PT  - JOURNAL ARTICLE
AU  - Timothy J.C. Tan
AU  - Zongjun Mou
AU  - Ruipeng Lei
AU  - Wenhao O. Ouyang
AU  - Meng Yuan
AU  - Ge Song
AU  - Raiees Andrabi
AU  - Ian A. Wilson
AU  - Collin Kieffer
AU  - Xinghong Dai
AU  - Kenneth A. Matreyek
AU  - Nicholas C. Wu
TI  - High-throughput identification of prefusion-stabilizing mutations in SARS-CoV-2 spike
AID  - 10.1101/2022.09.24.509341
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.09.24.509341
4099  - http://biorxiv.org/content/early/2022/09/26/2022.09.24.509341.short
4100  - http://biorxiv.org/content/early/2022/09/26/2022.09.24.509341.full
AB  - 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 established 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 integrated a fluorescence-based fusion assay, mammalian cell display technology, and deep mutational scanning. As a proof-of-concept, this method was applied to a region in the S2 domain that includes the first heptad repeat and central helix. Our results revealed that besides K986P and V987P, several mutations simultaneously improved expression and significantly lowered 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.Competing Interest StatementN.C.W., K.A.M. and T.J.C.T. have filed a provisional patent application with the University of Illinois covering the method described in this article. N.C.W. serves as a consultant for HeliXon. The authors declare no other competing interests.