Cross-neutralizing antibodies bind a SARS-CoV-2 cryptic site and resist circulating variants

Abstract

The emergence of numerous variants of SARS-CoV-2, the causative agent of COVID-19, has presented new challenges to the global efforts to control the COVID-19 pandemic. Here, we obtain two cross-neutralizing antibodies (7D6 and 6D6) that target Sarbecoviruses' receptor-binding domain (RBD) with sub-picomolar affinities and potently neutralize authentic SARS-CoV-2. Crystal structures show that both antibodies bind a cryptic site different from that recognized by existing antibodies and highly conserved across Sarbecovirus isolates. Binding of these two antibodies to the RBD clashes with the adjacent N-terminal domain and disrupts the viral spike. Both antibodies confer good resistance to mutations in the currently circulating SARS-CoV-2 variants. Thus, our results have direct relevance to public health as options for passive antibody therapeutics and even active prophylactics. They can also inform the design of pan-sarbecovirus vaccines.

Fig. 1. Screening and characterization of SARS-CoV-2 and SARS-CoV cross-reactive monoclonal antibodies.

a The immunization schemes of two different strategies: immunized with the SARS-CoV-2 spike protein alone (upper) or with a combination of the SARS-CoV-2 and SARS-CoV spike proteins and the MERS-CoV RBD (lower). b The number of lead antibodies with cross-activity in each round of screening. c Characterization of 5 and 10 monoclonal antibodies obtained by the first and second immunization strategies, respectively. The neutralizing titers (IC₅₀) based on the vesicular stomatitis virus pseudotyping system (VSV NAT) and the lentiviral virus pseudotyping system (LV NAT) were tested. ND means not detected. Asterisks indicate the top three monoclonal antibodies with excellent cross-neutralizing potency.

Fig. 2. Comprehensive characterization of representative neutralizing mAbs 7D6, 6D6, and 16D8.

a–c Neutralization of three mAbs by LV-SARS-CoV-2 (a), LV-SARS-CoV (b), and VSV-SARS-CoV-2 (c). d Neutralization activities of three mAbs to authentic SARS-CoV-2. e SPR kinetics and the affinities of three antibodies to S-2P and RBD proteins of both SARS-CoV-2 and SARS-CoV. f SPR-based blocking assays of the three mAbs perturbing the engagement of ACE2 to RBD. g Inter-blocking potentials of the three mAbs. Data in a, b, d are presented as mean values ± SEM. Source data are provided as a Source Data file.

Fig. 3. Crystal structures of 7D6 and 6D6 in complex with SARS-CoV-2 RBD.

a, d Overall structures of 7D6:RBD and 6D6:RBD. b, e CDRs of 7D6 (b) and 6D6 (e) involved in the interactions. RBDs and CDRs are shown as surface and cartoon, respectively. The contact surfaces of the heavy and light chains on the RBD are colored in orange and yellow, respectively; residues contacting both the heavy and light chains are colored in cyan. c, f Residues on the RBD involved in the interactions with 7D6 (c) and 6D6 (f) are indicated; shared residues are labeled in red. g Superimposition of the ACE2:RBD complex (PDB: 6M0J) and our immune complexes, revealing no competition between ACE2 and the mAbs. h Conservation analysis of critical residues in the binding epitopes for 7D6 (outlined in black) and 6D6 (outlined in green). SARS-CoV-2 (N = 2,216,094) and other SARS-related (N = 83) genes encoding for Sarbecovirus spike proteins were selected to calculate conservation. Deeper red indicates more conservation. i Sequence alignment of the RBDs of various Sarbecoviruses. Secondary structure distribution is indicated according to the RBD structure in 7D6:RBD co-crystal structure (PDB no. 7EAM). The plot was prepared by the online server (https://espript.ibcp.fr/ESPript/cgi- bin/ESPript.cgi). The footprints of ACE2, 7D6, and 6D6 are marked in cyan, pink, and green, respectively. j Interaction between the 7D6 heavy chain variable region and the RBD. The contact region on the RBD is colored purple, and HCDR1-3 are colored in green, cyan, and yellow, respectively. k Hydrogen bonds (dashed lines) between 7D6 and RBD. The electron density (2Fo−Fc) map of all residues is displayed on the contour level of 1σ above the mean value. Conserved residues between SARS-CoV-2 and SARS-CoV are shown in stick mode and in the same color scheme as in (j) for SARS-CoV-2 and in gray for SARS-CoV. l Interactions between the 6D6 CDRs and RBD. The contact region on the RBD is colored in purple; H0, HCDR2, HCDR3, LCDR2, and LCDR3 are colored in dark blue, cyan, yellow, pink, and orange, respectively. m Hydrogen bonds (dashed lines) between 6D6 and RBD.

