Potent neutralization of SARS-CoV-2 variants of concern by an antibody with an uncommon genetic signature and structural mode of spike recognition

Abstract

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages that are more transmissible and resistant to currently approved antibody therapies poses a considerable challenge to the clinical treatment of coronavirus disease (COVID-19). Therefore, the need for ongoing discovery efforts to identify broadly reactive monoclonal antibodies to SARS-CoV-2 is of utmost importance. Here, we report a panel of SARS-CoV-2 antibodies isolated using the linking B cell receptor to antigen specificity through sequencing (LIBRA-seq) technology from an individual who recovered from COVID-19. Of these antibodies, 54042-4 shows potent neutralization against authentic SARS-CoV-2 viruses, including variants of concern (VOCs). A cryoelectron microscopy (cryo-EM) structure of 54042-4 in complex with the SARS-CoV-2 spike reveals an epitope composed of residues that are highly conserved in currently circulating SARS-CoV-2 lineages. Further, 54042-4 possesses uncommon genetic and structural characteristics that distinguish it from other potently neutralizing SARS-CoV-2 antibodies. Together, these findings provide motivation for the development of 54042-4 as a lead candidate to counteract current and future SARS-CoV-2 VOCs.

Keywords: COVID-19; Delta variant; LIBRA-seq; SARS-CoV-2; antibody discovery; cryo-EM; monoclonal antibodies biology.

Graphical abstract

Figure 1

Identification and characterization of SARS-CoV-2 antibodies isolated using LIBRA-seq (A) Variable heavy gene usage (x axis) as a function of IgG⁺ B cells with a SARS-CoV-2 spike LIBRA-seq score (≥1) (y axis). The nine lead candidates are highlighted in purple. (B) Sequence characteristics and antigen specificity of nine lead candidate antibodies from a recovered COVID-19 donor. Percent identity is calculated at the nucleotide level, and CDR length and sequences are displayed at the amino acid level. LIBRA-seq scores for each antigen are displayed as a heatmap with a LIBRA-seq score of −2 displayed as light yellow, 0 as white, and 2 in purple; in this heatmap scores lower or higher than that range are shown as −2 and 2, respectively. ELISA binding data for SARS-CoV-2 S are displayed as a heatmap of the area under the curve (AUC) analysis calculated from Figure S1D. (C) RTCA VSV-SARS-CoV-2 neutralization by lead candidate antibodies. IC₅₀ values are calculated by non-linear regression analysis by GraphPad Prism software. Neutralization assays were performed in technical triplicate; data are represented as mean ± SD.

Figure 2

Antigenic characterization of antibody 54042-4 (A) ELISA binding values against SARS-CoV-2 subdomains are displayed as a heatmap of AUC values calculated from the data in Figure S2. Antibodies CR3022, 46472-6, and 46472-4 were used as positive controls for the RBD, NTD, and S2 antigens, respectively (Shiakolas et al., 2021; Yuan et al., 2020b). 3602-1707 was included as an influenza HA-specific negative control antibody (Setliff et al., 2019). (B) A biolayer interferometry sensogram that shows 54042-4 IgG binding to recombinant SARS-CoV-2 RBD-SD1. Binding data are depicted by the black lines and the best fit line of the data to a 1:1 binding model is shown in red. (C) SARS-CoV-2 spike/ACE2 inhibition ELISA is shown for 54042-4, SARS-CoV-2 antibody CR3022, and negative control HA-specific antibody 3602-1707. For each antibody, the ACE2 binding signal is depicted on the y axis, in comparison to ACE2-only binding to SARS-COV-2 spike is shown as a dotted line. ELISAs were performed in technical triplicate with 2 biological duplicates; data represented as mean ± SEM. (D) Competition ELISA of 54042-4 with antibodies COV2-2196, COV2-2130, and CR3022. Values in white indicate no competition (presence of competing antibody reduced reference antibody binding by less than 30%) and values in dark gray indicate competition (presence of competing antibody reduced reference antibody binding by more than 60%). Competition ELISA was performed in technical quadruplicate with 2 biological duplicates; data shown as mean of quadruplicate values.

Figure 3

Atomic resolution of 54042-4 binding mode to SARS-CoV-2 S (A) 3D reconstructions of side and top views of Fab 54042-4 bound to SARS-CoV-2 spike. (B) Focused refinement maps showing the 54042-4 epitope at the apex of the RBM in the down position (left). Top-down view of the 54042-4 epitope showing heavy and light chain contacts, as well as residues outside of the binding interface that are mutated in circulating VOCs (right). (C) The 54042-4 heavy chain binds to RBD residues 443–447 primarily through a network of hydrogen bonds involving CDRH2 and CDRH3 and hydrophobic contacts involving Ile32 of CDRH1. (D) The 54042-4 light chain contacts RBD residues 498–500 through a hydrogen bond between Thr500 and His92 of CDRL3 and hydrophobic contacts involving Phe30 and Tyr32 of CDRL1.

