Vaccine-based elicitation of broadly neutralizing antibodies holds great promise for preventing HIV-1 transmission. However, the key biophysical markers of improved antibody recognition remain uncertain in the diverse landscape of potential antibody mutation pathways, and a more complete understanding of anti–HIV-1 fusion peptide (FP) antibody development will accelerate rational vaccine designs. Here we survey the mutational landscape of the vaccine-elicited anti-FP antibody, vFP16.02, to determine the genetic, structural, and functional features associated with antibody improvement or fitness. Using site-saturation mutagenesis and yeast display functional screening, we found that 1% of possible single mutations improved HIV-1 envelope trimer (Env) affinity, but generally comprised rare somatic hypermutations that may not arise frequently in vivo. We observed that many single mutations in the vFP16.02 Fab could enhance affinity >1,000-fold against soluble FP, although affinity improvements against the HIV-1 trimer were more measured and rare. The most potent variants enhanced affinity to both soluble FP and Env, had mutations concentrated in antibody framework regions, and achieved up to 37% neutralization breadth compared to 28% neutralization of the template antibody. Altered heavy- and light-chain interface angles and conformational dynamics, as well as reduced Fab thermal stability, were associated with improved HIV-1 neutralization breadth and potency. We also observed parallel sets of mutations that enhanced viral neutralization through similar structural mechanisms. These data provide a quantitative understanding of the mutational landscape for vaccine-elicited FP-directed broadly neutralizing antibody and demonstrate that numerous antigen-distal framework mutations can improve antibody function by enhancing affinity simultaneously toward HIV-1 Env and FP.

基于疫苗的广泛中和抗体的诱导对于预防 HIV-1 传播具有很大的希望。然而，在潜在抗体突变途径的多样化景观中，改善抗体识别的关键生物物理标志物仍然不确定，而对抗 HIV-1 融合肽 (FP) 抗体开发的更全面了解将加速合理的疫苗设计。在这里，我们调查了疫苗引发的抗 FP 抗体 vFP16.02 的突变情况，以确定与抗体改进或适应性相关的遗传、结构和功能特征。使用位点饱和诱变和酵母显示功能筛选，我们发现 1% 的可能单突变提高了 HIV-1 包膜三聚体 (Env) 的亲和力，但通常包括罕见的体细胞超突变，这些突变在体内可能不会经常出现。我们观察到 vFP16.02 Fab 中的许多单突变可以增强对可溶性 FP 的亲和力 > 1,000 倍，尽管对 HIV-1 三聚体的亲和力改善更多且罕见。最有效的变体增强了对可溶性 FP 和 Env 的亲和力，突变集中在抗体框架区，与模板抗体的 28% 中和相比，实现了高达 37% 的中和宽度。改变重链和轻链界面角度和构象动力学，以及降低的 Fab 热稳定性，都与改善 HIV-1 中和广度和效力有关。我们还观察到平行的突变组，它们通过类似的结构机制增强了病毒中和作用。