Binding affinity landscapes of broadly neutralising antibodies

Over the past 20 years, several broadly neutralizing antibodies (bnAbs) effective against a range of influenza strains have been identified. While structural and biochemical analyses offer insight into their binding mechanisms, the evolutionary paths that lead to these bnAbs are less understood. We explored the landscapes of two key bnAbs, CR9114 and CR6261, by measuring the binding affinity over 100,000 antibody variants against a panel of antigens. We combined exhaustive mutational libraries to simulate all possible evolutionary routes leading to the bnAbs and in-vitro directed evolution for generating new antibody sequences and explore the evolutionary potential of thes bnAbs. We find that both broadly neutralising antibodies exhibits very different patterns of breadth: while many variants of CR6261 display moderate affinity to diverse antigens, those of CR9114 display appreciable affinity only in specific, nested combinations. We also observe extensive pairwise and higher order epistasis between mutations, allowing us to model and recreate plausible trajectory for both antibodies. Together, these features of the binding affinity landscapes strongly favor a sequential acquisition of breadth for CR9114, while CR6261 is less constrained. These results, if generalizable to other bnAbs, may explain the molecular basis for the widespread observation that sequential exposure favors greater breadth, and such mechanistic insight will be essential for predicting and eliciting broadly protective immune responses.