A monoclonal antibody against staphylococcal enterotoxin B superantigen inhibits SARS-CoV-2 entry in vitro

Abstract

We recently discovered a superantigen-like motif sequentially and structurally similar to a staphylococcal enterotoxin B (SEB) segment, near the S1/S2 cleavage site of the SARS-CoV-2 spike protein, which might explain the multisystem inflammatory syndrome (MIS-C) observed in children and the cytokine storm in severe COVID-19 patients. We show here that an anti-SEB monoclonal antibody (mAb), 6D3, can bind this viral motif at its polybasic (PRRA) insert to inhibit infection in live virus assays. The overlap between the superantigenic site of the spike and its proteolytic cleavage site suggests that the mAb prevents viral entry by interfering with the proteolytic activity of cell proteases (furin and TMPRSS2). The high affinity of 6D3 for this site originates from a polyacidic segment at its heavy chain CDR2. The study points to the potential utility of 6D3 for possibly treating COVID-19, MIS-C, or common colds caused by human coronaviruses that also possess a furin-like cleavage site.

Keywords: 6D3; COVID-19; MIS-C; TMPRSS2; cytokine storm; furin-cleavage site; neutralizing antibodies; staphylococcal enterotoxin B; superantigen; viral entry.

Graphical abstract

Figure 1

SARS-CoV-2 spike (S) glycoprotein structure, sequence alignment against other CoVs, and interaction sites observed in cryo-EM studies with neutralizing antibodies (A) SARS-CoV-2 S trimer in the pre-fusion state. Protomers 1 and 2 are in white and light blue, respectively, and protomer 3 is in spectral colors from blue (N-terminal domain, NTD; residues 1–305) to red (C terminus), except for the ₆₈₁PRRA₆₈₄ insert in magenta. The insert was modeled using SWISS-MODEL (Waterhouse et al., 2018). Each protomer's RBD (residues 331–524) can assume an up or down conformation in the respective receptor-bound and unbound state. (B) Sequence alignment of SARS-CoV-2 near the S1/S2 cleavage site against multiple bat and pangolin SARS-related strains and other HCoVs, adjusted following previous studies (Coutard et al., 2020; Zhou et al., 2020b). Viruses belonging to the same lineage are shown by the same color shade, and HCoVs that encode furin-like cleavage sites are highlighted in bold font. Note that the polybasic insert PRRA of SARS-CoV-2 S is not found in closely related SARS-like CoVs but exists in MERS and HCoVs HKU1 and OC43. The furin-like cleavage site is indicated by the blue-shaded box. (C) Side (left) and bottom (right) views of receptor (ACE2)- and antibody-binding sites observed in cryo-EM structures resolved for the S protein complexed with the ACE2 and/or various antibodies. The S trimer is shown in cartoons with a light blue protomer in the RBD-up conformation and gray and light orange protomers in the RBD-down conformation. Binding sites for ACE2 and antibodies C105 (Barnes et al., 2020), 2-4 (Liu et al., 2020), S309 (Pinto et al., 2020), H014 (Lv et al., 2020b), 4A8 (Chi et al., 2020), Ab23 (Cao et al., 2020b), and EY6A (Zhou et al., 2020a) are shown in space-filling surfaces in different colors (see the code in the inset). See Table 1 for additional details.

Figure 2

SEB-associated mAb 6D3 binds the furin cleavage site of SARS-CoV-2 S protein, potentially interfering with the S1/S2 cleavage by furin or TMPRSS2 (A and B) Binding pose of three SEB-neutralizing Abs (mAbs 6D3, 14G8, and 20B1) onto SEB. The diagram was generated by superposing the crystal structures (PDB: 4RGN and 4RGM) resolved for the complexes (Dutta et al., 2015). SEB is colored beige, with its SAg motif ₁₅₀TNKKKATVQELD₁₆₁ highlighted in blue space-filling. (B) Close-up view of the tight interaction between the acidic residues E50, D52, and D55 of the 6D3 heavy chain and four basic residues of SEB. (C–E) (C) Interface between 6D3 and SEB SAg motif. Heavy and light chains of 6D3 are green and cyan, respectively. (D) Overall and (E) close-up views of the complex model for S protein and anti-SEB mAb 6D3. The interfacial interactions engage the arginines in the PRRA insert. SARS-CoV-2 S interfacial residues include I210–Q218, N603–Q607, E654–Y660, and A688–I693, and the SAg motif residues Y674, T678–R683. 6D3 interfacial residues include A24–K33, E50, D52, S54, D55, Y57, N59, K74–T77, and A100–A104 in the heavy chain, and D1, I2, Q27, N31–F38, Y55, W56, and D97–Y100 in the light chain. The spike-6D3 complex was generated in silico using the S structure modeled with one RBD up (PDB: 6VSB).

