Cold sensitivity of the SARS-CoV-2 spike ectodomain

Abstract

The SARS-CoV-2 spike (S) protein, a primary target for COVID-19 vaccine development, presents its receptor binding domain in two conformations, the receptor-accessible 'up' or receptor-inaccessible 'down' states. Here we report that the commonly used stabilized S ectodomain construct '2P' is sensitive to cold temperatures, and this cold sensitivity is abrogated in a 'down' state-stabilized ectodomain. Our findings will impact structural, functional and vaccine studies that use the SARS-CoV-2 S ectodomain.The spike (S) protein of SARS-CoV-2 mediates receptor binding and cell entry and is a key target for vaccine development efforts. Stabilized S ectodomain constructs have been developed that mimic the native spike, bind the ACE-2 receptor 1,2 and present epitopes for neutralizing antibodies on their surface [3][4][5][6] . The so-called '2P' S ectodomain construct (2P S) comprises residues 1-1,208 of SARS-CoV-2 S and contains two proline (2P) substitutions in the C-terminal S2 domain designed to stabilize the prefusion S conformation; a C-terminal foldon trimerization motif and a mutation that abrogates the furin-cleavage site 1 (Fig. 1a). This and similar constructs have been widely used for structural biology and vaccine studies [1][2][3]7,8 . Purified S ectodomain proteins 9 are assessed for quality control by SDS-PAGE, size exclusion chromatography (SEC), differential scanning fluorimetry (DSF) 10 and negative-stain electron microscopy (NSEM). The last technique has been particularly informative because it reveals the structural integrity of individual molecules, allowing us to examine preparations that look similar by using bulk methods such as SDS-PAGE and SEC (Supplementary Fig. 1). The observed variability between preparations indicates a fragile S ectodomain, and measures to overcome the issue have been previously reported 11,12 .Here we link the apparent fragility of 2P S to its rapid denaturation on storage at 4 °C (Fig. 1b). We followed the structural, biophysical and antigenic properties of 2P S stored under different temperature conditions (Fig. 1, Extended Data Figs. 1 and 2, Supplementary Tables 1 and 2 and Supplementary Figs. 2-6). 2P S was produced in 293F cells at 37 °C and purified at room temperature within 6-8 h (Supplementary Fig. 1). We performed NSEM analysis of 2P S incubated at different temperatures (Fig. 1b and Extended Data Fig. 1d,e). Freshly prepared 2P S samples assessed on the same day they were purified showed on average 75% well-formed spikes, with characteristic kite-shaped morphology on NSEM micrographs

Elicitation of Potent Neutralizing Antibody Responses by Designed Protein Nanoparticle Vaccines for SARS-CoV-2

“…Consistent with previous reports, the monomeric RBD proved quite stable, yielding little change in appearance by SDS-PAGE (Figure S4A), mACE2-Fc and CR3022 binding (Figure S4B), or the ratio of UV/ vis absorption at 320/280 nm, a measure of particulate scattering (Figure S4C). As reported recently (Edwards et al, 2020;Hsieh et al, 2020), the S-2P trimer was unstable at 2 C-8 C, exhibiting clear signs of unfolding by nsEM even at early time points (Figure S3D; Data S2). Instead, S-2P maintained its structure considerably better at 22 C-27 C until the latest time point (28 days), when unfolding was apparent by nsEM and absorbance measurements indicated some aggregation (Figure S4C).…”

“…Accordingly, the diverse polyclonal Ab responses elicited by the RBD-12GS-I53-50 nanoparticle, targeting multiple distinct epitopes, might explain the magnitude of neutralization observed and should minimize the risk of selection or emergence of escape mutations (Davis et al, 2018;Lee et al, 2019). Finally, the high production yield of RBD-I53-50A components and the robust stability of the antigen-bearing RBD nanoparticles suggests that these will likely be more amenable to large-scale manufacturing than the SARS-CoV-2 S-2P trimer, which expresses poorly and is unstable (Edwards et al, 2020;Hsieh et al, 2020;McCallum et al, 2020).…”

A model for pH coupling of the SARS-CoV-2 spike protein open/closed equilibrium

“…Cold sensitivity of a spike protein construct is reduced when the protein is engineered to bias towards the closed/down trimer [28]. It is also significantly reduced when the pH is reduced from 7.4 to 6.0, although the molecular origin of this pH effect, and thus whether the D398 – R355 charge pair is involved, is unknown.…”

Enhancing the Prefusion Conformational Stability of SARS-CoV-2 Spike Protein Through Structure-Guided Design

“…Prefusion stabilization through the insertion of two proline mutations was shown to be successful in preventing postfusion transition in the SARS-CoV and MERS-CoV Spike proteins (22), and a similar SARS-CoV-2 Spike design serves as the antigen for the recent COVID-19 vaccines (which have shown up to 95% efficacy in clinical trials) (38,39). However, due to the limited stability and conformational heterogeneity of the SARS-CoV-2 Spike trimer observed here and elsewhere (34), further improvements are needed to expand use for other applications (such as a reagent for eliciting neutralizing antibodies) (3,4,29,(40)(41)(42).…”