A single residue change only differing by an atomic group can drive imprinting to influenza.

First described as original antigenic sin (OAS), which is deleterious, or now immune imprinting, which also accounts for beneficial effects, it is clear that immune responses to viruses tend to be biased by previous exposure to similar strains1,2. Various non-exclusive models for the basis of imprinting include that it results from unique features of childhood immunity3,4; it is driven by pre-existing serum antibodies via epitope masking5; or it occurs as a byproduct of residual memory following viral antigenic evolution5. To understand the basis and impact of imprinting from influenza, we characterized the B cell responses of young children upon consecutive first infections with divergent H1N1 and H3N2 influenza viruses. Here, we show that beyond being a primary response, there are no major phenotypic differences in the B cell response of children compared to that of adults. The distinct immunoglobulin variable (IgV) gene repertoire of influenza virus hemagglutinin (HA)-reactive B cells in children, along with increased cross-reactivity to past strains in adults, suggests significant homosubtypic imprinting in adults. As most B cells induced after consecutive infections with antigenically distant H1N1 and H3N2 are strain-specific, heterosubtypic imprinting is rare. However, these successive infections resulted in up to 6% of H1/H3 cross-reactive B cells, targeting the highly conserved central stalk epitope. These B cells express antibodies that are dominantly affected by imprinting with reduced affinity, neutralization potency, and breadth of activity. Mechanistically, H3 to H1 imprinting was caused by a single amino acid change (D46N), differing by just a carboxyl versus an amide atomic group on the central stalk epitope, resulting in a detrimental shift in the specificity of most H1/H3 cross-neutralizing B cells from seven children. We conclude that imprinting by influenza is most evident at the individual epitope level, where minor molecular differences can have a significant impact and need to be accounted for in epitope-targeting vaccine designs.