Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Vaccine Research Center Electron Microscopy Unit, Cancer Research Technology Program, Leidos Biomedical Research Inc, Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA.
J Biol Chem. 2021 Oct;297(4):101127. doi: 10.1016/j.jbc.2021.101127. Epub 2021 Aug 27.
The SARS-CoV-2 spike is the primary target of virus-neutralizing antibodies and critical to the development of effective vaccines against COVID-19. Here, we demonstrate that the prefusion-stabilized two-proline "S2P" spike-widely employed for laboratory work and clinical studies-unfolds when stored at 4 °C, physiological pH, as observed by electron microscopy (EM) and differential scanning calorimetry, but that its trimeric, native-like conformation can be reacquired by low pH treatment. When stored for approximately 1 week, this unfolding does not significantly alter antigenic characteristics; however, longer storage diminishes antibody binding, and month-old spike elicits virtually no neutralization in mice despite inducing high ELISA-binding titers. Cryo-EM structures reveal the folded fraction of spike to decrease with aging; however, its structure remains largely similar, although with varying mobility of the receptor-binding domain. Thus, the SARS-CoV-2 spike is susceptible to unfolding, which affects immunogenicity, highlighting the need to monitor its integrity.
SARS-CoV-2 的刺突蛋白是病毒中和抗体的主要靶点，对于开发针对 COVID-19 的有效疫苗至关重要。在这里，我们证明了预融合稳定的双脯氨酸“S2P”刺突蛋白——广泛用于实验室工作和临床研究——在 4°C 和生理 pH 下会展开，这可以通过电子显微镜 (EM) 和差示扫描量热法观察到，但它的三聚体、天然样构象可以通过低 pH 处理重新获得。储存约 1 周后，这种展开不会显著改变抗原特性；然而，长时间储存会降低抗体结合能力，而且即使诱导出高 ELISA 结合滴度，一个月大的刺突蛋白也几乎不能引起中和作用。冷冻电镜结构显示，随着时间的推移，刺突蛋白的折叠部分减少；然而，尽管受体结合域的流动性不同，其结构仍然大致相似。因此，SARS-CoV-2 的刺突蛋白容易展开，这会影响其免疫原性，强调了需要监测其完整性。
