Mutational fitness landscapes reveal genetic and structural improvement pathways for an HIV-1 broadly neutralizing antibody

Vaccine-based elicitation of broadly neutralizing antibodies holds great promisefor preventing HIV-1 transmission. However, the key biophysical markers ofimproved antibody recognition remain uncertain among the diverse landscape ofpotential antibody mutation pathways, and a more complete understanding ofanti-HIV-1 fusion peptide (FP) antibody development will accelerate rationalvaccine designs. Here we surveyed the mutational landscape of a vaccineelicitedanti-FP antibody, vFP16.02 to determine the genetic, structural, andfunctional features that are associated with antibody improvement or fitness.Using site-saturation mutagenesis and yeast display functional screening, wefound that 1.0% of possible single mutations improved HIV-1 envelope (Env)trimer affinity, but these generally comprised rare somatic hypermutations thatmay not arise frequently in vivo. We observed that many single mutations couldenhance soluble FP affinity >1,000-fold, although affinity improvements againstthe HIV-1 trimer were more measured and rare. The most potent variantsenhanced affinity to both soluble FP and Env, were concentrated in antibodyframework regions, and achieved up to 37% neutralization breadth. Alteredheavy and light chain interface angles and conformational dynamics, as well asreduced Fab thermal stability, were associated with improved HIV-1neutralization breadth and potency. We also observed parallel sets of mutationsthat enhanced viral neutralization through similar structural mechanisms. Thesedata provide a quantitative understanding of the mutational landscape for an FPdirectedbroadly neutralizing antibody, and demonstrate that numerous antigendistalframework mutations can improve antibody function by enhancing affinitysimultaneously towards HIV-1 Env and FP.

基于疫苗诱导广谱中和抗体（broadly neutralizing antibodies）的策略，在预防人类免疫缺陷病毒1型（HIV-1）传播方面展现出巨大前景。然而，在纷繁复杂的潜在抗体突变通路图景中，提升抗体识别能力的关键生物物理标志物仍未明确；而更全面地阐明抗HIV-1融合肽（fusion peptide, FP）抗体的发育机制，将加速理性疫苗设计的进程。本研究针对疫苗诱导的抗FP抗体vFP16.02的突变图景展开系统性调研，旨在明确与抗体功能优化或适配性相关的遗传、结构及功能特征。通过位点饱和诱变（site-saturation mutagenesis）与酵母展示（yeast display）功能筛选，我们发现仅1.0%的潜在单突变可提升HIV-1包膜（envelope, Env）三聚体的结合亲和力，但这类突变大多属于罕见的体细胞超突变（somatic hypermutations），在体内可能难以高频发生。我们观察到，诸多单突变可使可溶性FP的结合亲和力提升超1000倍，不过针对HIV-1三聚体的亲和力提升则相对有限且较为罕见。效力最强的变异株可同时提升对可溶性FP与Env的亲和力，且主要集中于抗体框架区（framework regions），其中和广度最高可达37%。重链与轻链界面夹角的改变、构象动力学变化，以及抗原结合片段（Fragment antigen-binding, Fab）热稳定性的降低，均与HIV-1中和广度与效力的提升相关。我们还发现了多组平行突变，它们可通过相似的结构机制增强病毒中和能力。本研究数据为靶向FP的广谱中和抗体的突变图景提供了定量认知，并证实诸多抗原远端的框架区突变，可通过同时增强对HIV-1 Env与FP的亲和力来优化抗体功能。