Project Summary While a universal influenza vaccine, providing life-long protective immunity against all current and future drifted and shifted subtypes of influenza virus after 2 or 3 doses, would be a game-changing solution to reducing the global burden of influenza and its associated morbidity and mortality, other approaches to solving this problem are urgently needed. There is still much to learn about immune responses to respiratory pathogens, such as influenza virus, and vaccine approaches that drive life-long immunity to respiratory viruses have yet to be demonstrated in humans. Therefore, we propose to develop a broadly protective influenza booster vaccine that can be administered, either seasonally or during pandemics, to individuals with prior exposures to either natural infection or seasonal vaccination. Given the non-uniform influenza immune history in a given population of individuals, it is likely that booster vaccines will need to be customized either at the individual or regional level, based on local influenza virus epidemiology or vaccine uptake. Nucleic acid vaccine platforms provide the ideal framework for such personalized-medicine approaches, due to the flexibility of development, as typified by the ongoing COVID-19 pandemic. As proof-of-concept, here we propose to apply our clinical-stage replicating RNA vaccine platform to develop a booster vaccine targeting the conserved hemagglutinin stem and nucleoprotein of group 1 influenza viruses and evaluating immunogenicity and efficacy against heterologous group 1 influenza virus infections in mouse and ferret models of pre-existing influenza virus immunity. These data will inform the feasibility of such an approach and characterize what types of pre-existing immunity, in terms of anti-hemagglutinin antibody specificity and magnitude, are required for booster vaccine efficacy. As these pre-existing antibody criteria are easily assessed in humans, it is likely that a personalized approach to influenza booster vaccination is achievable.