Influenza viruses cause seasonal epidemics and occasional pandemics that inflict significant global morbidity and mortality. The threat of emerging influenza infections has stimulated development of vaccines to induce broadly protective and durable immunity. Technology that may boost immunogenicity safely beyond levels afforded by currently used adjuvants is critical to advancing vaccine design and development. While research over several decades has identified broad peptidoglycan (PGN) immunostimulatory properties with potential application as an adjuvant, technical, regulatory, and development challenges have prevented development of native PGN for vaccine applications. Teichos Laboratories, LLC (Teichos) proprietary technology produces a platform of macromolecular immunostimulatory biologics by chemoenzymatic total synthesis of authentic PGN core structure, termed sPGN. Preliminary results with a prototype sPGN, TL-001, support its utility as a vaccine adjuvant. TL-001 is a single-strand, uncrosslinked, macromolecular (ca. 20 – 200 kDa) sPGN produced by robust, scalable, and flexible total synthesis from commercially available small molecules, standard reagents, and a single enzyme. Therefore, TL-001, like all sPGN, is produced free of the adventitious, and often potent, contaminant immune reactive materials that accompany similar macromolecules isolated from microorganisms. In a laboratory vaccination model, low dose TL-001 induces a robust response that is mediated through TLR2 and NOD2 receptors to stimulate coordinated innate and adaptive immune responses through two synergistic layers of signalling that amplify antibody production and T cell activation. Neither toxicity nor reactogenicity were observed when high dose sPGN was administered to laboratory animals. This proposal seeks support to advance TL-001 from discovery (preliminary data) to development phase (application to disease prevention studies) by conducting proof-of- principle experiments to validate development as a human influenza vaccine adjuvant. We will produce a test lot of TL-001 that will be evaluated to define the role of TLR2 and other cellular mechanisms in mediating the immunostimulatory responses in human monocytes and macrophages. We will characterize the antibody and T cell responses to TL-001 using an influenza H1 subunit vaccination model in mice. Anticipated technical success in the proposed studies will help support Phase II assessments of TL-001 safety, protection in lethal influenza challenge models, alternate routes of administration, and formulation development.