Contribution of single mutations to selected SARS-CoV-2 emerging variants spike antigenicity

Abstract

Towards the end of 2020, multiple variants of concern (VOCs) and variants of interest (VOIs) have arisen from the original SARS-CoV-2 Wuhan-Hu-1 strain. Mutations in the Spike protein are highly scrutinized for their impact on transmissibility, pathogenesis and vaccine efficacy. Here, we contribute to the growing body of literature on emerging variants by evaluating the impact of single mutations on the overall antigenicity of selected variants and their binding to the ACE2 receptor. We observe a differential contribution of single mutants to the global variants phenotype related to ACE2 interaction and antigenicity. Using biolayer interferometry, we observe that enhanced ACE2 interaction is mostly modulated by a decrease in off-rate. Finally, we made the interesting observation that the Spikes from tested emerging variants bind better to ACE2 at 37°C compared to the D614G variant. Whether improved ACE2 binding at higher temperature facilitates emerging variants transmission remain to be demonstrated.

Keywords: ACE2; COVID-19; Coronavirus; RBD; SARS-CoV-2; Spike glycoproteins; Temperature; Vaccines; Variants of concern.

Fig. 1

Evaluation hACE2 Fc binding to SARS-CoV-2 Spike variants. HEK 293T cells were transfected to express the indicated SARS-CoV-2 Spike variants. Two days post transfection, cells were stained with ACE2-Fc or with CV3-25 Ab as Spike expression control and analyzed by flow cytometry. ACE2-Fc binding to (A) full Spikes variants or the (B) B.1.1.7, (C) B.1.351, (D) P.1, (E) B.1.429, and (F) B.1.526 Spike and its corresponding single mutations are presented as a ratio of ACE2 binding to D614G Spike normalized to CV3-25 binding. Error bars indicate means ± SEM. Statistical significance has been performed using Mann-Whitney U test according to normality analysis (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Fig. 2

Kinetic Analysis of RBD interaction to hACE2 Binding by Biolayer Interferometry. Association of the different RBD proteins to sACE2 was carried out for 180s at various concentrations in a two-fold dilution series from 500 nM to 31.25 nM prior to dissociation for 300s for (A) WT, (B) N501Y, (C) K417 N, (D) K417T, (E) E484K, and (F) L452R. Curve fitting was performed using a 1:1 binding model in the ForteBio data analysis software. Calculation of on-rates (K_a), off-rates (K_dis), and affinity constants (K_D) was computed using a global fit applied to all data. Raw data are presented in blue and fitting models are in red. Results are summarized in Table S1.

Fig. 3

Evaluation of the impact of temperature on Spike-ACE2 interaction. HEK 293T cells were transfected with the indicated SARS-CoV-2 Spike variants. Two days post transfection, cells were stained with ACE2-Fc or with CV3-25 Ab as Spike expression control at 4°C or 37°C and analyzed by flow cytometry. ACE2-Fc binding to the different Spike variants are presented as a ratio of ACE2 binding to D614G Spik, normalized to CV3-25 binding at 37°C (red) or at 4°C (blue). Statistical analyses were used to compare each Spike at 4°C vs 37°C (black) or to compare variants Spike to D614G at 37°C (red) or at 4°C (blue). Fold changes of ACE2 binding at 4°C vs 37°C for each Spike is shown in black. Error bars indicate means ± SEM. Statistical significance has been performed using Mann-Whitney U test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, ns; non-significant).

Fig. 4

Recognition of SARS-CoV-2 Spike variants and single mutants by plasma from vaccinated SARS-CoV-2 naïve individuals. HEK 293T cells were transfected with the indicated SARS-CoV-2 spike variants. Two days post transfection, cells were stained with 1:250 dilution of plasma collected from naïve vaccinated individuals (n = 3–5) or with CV3-25 Ab as control and analyzed by flow cytometry. Plasma recognition of (A) full Spike variants (B) B.1.1.7, (C) B.1.351, (D) P.1, (E) B.1.429, (F) B.1.526 Spike and variant-specific Spike single mutations are presented as ratio of plasma binding to D614G Spike normalized CV3-25 binding. Error bars indicate means ± SEM. Statistical significance has been performed using Mann-Whitney U test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Fig. 5

Recognition of SARS-CoV-2 Spike variants and single mutants by plasma from vaccinated previously-infected individuals. HEK293T cells were transfected with SARS-CoV-2 full Spike variants and stained with plasma collected 3 weeks post-first dose vaccinated previously infected individuals (n = 3– 5) or with CV3-25 Ab and analyzed by flow cytometry. Plasma recognition of (A) full Spike variants or the (B) B.1.1.7, (C) B.1.351, (D) P.1, (E) B.1.429, (F) B.1.526 Spikes and Spikes with their corresponding single mutations are presented as a ratio of plasma binding to D614G Spike normalized with CV3-25 binding. Error bars indicate means ± SEM. Statistical significance has been performed using Mann-Whitney U test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Fig. 6

Neutralization of SARS-CoV-2 Spike variants by plasma from previously infected vaccinated individuals. Neutralizing activity of previously infected vaccinated individuals against pseudoviruses bearing the SARS-CoV-2 Spike variants were assessed. Pseudoviruses with serial dilutions of plasma were incubate for 1 h at 37°C before infecting 293T-ACE2 cells. ID₅₀ against pseudoviruses were calculated by a normalized non-linear regression using GraphPad Prism software. Detection limit is indicated in the graph (ID₅₀ = 50). Statistical significance has been performed using Mann-Whitney U test (*p < 0.05).