A potent human monoclonal antibody with pan-neutralizing activities directly dislocates S trimer of SARS-CoV-2 through binding both up and down forms of RBD

Figures

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  Fig. 1. 35B5 and 32C7 protect against authentic SARS-CoV-2 virus. a, b Affinity analysis of 35B5 (a) and 32C7 (b) binding to immobilized SARS-CoV-2 RBD by using SPR. The experimental data were calculated using a 1:1 binding model. c, d In vitro neutralizing activity of 35B5 (c) and 32C7 (d) against authentic SARS-CoV-2. The mixture of SARS-CoV-2 and serially diluted 35B5 or 32C7 were added to Vero E6 cells. After 48 h, IC50 values were calculated by fitting the viral RNA copies from serially diluted mAb to a sigmoidal dose–response curve. e Schematic diagram of 35B5 and 32C7 treatment in vivo. Six hours after infection with 4 × 104 PFU SARS-CoV-2, the hACE2 mice received a single dose of 35B5 or 32C7 with 20 mg/kg or no mAb treatment (mock). At day 5 post infection, lung tissues were collected for viral burden assessment, cytokine/chemokine assay and histological analysis. f Viral titers in the lungs were measured by qRT-PCR and presented as RNA copies per milliliter of lung abrasive fluid. g Gene expressions of cytokines and chemokines in the lungs were determined by qPT-PCR. h Histopathological analysis of lung tissues. The data are representative of at least two independent…

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  Fig. 2. Neutralizing activity of 35B5 against SARS-CoV-2 VOCs. a, b Neutralizing activity of 35B5 (a) and 32C7 (b) against authentic SARS-CoV-2 viruses, including WT strain, D614G variant, B.1.351 variant, and B.1.617.2 variant. Mixture of each SARS-CoV-2 strain and serially diluted 35B5 or 32C7 were added to Vero E6 cells. After 48 h, IC50 values were calculated by fitting the viral RNA copies from serially diluted mAb to a sigmoidal dose–response curve. c, d FFA analysis of 35B5 (c) and 32C7 (d) against WT SARS-CoV-2, D614G variant, B.1.351 variant, and B.1.617.2 variant. Virus cultures were serially diluted and added to Vero E6 cells. After 24 h, the foci were visualized by using HRP-conjugated polyclonal antibodies targeting SARS-CoV-2 nucleocapsid protein in Vero E6 cells and counted with a ELISPOT reader. e Schematic diagram of 35B5 treatment in vivo. Four hours after infection with 4 × 104 PFU SARS-CoV-2 D614G or B.1.351 or B.1.617.2, the hACE2 mice received a single dose of 35B5 with 30 mg/kg or no mAb treatment (mock). At day 5 post infection, lung tissues were collected for viral burden assessment. f Viral titers in the lungs were measured by qRT-PCR and presented as RNA copies…

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  Fig. 3. Cryo-EM structures of the spike protein S-6P complexed with 35B5 Fab. a The structures of the S-6P-35B5 Fab complex in three states. The S trimer is represented as surface. 35B5 Fab is shown in cartoon and colored in purple. The “down”, “up”, and “releasing” RBD domains are colored in blue, yellow, and red, respectively. The NTD domain of the S trimer is colored in deep gray. The SD1, SD2, and S2 domains are colored in light gray. The gap caused by 35B5 Fab between the “releasing” RBD and NTD domains is highlighted with dashed lines. b Interactions of 35B5 Fab with “up” RBD. The interacting residues within 4 Å in RBD are colored in cyan. The RBD-interacting regions CDRH2, CDRH3, FRH1 and FRH3 of 35B5 Fab are colored in yellow, blue, orange, and red, respectively. c The 35B5 epitope on RBD. The epitope residues are labeled as indicated. d–f Detailed interactions of the CDR (d) and FR regions (e, f) of 35B5 Fab with RBD. Hydrogen-bond and salt-bridge interactions are shown as blue and orange dashed lines, respectively

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  Fig. 4. Structural basis and neutralizing mechanism for the broad neutralizing activities of 35B5 to the SARS-CoV-2 variants. a Structural comparisons of the 35B5 Fab- “up” RBD interactions with those of the previously identified Class 2 and 3 neutralizing antibodies. 35B5 Fab and the Fab regions of the representative antibodies CV07-270 and REGN10987 from Class 2 and 3, respectively, are illustrated in cartoon and colored as indicated. RBD is represented as surface in yellow. b Comparison of the epitope of 35B5 with those of the representative neutralizing antibodies of Class 2 and 3 on RBD. The 35B5 epitope is colored in cyan. The epitopes of CV07-270 (Class 2) and REGN10987 (Class 3) are labeled using red and black lines, respectively. c Mapping of the frequently mutated residues of the spike protein in the recent SARS-CoV-2 variant in the 35B5 Fab-RBD structure. RBD and 35B5 Fab are shown in cartoon and colored in yellow and purple, respectively. The frequently mutated residues in RBD in the recent SARS-CoV-2 variant are highlighted with green spheres. d Interactions of 35B5 Fab with the “down” RBD. 35B5 Fab, the “down” RBD, and NTD domains are colored in purple, cyan, and gray,…

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  Fig. 5. Interactions of 35B5 Fab with RBD. a Mutations in the RBD of reported SARS-CoV-2 variants of concern or interest. b–g The residues E484 (b) and L452 (d) are not involved in the interactions of RBD with 35B5 Fab. E484 is more than 4.2 Å away from 35B5 R84, which forms cation–π interaction with RBD F490. The E484K mutation in RBD does not interact with 35B5 R84 through molecular modeling (c). The L452Q modeling suggests none interactions formed at this site with 35B5 (f). In structural modeling, the L452R mutation (e) and F490S mutation (g) in RBD generate potential additional interactions with 35B5 Fab. Potential hydrogen bonds are shown as dashed lines