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.

Extended Data Fig. 1. SDS-PAGE and NSEM analysis of SARS-CoV-2 2PS ectodomain incubated at different temperatures.

(a) SEC profile, (b) SDS-PAGE analysis (lane 1= molecular weight marker, lane 2= reducing conditions, lane 3= non-reducing conditions) and (c) DSF profile of a freshly purified sample of the 2P spike. (d) NSEM micrograph of 2P spike after storage at 22 °C for one week. (e) NSEM micrograph of 2P spike after storage at 4 °C for one week followed by recovery at 37 °C for 6 days. The circle indicates spike aggregation visible in the micrograph.

Extended Data Fig. 2. Changes in antigenicity of SARS-CoV-2 2PS ectodomain incubated at different temperatures.

(a) Antibody CR3022 IgG (left) and 2G12 IgG (right) binding to two independently purified lots of 2P spike stored at different temperatures measured by SPR. Data for spike samples measured after a 1-week incubation at 37, 22, and 4 °C, are shown in blue, green, and red respectively; sample stored 1 week at 4 °C and then incubated for 6 hours at 37 °C shown in cyan. During the SPR run the sample chamber was maintained at temperatures of 37 °C, 22 °C and 8 °C, for the 37 °C, 22 °C and 4 °C incubated samples, respectively. The binding experiments were carried out at 25 °C. The bar graphs in Fig. 1d show data from Lot # 025MFK. The schematics shows the assay format. (b) ELISA binding profiles showing binding of ACE-2 receptor ectodomain, RBD-directed antibody CR3022, and S2-directed, glycan-reactive antibody, 2G12. The same color scheme was used as for the SPR experiment. The black line indicates a freshly purified 2P spike sample that was flash frozen, then thawed and incubated for 20 min at 37 °C. The schematics show the ELISA format. The measurements in the top row were done in a format where antibody was coated on the plate and the measurements in the bottom row were done in a format where spike was captured on a strep-coated plate (see Methods).

Extended Data Fig. 3. Antigenic response of SARS-CoV-2 2PS ectodomain incubated at different temperatures to antibodies elicited from convalescent patient sera.

(a) SPR binding profiles showing binding of (left) RBD-directed antibody, DH1179 and (right) S2-directed antibody, DH1189.1 to spike samples incubated for 1 week at either 37 °C (blue), 22 °C (green) or 4 °C (red). Binding to spike sample first incubated at 4 °C for 1 week, then moved to 37 °C for 3 hours prior to the experiment is shown in cyan. (b) ELISA binding profiles showing binding of RBD-directed antibody, DH1179, S2-directed antibody, DH1189.1, and influenza HA-directed antibody CH65 (control) to spike samples incubated at different temperatures (same color scheme as in panel a). The black line indicates a freshly purified 2P spike sample that was flash-frozen, then thawed and incubated for 20 min at 37 °C.

Figure 1.

Temperature-dependence of the SARS-CoV-2 S ectodomain. (a) Schematic of the SARS-CoV-2 spike (top, S) and a stabilized, furin cleavage-deficient, soluble ectodomain construct (bottom, 2P). (b) Representative NSEM micrographs of 2P S samples: freshly prepared (left), after storage at 4 °C for 7 days (middle), or stored at 4 °C for 7 days followed by a 3-hour incubation at 37 °C (right). Bar graph summarizes results from NSEM analyses of 2P S samples stored under different conditions. Data shown are mean and range (for N=2) or mean and s.e.m. (from N = 3–7) independent experiments with different protein lots; exact N at the top of each bar. (c) Left, DSF profiles following changes in protein intrinsic fluorescence (expressed as ratio between fluorescence at 350 nm and 330 nm) upon applying a thermal ramp. Maxima and minima indicate inflection temperatures, Ti. For each storage condition (color coded as shown in 1d), 5 overlaid curves (technical replicates) are shown. Right. DSC profiles, shown as 2 overlaid curves of technical replicates. (d) Antibody binding to 2P S stored at different temperatures, measured by SPR; antibodies were CR3022 IgG (left) and 2G12 IgG (right). During the SPR run, the sample chamber was maintained at 37 °C, 22 °C or 8 °C, for samples that had been stored at 37 °C, 22 °C or 4 °C, respectively. The binding experiments were carried out at 25 °C. Data are mean and s.e.m. of three technical replicates, and are representative of at least 5 independent experiments, using separate protein lots (two independent repeats are shown in Extended Data Figure 2).

Figure 2.

Engineered SARS-CoV-2 spike variant, rS2d-HexaPro, is resistant to temperature-dependent structural changes. (a) Left, structures of HexaProshowing a 1-RBD-up conformation (PDB 6XKL) andrS2d (PDB 6X29) showing an all-RBD-down conformation. Middle, SDS-PAGE analysis of HexaPro and rS2d-HexaPro samples, under reducing (R) or non-reducing (NR) conditions. Right, bar graph showing protein yields for rS2d and rS2d-HexaPro produced in 293F or CHO cells. (b) Left, representative NSEM micrograph from a preparation of rS2d-Hexapro (top) and 2D class averages (bottom). Middle, 3D reconstruction of rS2d-Hexapro. Right, bar graph summarizing results from NSEM on S ectodomain variants stored at different temperatures. Data are mean and range (for N=2) or s.e.m. (for N ≥ 3) for independent experiments with different protein lots; exact N indicated at the top of each bar. (c) DSF profiles for Hexapro (left) and rS2d-Hexapro (right). For each storage condition, 3–4 overlaid curves (technical replicates) are shown. (d) Binding of 2G12 and ACE-2 to Hexapro (left) and rS2d-Hexapro (right) measured by ELISA.