zed below. 1. Orthopoxvirus pathogenesis A goal of EVPS is to understand virus-host interactions and exploit them for countermeasure development. Here, we sought to use a systems kinomics approach to characterize the host cell response to MPXV strains of varying virulence and identify host targets for development of therapeutics. MPXV is comprised of two clades: Congo Basin MPXV, with an associated case fatality rate of 10%, and Western African MPXV, which is associated with less severe infection and minimal lethality. We thus postulated that Congo Basin and West African MPXV would differentially modulate host cell responses and, as many host responses are regulated through phosphorylation independent of transcription or translation, we employed systems kinomics with peptide arrays to investigate these responses. We demonstrated that Congo Basin MPXV infection selectively downregulates host responses as compared to West African MPXV, including growth factor- and apoptosis-related responses. These results were confirmed using FACS analysis demonstrating that West African MPXV infection resulted in a significant increase in apoptosis in human monocytes as compared to Congo Basin MPXV. Further, differentially phosphorylated kinases were identified through comparison of our MPXV data sets and validated as potential targets for pharmacological inhibition of Congo Basin MPXV infection, including increased Akt S473 phosphorylation and decreased p53 S15 phosphorylation. Inhibition of Akt S473 phosphorylation resulted in a significant decrease in Congo Basin MPXV virus yield (261-fold) but did not affect West African MPXV. In addition, treatment with staurosporine, an apoptosis activator resulted in a 49-fold greater decrease in Congo Basin MPXV yields as compared to West African MPXV. Thus, using a systems kinomics approach, our investigation demonstrated that West African and Congo Basin MPXV differentially modulate host cell responses, and we identified potential host targets of therapeutic interest. We have initiated a similar systems kinomics analysis of Ebola virus infection of monocytes and epithelial cells. We have also continued our studies of monkeypox pathogenesis in nonhuman primates. In FY2012, we performed a statistical analysis of previously acquired data from NHPs infected with MPXV to attempt to identify biomarkers that associate with disease severity. We applied a novel statistical technique, Backward Matched Longitudinal Analysis, to several clinical and immunological parameters measured during MPXV infection of NHPs. Our analysis identified that high concentrations of MCP-1 were strongly associated with non-survival, while higher viremia and IL-1ra and G-CSF concentrations were weakly associated with non-survival. Increased IFN-gamma and RANTES concentrations were weakly associated with survival. These studies have defined targets for further study in investigations of viral pathogenesis, disease staging, and countermeasure development. In collaboration with NIAID Integrated Research Facility investigators, we also identified an additional biomarker that can be used to monitor disease progression;poxvirus antigen staining of monocytes and granulocytes. We found that NHPs with positive detection of poxvirus antigens in monocytes and granulocytes by flow cytometric staining succumbed to virus infection and that early positive pox staining is a strong predictor for lethality. Ten NHPs displayed positive pox-staining and all 10 NHPs reached moribund endpoint. In contrast, none of three NHPs that survived the challenge showed apparent virus staining in their immune cells. Furthermore, data from a CPXV study also demonstrated that 6/9 NHPs showed positive pox-staining and all 6 NHPs reached moribund endpoint. In contrast, the three survivors were pox-staining negative. Thus, we conclude that monitoring of virus infection of immune cells can be used as a biomarker to predict the prognosis of virus infection as early as day 2 post-infection. Finally, we have further explored the use of FDG-PET/CT imaging to monitor MPXV infection in NHPs. Six rhesus macaques were infected intravenously with a sublethal dose of MPXV. Three animals were treated with a dose of cidofovir (CDV) that has been shown to protect against MPXV, while three animals served as the untreated control group. CDV-treated NHPs survived and had a 1,000-fold reduction in viral load. Two NHPs in the untreated control group succumbed and one survived. PET imaging showed that monkeypox infection lead to an increase in 18-FDG uptake in the bone marrow in all six animals early in infection (day 3/4 post inoculation). However, the 2 moribund NHPs had significantly higher uptake of 18-FDG in the bone marrow (SUV mean 4.2) than the 4 surviving NHPs (SUV mean 2.9). Furthermore, nonsurvivors also had significantly higher FDG uptake in axillary lymph nodes at day 3/4 post-infection. Thus, analysis of 18-FDG uptake and LN size resulted in identification of two patterns that differed between moribund and surviving NHPs. These studies indicate that analysis of structural and functional changes of LNs and bone marrow during infection may be a useful in vivo approach to monitor monkeypox pathogenesis. 2. Bivalent vaccines that confer protection against rabies and Ebola virus We have previously 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 ZEBOV GP induced humoral immunity against each virus and conferred protection from both lethal RABV and EBOV challenge in mice. In FY12, we further characterized the immunogenicity and safety of these vaccine candidates in mice. Both live and killed vaccines induced primary EBOV GP-specific T-cells and a robust recall response as measured by interferon-gamma ELISPOT assay. In addition to cellular immunity, an effective filovirus vaccine will likely require a multivalent humoral immune response against multiple virus species. As a proof-of-principle experiment, we demonstrated that inactivated RV-GP could be formulated with another inactivated RABV vaccine expressing the nontoxic fragment of botulinum neurotoxin A heavy chain without a reduction in immunity to each component. Finally, we demonstrated that humoral immunity to GP could be induced by immunization of mice with inactivated RV-GP in the presence of pre-existing immunity to RABV. The ability of these novel vaccines to induce strong humoral and cellular immunity indicates that they should be further evaluated in additional animal models of infection. We also further characterized the pathogenesis of the live-attenuated RABV/EBOV vaccine candidates in mice in an effort to define their growth properties and potential for safety. We are considering live vaccines for use in wildlife. RABV vaccines expressing GP (RV-GP) or a replication-deficient derivative with a deletion of the RABV G gene (RVdelG-GP) are both avirulent after intracerebral inoculation of adult mice. Furthermore, RVdelG-GP is completely avirulent upon intracerebral inoculation of suckling mice unlike parental RABV vaccine or RV-GP. Analysis of RVdelG-GP in the brain by quantitative PCR, determination of virus titer, and immunohistochemistry indicated greatly restricted virus replication. In summary, our findings indicated that RV-GP retains the attenuation phenotype of the live-attenuated RABV vaccine, and RVdelG-GP would appear to be an even safer alternative for use in wildlife or consideration for human use.
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