Antibodies with potent and broad neutralizing activity against antigenically diverse and highly transmissible SARS-CoV-2 variants

Abstract

The emergence of highly transmissible SARS-CoV-2 variants of concern (VOC) that are resistant to therapeutic antibodies highlights the need for continuing discovery of broadly reactive antibodies. We identify four receptor-binding domain targeting antibodies from three early-outbreak convalescent donors with potent neutralizing activity against 12 variants including the B.1.1.7 and B.1.351 VOCs. Two of them are ultrapotent, with sub-nanomolar neutralization titers (IC50 <0.0006 to 0.0102 μ g/mL; IC80 < 0.0006 to 0.0251 μ g/mL). 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 potential means to mitigate resistance development. These results define the basis of therapeutic cocktails against VOCs and suggest that targeted boosting of existing immunity may increase vaccine breadth against VOCs.

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

(A) Final flow cytometry sorting gate of CD19+/CD20+/IgG+ or IgA+ PBMCs for four convalescent subjects (Subjects 1–4). Shown is the staining for RBD-SD1 BV421, S1 BV786 and S-2P APC or Ax647. Cells were sorted using indicated sorting gate (pink) and percent positive cells that were either RBD-SD1, S1 or S-2P positive is shown for each subject. (B) Gross binding epitope distribution was determined using an MSD-based ELISA testing against RBD, NTD, S1, S-2P or HexaPro. S2 binding was inferred by S-2P or HexaPro binding without binding to other antigens. Indeterminant epitopes showed a mixed binding profile. Total number of antibodies (i.e., 200) and absolute number of antibodies within each group is shown. (C) Lentivirus particles pseudotyped with WA-1 spike were used to test the neutralization capacity of the indicated antibodies (n=3). (D) Live virus neutralization assay for A23–58.1 (n=2), A19–46.1 (n=2), A19–61.1 (n=2) and B1–182.1 (n=3). (E) Table showing antibody binding target, IC₅₀ for pseudovirus and live virus neutralization and Fab:S-2P binding kinetics (n=2) for the indicated antibodies. (F) Biolayer interferometry-based epitope binning experiment. Competitor antibody (y-axis) is bound to S-2P prior to incubation with the analyte antibody or ACE2 protein (x-axis) as indicated and percent competition range bins are shown as red (>=75%), orange (60–75%) or white <60%) (n=2). mAb114 is an anti-Ebola glycoprotein antibody and is included as a negative control (37) (G) 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 while A23–58.1 and B1–182.1 bind to RBD in the up position. Representative classes were shown with 2 Fabs bound, though stoichiometry at 1 to 3 were observed.

Fig. 2.. Neutralization and binding activity against spike proteins from circulating variants.

(A) Table showing domain and mutations relative to WA-1 for each of the 13 variants tested in panels B-C. (B) Spike protein variants were expressed on the surface of HEK293T cells and binding to the indicated antibody was measured using flow cytometry. Data is shown as Mean Fluorescence intensity (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 250%) to white (no change, 100%) to blue (no binding, 0%). Black indicates binding was not tested. (C) IC50 and IC80 values for the indicated antibodies against WA-1 and the 12 variants shown in (A). Ranges are indicated by colors white (>10 μg/mL), light blue (1–10 μg/mL), yellow (0.1–1 μg/mL), orange (0.05–0.1 μg/mL), red (0.01–0.05 μg/mL), maroon (0.001–0.01 μg/mL) and purple (<0.001 μg/mL). Black indicates a variant that was not tested. (D) Location of spike protein variant mutations on the spike glycoprotein for B.1.1.7 (left) and B.1.351 (right). P681H is not resolved in the structure and therefore its location is not noted in B.1.1.7.

Fig. 3.. Structural basis of A23–58.1 binding.

(A) Cryo-EM structure of A23–58.1 Fab in complex with SARS-CoV-2 HexaPro spike. Overall density map is shown to the left with protomers colored light green, gray and cyan. One of the A23–58.1 Fab bound to the RBD is shown in orange and blue. Structure of the RBD and A23–58.1 after local focused refinement was shown to the right. The heavy chain CDRs are colored brown, salmon and orange for CDR H1, CDR H2 and CDR H3, respectively. The light chain CDRs are colored marine blue, light blue and purple blue for CDR L1, CDR L2 and CDR L3, respectively. The contour level of Cryo-EM map is 5.7σ. (B) 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 with a yellow border (left, viewing from antibody to RBD). RBD mutations in current circulating SARS-CoV-2 variants are colored red. Lys417 and Glu484 are located at the edge of the epitope. The tip of the RBD binds to a cavity formed by the CDRs (right, viewing down to the cavity). Interactions between aromatic/hydrophobic residues are prominent at the lower part of the cavity. Hydrogen bonds at the rim of the cavity are marked with dashed lines. RBD residues were labeled with italicized font. (C) Paratope of A23–58.1. Sequences of B1–182.1 and S2E12 were aligned with variant residues underlined. Paratope residues for A23–58.1 and S2E12 were highlighted in green and light brown, respectively. (D) Epitope of A23–58.1 on RBD. Epitope residues for different RDB-targeting antibodies are marked with * under the RBD sequence. (E) Comparison of binding modes of A23–58.1 and REGN10933. One Fab is shown to bind to the RBD on the spike. The shift of the binding site to the saddle of RBD encircled Lys417, Glu484 and Tyr453 inside the REGN10933 epitope (violet), explaining its sensitivity to the K417N, Y453F and E484K mutations. (F) Comparison of binding modes of A23–58.1 and LY-CoV555. One Fab is shown to bind to the RBD on the spike. Glu484 is located in the middle of LY-CoV555 epitope (light orange), explaining its sensitivity to the E484K mutation.

Fig. 4.. Critical binding residue determination and mitigation of escape risk using dual antibody combinations

(A) The indicated Spike protein mutations predicted by structural analysis were expressed on the surface of HEK293T cells and binding to the indicated antibody was measured using flow cytometry. Data is shown as Mean Fluorescence intensity (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 250%) to white (no change, 100%) to blue (no binding, 0%). (B) IC₅₀ and IC₈₀ values for the indicated antibodies against WA-1 and the 10 mutations. Ranges are indicated by colors white (>10 μg/mL), light blue (1–10 μg/mL), yellow (0.1–1 μg/mL), orange (0.05–0.1 μg/mL), red (0.01–0.05 μg/mL), maroon (0.001–0.01 μg/mL) and purple (<0.001 μg/mL). (C) 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 (Figure S6) after 48–72 hours. Total supernatant RNA was harvested and viral genomes shotgun sequenced to determine the frequency of amino acid changes. Shown are the spike protein amino acid/position change and frequency as a logo plot. (D) Negative stain 3D reconstruction of the ternary complex of spike with Fab B1–182.1 and A19–46.1 (left) or A19–61.1 (right). (E) rcVSV SARS-CoV-2 was incubated with increasing concentrations (1.3e-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 and a dashed line is used to indicate maximum antibody concentration was reached in subsequent rounds.