Ultrapotent antibodies against diverse and highly transmissible SARS-CoV-2 variants

Abstract

The emergence of highly transmissible SARS-CoV-2 variants of concern (VOCs) that are resistant to therapeutic antibodies highlights the need for continuing discovery of broadly reactive antibodies. We identified four receptor binding domain-targeting antibodies from three early-outbreak convalescent donors with potent neutralizing activity against 23 variants, including the B.1.1.7, B.1.351, P.1, B.1.429, B.1.526, and B.1.617 VOCs. Two antibodies are ultrapotent, with subnanomolar neutralization titers [half-maximal inhibitory concentration (IC₅₀) 0.3 to 11.1 nanograms per milliliter; IC₈₀ 1.5 to 34.5 nanograms per milliliter). We define the structural and functional determinants of binding for all four VOC-targeting antibodies and show that combinations of two antibodies decrease the in vitro generation of escape mutants, suggesting their potential in mitigating resistance development.

Isolation and characterization of convalescent donor antibodies that effectively neutralize emerging SARS-CoV-2 VOCs.

Antibodies isolated from donors infected with ancestral SARS-CoV-2 viruses showed ultrapotent neutralization of emerging VOCs. The two most potent antibodies shared usage of the IGHV1-58 gene and targeted the RBD with minimal contact to VOC mutational hotspots. Cocktails of antibodies with complementary binding modes suppressed antibody escape.

Fig. 1.. Identification and classification of highly potent antibodies from convalescent SARS-CoV-2 subjects.

(A) Sera from 22 convalescent subjects were tested for neutralizing (y axis, ID₅₀) and binding antibodies (x axis, S-2P ELISA AUC), and four subjects—A19, A20, A23, and B1 (colored) with both high neutralizing and binding activity against the WA-1—were selected for antibody isolation. (B) Final flow cytometry sorting gate of CD19⁺/CD20⁺/IgG⁺ or IgA⁺ PBMCs for four convalescent subjects (A19, A20, A23, and B1). Shown is the staining for RBD-SD1 BV421, S1 BV786, and S-2P APC or Ax647. Cells were sorted by using indicated sorting gate (pink), and percent of positive cells that were either RBD-SD1-, S1-, or S-2P-– positive is shown for each subject. (C) Gross binding epitope distribution was determined by using an MSD-based ELISA testing against RBD, NTD, S1, S-2P, or HexaPro. S2 binding was inferred from S-2P or HexaPro binding without binding to other antigens. Indeterminant epitopes showed a mixed binding profile. Total number of antibodies (200) and absolute number of antibodies within each group is shown. (D) Neutralization curves by using WA-1 spike pseudotyped lentivirus and live virus neutralization assays to test the neutralization capacity of the indicated antibodies (n = 2 to 3 replicates). (E) Table showing antibody binding target, IC₅₀ for pseudovirus and live virus neutralization, and Fab:S-2P binding kinetics (n = 2 replicates) for the indicated antibodies. (F) SPR-based epitope binning experiment. Competitor antibody (y axis) is bound to S-2P before incubation with the analyte antibody (x axis) as indicated, and percent competition range bins are shown as red (>75%), orange (60 to 75%), or white (<60%) (n = 2 replicates). Negative control antibody is anti-Ebola glycoprotein antibody mAb114 (37). (G) Competition of ACE2 binding. The indicated antibodies (y axis) complete binding of S-2P to soluble ACE2 protein by using biolayer interferometry [left column, percent competition (>75% shown as red, <60% as white)] or to cell surface–expressed ACE2 by using cell-surface staining (right column, EC₅₀ at ng/ml shown). (H) Negative-stain 3D reconstructions of SARS-CoV-2 spike and Fab complexes. A19-46.1 and A19-61.1 bind to RBD in the down position, whereas A23-58.1 and B1-182.1 bind to RBD in the up position. Representative classes were shown with two Fabs bound, although stoichiometry at one to three Fabs was observed.

Fig. 2.. Antibody binding and neutralization of VOCs or VOIs.

