Over the past two decades, several broadly neutralizing antibodies (bnAbs) that confer protection against diverse influenza strains have been isolated. Structural and biochemical characterization of these bnAbs has provided molecular insight into how they bind distinct antigens. 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 bnAbs (CR9114, 16 heavy-chain mutations; CR6261, 11 heavy-chain mutations), reconstructing all possible evolutionary intermediates back to the unmutated germline sequences. We find that these two libraries exhibit strikingly 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. 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 CR6261 and different for CR9114. Together, these features of the binding affinity landscapes strongly favor sequential acquisition of affinity to diverse antigens for CR9114, while the acquisition of breadth to more similar antigens for 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.

中文翻译：

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结合亲和力景观限制了广泛中和抗流感抗体的进化

在过去的 20 年里，已经分离出几种可以保护多种流感病毒株的广泛中和抗体 (bnAbs)。这些 bnAb 的结构和生化表征为它们如何结合不同的抗原提供了分子见解。然而，我们对导致 bnAb 的进化途径以及如何最好地引发它们的理解仍然有限。在这里，我们测量了两种天然分离的流感 bnAb（CR9114，16 个重链突变；CR6261，11 个重链突变）的组合完整突变文库的平衡解离常数，将所有可能的进化中间体重建回未突变的种系序列。我们发现这两个库表现出截然不同的广度模式：虽然 CR6261 的许多变体对不同抗原表现出中等亲和力，但 CR9114 的变体仅在特定的嵌套组合中表现出可观的亲和力。通过检查突变之间广泛的成对和高阶上位性，我们发现具有强协同相互作用的关键位点对于 CR6261 的抗原高度相似，而对于 CR9114 则不同。总之，结合亲和力景观的这些特征强烈有利于 CR9114 对不同抗原的亲和力的连续获得，而 CR6261 对更相似抗原的广度的获得较少受到限制。如果将这些结果推广到其他 bnAb，则可以解释广泛观察的分子基础，即顺序暴露有利于更大的广度，