Effectiveness of pyronaridine-artesunate against Plasmodium malariae, Plasmodium ovale spp, and mixed-Plasmodium infections: a post-hoc analysis of the CANTAM-Pyramax trial.

BACKGROUND: High-quality evidence for the therapeutic efficacy and effectiveness of antimalarials for infections caused by Plasmodium malariae, Plasmodium ovale spp, and mixed-Plasmodium infections is scarce. In this study, we aimed to analyse the efficacy of pyronaridine-artesunate for the treatment of non-falciparum and mixed-species Plasmodium infections from a large phase 3b/4 clinical trial in central Africa. METHODS: This post-hoc analysis was done in a random subset of samples from two sites (in the Democratic Republic of the Congo and in Gabon) of the CANTAM-Pyramax trial assessing pyronaridine-artesunate therapy. We randomly selected paired dried blood spot samples from day 0 and day 28 (or unforeseen visit) and analysed them by quantitative PCR for mixed Plasmodium infections or non-falciparum mono-infections. Day 28 (or unforeseen visit) samples positive for non-falciparum malaria were re-assessed by microscopy to identify microscopic versus submicroscopic infections. Analyses were done on two sample sets: a per-protocol set and an intention-to-treat set. FINDINGS: Among 1502 randomly selected samples, 192 (12·8%) showed mixed-Plasmodium infections or non-falciparum mono-infections. We did not detect P vivax in the samples. For both the per-protocol and intention-to-treat sets, the overall day 28 cure rates for P malariae, P ovale curtisi, and P ovale wallikeri were 96·3% or higher (95% CIs from 81·0-99·9 to 95·7-100). Cure rates were consistently high in P malariae (99·2%, 95·7-100) and P ovale spp (97·9%, 88·7-99·9, for P ovale curtisi and 96·3%, 81·0-99·9, for P ovale wallikeri) infections. INTERPRETATION: This post-hoc analysis provides important evidence supporting the high efficacy of pyronaridine-artesunate against mono-infections with P malariae, P ovale curtisi, or P ovale wallikeri and mixed-Plasmodium infections in a real-world setting. FUNDING: Medicines for Malaria Venture.

Heterologous protection against malaria by a simple chemoattenuated PfSPZ vaccine regimen in a randomized trial.

Immunization with Plasmodium falciparum (Pf) sporozoites under chemoprophylaxis (PfSPZ-CVac) is the most efficacious approach to malaria vaccination. Implementation is hampered by a complex chemoprophylaxis regimen and missing evidence for efficacy against heterologous infection. We report the results of a double-blinded, randomized, placebo-controlled trial of a simplified, condensed immunization regimen in malaria-naive volunteers (EudraCT-Nr: 2018-004523-36). Participants are immunized by direct venous inoculation of 1.1 × 105 aseptic, purified, cryopreserved PfSPZ (PfSPZ Challenge) of the PfNF54 strain or normal saline (placebo) on days 1, 6 and 29, with simultaneous oral administration of 10 mg/kg chloroquine base. Primary endpoints are vaccine efficacy tested by controlled human malaria infection (CHMI) using the highly divergent, heterologous strain Pf7G8 and safety. Twelve weeks following immunization, 10/13 participants in the vaccine group are sterilely protected against heterologous CHMI, while (5/5) participants receiving placebo develop parasitemia (risk difference: 77%, p = 0.004, Boschloo's test). Immunization is well tolerated with self-limiting grade 1-2 headaches, pyrexia and fatigue that diminish with each vaccination. Immunization induces 18-fold higher anti-Pf circumsporozoite protein (PfCSP) antibody levels in protected than in unprotected vaccinees (p = 0.028). In addition anti-PfMSP2 antibodies are strongly protection-associated by protein microarray assessment. This PfSPZ-CVac regimen is highly efficacious, simple, safe, well tolerated and highly immunogenic.

Ivermectin for causal malaria prophylaxis: a randomised controlled human infection trial.