(A) Table showing domain and mutations relative to WA-1 for each of the 10 variants tested in (B) and (C). (B) Spike protein variants were expressed on the surface of HEK293 T cells, and binding to the indicated antibody was measured with flow cytometry. Data are shown as MFI normalized to the MFI for the same antibody against the D614G parental variant. Percent change is indicated by a color gradient from red (increased binding, Max 500%) to white (no change, 100%) to blue (no binding, 0%). (C) IC₅₀ and IC₈₀ values for the indicated antibodies against 10 variants shown in (A). Ranges are indicated with white (>10,000 ng/ml), light blue (>1000 to ≤10,000 ng/ml), yellow (>100 to ≤1000 ng/ml), orange (>50 to ≤100 ng/ml), red (>10 to ≤50 ng/ml), maroon (>1 to ≤10 ng/ml), and purple (≤1 ng/ml). (D) Location of spike protein variant mutations on the spike glycoprotein for B.1.1.7, B.1.351, B.1.429, P.1 v2, B.1.617.1, and B.1.617.2. P681 and V1176 are not resolved in the structure, and therefore their locations are not noted in B.1.1.7 and P.1 v2.

Fig. 3.. Structural basis of binding and neutralization for antibodies A23-58.1 and B1-182.1.

(A) Cryo-EM structure of A23-58.1 Fab in complex with SARS-CoV-2 HexaPro spike. (Left) Overall density map. Protomers are light green, gray, and cyan. One of the A23-58.1 Fab bound to the RBD is shown in orange and blue. (Right) Structure of the RBD and A23-58.1 after local focused refinement. The heavy-chain CDRs are brown, salmon, and orange for CDR H1, CDR H2, and CDR H3, respectively. The light-chain CDRs are marine blue, light blue, and purple blue for CDR L1, CDR L2, and CDR L3, respectively. The contour level of the cryo-EM map is 5.7σ. (B) Cryo-EM structure of B1-182.1 Fab in complex with SARS-CoV-2 HexaPro spike. (Left) Overall density map. Protomers are light green, gray, and cyan. One of the B1-182.1 Fab bound to the RBD is shown in salmon and light blue. (Right) Structure of the RBD and B1-182.1 after local focused refinement. The heavy-chain CDRs are brown, deep salmon, and orange for CDR H1, CDR H2, and CDR H3, respectively. The light-chain CDRs are marine blue, slate, and purple blue for CDR L1, CDR L2, and CDR L3, respectively. The contour level of the cryo-EM map is 4.0σ. (C) Interaction between A23-58.1 and RBD. All CDRs were involved in binding of RBD. Epitope of A23-58.1 is shown in bright green surface. RBD mutations in current circulating SARS-CoV-2 variants are red. K417 and E484 are located at the edge of the epitope. (D) Interaction details at the antibody-RBD interface. The tip of the RBD binds to a crater formed by the CDRs (shown viewing down to the crater). Interactions between aromatic and hydrophobic residues are prominent at the lower part of the crater. Hydrogen bonds at the rim of the crater are indicated with dashed lines. RBD residues are indicated with italicized font. (E) Paratopes of A23-58.1, B1-182.1, S2E12 (PDB ID: 7K45), and COVOX253 (PDB ID: 7BEN) from the same germline. Sequences of B1-182.1, S2E12, and COVOX253 were aligned with variant residues underlined. Paratope residues for A23-58.1, B1-182.1, S2E12, and COVOX253 were highlighted in green, dark green, light brown, and light orange, respectively.

Fig. 4.. Binding modes of A23-58.1 and B1-182.1 enable neutralization to VOCs.

