Hidden complexity of glycan-dependent broadly neutralizing HIV antibodies uncovered by digital panning and native trimer

Precursors and intermediates of HIV-1 broadly neutralizing antibodies (bNAbs) are the key to Bcell-lineage immunogen design. In this study, we analyzed the antibody libraries from donor 17, the source of glycan-dependent PGT121-class bNAbs, using a native, prefusion-optimized trimer probe and the “digital panning” method, which combines 900bp deep sequencing and H/L-paired antibodyomics. In addition to single-chain variable fragments (scFvs) resembling bNAbs, digitalpanning also identified early bNAb intermediates with notable binding affinities for the native trimer and broad neutralization. By examining mouse sera from our previous immunization, we further demonstrated that a scaffolded trimer, without mutations to the glycan shield, could elicit robust responses to the N332 supersite that were blocked by donor-derived bNAb intermediates. Our study thus revealed the hidden lineage complexity of the PGT121 class, which will facilitate future vaccine development targeting the N332 supersite, while digital panning provides a useful new tool for identifying rare bNAb intermediates.

HIV-1广谱中和抗体（broadly neutralizing antibodies，bNAbs）的前体与中间态抗体，是B细胞谱系免疫原设计的关键靶点。本研究以一株糖基依赖型PGT121类广谱中和抗体的供体17为研究对象，采用天然预融合优化（prefusion-optimized）三聚体探针，结合900bp深度测序与轻重链配对抗体组学（H/L-paired antibodyomics）的数字淘选（digital panning）方法，对其抗体库进行了系统分析。除筛选到与广谱中和抗体结构相似的单链可变片段（single-chain variable fragments，scFvs）外，数字淘选还鉴定出对天然三聚体具有显著结合亲和力且具备广谱中和活性的早期广谱中和抗体中间态。通过分析本团队此前免疫实验中小鼠的血清样本，本研究进一步证实，一种未对糖基化屏蔽进行突变的支架化三聚体，可诱导针对N332超位点的强烈免疫应答，且该应答可被供体来源的广谱中和抗体中间态所阻断。综上，本研究揭示了PGT121类抗体隐藏的谱系复杂性，这将为未来靶向N332超位点的疫苗开发提供重要助力；同时，数字淘选方法也为稀有广谱中和抗体中间态的鉴定提供了一种极具应用价值的新工具。