Figure 4

Sequence and structural comparison of 54042-4 to known SARS-CoV-2 antibodies (A) Amino acid CDRH3 identity to 54042-4 (x axis) is plotted against CDRL3 identity to 54042-4 (y axis) for paired heavy and light chain sequences obtained from the CoV-AbDab database (Raybould et al., 2021). Antibodies using the same heavy and light chain germline gene as 54042-4 (IGHV2-5 and IGKV1-39) are shown in light blue. Antibodies using the IGHV2-5 heavy chain gene and a non-IGKV1-39 light chain gene are shown in orange. Additionally, antibodies using a non-IGHV2-5 heavy chain gene and the IGKV1-39 light chain gene, with CDRH3 or CDRL3 identity to 54042-4 of at least 50%, are highlighted in purple. Finally, antibodies that do not use IGHV2-5 or IGKV1-39, but that have at least 50% identity to CDRH3 or CDRL3 of 54042-4, are shown in gray. (B) Pearson correlation of epitopes of known SARS-CoV-2 antibodies (Table S2) in comparison to 54042-4 antibody, with the six antibodies showing a statistically significant (p < 0.05) positive correlation highlighted in red. (C) Heatmap (top) depicting buried surface area (Ų) at the SARS-CoV-2 RBD interface for the six antibodies with highest epitope correlations with 54042-4. Bar graph (bottom) showing the frequency (%) of substitutions at each given residue position in log scale, with a dashed line at 1% and residue positions with a frequency greater than 1% highlighted in red. (D) Distinct angles of approach of antibodies 54042-4 (heavy chain: orange, light chain: white), REGN10987 (heavy chain: blue, light chain: white) (PDB id: 6XDG), 2-7 (heavy chain: pink, light chain: white) (PDB id: 7LSS), and LY-CoV1404 (heavy chain: purple, light chain: white) (PDB:7MMO) to the SARS-CoV-2 RBD (green). (E) Structural comparison of CDRH1, 2, and 3 of antibodies 54042-4 and 2-7. CDRH1 of 2-7 extends further than 54042-4, forming additional contacts with Thr345 and Arg346 of the RBD (left). The CDRH2 region of 2-7 approaches at a different angle, but maintains RBD contacts via Asp56 and Arg58 (center). The CDRH3 contacts of 2-7 and 54042-4 are divergent, with unique CDRH3 residues and RBD interactions (right).

Figure 5

Functional characterization of antibody 54042-4 (A) Binding data of 54042-4 antibody to a shotgun alanine mutagenesis screening library of the SARS-CoV-2 RBD (Wuhan-Hu-1 strain). Residues displayed are the alanine substitutions that resulted in the biggest loss of binding to 54042-4 yet still retained signal with the RBD antibody control. Data are represented as an average of at least two replicate measurements, and shown with ranges (half of the maximum minus minimum values from replicates). (B) RTCA Neutralization of VSV SARS-CoV-2 chimera variants harboring specific substitutions. Cell sensograms are shown in boxes corresponding to mutations indicated in each row. Columns (from left to right) are each chimera treated with COV2-2381, 54042-4 and virus only control. Neutralization of 54042-4 of USA-WA1 strain and cells only are indicated on the right. COV2-2381 was chosen as a positive control due to its distinct epitope footprint from the selected substitutions. RTCA neutralization was performed in technical duplicate conditions; only one sensogram profile per condition is shown. (C) 54042-4 epitope residues (non-zero buried surface area on SARS-CoV-2 RBD) with their associated % conservation (the percentage of deposited sequences containing the highest-frequency amino acid at that position) in the GISAID database. The only 54042-4 epitope residue with a % conservation of less than 99%, Asn439, is highlighted in red. (D) ELISA AUC of 54042-4, CR3022, and an influenza HA-specific negative control antibody 3602-1707 calculated from the data in Figures S4A and S4B. AUC is displayed as a heatmap with a value of 0.02 corresponding to white, 50% maximum as light-purple, and maximum (42.41) AUC as purple. (E) Authentic SARS-CoV-2 % neutralization of USA-WA1, Alpha, Beta, Delta, and Gamma strains (y axis) is depicted as a function of antibody concentration (x axis). Also shown are the respective IC₅₀ and IC₈₀ values for 54042-4 neutralization against each variant. Neutralization assays were performed with 2 technical duplicates; data represented as mean ± SD.