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. The Marburg-rabies construct has demonstrated safety, immunogenicity, and efficacy in mice. The Marburg-rabies construct was efficacious in both nonhuman primates and guinea pigs. The RABV-MERS vaccine has demonstrated efficacy in an improved murine model of MERS through collaboration with Dr. Ralph Baric. We have also performed an efficacy experiment in the alpaca (a camelid) model of MERS-CoV, the data suggest that vaccination of camels may prevent spread of MERS to humans. The RABV-Lassa virus vaccine candidate was efficacious in guinea pigs and evaluation in the nonhuman primates is scheduled to start in the winter of 2019. 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 18Fluoromisonidazole 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. The data indicate that mouse-adapted MARV causes a lethal disease in guinea pigs that shares basic histopathological features of human Marburg virus disease and can be used to evaluate countermeasure efficacy and that changes in 18-Fluorodeoxyglucose uptake correspond to disease severity. Experiments are underway that incorporate 18-Fluorodeoxyglucose -PET imaging into efficacy experiments to monitor lymphoid changes over time. 3. Filovirus Molecular Virology Previously, we have resolved the secondary structure of the EBOV 3E-5E minigenome by selective hydroxyl acylation by primer extension (SHAPE) and identified host proteins that interact with the EBOV trailer from cell lines of varying permissivity. In cell lines with low permissivity viral-RNA sensing proteins involved in innate immunity were identified as acting with the viral genomic RNA, in contrast to permissive cells in which proteins associated with RNA stability for translation were identified. 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. Preliminary data suggests that interfering with RNA directly impacts viral transcription and replication dynamics. 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 as are the iPSCs, however astrocytes are productively infected and may act as a reservoir for EBOV. 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. Confocal microscopy data supports that NP will inhibit TLR7 trafficking within cells. 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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