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2012 Research Grant Program Winning Abstract Innate Immune Response to Ebolavirus Infection By Elizabeth Fritz Ebolavirus (EBOV) and Marburgvirus (MARV) are single-stranded, negative-sense RNA viruses of the Filoviridae family recognized for their impressive lethality. Filoviruses are causative agents of viral hemorrhagic fever (VHF) characterized by hypotension, inflammation, lymphopenia, thrombocytopenia, coagulation disorders, hemorrhage, and a rampant cytokine-storm response resulting in multi-organ failure and death. There is one species of MARV, Lake Victoria marburgvirus, whereas there are five distinct EBOV species: Sudan ebolavirus (SEBOV), Zaire ebolavirus (ZEBOV), Ivory Coast ebolavirus (ICEBOV), Bundibugyo ebolavirus (BEBOV), and Reston ebolavirus (REBOV). Mortality rates are approximately 40 to 90%, depending on the virus, with ZEBOV and MARVAngola being the most virulent. There are no FDA-approved vaccines or therapeutics to combat EBOV or MARV infection, and we still lack an understanding of the host’s innate immune response to these Category A Priority Pathogens. Antiviral host-response elements are affected by filovirus infection: the interferon (IFN) response, natural killer (NK cells), macrophages, and dendritic cells (DCs). Macrophages and DCs are primary targets of filovirus infection. DCs are potent antigenpresenting cells that capture foreign antigens for uptake and processing to target secondary lymphoid tissues for stimulation of T and B cells. The latter process occurs by antigen presentation, co-stimulation via cell surface expression of CD-40, CD86, and CD80, and DC cytokines (i.e. IL-2, IL-12, IL-10, and TNF-alpha). Immature DCs display low MHC class II and co-stimulatory molecule surface expression, whereas high expression of MHC II, CD80, CD86, CD40, DC83, and IL-12 categorizes mature DCs. MARV may affect DC maturation and MHC class II antigen presentation, as we observed low surface expression of HLA-DR in MARV-positive cells of infected nonhuman primates (NHPs). Plasmacytoid DCs in the spleens of these animals showed low HLA-DR surface staining. Filovirus envelope glycoproteins bind to DC-SIGN, and diminished DC-SIGN expression was noted in the spleens and lymph nodes of MARVinfected animals. This suggests the virus causes a loss of DCs in lymphoid tissues and/or affects DC-SIGN surface expression and maturation. A report found that the EBOV VP35 viral protein impedes the maturation and function of EBOV-stimulated DCs, with decreased CD4+ cell activation. DCs and NK cells are in close proximity in secondary lymphoid tissues. NK cells are activated by DC cytokines (IL-2, IL-12, and IL-18), and DC IL-15 is crucial for NK cell development, priming, function, and survival. NK cells co-stimulate T and B cells by surface expression of CD40L, OX40, CD86, and CD70. Two activating killer immunoglobulin-like receptor (KIR) genes, KIR2DS1 and KIR2DS3, were correlated with lethal EBOV infection. KIRs are NK cell receptors that control NK function following specific interactions with MHC class I EBOV-infected NHPs showed a depletion of NK cells and CD8+ T cells soon after infection. NKp30 up-regulation was linked to potent NK cytolytic activity against DCs infected with MARV virus-like particles (VLPs). We will study the DC and NK cell response to EBOV infection in vitro with the objective of elucidating the innate immune response to EBOV. We hypothesize that filoviruses immediately target DCs and NK cells allowing rampant viral replication/spread resulting in an immunosuppressive state incapable of battling infection. These studies will be conducted in a biosafety level-4 (BSL-4) laboratory equipped with a BD FACSCanto™ II instrument with BD FACSDiva™ software. Aim 1: Determine DC and NK cell responses to EBOV. We identified changes in DCs (HLA-DRlo and DC-SIGNlo) and depletion of NK cells following filovirus infection. To determine the effect EBOV has on cellular maturation, activation, and function, DCs and NK cells will be purified from peripheral blood mononuclear cells (PBMCs), checked for purity using BD fluorochrome-conjugated antibodies (lineage cocktail, CD123, CD11c, HLA-DR, CD3, CD8, CD16, CD56) and cultured following standard techniques. DCs will be infected with ZEBOV, a highly pathogenic virus in humans, or REBOV, a nonpathogenic virus in humans, and stained for cellular markers by using BD fluorochrome-conjugated antibodies (CD123, CD11c, HLA-DR, CD209, CD86, CD80, CD40) following a time course of infection (8, 24, 48, and 72 hours). Standard protocols will apply for using BD Pharmingen™ stain and BD Cytofix™ fixation buffers. In parallel, DC cytokines (IL-2, IL-10, IL-12, IL-6, IL-18, IL-15, TNF-alpha, IFN-alpha/beta, IFNgamma) will be measured by intracellular cytokine staining by using standard protocols (BD GolgiPlug™ and BD Cytofix/Cytoperm™ buffer) and standard ELISAs for secreted protein. EBOV-infected DCs will be co-cultured with homologous donor NK cells to determine altered activation and function by using BD fluorochrome-conjugated antibodies against cell surface and intracellular targets (NKG2D, NKp46, NKp30, CD16, CD3, CD56, CD8, Granzyme B, IFN-gamma) and following standard protocols as above. Aim 2: Determine DC and NK cell signaling pathways affected by EBOV. EBOV and MARV are known to interfere with host cell signal transduction pathways, especially that of STAT phosphorylation. We will examine the effect of ZEBOV and REBOV on DCs and NK cell signal transduction and apoptosis, and correlate with Aim 1 findings. We will use BD Phosflow™ and apoptosis reagents (ERK, p38, MEK1, TBK1, STATs, Annexin V, APO-BRDU kit) to examine signal transduction and apoptosis in DCs and co-cultured NK cells, following a time course of viral infection (0.5, 1.5, 4.0, 8.0, 12.0, and 24 hours). Western blot analysis will be used for additional targets and confirm flow cytometry findings. The BD Biosciences Research Grant Program aims to reward and enable important research by providing vital funding for scientists pursuing innovative experiments to advance the scientific understanding of disease. Visit bdbiosciences.com/grant to learn more and apply online.