bound material after incubation of GST-PfCK1 agarose beads with soluble His-PfCK2α; lane 3: soluble His-PfCK2α control. <b>(C) Recombinant PfCK1 and PfRON3 interact <i>in vitro</i>.</b> GST-PfCK1 was incubated with His-PfRON3 and complexes containing the CK1 GST-tagged protein were then purified using glutathione agarose beads. The His-tagged proteins were detected by Western blot analysis using an anti-His antibody and the corresponding Coomassie blue-stained gels are shown. Lane 1: bound material after incubation of GST agarose beads with soluble His-PfRON3; lane 2: bound material after incubation of GST-PfCK1 agarose beads with soluble His-PfRON3; lane 3: soluble His-PfRON3 control.</p

Label-free quantitative analysis of PfCK1 interacting proteins.

<p>Volcano plot representing the logarithmic ratio of protein LFQ intensities in the CK1/3D7 experiments plotted against negative logarithmic p-values of the <i>t</i> test performed from triplicates (FDR threshold = 0.1, S0 = 0.5). A hyperbolic curve separates specific CK1-interacting proteins (dark blue dots) from background (light blue dots). PfCK1 and PfRON3 are highlighted with a red square and a red triangle respectively. (B) Distribution of potential PfCK1 interactors across metabolic processes. Histogram representing the number of potential PfCK1 interactors distributed among the various metabolic processes described in the Metabolic Pathways of Malaria Parasites website (<a href="http://sites.huji.ac.il/malaria/" target="_blank">http://sites.huji.ac.il/malaria/</a>). The total number of proteins in the most represented pathway is represented in brackets. (C) Distribution of potential PfCK1 interactors across specific pathways in the transcription process. The histogram represents the number of potential PfCK1 interactors across the various pathways present in the “Transcription” process.</p

PfCK1 expression and kinase activity.

<p>(A) PfCK1 protein was detected by western blot using a peptide-derived anti-PfCK1 antibody. R: Rings; T: trophozoites; S: Schizonts; G: Gametocytes. Detection of 2-Cys-peroxiredoxin was used as a loading control (bottom panel). (B) Casein kinase activity immunoprecipitated with an anti-PfCK1 antibody from parasite extracts from a mixed asexual culture. Kinase assays were set up with casein as a substrate. Lane 1 (“-“), no immunoprecipitated added; lane 2 (“Preim”); immunoprecipitate obtained with a pre-immune serum lane 3 (“CK1”), immunoprecipitate obtained with the anti-PfCK1 antiserum. Reactions were subsequently analysed by SDS-PAGE and autoradiography. (C) Endogenously GFP tagged PfCK1. Western blot of parasite extracts from wild-type 3D7 parasites and parasites expressing GFP-tagged PfCK1 from the endogenous locus using anti-PfCK1, anti-GFP and anti-2-Cys-peroxiredoxin antibodies. (D) Kinase activity after immunoprecipitation from parasite extracts using an anti-GFP antibody. Kinase assays were set up with casein as a substrate. Lane 1, no immunoprecipitated added; lane 2, immunoprecipitate obtained from wild-type 3D7 parasites; lane 3, immunoprecipitate from parasites expressing GFP-tagged PfCK1 from the endogenous locus. Reactions were subsequently analysed by SDS-PAGE and autoradiography. (E) Endogenously His tagged PfCK1. Western Blot of parasite extracts from wild-type 3D7 parasites and parasites expressing His-tagged PfCK1 from the endogenous locus using anti-His and anti-PfCK1 antibodies. (F) Kinase activity after immunoprecipitation from parasite extracts using an anti-His antibody. Kinase assays were set up with casein as a substrate. Lane 1, immunoprecipitate obtained from wild-type 3D7 parasites; lane 2, immunoprecipitate from parasites expressing His-tagged PfCK1 from the endogenous locus. Reactions were subsequently analysed by SDS-PAGE and autoradiography.</p