SARS-CoV-2 S2P spike ages through distinct states with altered immunogenicity

Olia, Adam S.; Tsybovsky, Yaroslav; Chen, Steven J.; Liu, Cuiping; Nazzari, Alexandra F.; Ou, Li; Wang, Lingshu; Kong, Wing-Pui; Leung, Kwan; Liu, Tracy; Stephens, Tyler; Teng, I-Ting; Wang, Shuishu; Yang, Eun Sung; Zhang, Baoshan; Zhang, Yi; Zhou, Tongqing; Mascola, John R.; Kwong, Peter D.

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 one 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, though 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.