OBJECTIVE: Ivermectin is safe and widely used for treating helminth infections. It also kills arthropods feeding on treated subjects, including malaria vectors. Thus, ivermectin mass drug administration as an additional tool for malaria control is being evaluated by WHO. As in vitro data, animal experiments and epidemiological observations suggest that ivermectin has a direct effect on the liver stages of the malaria parasite, this study was designed to assess the prophylactic effect of ivermectin on Plasmodium falciparum controlled human malaria infection. METHODS: A total of 4 volunteers were randomised to placebo, and 8 volunteers were randomised to receive ivermectin 0.4 mg/kg, orally, once 2 h before being experimentally infected intravenously with 3200 P. falciparum sporozoites. The primary endpoint was time to parasitaemia detected by positive thick blood smear; RT-qPCR was performed in parallel. RESULTS: All but one volunteer became thick blood smear positive between day 11 and day 12 after infection, and there was no significant effect of ivermectin on parasitaemia. CONCLUSION: Ivermectin - at the dose used - has no clinically relevant activity against the pre-erythrocytic stages of P. falciparum.

OBJECTIF: L'ivermectine est sûr et largement utilisé pour traiter les helminthiases. Il tue également les arthropodes se nourrissant sur les sujets traités, y compris les vecteurs du paludisme. Ainsi, l'administration en masse d'ivermectine en tant qu'outil supplémentaire de lutte contre le paludisme est actuellement évaluée par l'OMS. Comme les données in vitro, les expériences sur animaux et les observations épidémiologiques suggèrent que l'ivermectine a un effet direct sur les stades hépatiques du parasite du paludisme, cette étude a été conçue pour évaluer l'effet prophylactique de l'ivermectine sur l'infection paludéenne humaine par Plasmodium falciparum contrôlée. MÉTHODES: Quatre volontaires ont été randomisés pour un placebo et 8 volontaires ont été randomisés pour recevoir de l'ivermectine à 0,4 mg/kg en une fois par voie orale, 2 heures avant d'être expérimentalement infectés par voie intraveineuse avec 3.200 sporozoïtes de P. falciparum. Le critère d'évaluation principal était le temps à la parasitémie détectée par un frottis sanguin épais positif. Une RT-qPCR a été réalisée en parallèle. RÉSULTATS: Tous les volontaires sauf un sont devenus positifs pour les frottis sanguins épais entre le jour 11 et le jour 12 après l'infection et il n'y avait aucun effet significatif de l'ivermectine sur la parasitémie. CONCLUSION: L'ivermectine - à la dose utilisée - n'a aucune activité cliniquement pertinente contre les stades pré-érythrocytaires de P. falciparum.

First-in-human, Randomized, Double-blind Clinical Trial of Differentially Adjuvanted PAMVAC, A Vaccine Candidate to Prevent Pregnancy-associated Malaria.

BACKGROUND: Malaria in pregnancy has major impacts on mother and child health. To complement existing interventions, such as intermittent preventive treatment and use of impregnated bed nets, we developed a malaria vaccine candidate with the aim of reducing sequestration of asexual "blood-stage" parasites in the placenta, the major virulence mechanism. METHODS: The vaccine candidate PAMVAC is based on a recombinant fragment of VAR2CSA, the Plasmodium falciparum protein responsible for binding to the placenta via chondroitin sulfate A (CSA). Healthy, adult malaria-naive volunteers were immunized with 3 intramuscular injections of 20 µg (n = 9) or 50 µg (n = 27) PAMVAC, adjuvanted with Alhydrogel or glucopyranosyl lipid adjuvant in stable emulsion (GLA-SE) or in a liposomal formulation with QS21 (GLA-LSQ). Allocation was random and double blind. The vaccine was given every 4 weeks. Volunteers were observed for 6 months following last immunization. RESULTS: All PAMVAC formulations were safe and well tolerated. A total of 262 adverse events (AEs) occurred, 94 (10 grade 2 and 2 grade 3) at least possibly related to the vaccine. No serious AEs occurred. Distribution and severity of AEs were similar in all arms. PAMVAC was immunogenic in all participants. PAMVAC-specific antibody levels were highest with PAMVAC-GLA-SE. The antibodies inhibited binding of VAR2CSA expressing P. falciparum-infected erythrocytes to CSA in a standardized functional assay. CONCLUSIONS: PAMVAC formulated with Alhydrogel or GLA-based adjuvants was safe, well tolerated, and induced functionally active antibodies. Next, PAMVAC will be assessed in women before first pregnancies in an endemic area. CLINICAL TRIALS REGISTRATION: EudraCT 2015-001827-21; ClinicalTrials.gov NCT02647489.

