The interactions of Toll-like receptors (TLR) with their cognate ligands, Pathogen-Associated Molecular Patterns (PAMPs), trigger the immediate response of the innate immune system and set off a cascade of events that influence the adaptive immune response. PAMP:antigen fusion protein vaccines target the antigen to the appropriate accessory cell population and trigger both antigen-specific and critical costimulatory signals in the same cell. At the present time, only a handful of polypeptide PAMPs has been identified. We propose to discover and develop novel polypeptide TLR ligands that are highly specific for individual TLRs and incorporate these ligands into vaccines for use against infectious diseases that pose a public health and national defense threat. The phage display technology will be used to screen a large number (>10) of random or biased peptides for binding to cells over-expressing the TLR of interest. Biased peptide libraries will be based on sequences of known polypeptide TLR ligands, such as bacterial flagellin (TLR5 ligand) or measles virus HA protein (TLR2 ligand). Phages displaying TLR-binding peptides will be enriched by 3 to 6 rounds of panning on mammalian cells expressing the TLR of interest. Relevant biological activity of the enriched phage will be confirmed in NF-kappaB-dependent luciferase reporter assays in a panel of mammalian cell lines expressing different TLRs. Novel TLR-binding peptides will be expressed as histidine-tagged fusion proteins with model antigens (e.g., LLO and p60 from Listeria monocytogenes) and purified by state of the art biochemical techniques. Purified recombinant proteins will be formulated in phosphate-buffered saline for administration to mice without conventional adjuvants. Immunized mice will be challenged with the appropriate infectious agent (such as L. monocytogenes). Analysis of cytotoxic T-lymphocyte responses, antibody responses, and cytokine profiles induced by triggering different TLRs will help elucidate the mechanisms of action of different TLR ligands. The goal of the program is to identify, characterize, and express polypeptide ligands for known TLR, and use the ligands as tools to develop a better understanding of the immunological sequelae associated with triggering each TLR. Such studies could lead to the generation of very efficacious and cost-effective vaccines.