Influenza virus infections cause significant morbidity and mortality in the US and world. Since the current inactivated virus vaccines do not induce protective immunity to emerging strains of influenza virus, new vaccines are urgently needed. Unfortunately, the current inactivated virus vaccines do not contain adjuvant and do not induce lasting cellular immunity. Since the only adjuvant approved for use in humans is alum and alum enhances type 2 immune responses that are often detrimental for lung function, it is important to identify new, safe adjuvants that can be used in an influenza vaccine. Most commonly used experimental adjuvants are pathogen-derived and enhance immune responses by triggering Toll-like receptors (TLRs) which induce inflammation and tissue damage and activate dendritic cells. However, other molecules, unrelated to TLRs, also have the potential to modulate immune responses. One such molecule is the ecto-enzyme CD38 which catabolizes extracellular NAD, released by damaged and dying cells, to produce Ca2+mobilizing metabolites that modulate chemokine receptor signaling. CD38 and its substrate, NAD, are required for leukocyte migration and are also important for the generation of functional effector T cells. Interestingly, NAD, when co-administered with protein antigens and subthreshold amounts of adjuvant, significantly enhances T cell dependent immune responses. Based on these data, we hypothesize that NAD functions as a "co-adjuvant" to enhance innate and adaptive immune responses and that CD38 and extracellular NAD represent a novel class of immune modulators that are distinct from the TLRs. To test this hypothesis, we will first determine how NAD mediates its co-adjuvant properties (Aim 1). In Aim 2, we will determine how extracellular NAD regulates immune responses to influenza virus and in Aim 3, we will test the efficacy of a NAD-containing vaccine in enhancing immune protection to challenge infection with a new serotype of influenza virus. These experiments will facilitate the development of a new class of co-adjuvants that can be used safely in humans to improve cell-mediated immune protection to pathogenic intracellular organisms like influenza. Relevance to public health. Infections with influenza virus cause significant morbidity and mortality in the United States. In a typical year, approximately 100,000 peoples are hospitalized in the US due to influenza and 20,000-35,000 people die from the viral infection or secondary bacterial infections. Furthermore, the new emerging strains of influenza, such as the highly virulent H5N1 variant that appeared in Hong Kong in 1997, have the potential to trigger a worldwide pandemic with devastating social and economic consequences. Thus, it is one of NIAID's highest priorities (NOT-AI-05-013) that we develop more effective influenza vaccines that are protective against a wide array of different viral serotypes.