Determinants of Spike infectivity, processing, and neutralization in SARS-CoV-2 Omicron subvariants BA.1 and BA.2

Abstract

SARS-CoV-2 Omicron rapidly outcompeted other variants and currently dominates the COVID-19 pandemic. Its enhanced transmission and immune evasion are thought to be driven by numerous mutations in the Omicron Spike protein. Here, we systematically introduced BA.1 and/or BA.2 Omicron Spike mutations into the ancestral Spike protein and examined the impacts on Spike function, processing, and susceptibility to neutralization. Individual mutations of S371F/L, S375F, and T376A in the ACE2-receptor-binding domain as well as Q954H and N969K in the hinge region 1 impaired infectivity, while changes to G339D, D614G, N764K, and L981F moderately enhanced it. Most mutations in the N-terminal region and receptor-binding domain reduced the sensitivity of the Spike protein to neutralization by sera from individuals vaccinated with the BNT162b2 vaccine and by therapeutic antibodies. Our results represent a systematic functional analysis of Omicron Spike adaptations that have allowed this SARS-CoV-2 variant to dominate the current pandemic.

Keywords: BA.1; BA.2; COVID-19; Omicron; SARS-CoV-2; Spike protein; neutralization; variant evolution.

Graphical abstract

Figure 1

Features of Omicron BA.1 and BA.2 VOCs (A) Radial phylogenetic tree of representative SARS-CoV-2 strains (n = 2,793 genomes, sampled between December 2019 and June 2022), scaled according to their divergence, compared with the Wuhan Hu-1 sequence. Retrieved from Nextstrain on June 24, 2022 (https://nextstrain.org/ncov/open/global/6m?l=radial) and modified. Color coding according to VOCs as indicated. (B) Frequencies of SARS-CoV-2 Delta, BA.1, and BA.2 sequences in data from GenBank from September 2021 to June 2022. Scaled to 100%. Retrieved and modified from Nextstrain on June 24, 2022. Blue, Delta VOC; green, BA.1; light green, BA.2; yellow, BA.4; orange, BA.5; and red, BA.2.12.1. (C) Overview of the SARS-CoV-2 spike structure (downloaded from PDB: 7KNB) and localization of amino acid changes that are shared between BA.1 and BA.2 or specific for BA.1 or BA.2 as indicated. S1 (orange), S2 (blue), ACE2 (gray), mutations (red), BA.1-specific deletions (blue), BA.2-specific deletions (yellow). (D) Schematic depiction of the SARS-CoV-2 spike, its domains, and amino acid alterations in Omicron BA.1 (green) and BA.2 (light green) VOCs compared with the Wuhan Hu-1 sequence. S1 subunit: N-terminal domain, NTD (purple); receptor-binding domain, RBD (orange). Receptor-binding motif, RBM (dark orange). S2 subunit: fusion peptide, FP (blue); heptad repeat 1, HR1 (dark blue); central helix, CH; connector domain, CD; heptad repeat 2, HR2; transmembrane domain, TM (blue).

Figure 2

Impact of mutations in Omicron on Spike-mediated infection (A) Automatic quantification of infection events of CaCo-2 cells transduced with VSVΔG-GFP pseudotyped with SARS-CoV-2 Hu-1 (gray), Delta (blue), BA.1 (green), BA.2 (light green), or indicated mutant S proteins. The localization of each mutation in S is indicated by color. S1: NTD (purple), RBD (orange), RBM (dark orange), and others (light orange). S2: HR1 (dark blue) and others (light blue). Bars represent the mean of three independent experiments (± SEM). Statistical significance was tested by one-way ANOVA. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001. (B) Infection kinetics of CaCo-2 cells infected by VSVpp containing the indicated mutant S proteins. Infected GFP+ cells were automatically quantified over a period of 22 h. See also Figure S1.