Fig. 4. Mutation resistance of 7D6 and 6D6 and comparison of the 7D6/6D6 site with other binding modes of known RBD nAbs.

a Predominant mutations on the RBD in the SARS-CoV-2 variants. None of these mutations occur within the 7D6/6D6 site. b Binding reactivities of 7D6, 6D6 against the RBD mutants and their neutralizing activities against pseudotyped LVs and authentic virus of the major SARS-CoV-2 variant(s). Three convalescent sera and nAb REGE10933 served as control. c Superimposed structures of S trimers (shown as gray surface) and three classes of nAb-RBD complexes, the bound RBD were highlighted in dark gray. The epitopes of the six representative nAbs were shown on RBD separately. nAbs belonging to the same class (with similar epitopes or binding modes) are listed in the corresponding color. The underlined antibodies indicate cross-reactive antibodies and the asterisks indicate cross-neutralizing antibodies. d Superimposed structures of the Class 3 cross-reactive nAbs (CR3022, Ey6A, S304, H014, VHH-72, and COVA1-16), Class 1 cross-reactive nAb (S309, 7D6, and 6D6), showing three different binding orientations. Both two classes of nAbs bind epitopes without overlapping the RBM. e Comparison of 7D6, 6D6 and antibodies clinically used under EUA. REGN10933 + REGN10987 developed by Regeneron Pharmaceuticals Inc., LY-CoV16 (CB6) + LY-CoV555 from Eli Lilly & Company and Junshi Biosciences Inc., VIR-7831(S309) from Vir Biotechnology and GlaxoSmithKline group of companies (GSK). Source data are provided as a Source Data file.

Fig. 5. Structural analyses reveal the spatial clash induced by 7D6 and 6D6 binding.

a, e, i, m Epitope locations of 7D6 and 6D6 on the trimeric spike (PDB: 6zgg) when the RBD is in its open (blue) or closed (cyan) states (PDB:6vxx). 7D6 are colored in salmon and 6D6 in pale green. b, f, j, n Superimposition of the 7D6:RBD or 6D6:RBD complex and the open or closed RBD on the trimeric spike. Binding of 7D6 or 6D6 to either the open or closed state of the RBD would cause clashing (indicated by the red dash circles) with the neighboring NTD. c, g, k, o A close-up view of the clash of 7D6 and 6D6 on RBD. d, h, l, p Cryptic feature of the epitopes of 7D6 and 6D6 is shown on RBD in surface mode with a neighboring NTD rendered as semi-transparent surface, with close-up views of (b), (f), (j), (n), respectively.

Fig. 6. Neutralization mechanism revealed by biochemical and cryo-EM analyses.

a HPLC profiles of 7D6 and 6D6 binding to the S trimer. The black peak (arrow) indicates depolymerization of the spike. b SDS-PAGE analysis of the fractions harvested from the gel filtration chromatography of the mixture of S-2P and 7D6 or 6D6 Fab. The fractions are the peaks indicated by arrows in HPLC profile. c Cryo-EM micrographs (upper) and 2D analysis (lower) of S-2P and its immune complexes. Scale bar = 50 nm. d Shedding of S1 by IgG- and Fab forms of nAbs measured at 293 T cell-surface decorated with SARS-CoV-2 wild-type S protein by flow cytometry. e Two possible mechanisms of 7D6/6D6-mediated neutralization. First, 7D6/6D6 destabilizes SARS-CoV-2 spikes on the virus surface and the disordered spikes lose ability to engage ACE2 receptor. Or second, 7D6/6D6 binding triggers shedding off S1 moieties from S trimers, rendering viruses non-infectious, even though the free 7D6/6D6-bound S1 moieties could still engage ACE2. The experiments in (b) and (c) were performed twice with similar results. Source data are provided as a Source Data file.