Figure 3

Glycosylation of SARS-CoV-2 spike at N603 does not block mAb 6D3 binding and may even assist in binding the mAb (A) Computationally modeled SARS-CoV-2 glycosylated spike. The three monomers of the spike protein are shown in blue, red, and orange surface representations, with their SAg region (residues 661–685) colored yellow. High-mannose N-glycans are shown in colored (small) spheres. (B) Structural alignment of computationally modeled 6D3-spike protein complex onto the glycosylated spike. No spatial clash was observed. The inset shows a close-up view.

Figure 4

Monoclonal antibody 6D3 prevents SARS-CoV-2 infection 6D3 or isotype control antibodies (at indicated concentrations) were incubated with virus (100 plaque-forming units/well) for 1 h at room temperature before addition to Vero-E6 cells (5 × 10³ cells/well). Forty-eight hours post-infection the cells were fixed and stained for dsRNA or SARS-CoV-2 spike protein. (A) Quantification of the percentage of infected cells per well by dsRNA staining. (B) Representative fluorescence images of 6D3-mediated inhibition of virus infection (dsRNA). (C) Quantification of the percentage of infected cells per well by spike staining. (D) Representative fluorescence images of 6D3-mediated inhibition of virus infection (spike). Data were analyzed by t test (6D3 versus isotype control) with multiple testing correction (false discovery rate). Data are presented as the mean ± standard error of the mean. n = 3 technical replicates. Data are representative of three independent experiments. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. See also Figure S1 for detailed results as a function of 6D3 concentration.

Figure 5

Binding poses of human proteases TMPRSS2 and furin to SARS-CoV-2 S protein (A and B) Structural models for the SARS-CoV-2 S protein complexed with (A) TMPRSS2 and (B) furin, obtained from docking simulations followed by refinements. An overview (left) and a zoomed-in view (right) are shown in each case. The arginines in the S1/S2 loop P₆₈₁RRARS₆₈₆ are shown in different shades of blue, and their interaction partners (acidic residues) in the proteases are shown in red. Spheres (right) highlight the R685↑S686 peptide bond. The TMPRSS2 catalytic triad residues are S441 (yellow), H296 (green), and D345 (dark red). Their counterparts in furin are S368, H194, and D153. Note the short distance between the carbonyl carbon of R685 and the hydroxyl oxygen of S441 of TMPRSS2 (3.5 Å) or S368 of furin (3.1 Å). Black dashed lines show interfacial polar contacts and salt bridges, and those including the S1/S2 loop arginines are highlighted by ellipses.

Figure 6

Polyacidic residues in CDR2 of the mAbs 6D3 heavy chain play a major role in blocking the furin-like cleavage site of SARS-CoV-2 S protein (A) Multiple sequence alignment of the VH domain of anti-SEB Abs (6D3, 14G8, and 20B1) and anti-SARS-CoV-2 S Abs (see the names on the left). The residue ranges of the three CDRs are CDR1, residues 25 to 32; CDR2, 51 to 58; and CDR3, 100 to 116 (Chi et al., 2020), as indicated by the blue bars. (B) Overall and close-up views of the complex and interfacial interaction of the spike protein complexed with 6D3 antibody. Note that three acidic residues from CDR2 interact with the basic residues R682, R683, and R685 of the S protein. The complex was generated in silico using the SARS-CoV-2 S structure with all three RBDs in the down conformer (PDB: 6VXX). (C) Same as (B), repeated for the human cold virus HCoV-OC43 S protein. The complex was generated in silico using the HCoV-OC43 S structure with all three RBDs in the down conformer (PDB: 6NZK). HCoV-OC43 encodes an S1/S2 furin-like cleavage site at ₇₅₄RRAR↑G₇₅₈. Note that three acidic residues from CDR2 interact with R754, R755, and R757 in HCoV-OC43 S protein. The residues belonging to the Abs are labeled in regular font and those of the S protein in bold in both (B) and (C).