Abstract
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.
Highlights
As is the general case for type-1 fusion proteins, the mature spike is embedded in the viral membrane in a metastable prefusion conformation, which undergoes large structural rearrangements as it transitions to its postfusion form, a transition that facilitates merging of viral and target cell membranes and virus entry into the cell
Differential scanning calorimetry (DSC) showed that storage at 4 C in phosphate buffered saline (PBS) for 8 days resulted in a drop of melting temperature from 65.1 C for freshly purified protein to 47.9 C, the overall energy of unfolding, as calculated from the peak area, remained similar at 250 kcal/mol (Fig. 1C)
As most known SARS-CoV-2 neutralizing antibodies target the receptorbinding domain (RBD) or Nterminal domain (NTD) regions of the spike, the Bio-Layer Interferometry (BLI) results indicate that the high ELISA titers, in combination with lack of neutralization from sera of 30-day aged S2P immunized mice, could be attributed to elicited antibody responses being directed to nonneutralizing epitopes
Summary
As is the general case for type-1 fusion proteins, the mature spike is embedded in the viral membrane in a metastable prefusion conformation, which undergoes large structural rearrangements as it transitions to its postfusion form, a transition that facilitates merging of viral and target cell membranes and virus entry into the cell. Unfolding of SARS-CoV-2 S2P spike determine the stability of the protein as a function of storage time.
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