Binding affinity landscapes constrain the evolution of broadly neutralizing anti-influenza antibodies

Over the past two decades, several broadly neutralizing antibodies (bnAbs) that confer protection against diverse influenza strains have been isolated1,2. Structural and biochemical characterization of these bnAbs has provided molecular insight into how they bind distinct antigens1. However, our understanding of the evolutionary pathways leading to bnAbs, and thus how best to elicit them, remains limited. Here, we measure equilibrium dissociation constants of combinatorially complete mutational libraries for two naturally isolated influenza bnAbs3-5 (CR-9114, 16 mutations; CR-6261, 11 mutations), reconstructing all possible intermediates back to the unmutated germline sequences. We find that these two libraries exhibit strikingly different patterns of breadth: while many variants of CR-6261 display moderate affinity to diverse antigens, those of CR-9114 display appreciable affinity only in specific, nested combinations. By examining the extensive pairwise and higher-order epistasis between mutations, we find key sites with strong synergistic interactions that are highly similar across antigens for CR-6261 and different for CR-9114. Together, these features of the binding affinity landscapes strongly favor sequential acquisition of affinity to diverse antigens for CR-9114, while the acquisition of breadth to more similar antigens for CR-6261 is less constrained. These results, if generalizable to other bnAbs, may explain the molecular basis for the widespread observation that sequential exposure favors greater breadth6-8, and such mechanistic insight will be essential for predicting and eliciting broadly protective immune responses.