High-throughput identification of prefusion-stabilizing mutations in SARS-CoV-2 spike.
High-throughput identification of prefusion-stabilizing mutations in SARS-CoV-2 spike.
复制标题
高通量鉴定 SARS-CoV-2 刺突中的融合前稳定突变。
DOI：
10.1101/2022.09.24.509341
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Wu,NicholasC
中科院分区：
文献类型：
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作者：
Tan,TimothyJC;Mou,Zongjun;Lei,Ruipeng;Ouyang,WenhaoO;Yuan,Meng;Song,Ge;Andrabi,Raiees;Wilson,IanA;Kieffer,Collin;Dai,Xinghong;Matreyek,KennethA;Wu,NicholasC
设计融合前稳定的SARS-CoV-2加标物对于COVID-19疫苗的有效性至关重要。美国的所有COVID-19疫苗都编码具有K986 P/V987 P突变的刺突，以稳定其融合前构象。然而，工程化融合前稳定的刺突免疫原的当代方法涉及繁琐的实验工作，并且严重依赖于结构信息。在这里，我们建立了一个系统的和公正的方法来识别突变，同时提高表达和稳定的融合前构象的SARS-CoV-2刺突。我们的方法集成了基于荧光的融合测定、哺乳动物细胞展示技术和深度突变扫描。作为概念验证，我们将这种方法应用于S2结构域中的一个区域，该区域包括第一个七肽重复序列和中央螺旋。我们的研究结果表明，除了K986 P和V987 P，几个突变同时提高表达，并显着降低融合的穗。由于融合前稳定化是病毒免疫原设计的常见挑战，这项工作将有助于加速针对不同病毒的疫苗开发。
Designing prefusion-stabilized SARS-CoV-2 spike is critical for the effectiveness of COVID-19 vaccines. All COVID-19 vaccines in the US encode spike with K986P/V987P mutations to stabilize its prefusion conformation. However, contemporary methods on engineering prefusion-stabilized spike immunogens involve tedious experimental work and heavily rely on structural information. Here, we establish a systematic and unbiased method of identifying mutations that concomitantly improve expression and stabilize the prefusion conformation of the SARS-CoV-2 spike. Our method integrates a fluorescence-based fusion assay, mammalian cell display technology, and deep mutational scanning. As a proof-of-concept, we apply this method to a region in the S2 domain that includes the first heptad repeat and central helix. Our results reveal that besides K986P and V987P, several mutations simultaneously improve expression and significantly lower the fusogenicity of the spike. As prefusion stabilization is a common challenge for viral immunogen design, this work will help accelerate vaccine development against different viruses.