(A) Mapping of epitopes of A23-58.1, B1-182.1, and other antibodies on RBD. Epitope residues for different RBD-targeting antibodies are marked with an asterisk under the RBD sequence. (B) Comparison of binding modes of A23-58.1 and B1-182.1. (Left) Analysis indicated that axis of Fab B1-182.1 is rotated 6° from that of A23-58.1. (Right) This rotation resulted in a slight shift of the epitope of B1-182.1 on RBD, which reduced its contact to E484. RBD mutations of concern are red, the epitope surface of B1-182.1 is dark green, and the borders of ACE2-binding site and A23-58.1 epitope are yellow and olive, respectively. (C) Comparison of binding modes of A23-58.1, CB6, and REGN10933. For clarity, one Fab is shown to bind to the RBD on the spike. The shift of the binding site to the saddle of RBD encircled K417, E484, and Y453 inside the CB6 (black line) and REGN10933 epitopes (violet surface), explaining their sensitivity to the K417N, Y453F, and E484K mutations. (D) Comparison of binding modes of A23-58.1 and LY-CoV555. (Left) One Fab is shown to bind to the RBD on the spike. (Top right) E484 is located inside the LY-CoV555 epitope. (Bottom right) E484K/Q mutation abolishes critical contacts between RBD and CDR H2 and CDR L3; moreover, E484K/Q and L452R cause potential clashes with heavy chain of LY-CoV555, explaining its sensitivity to the E484K/Q and L452R mutations. (E) IGHV1-58–derived antibodies target a supersite with minimal contacts to mutational hotspots. Supersite defined by common atoms contacted by the IGHV1-58–derived antibodies (A23-58.1, B1-182.1, S2E12, and COVOX253) on RBD is indicated with the green line. Boundaries of the ACE2-binding site and epitopes of class I, II, and III antibodies represented by C102 (PDB ID 7K8M), C144 (PDB ID 7K90), and C135 (PDB ID 7K8Z) are indicated with yellow, pink, light orange, and blue boundary lines, respectively.

Fig. 5.. Critical binding residues for antibodies A23-58.1 and B1-182.1.

(A) The indicated spike protein mutations predicted with structural analysis were expressed on the surface of HEK293 T cells, and binding to the indicated antibody was measured with flow cytometry. Data are shown as MFI normalized to the MFI for the same antibody against the WA-1 parental binding. Percent change is indicated by a color gradient from red (increased binding, max 200%) to white (no change, 100%) to blue (no binding, 0%). (B) IC₅₀ and IC₈₀ values for the indicated antibodies against WA-1 and the nine spike mutations. Ranges are indicated with white (>10,000 ng/ml), light blue (>1000 to ≤10,000 ng/ml), yellow (>100 to ≤1000 ng/ml), orange (>50 to ≤100 ng/ml), red (>10 to ≤50 ng/ml), and maroon (>1 to ≤10 ng/ml).

Fig. 6.. Mitigation of escape risk by using dual antibody combinations.

(A) Replication competent vesicular stomatitis virus (rcVSV) whose genome-expressed SARS-CoV-2 WA-1 was incubated with serial dilutions of the indicated antibodies and wells with cytopathic effect (CPE) were passaged forward into subsequent rounds (fig. S8) after 48 to 72 hours. Total supernatant RNA was harvested, and viral genomes were shotgun sequenced to determine the frequency of amino acid changes. Shown are the spike protein amino acid and position change and frequency as a logo plot. Amino acid changes observed in two independent experiments are indicated in blue and green letters. (B) The indicated spike protein mutations predicted with structural analysis (Fig. 3) or observed with escape analysis (Fig. 6A) were expressed on the surface of HEK293 T cells, and binding to the indicated antibody was measured with flow cytometry. Data are shown as MFI normalized to the MFI for the same antibody against the (left) WA-1 or (right) D614G parental binding. Percent change is indicated with a color gradient from red (increased binding, max 200%) to white (no change, 100%) to blue (no binding, 0%). (C) IC₅₀ and IC₈₀ values for the indicated antibodies against WA-1 and the mutations predicted with structural analysis (Fig. 3) or observed with escape analysis (Fig. 6A). Ranges are indicated with white (>10,000 ng/ml), light blue (>1000 to ≤10,000 ng/ml), yellow (>100 to ≤1000 ng/ml), orange (>50 to ≤100 ng/ml), red (>10 to ≤50 ng/ml), and maroon (>1 to ≤10 ng/ml). (D) Negative-stain 3D reconstruction of the ternary complex of spike with Fab B1-182.1 and (left) A19-46.1 or (right) A19-61.1. (E) rcVSV SARS-CoV-2 was incubated with increasing concentrations (1.3 × 10^–4 to 50 μg/ml) of either single antibodies (A19-46.1, A19-61.1, and B1-182.1) and combinations of antibodies (B1-182.1/A19-46.1 and B1-182.1/A19-61.1). Every 3 days, wells were assessed for CPE, and the highest concentration well with the >20% CPE was passaged forward onto fresh cells and antibody-containing media. Shown is the maximum concentration with >20% CPE for each of the test conditions in each round of selection. Once 50 μg/ml has been reached, virus was no longer passaged forward.