1. Bivalent vaccines that confer protection against rabies and other viruses We have been developing a rabies virus based vaccine that expresses the glycoprotein (GP) from ebolavirus, marburgvirus and sudanvirus, the GPC from lassa virus, and the Spike protein from MERS-CoV. Previously we developed (a) replication-competent, (b) replication-deficient, and (c) chemically inactivated rabies virus (RABV) vaccines expressing Zaire ebolavirus (EBOV) glycoprotein (GP) using a reverse genetics system based on the SAD B19 RABV wildlife vaccine in collaboration with Matthias Schnell of Thomas Jefferson University. Immunization with live or inactivated RABV vaccines expressing EBOV GP induced cellular and humoral immunity against each virus and conferred protection from both lethal RABV and EBOV challenge in mice. We have continued our evaluation of the RABV based EBOV vaccine and have found that an adjuvant improves efficacy of the inactivated vaccine in rhesus and cynomolgus macaque challenge models. A clinical trial lot of the rabies-ebolavirus has been produced and evaluated in NHPs. The Marburg-rabies construct has demonstrated immunogenicity and efficacy in mice. Efficacy in the nonhuman primate model of Marburg virus is being pursued. The RABV-MERS vaccine has demonstrated immunogenicity and efficacy in mice. An additional efficacy test is underway with an improved murine model of MERS. An immunogenicity experiment in camels is currently being planned. The RABV-Lassa virus vaccine candidate has demonstrated immunogenicity and safety in mice. Efficacy testing in guinea pigs in nonhuman primates is scheduled to start in fall of 2017 and Spring of 2018, respectively. 2. Filovirus nonhuman primate model of human disease. EVPS is currently developing PET/CT to investigate pathogenesis of EBOV infection in NHPs. We have evaluated 18Fluorodeoxyglucose as a marker of increased glucose metabolism as a marker of inflammation 18Fluoro-Albumin as a hemodynamic marker, and 18Fluromisonidazole as a marker of hypoxia. We observed increased metabolism in the spleen, liver, bone marrow and draining lymph nodes that progressed rapidly and plateaued 5 days post-infection and rapidly decreased as subjects met endpoint criteria. 18-fluoro-Albumin imaging indicated congestion in the bone marrow and spleen. 18Fluoromisonidazole imaging has identified putative areas of hypoxia in the liver and indications of acute tubular necrosis in the kidneys. Manuscripts describing these data are currently in development. As evidence by these experiments, PET/CT imaging can be used to evaluate disease progression in real-time, thus establishing biomarkers that can be used to validate potential countermeasures and increase understanding of pathogenesis. EVPS is also developing a guinea pig model of Marburg virus (MARV) disease. Preliminary experiments indicate that mouse-adapted MARV causes a lethal disease in guinea pigs. Experiments are underway to determine mechanisms of pathogenesis in the model and evaluate countermeasures incorporating FDG-PET imaging and a PET radiotracer for apoptosis to monitor pathogenic processes in real-time. 3. Filovirus Molecular Virology We have resolved the secondary structure of the EBOV 3E-5E minigenome by selective hydroxyl acylation by primer extension (SHAPE). Previously, we identified host proteins that interact with the EBOV trailer. Specifically, HSC70 binds to a specific motif within the EBOV trailer and mutational analysis, chemical inhibition targeting HSC70, and reverse genetics have demonstrated a role for HSC70 in the virus lifecycle. We are currently identifying host proteins that interact with the EBOV NCRs from cell lines of varying permissivity to EBOV infection to identify therapeutic targets as well as establish mechanisms of filovirus lifecycle regulation. As a further step in investigating the role of RNA secondary structures as regulators of the virus lifecycle we are using a yeast based system to evolve RNA binding proteins that target specific RNA motifs in collaboration with Drs. LeGrice and McNaughton. Persistence of EBOV RNA in the cerebral spinal fluid of survivors has led to the possibility that the virus can establish infection in the CNS. In one scenario, a health care worker who survived EBOV infection developed encephalitis months after clearing disease and infectious virus was isolated from the CSF. This case demonstrates that persistent infection of the CNS is possible. To determine if persistent infection of CNS cell types can be established and characterized, we are collaborating with Avindra Nath of NIMS. We are establishing the dynamics of EBOV infection of inducible pluripotent stem cells (iPSCs) of neuronal origin. Traditional neuronal tissue culture is refractory to EBOV infection, and the iPSCs provide the possibility to explore cell types that more closely reflect tissue. Role of NP in EBOV pathogenesis. Changes in NP and VP24 necessary are necessary for adaptation of EBOV to mice, therefore it was of interest to determine what host-factors NP interacts with to modulate infection. We performed a yeast 2-hybrid assay to identify host proteins from murine macrophages that interact with NP. We identified TLR translocator proteins and confirmed the interaction by immunofluorescence. A TLR reporter system found that NP specifically blocks TLR7 signaling, TLR7 specifically recognizes single-stranded RNA to trigger the innate cellular responses and apoptosis in macrophages. The data suggest that NP interaction with the TLR translocator likely provides the first hit against the innate response until VP35, VP24 concentrations accumulate to further suppress innate response.
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