Single injection recombinant vesicular stomatitis virus vaccines protect ferrets against lethal Nipah virus disease

Background

Nipah virus (NiV) is a highly pathogenic zoonotic agent in the family Paramyxoviridae that is maintained in nature by bats. Outbreaks have occurred in Malaysia, Singapore, India, and Bangladesh and have been associated with 40 to 75% case fatality rates. There are currently no vaccines or postexposure treatments licensed for combating human NiV infection.

Methods and results

Four groups of ferrets received a single vaccination with different recombinant vesicular stomatitis virus vectors expressing: Group 1, control with no glycoprotein; Group 2, the NiV fusion protein (F); Group 3, the NiV attachment protein (G); and Group 4, a combination of the NiV F and G proteins. Animals were challenged intranasally with NiV 28 days after vaccination. Control ferrets in Group 1 showed characteristic clinical signs of NiV disease including respiratory distress, neurological disorders, viral load in blood and tissues, and gross lesions and antigen in target tissues; all animals in this group succumbed to infection by day 8. Importantly, all specifically vaccinated ferrets in Groups 2-4 showed no evidence of clinical illness and survived challenged. All animals in these groups developed anti-NiV F and/or G IgG and neutralizing antibody titers. While NiV RNA was detected in blood at day 6 post challenge in animals from Groups 2-4, the levels were orders of magnitude lower than animals from control Group 1.

Conclusions

These data show protective efficacy against NiV in a relevant model of human infection. Further development of this technology has the potential to yield effective single injection vaccines for NiV infection.

     

Emergent growth cone responses to combinations of Slit1 and Netrin 1 in thalamocortical axon topography

How guidance cues are integrated during the formation of complex axonal tracts remains largely unknown. Thalamocortical axons (TCAs), which convey sensory and motor information to the neocortex, have a rostrocaudal topographic organization initially established within the ventral telencephalon [1-3]. Here, we show that this topography is set in a small hub, the corridor, which contains matching rostrocaudal gradients of Slit1 and Netrin 1. Using in vitro and in vivo experiments, we show that Slit1 is a rostral repellent that positions intermediate axons. For rostral axons, although Slit1 is also repulsive and Netrin 1 has no chemotactic activity, the two factors combined generate attraction. These results show that Slit1 has a dual context-dependent role in TCA pathfinding and furthermore reveal that a combination of cues produces an emergent activity that neither of them has alone. Our study thus provides a novel framework to explain how a limited set of guidance cues can generate a vast diversity of axonal responses necessary for proper wiring of the nervous system.     

Durability of a Vesicular Stomatitis Virus-Based Marburg Virus Vaccine in Nonhuman Primates

The filoviruses, Marburg virus (MARV) and Ebola virus, causes severe hemorrhagic fever with high mortality in humans and nonhuman primates. A promising filovirus vaccine under development is based on a recombinant vesicular stomatitis virus (rVSV) that expresses individual filovirus glycoproteins (GPs) in place of the VSV glycoprotein (G). These vaccines have shown 100% efficacy against filovirus infection in nonhuman primates when challenge occurs 28–35 days after a single injection immunization. Here, we examined the ability of a rVSV MARV-GP vaccine to provide protection when challenge occurs more than a year after vaccination. Cynomolgus macaques were immunized with rVSV-MARV-GP and challenged with MARV approximately 14 months after vaccination. Immunization resulted in the vaccine cohort of six animals having anti-MARV GP IgG throughout the pre-challenge period. Following MARV challenge none of the vaccinated animals showed any signs of clinical disease or viremia and all were completely protected from MARV infection. Two unvaccinated control animals exhibited signs consistent with MARV infection and both succumbed. Importantly, these data are the first to show 100% protective efficacy against any high dose filovirus challenge beyond 8 weeks after final vaccination. These findings demonstrate the durability of VSV-based filovirus vaccines.     