Antagonistic PCP Signaling Pathways in the developing Drosophila eye.

In Planar cell polarity (PCP), cells coordinately polarize their cytoskeletons within the plane of the epithelium in which they lie. In most insect epithelia this is indicated by the coordinated projections of the hairs secreted by the ectodermal cells. PCP of this form has been effectively studied in Drosophila, but it has proven difficult to achieve an integrated description of the roles played by the various proteins. In the insect eye, PCP is not evident as the polarization of individual cells, but as the asymmetric arrangements of the cells of the ommatidia. This different form of PCP allows different studies to be performed, and using this system we have detected the action of two antagonistic signaling pathways. Even though antagonistic, the two pathways synergize and cooperate to ensure that the correct arrangement of the cells is achieved. The cooperative use of antagonistic signaling pathways occurs in the polarization of chemotacting cells, and we discuss the possibility that a similar molecular principle may underlie PCP.

Heterotrimeric Go protein links Wnt-Frizzled signaling with ankyrins to regulate the neuronal microtubule cytoskeleton.

Drosophila neuromuscular junctions (NMJs) represent a powerful model system with which to study glutamatergic synapse formation and remodeling. Several proteins have been implicated in these processes, including components of canonical Wingless (Drosophila Wnt1) signaling and the giant isoforms of the membrane-cytoskeleton linker Ankyrin 2, but possible interconnections and cooperation between these proteins were unknown. Here, we demonstrate that the heterotrimeric G protein Go functions as a transducer of Wingless-Frizzled 2 signaling in the synapse. We identify Ankyrin 2 as a target of Go signaling required for NMJ formation. Moreover, the Go-ankyrin interaction is conserved in the mammalian neurite outgrowth pathway. Without ankyrins, a major switch in the Go-induced neuronal cytoskeleton program is observed, from microtubule-dependent neurite outgrowth to actin-dependent lamellopodial induction. These findings describe a novel mechanism regulating the microtubule cytoskeleton in the nervous system. Our work in Drosophila and mammalian cells suggests that this mechanism might be generally applicable in nervous system development and function.

The phosphoinositide-associated protein Rush hour regulates endosomal trafficking in Drosophila.

Endocytosis regulates multiple cellular processes, including the protein composition of the plasma membrane, intercellular signaling, and cell polarity. We have identified the highly conserved protein Rush hour (Rush) and show that it participates in the regulation of endocytosis. Rush localizes to endosomes via direct binding of its FYVE (Fab1p, YOTB, Vac1p, EEA1) domain to phosphatidylinositol 3-phosphate. Rush also directly binds to Rab GDP dissociation inhibitor (Gdi), which is involved in the activation of Rab proteins. Homozygous rush mutant flies are viable but show genetic interactions with mutations in Gdi, Rab5, hrs, and carnation, the fly homologue of Vps33. Overexpression of Rush disrupts progression of endocytosed cargo and increases late endosome size. Lysosomal marker staining is decreased in Rush-overexpressing cells, pointing to a defect in the transition between late endosomes and lysosomes. Rush also causes formation of endosome clusters, possibly by affecting fusion of endosomes via an interaction with the class C Vps/homotypic fusion and vacuole protein-sorting (HOPS) complex. These results indicate that Rush controls trafficking from early to late endosomes and from late endosomes to lysosomes by modulating the activity of Rab proteins.