Figure 3

Expression and processing of Spike proteins containing mutations present in the Omicron BA.1 and BA.2 VOCs (A) The upper panels show exemplary immunoblots of whole cells lysates (WCLs) and VSVpp containing supernatants of HEK293T cells transfected with vectors expressing the Hu-1, BA.1, BA.2, or mutant SARS-CoV-2 S proteins and infected with VSVΔG-GFP. Blots were stained with anti-V5 (Spike), anti-ß-actin, and anti-VSV-M protein. Lower panels: expression levels of uncleaved, full-length Spike protein (S, gray bars) and the S2 subunit (bars colored according to the corresponding domains, as shown in Figure 1D) were quantified. The results show mean values (±SEM) obtained from three independent experiments. (B) Correlation of the S2 expression/incorporation and S2/S processing of the parental S Hu-1 or indicated mutant S proteins in cells and supernatants, with the corresponding pseudotype infection rates. The correlation coefficients ( r values) and two-tailed p values are provided. See also Figure S2.

Figure 4

Functional relevance of S371L/F, S373P, and S375F changes in the Spike protein (A) Phylogenetic tree of Delta and Omicron BA.1 and BA.2 strains. Amino acids at position 371, 373, and 375 are indicated; all other SARS-CoV-2 variants almost invariantly contain three serines at these positions. Color coding as indicated according to VOC. Retrieved and modified from Nextstrain on April 7, 2022. (B) Close-up view of the region encompassing the mutations S371L, S373P, and S375F and the surrounding region. Downloaded from PDB: 7KNB, 7TGW, or 7WKA as indicated. (C) Automatic quantification of infection events of CaCo-2 cells transduced with VSVΔG-GFP pseudotyped with SARS-CoV-2 Hu-1 or indicated combined mutations. Bars represent the mean of three independent experiments (±SEM). Statistical significance was tested by one-way ANOVA. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001. (D) Immunofluorescence images of HEK293T cells expressing the parental S Hu-1, the BA.1-specific SSSxLPF, or the BA.2-specific SSSxFPF mutations. Scale bars, 10 μm. See also Figure S3.

Figure 5

Impact of mutations in Omicron Spike on cell-to-cell fusion and ACE2 interaction (A) Representative fluorescence microscopy images of HEK293T cells expressing parental Hu-1 or indicated mutant S proteins, human ACE2, and GFP (green). Scale bar, 125 μm. (B) Automatic quantification of syncytia formation of HEK293T cells expressing parental Hu-1 or indicated mutant S proteins and human ACE2. Bars represent the mean of three independent experiments (±SEM). Statistical significance was tested by two-tailed Student’s t test with Welch’s correction. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001. (C) Overview on the SARS-CoV-2 post-fusion spike structure (downloaded from PDB: 6M3W) and comparative ReaxFF simulation of the mutation L981F. (D) Binding of the indicated Hu-1 and mutant S proteins to ACE2 binding using whole-cell lysates of transfected HEK293T. Bars represent the mean of three independent experiments (±SEM). Statistical significance was tested by one-way ANOVA. ^∗p < 0.05; ^∗∗p < 0.001. See also Table S1.

Figure 6

Impact of mutations in the Omicron Spike on serum neutralization (A) Neutralization of VSVpp carrying the indicated wild-type and mutant S proteins by sera obtained from five BNT/BNT-vaccinated individuals compared with the untreated control (set to one). Shown are mean values obtained for the five sera, each tested in two technical replicates. (B) Changes in TCID₅₀ values obtained for neutralization of the indicated mutant S proteins by sera from five vaccinated individuals relative to those obtained for the Hu-1 S. Solid red bars indicate mean values (±SEM) for the five sera and open black squares the average infectivity of the respective S-containing VSVpp shown in Figure 2A. See also Table S2.

Figure 7

Impact of mutations in the Omicron Spike on neutralization by therapeutic Abs (A) Schematic depiction of SARS-CoV-2 Spike domains, interaction sites of therapeutic antibodies, and resistance-conferring amino acid alterations highlighted in red. (B) Close-up view of neutralizing antibodies binding the SARS-CoV-2 Spike (PDB: 6XDG or 7L3N as indicated) and automated quantification of GFP fluorescence of Caco-2 cells infected with VSVΔG-GFP pseudotyped with the indicated S variants. VSVpp were pre-treated (30 min, 37°C) with the indicated amounts of imdevimab, bamlanivimab or casivirimab. Lines represent the mean of three independent experiments. IC₅₀ values indicate Ab concentrations (μg/mL) required to reduce pseudoparticle infection by 50%.