PROJECT SUMMARY Age-related decline in immunity and chronic inflammation significantly contribute to the incidence and susceptibility of the elderly to serious vaccine-preventable conditions including influenza, pneumonia, and herpes zoster. Immune function wanes in all adults as they age into their fifth decade and beyond. Current vaccines are not clinically effective and have to be administered with adjuvants to improve efficacy. Studies comparing vaccine responses in older and younger adults have shown that adjuvant-related inflammation is detrimental to vaccine efficacy in older adults. Therefore, platforms that can improve immune responses without overt inflammation are attractive for vaccine development for the elderly. We are interested in the development of peptide nanofibers as vaccine delivery vehicles for the elderly. These nanofibers trigger autophagy, which can induce an immune response without the use of inflammation-inducing adjuvants. In the current project, we will assess the toxicity and clearance of peptide nanofiber vaccines using yeast models and assess their immunogenicity and efficacy in aged mice using an influenza model. In aim 1, we will express peptide nanofibers in yeast with a GFP tag along with relevant controls to assess toxicity. We will vary the repeat length and expression conditions and confirm expression via immunoblotting. We will also analyze the cells using microscopy to confirm that cell morphology is not altered and assess clearance of the nanofibers over time. Also, the peptide nanofibers will be expressed in autophagy-deficient yeast and clearance of puncta over time will be followed using microscopy. We will also conduct a genome-wide screen to determine if peptide nanofibers engage and interact with other proteins in the cell that might provide insight into the mechanisms of action. In aim 2, we will assess the ability of peptide nanofiber vaccines to elicit robust cellular immunity and protect against lethal influenza infection using aged mice. Following a systemic prime and intranasal boost with the nanofiber vaccines bearing the model antigen OVA, we will investigate effector responses, short-term memory, and long- term memory using flow cytometry. Inflammatory response at the injection site will be assessed using footpad thickness and immunohistochemistry. Protection will be assessed in vaccinated mice following infection with a lethal dose of transgenic influenza A virus expressing the OVA epitope. Completion of the proposed work will leverage peptide nanofiber vaccines as an attractive platform for generating protective immune responses in the elderly with minimal inflammation.