Yellow submarine of the Wnt/Frizzled signaling: submerging from the G protein harbor to the targets.

The Wnt/Frizzled signaling pathway plays multiple functions in animal development and, when deregulated, in human disease. The G-protein coupled receptor (GPCR) Frizzled and its cognate heterotrimeric Gi/o proteins initiate the intracellular signaling cascades resulting in cell fate determination and polarization. In this review, we summarize the knowledge on the ligand recognition, biochemistry, modifications and interacting partners of the Frizzled proteins viewed as GPCRs. We also discuss the effectors of the heterotrimeric Go protein in Frizzled signaling. One group of these effectors is represented by small GTPases of the Rab family, which amplify the initial Wnt/Frizzled signal. Another effector is the negative regulator of Wnt signaling Axin, which becomes deactivated in response to Go action. The discovery of the GPCR properties of Frizzled receptors not only provides mechanistic understanding to their signaling pathways, but also paves new avenues for the drug discovery efforts.

The trimeric G protein Go inflicts a double impact on axin in the Wnt/frizzled signaling pathway.

The Wnt/Frizzled signaling pathway plays crucial roles in animal development and is deregulated in many cases of carcinogenesis. We and others have previously demonstrated that Frizzled proteins initiating the intracellular signaling are typical G protein-coupled receptors and rely on the trimeric G protein Go for Wnt transduction in Drosophila. However, the mode of action of Go and its interplay with other transducers of the pathway such as Dishevelled and Axin remained unclear. Here we show that the alpha-subunit of Go directly acts on Axin, the multidomain protein playing a negative role in the Wnt signaling. G alpha o physically binds Axin and re-localizes it to the plasma membrane. Furthermore, G alpha o suppresses Axin's inhibitory action on the Wnt pathway in Drosophila wing development. The interaction of G alpha o with Axin critically depends on the RGS domain of the latter. Additionally, we show that the betagamma-component of Go can directly bind and recruit Dishevelled from cytoplasm to the plasma membrane, where activated Dishevelled can act on the DIX domain of Axin. Thus, the two components of the trimeric Go protein mediate a double-direct and indirect-impact on different regions of Axin, which likely serves to ensure a robust inhibition of this protein and transduction of the Wnt signal.

Windei, the Drosophila homolog of mAM/MCAF1, is an essential cofactor of the H3K9 methyl transferase dSETDB1/Eggless in germ line development.

The epigenetic regulation of gene expression by the covalent modification of histones is a fundamental mechanism required for the proper differentiation of germ line cells during development. Trimethylation of histone 3 lysine 9 (H3K9me3) leads to chromatin silencing and the formation of heterochromatin by recruitment of heterochromatin protein 1 (HP1). dSETDB1/Eggless (Egg), the ortholog of the human methyltransferase SETDB1, is the only essential H3K9 methyltransferase in Drosophila and is required for H3K9 trimethylation in the female germ line. Here we show that Windei (Wde), the Drosophila homolog of mouse mAM and human MCAF1, is an essential cofactor of Egg required for its nuclear localization and function in female germ line cells. By deletion analysis combined with coimmunoprecipitation, we have identified the protein regions in Wde and Egg that are necessary and sufficient for the interaction between the two proteins. We furthermore identified a region of Egg that gets covalently modified by SUMOylation, which may facilitate the formation of higher order chromatin-modifying complexes. Together with Egg, Wde localizes to euchromatin, is enriched on chromosome 4, and binds to the Painting of fourth (POF) protein. Our data provide the first genetic and phenotypic analysis of a mAM/MCAF1 homolog in a model organism and demonstrate its essential function in the survival of germ line cells.

PP2A antagonizes phosphorylation of Bazooka by PAR-1 to control apical-basal polarity in dividing embryonic neuroblasts.