Analytical Performance Characteristics of the Cepheid GeneXpert Ebola Assay for the Detection of Ebola Virus

BackgroundThe recently developed Xpert® Ebola Assay is a novel nucleic acid amplification test for simplified detection of Ebola virus (EBOV) in whole blood and buccal swab samples. The assay targets sequences in two EBOV genes, lowering the risk for new variants to escape detection in the test. The objective of this report is to present analytical characteristics of the Xpert® Ebola Assay on whole blood samples.ConclusionIn summary, we found the Xpert® Ebola Assay to have high analytical sensitivity and specificity for the detection of EBOV in whole blood. It offers ease of use, fast turnaround time, and remote monitoring. The test has an efficient viral inactivation protocol, fulfills inclusivity and exclusivity criteria, and has specimen stability characteristics consistent with the need for decentralized testing. The simplicity of the assay should enable testing in a wide variety of laboratory settings, including remote laboratories that are not capable of performing highly complex nucleic acid amplification tests, and during outbreaks where time to detection is critical.     

Induction of the Cytoprotective Enzyme Heme Oxygenase-1 by Statins Is Enhanced in Vascular Endothelium Exposed to Laminar Shear Stress and Impaired by Disturbed Flow

In addition to cholesterol-lowering properties, statins exhibit lipid-independent immunomodulatory, anti-inflammatory actions. However, high concentrations are typically required to induce these effects in vitro, raising questions concerning therapeutic relevance. We present evidence that endothelial cell sensitivity to statins depends upon shear stress. Using heme oxygenase-1 expression as a model, we demonstrate differential heme oxygenase-1 induction by atorvastatin in atheroresistant compared with atheroprone sites of the murine aorta. In vitro, exposure of human endothelial cells to laminar shear stress significantly reduced the statin concentration required to induce heme oxygenase-1 and protect against H₂O₂-mediated injury. Synergy was observed between laminar shear stress and atorvastatin, resulting in optimal expression of heme oxygenase-1 and resistance to oxidative stress, a response inhibited by heme oxygenase-1 small interfering RNA. Moreover, treatment of laminar shear stress-exposed endothelial cells resulted in a significant fall in intracellular cholesterol. Mechanistically, synergy required Akt phosphorylation, activation of Kruppel-like factor 2, NF-E2-related factor-2 (Nrf2), increased nitric-oxide synthase activity, and enhanced HO-1 mRNA stability. In contrast, heme oxygenase-1 induction by atorvastatin in endothelial cells exposed to oscillatory flow was markedly attenuated. We have identified a novel relationship between laminar shear stress and statins, demonstrating that atorvastatin-mediated heme oxygenase-1-dependent antioxidant effects are laminar shear stress-dependent, proving the principle that biomechanical signaling contributes significantly to endothelial responsiveness to pharmacological agents. Our findings suggest statin pleiotropy may be suboptimal at disturbed flow atherosusceptible sites, emphasizing the need for more specific therapeutic agents, such as those targeting Kruppel-like factor 2 or Nrf2.The efficacy of 3-hydroxy-3-methylglutaryl-coenzyme A reductase antagonists (statins) in reducing low density lipoprotein cholesterol, cardiovascular morbidity, and mortality is widely recognized (1). The observation that beneficial actions of statins on vascular function are detectable prior to any fall in serum cholesterol, extend to normocholesterolemic patients and exceed those of other lipid-lowering drugs despite comparable falls in total cholesterol (2, 3), suggest the existence of low density lipoprotein-cholesterol-independent effects (4, 5). Judging from in vitro studies, these may include immunomodulatory, anti-inflammatory, anti-adhesive, anti-thrombotic, and cytoprotective actions (6). However, the experimental work demonstrating these pleiotropic effects has predominantly used statin concentrations exceeding those achieved by therapeutic dosing, raising questions concerning clinical relevance (4).Heme oxygenase-1 (HO-1)² acts as the rate-limiting factor in the catabolism of heme into biliverdin, releasing free iron and carbon monoxide (CO). Biliverdin is subsequently converted to bilirubin by biliverdin reductase, whereas intracellular iron induces expression of heavy chain-ferritin and the opening of Fe²⁺ export channels (7). The biologic activity of HO-1 represents an important adaptive response in cellular homeostasis, as revealed by widespread inflammation and persistent endothelial injury in human HO-1 deficiency (8).Expression of HO-1 in atherosclerotic lesions, and its ability to inhibit vascular smooth muscle cell proliferation, exert anti-inflammatory, antioxidant, and antithrombotic effects, suggests a protective role during atherogenesis (9, 10). HMOX1 promoter polymorphisms affecting HO-1 expression may influence susceptibility to intimal hyperplasia and coronary artery disease, whereas a low serum bilirubin constitutes a cardiovascular risk factor (11). Moreover, overexpression of HO-1 inhibited atherogenesis, whereas Hmox1^−/− mice bred onto an ApoE^−/− background developed more extensive and complex atherosclerotic plaques (12, 13).Recent interest has focused on the therapeutic potential of HO-1 and its products, with probucol, statins, rapamycin, nitric oxide donors, and aspirin being shown to induce HO-1 (reviewed in Ref. 10). Indeed, induction of HO-1 may represent an important component of the vasculoprotective profile of statins, with simvastatin, atorvastatin, and rosuvastatin variously shown to increase HMOX1 promoter activity and mRNA levels, to induce enzyme activity and increase antioxidant capacity in human endothelial cells (EC) (14–18). However, induction of HO-1 in vascular EC in vivo has not yet been demonstrated.Vascular endothelium exposed to unidirectional, pulsatile laminar shear stress (LSS) >10 dynes/cm² is relatively protected against atherogenesis. LSS increases nitric oxide (NO) biosynthesis, prolongs EC survival, and generates an anticoagulant, anti-adhesive cell surface. In contrast, endothelium exposed to disturbed blood flow, with low shear reversing or oscillatory flow patterns, such as that located at arterial branch points and curvatures, is atheroprone. Thus endothelial cells exposed to disturbed blood flow exhibit reduced levels of endothelial nitric-oxide synthase (eNOS), increased apoptosis, oxidative stress, permeability to low density lipoprotein, and leukocyte adhesion (19).The atheroprotective influence of unidirectional LSS and the overlap between these actions and those of statins led us to hypothesize that LSS increases endothelial responsiveness to statins. We demonstrate for the first time that treatment of mice with atorvastatin induces HO-1 expression in the aortic endothelium and that this occurs preferentially at sites exposed to LSS. In vitro, pre-conditioning human EC with an atheroprotective, but not an atheroprone waveform, significantly reduces the concentration of atorvastatin required to enhance HO-1-mediated cytoprotection against oxidant-induced injury. A synergistic relationship between LSS and statins is revealed, resulting in maximal Akt phosphorylation and dependence upon eNOS, Kruppel-like factor 2 (KLF2), and NF-E2-related factor-2 (Nrf2) activation.     

