Cold sensitivity of the SARS-CoV-2 spike ectodomain

Abstract

SummaryThe impact of COVID-19 and the urgency to develop a vaccine against the SARS-CoV-2 virus cannot be overstated. The viral fusion spike (S) protein ectodomain is the primary target for vaccine development. Here we report an unexpected cold sensitivity of a stabilized SARS-CoV-2 ectodomain construct currently being widely used for immunogen design. We found that when stored at 22 or 37 °C for 1 week, the S-protein displayed well-ordered trimeric spikes by negative stain electron microscopy. However, storage at 4 °C reduced the trimeric spikes to <10%, accompanied by decreased stability and enhanced exposure of the ACE-2 receptor binding site. Well-formed S particles could be recovered from cold-stored samples by a brief incubation at 37 °C. Our results will have broad impact on structural, functional and vaccine studies using the SARS-CoV-2 S ectodomain.HighlightsSARS-CoV-2 S ectodomain construct, widely used for vaccine studies, exhibits cold sensitivity.Negative stain electron microscopy shows disintegration of spike structure upon storage at 4 °C.Differential scanning calorimetry measurements confirm destabilization by cold.Cold storage alters antigenicity of SARS-CoV-2 spike.Brief incubation at 37 °C restored spike integrity after cold-storage.

D614G mutation alters SARS-CoV-2 spike conformational dynamics and protease cleavage susceptibility at the S1/S2 junction

“…This finding is in contrast to previous observations for MERS and the SARS-CoV-1 ectodomains, which showed a mixture of the prefusion and postfusion conformations unless the PP mutation was included (Pallesen et al, 2017). Binding of S-GSAS and S-GSAS/PP measured by ELISA to ACE2 and CR3022 (Edwards et al, 2020), both requiring an RBD-up conformation, Ab712199 and Ab511584, two antibodies isolated from a COVID-19 convalescent donor with epitopes mapping to the ACE2 binding site and S2 domain respectively, and 2G12, binding to a quaternary S2 glycan epitope, were all nearly identical demonstrating that both constructs showed similar antigenic behavior ( Figure 2D). Using differential scanning fluorimetry to measure the spike thermostability, we found the S-GSAS and S-GSAS/PP ectodomains showed similar melting temperatures (Figure 2E).…”

“…SEC and SDS-PAGE profiles were similar to the S-GSAS and S-GSAS/D614G proteins confirming well-folded and homogeneous spike preparations (Figure 5A, B). NSEM micrographs showed characteristic kite-shaped particles (Edwards et al, 2020) for the pre-fusion S protein, and 2D-classification of particles from NSEM revealed well folded spikes, further confirming that S-HRV3C spikes retained the overall fold and structure of the S-GSAS spikes (Figure 5C, D).…”

“…Similar to the S-GSAS/PP construct (Edwards et al, 2020;Henderson et al, 2020;Wrapp et al, 2020), S-GSAS showed 80-90% intact prefusion spike trimers by negative stain electron microscopy (NSEM) ( Figure 2C). This finding is in contrast to previous observations for MERS and the SARS-CoV-1 ectodomains, which showed a mixture of the prefusion and postfusion conformations unless the PP mutation was included (Pallesen et al, 2017).…”

“…ELISA assays Spike samples were pre-incubated at different temperatures then tested for antibody-or ACE-2binding in ELISA assays as previously described (Edwards et al, 2020). Assays were run in two formats.…”

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 2-8°C, exhibiting clear signs of unfolding by nsEM even at early time points ( Figure S3D and Supplementary Item 2). It maintained its structure considerably better at 22-27°C until the latest time point (28 days), when unfolding was apparent by nsEM and UV/vis indicated some aggregation ( Figure S4C).…”