Stabilizing the closed SARS-CoV-2 spike trimer

TLDR

The SARS‑CoV‑2 spike protein is a key vaccine target, but its intrinsic instability causes premature refolding to a post‑fusion state, reducing immunogenicity and prefusion trimer yields. The study aims to design soluble spike trimers with higher yields and stability through point mutations and disulfide bridges. Using structure‑based design, the authors introduced single point mutations and disulfide bridges to create soluble spike trimers with improved expression and stability. The authors identified stability‑critical regions (HR1, SD1, SD2‑614), engineered a minimal set of inter‑protomer mutations to produce an S‑closed variant with 6.4‑fold higher expression and no heterologous trimerization domain, and confirmed a predominantly closed prefusion conformation by cryo‑EM, providing a highly stable, well‑producing spike protein that will aid vaccine development and diagnostics.

Abstract

Abstract The trimeric spike (S) protein of SARS-CoV-2 is the primary focus of most vaccine design and development efforts. Due to intrinsic instability typical of class I fusion proteins, S tends to prematurely refold to the post-fusion conformation, compromising immunogenic properties and prefusion trimer yields. To support ongoing vaccine development efforts, we report the structure-based design of soluble S trimers with increased yields and stabilities, based on introduction of single point mutations and disulfide-bridges. We identify regions critical for stability: the heptad repeat region 1, the SD1 domain and position 614 in SD2. We combine a minimal selection of mostly interprotomeric mutations to create a stable S-closed variant with a 6.4-fold higher expression than the parental construct while no longer containing a heterologous trimerization domain. The cryo-EM structure reveals a correctly folded, predominantly closed pre-fusion conformation. Highly stable and well producing S protein and the increased understanding of S protein structure will support vaccine development and serological diagnostics.