SARS-CoV-2 Spike Does Not Possess Intrinsic Superantigen-like Inflammatory Activity

Abstract

Multisystem inflammatory syndrome in children (MIS-C) is a rare hyperinflammatory disease occurring several weeks after SARS-CoV-2 infection. The clinical similarities between MIS-C and the toxic shock syndrome, together with the preferential expansion of T cells with a T-cell receptor variable β chain (TCRVβ) skewing, suggested a superantigen theory of MIS-C. For instance, recent in silico modelling evidenced the presence of a highly conserved motif within SARS-CoV-2 spike protein similar in structure to the superantigenic fragment of staphylococcal enterotoxin B (SEB). However, experimental data on the superantigenic activity of the SARS-CoV-2 spike have not yet been provided. Here, we assessed the superantigenic activity of the SARS-CoV-2 spike by analysing inflammatory cytokine production in both Jurkat cells and the peripheral blood CD4⁺ T cells stimulated with the SARS-CoV-2 spike or SEB as a control. We found that, unlike SEB, the SARS-CoV-2 spike does not exhibit an intrinsic superantigen-like activity.

Keywords: CD4+ T cells; MIS-C; SARS-CoV-2 spike; SEB; superantigen.

Figure 1

SARS-CoV-2 spike does not stimulate inflammatory pathways in Jurkat cells expressing CD28 and TCRVb3.1. (a–c) CH7C17 Jurkat cells expressing CD28 and TCRVβ3.1 (n = 5) were cultured for 24 h with medium alone (Med) or 1 μg mL⁻¹ of SEB or 1 μg mL⁻¹ SARS-CoV-2 spike in the absence (Ctr) or presence of 5-3.1/B7 cells expressing human HLA-DR1 and B7.1/CD80 molecules. TNF-α (a), IL-2 (b) and IL-8 (c) secretion in culture supernatants was measured by ELISA. Data show the mean ± SEM, and statistical significance was calculated by one-way ANOVA. (d–f) Jurkat cells (n = 5) were transfected with 5 μg GFP together with 10 μg NF-AT-luciferase (Luc) (d), or 10 μg AP-1-Luc (e) or 2 μg NF-κB-Luc (f) constructs and then unstimulated (Ctr) or stimulated for 6 h with SEB or SARS-CoV-2 spike alone or in the presence of 5-3.1/B7 cells. The results were expressed as fold inductions (F.I.) over the basal level of luciferase activity in unstimulated cells after normalisation to GFP values. Bars show mean ± SEM of five independent experiments. Statistical significance was calculated by comparing each group with unstimulated cells (Ctr) cultured with medium alone (med) or between conditions indicated by the arcs by one-way ANOVA. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001, (****) p < 0.0001. NS = not significant.

Figure 2

SARS-CoV-2 spike does not stimulate inflammatory cytokine production in peripheral blood CD4⁺ T cells. (a) Representative FACS analysis of human CD4⁺ T cells isolated from the peripheral blood of HD stained with anti-CD4-FITC plus anti-CD28-PE or anti-HLA-DR-PE. (b) The percentage of human CD4⁺ T cells from HD (n = 5) expressing CD28, HLA-DR, B7.1/CD80 or B7.2/CD86 was calculated. The results express the mean percentage of positive cells ± SEM. (c–f) Peripheral blood CD4⁺ T cells from HD (n = 10) were cultured for 24 h with medium alone (Med) or SEB or SARS-CoV-2 spike in the absence (Ctr) or presence of 5-3.1/B7 cells. TNF-α (c), IL-6 (d), IL-2 (e) and IFN-γ (f) secretion in culture supernatant was measured by ELISA. Data show the mean ± SEM. Statistical significance was calculated by comparing each condition with unstimulated cells (Ctr) cultured with medium alone (med) or between conditions indicated by the arcs by one-way ANOVA. (g–j) Peripheral blood CD4⁺ T cells from HD (n = 10) were stimulated for 24 h with native SARS-CoV-2 spike or heat-denatured SARS-CoV-2 spike (boiled) in the absence (Ctr) or presence of 5-3.1/B7 cells. TNF-α (g), IL-6 (h), IL-2 (i) and IFN-γ (j) levels were measured by ELISA. Data show the mean ± SEM. Statistical significance was calculated by Mann–Whitney test. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001, (****) p < 0.0001. NS = not significant.

Figure 3

SARS-CoV-2 spike does not co-stimulate inflammatory cytokine production in anti-CD3 activated CD4⁺ T cells. (a–c) Peripheral blood CD4⁺ T cells from HD (n = 5) were cultured for 24 h with isotype control Abs (Ig) or 2 μg mL⁻¹ crosslinked anti-CD3 Abs (UCHT1) or anti-CD3 plus anti-CD28 (CD28.2) Abs or anti-CD3 Abs plus SARS-CoV-2 spike. TNF-α (a), IL-2 (b) and IFN-γ (c) secretions in culture supernatant were measured by ELISA. Data show the mean ± SEM. Statistical significance was calculated by comparing each condition with isotype control Abs (Ig) or between conditions indicated by the arcs by one-way ANOVA. (**) p < 0.01, (****) p < 0.0001. NS = not significant.

Figure 4

SARS-CoV-2 spike does not stimulate inflammatory cytokine production in CD4⁺ or CD8⁺ T cells stimulated with HeLa cells expressing HLA-A*0201. (a–f) Peripheral blood CD4⁺ T and CD8⁺ T cells from HD (n = 5) were cultured for 24 h with medium alone (Ctr) or SEB (a,c,e) or SARS-CoV-2 spike (b,d,f) in the presence of 5-3.1/B7 cells or HeLa cells expressing HLA-A*0201. TNF-α (a,b), IL-2 (c,d) and IFN-γ (e,f) secretion in culture supernatant was measured by ELISA. Data show the mean ± SEM. Statistical significance was calculated by comparing each condition with unstimulated cells cultured with medium alone (Ctr) or between conditions indicated by the arcs by one-way ANOVA. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001, (****) p < 0.0001. NS = not significant.

Figure 5

Structural prediction of the putative interaction between SARS-CoV-2 spike and TCR. (a) Structure superposition of SEB superantigenic fragment (150–161; blue) and the sequence of SARS-CoV-2 spike near the PRRARS insert (661–685; dark grey and red). (b) Top-1 cluster representative of SARS-CoV-2 spike and the human TRAV27/TRBV19 [61] complex, as predicted by ClusPro 2.0. The SARS-CoV-2 spike trimer is shown in grey, with the interacting chain as cartoons and the others as surface. The αβTCR structure (PDB: 2XN9) is represented as green (TCR α-chain) and cyan (TCR β-chain) cartoons. The PRRARS insert of the spike protein is coloured in red. The epitope that is recognised is coloured yellow. The number of representative structures within the cluster and the mean energy is shown in parenthesis.