Project Summary Antigenic variation and viral evolution have thwarted traditional influenza vaccination strategies. The development of a “universal influenza vaccine” is a goal of current influenza research. The definition of “universal” should include a vaccine that can induce broad immunity a) within circulating H1 and H3 subtypes, b) across subtypes (heterosubtypic) and c) pre-pandemic. A stepwise approach to accomplishing these criteria are goals of this proposal. The broad protection afforded by a universal influenza vaccine will likely come from immunogens that elicit humoral responses targeting conserved epitopes on the viral hemagglutinin (HA), such as the receptor binding site (RBS) and the “stem”. The focus of this proposal is on the former. The RBS coordinates sialic acid using conserved “core” residues and the surrounding periphery limits the range of protective responses. We will use structure- guided resurfacing to graft the RBS from circulating H1 and H3 HAs onto exotic, non-circulating HAs. These “acceptor” HAs will serve as molecular scaffolds to present the conserved H1 and H3 RBS core, but with a heterologous periphery. This approach will force elicited responses to maintain core contacts, while adapting to the foreign periphery and thus broadening the response. We will design chimeric, disulfide-stabilized, head-only trimers that present the grafted H1 and H3 RBS. Trimerization will conceal neo-epitopes present on monomers, and the exotic, acceptor HAs will remove epitopes targeted by strain-specific responses in immune-experienced individuals. The designed immunogens will be tested for in vivo efficacy. Our strategies will provide candidate immunogens for a universal flu vaccine, by exploiting the immunogenicity of the conserved RBS. They will also be applicable to other rapidly evolving pathogens and to contexts in which preexisting immunity within the population must be factored into vaccine design.