Developmental Upregulation of Ephrin-B1 Silences Sema3C/Neuropilin-1 Signaling during Post-crossing Navigation of Corpus Callosum Axons

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NMR lipid profile of Agaricus bisporus

Lipids extracted from freeze dried and powdered cultivated Agaricus bisporus by the Bligh and Dwyer method, were subjected to 1D-proton and 2D-COSY NMR analysis. The diacylglycerophospholipids, mono-, di- and tri-glycerides, ether lipids, sphingolipids and steroidal lipids were studied qualitatively and quantitatively. Our findings suggested that (a) ethanolamines and cholines were the predominant diacylphospholipids, (b) sterols, mainly ergosterol, were present in relatively large quantities and (c) the phospholipid fatty acid composition consisted almost exclusively of linoleic acid. This type of detailed data on lipid composition was accurately and rapidly obtained in one step, without chemical modification of the sample. Additional information on four classes of lipid, including their fatty acid composition was obtained after separating the total lipid extract by NH₂-aminopropyl Certify II Bond Elut solid phase chromatography and analysing the NMR spectra of each class of lipids. The results demonstrated the potential of the method for the study of plant metabolism, development and taxonomy.     

Interleukin-2 induces rapid phosphorylation of an 85 kilodalton protein in permeabilized lymphocytes

Lymphocytes were induced to express receptors for interleukin-2 by stimulation for 5 days with phytohaemagglutinin and subsequently permeabilized by treatment with L-alpha-lysophosphatidylcholine. Phosphorylation of an 85 kDa protein was stimulated when these cells were treated with interleukin-2 or with an antibody directed against the interleukin-2 receptor.