Bazooka/Par-3 (Baz) is a key regulator of cell polarity in epithelial cells and neuroblasts (NBs). Phosphorylation of Baz by PAR-1 and aPKC is required for its function in epithelia, but little is known about the dephosphorylation mechanisms that antagonize the activities of these kinases or about the relevance of Baz phosphorylation for NB polarity. We found that protein phosphatase 2A (PP2A) binds to Baz via its structural A subunit. By using phospho-specific antibodies, we show that PP2A dephosphorylates Baz at the conserved serine residue 1085 and thereby antagonizes the kinase activity of PAR-1. Loss of PP2A function leads to complete reversal of polarity in NBs, giving rise to an "upside-down" polarity phenotype. Overexpression of PAR-1 or Baz, or mutation of 14-3-3 proteins that bind phosphorylated Baz, causes essentially the same phenotype, indicating that the balance of PAR-1 and PP2A effects on Baz phosphorylation determines NB polarity.

Mars, a Drosophila protein related to vertebrate HURP, is required for the attachment of centrosomes to the mitotic spindle during syncytial nuclear divisions.

The formation of the mitotic spindle is controlled by the microtubule organizing activity of the centrosomes and by the effects of chromatin-associated Ran-GTP on the activities of spindle assembly factors. In this study we show that Mars, a Drosophila protein with sequence similarity to vertebrate hepatoma upregulated protein (HURP), is required for the attachment of the centrosome to the mitotic spindle. More than 80% of embryos derived from mars mutant females do not develop properly due to severe mitotic defects during the rapid nuclear divisions in early embryogenesis. Centrosomes frequently detach from spindles and from the nuclear envelope and nucleate astral microtubules in ectopic positions. Consistent with its function in spindle organization, Mars localizes to nuclei in interphase and associates with the mitotic spindle, in particular with the spindle poles, during mitosis. We propose that Mars is an important linker between the spindle and the centrosomes that is required for proper spindle organization during the rapid mitotic cycles in early embryogenesis.

Trimeric G protein-dependent signaling by Frizzled receptors in animal development.

Receptors of the Frizzled family transduce important signals during animal development and are conserved from sponges to humans. Frizzled receptors belong to the superfamily of G protein-coupled receptors (GPCRs), but until recently were considered G protein-independent in their signaling. In the present article we review the extensive knowledge demonstrating the functions of trimeric G proteins in Frizzled signal transduction in vertebrates and lower animals. Other structural and functional similarities of Frizzled receptors and the GPCRs are also discussed.

Perlecan and Dystroglycan act at the basal side of the Drosophila follicular epithelium to maintain epithelial organization.

Dystroglycan (Dg) is a widely expressed extracellular matrix (ECM) receptor required for muscle viability, synaptogenesis, basementmembrane formation and epithelial development. As an integral component of the Dystrophin-associated glycoprotein complex, Dg plays a central role in linking the ECM and the cytoskeleton. Disruption of this linkage in skeletal muscle leads to various types of muscular dystrophies. In epithelial cells, reduced expression of Dg is associated with increased invasiveness of cancer cells. We have previously shown that Dg is required for epithelial cell polarity in Drosophila, but the mechanisms of this polarizing activity and upstream/downstream components are largely unknown. Using the Drosophila follicle-cell epithelium (FCE) as a model system, we show that the ECM molecule Perlecan (Pcan) is required for maintenance of epithelial-cell polarity. Follicle cells that lack Pcan develop polarity defects similar to those of Dg mutant cells. Furthermore, Dg depends on Pcan but not on Laminin A for its localization in the basal-cell membrane, and the two proteins bind in vitro. Interestingly, the Dg form that interacts with Pcan in the FCE lacks the mucin-like domain, which is thought to be essential for Dg ligand binding activity. Finally, we describe two examples of how Dg promotes the differentiation of the basal membrane domain: (1) by recruiting/anchoring the cytoplasmic protein Dystrophin; and (2) by excluding the transmembrane protein Neurexin. We suggest that the interaction of Pcan and Dg at the basal side of the epithelium promotes basal membrane differentiation and is required for maintenance of cell polarity in the FCE.