Abstract Project 4 The goals of this component of a continuing Program Project are to advance understanding of the human humoral immune response to influenza virus infection and vaccination and to apply that understanding to immunogen design for next generation vaccines. To contribute to those goals, this Project has the following four Aims. (1) Analyze the distribution of neuraminidase (NA) epitopes recognized by memory B cells in recently vaccinated individuals, as a foundation both for analysis of the epitope- dependent response to NA and for design of immunogens in Project 3. (2) Carry out a broad analysis of the way in which the response to influenza vaccination depends on the age of the subjects and hence the extent of antigenic experience with influenza and the subtype responsible for immune imprinting. We will sample the accessible components of the human B-cell repertoire from several representatives of distinct age groups and map their epitopes by binding and, for representatives of all epitopic regions across the hemagglutinin molecule, by cryo-EM structure determination. This component will include influenza NA, but focus largely on hemagglutinin (HA). (3) Collaborate with Project 3 in three areas: studies of the influence of T-cell epitope diversity on humoral responses to designed HA immunogens, design of NA immunogens, and modeling the virus-antibody co-evolutionary "arms race" with directed evolution in the laboratory. (4) Determine structures of selected antibody:antigen complexes, to support efforts in Projects 1 and 2.