Influenza viruses are highly contagious human pathogens that infect up to 500 million people annually and the emergence of highly pathogenic variants with pandemic potential represents a major public health concern. An urgent global need exists to develop new and improved therapeutic strategies to control this infection, and development of antiviral agents that stimulate natural host antiviral response and function as vaccine adjuvants are important therapeutic goals. Because the viral RNA-RIG-I interaction is the initial trigger of the innate and adaptive immune response to infection, an attractive strategy for the development of broad spectrum antiviral therapy involves the use of natural or synthetic 5'triphosphate containing viral RNAs (5'pppRNA) that activate the innate immune response via the RIG-I like receptors (RLR). Recent experiments from our laboratory have highlighted the therapeutic potential of RIG-I agonists, with the capacity to block replication of diverse human pathogenic viruses (Goulet et al, 2013; Olagnier et al 2013, submitted). It is our hypothesis that stimulation of multiple innate antiviral and inflammatory pathways by 5'pppRNA activation of RIG-I provides a broad-spectrum protective effect against highly pathogenic influenza virus infection and is effective when used as an adjuvant in influenza vaccine development. Our first aim is to evaluate the ability of optimized RIG-I agonists to induce a strong cell-specific immune response in human dendritic cells. Our second aim is to optimize the dose and route of RIG-I agonist administration in vivo and to evaluate the cell-specific response associated with protection from human pathogenic influenza virus. A third aim of this study will determine the ability of 5'pppRNA to boost immune responsiveness and reduce antigenic dose in mice when combined with influenza virus like particles (VLP) vaccines. In these studies, human immune cells, as well as murine models of influenza pathogenesis will be used to examine the effect of RIG-I agonists on the outcome of infection. Biochemical and molecular techniques (such as high throughput BioMark qPCR, siRNA knockdown, flow cytometry, cytokine multiplex assay) will be used to investigate downstream antiviral signaling by 5'pppRNA in human DC. The efficacy of 5'pppRNA as an antiviral agent and adjuvant will be examined in survival studies in vivo; techniques including virus titration and qPCR, histopathological analysis, cytokine multiplex assay, flow cytometry and evaluation of the humoral and cellular adaptive immune response will measure the magnitude of the prophylactic effect of the RNA agonist. The adjuvant potential of 5'pppRNA will also be evaluated in vivo in combination with the COBRA-VLP vaccines. This translational study builds upon the mechanistic knowledge of RIG-I signaling and applies it to the discovery, evaluation, and development of a potent adjuvant to augment vaccine efficacy.