Deep repertoire mining uncovers ultra-broad coronavirus neutralizing antibodies targeting multiple spike epitopes

Abstract

The development of vaccines and therapeutics that are broadly effective against known and emergent coronaviruses is an urgent priority. We screened the circulating B cell repertoires of COVID-19 survivors and vaccinees to isolate over 9,000 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific monoclonal antibodies (mAbs), providing an expansive view of the SARS-CoV-2-specific Ab repertoire. Among the recovered antibodies was TXG-0078, an N-terminal domain (NTD)-specific neutralizing mAb that recognizes diverse alpha- and beta-coronaviruses. TXG-0078 achieves its exceptional binding breadth while utilizing the same VH1-24 variable gene signature and heavy-chain-dominant binding pattern seen in other NTD-supersite-specific neutralizing Abs with much narrower specificity. We also report CC24.2, a pan-sarbecovirus neutralizing antibody that targets a unique receptor-binding domain (RBD) epitope and shows similar neutralization potency against all tested SARS-CoV-2 variants, including BQ.1.1 and XBB.1.5. A cocktail of TXG-0078 and CC24.2 shows protection in vivo, suggesting their potential use in variant-resistant therapeutic Ab cocktails and as templates for pan-coronavirus vaccine design.

Keywords: CP: Immunology; CP: Microbiology.

Figure 1.. Discovery of an ultra-broad neutralizing antibody targeting the NTD

(A) Overview of the multiplexed antigen screening method. (B) Binding specificities of recombinantly expressed mAbs from donor 531 (d531). Each arc segment corresponds to a single mAb, and the ring corresponding to each spike antigen is colored if bound by the mAb. The centermost band is colored dark gray if the mAb neutralizes SARS-CoV-2 and light gray if it is non-neutralizing. (C) Letter-value plot of mAb affinity for WA1 spike comparing neut (n = 106) versus non-neut (n = 132) groups. The center line on each box represents the median. (D) Correlation between binding affinity (KD) and neutralization (IC₅₀) for the WA1 strain of SARS-CoV-2. The linear regression line is shown, as well as the p value indicating a significant positive slope. (E) Violin plot of heavy-chain variable (VH) germline gene identity. 100% identity indicates no somatic mutation. (F) Isotype distribution of WA1 nAbs and non-nAbs. The total number of mAbs in each category (neut and non-neut) is shown at the top of the respective bar. (G) Epitope binning by competitive binding inhibition. For each mAb pair, a darkly filled circle indicates strong competition, a lightly filled circle indicates weak competition, and no circle indicates no competition. Points are colored according to their respective epitope bin. (H) Neutralization potency against SARS-CoV-2 of nAbs in each epitope bin. Bins are colored as in (G). (I) Upset plot showing cross-reactivity binding and neutralization patterns among epitope-binned mAbs. The broadest cross-reactivity group, which bind all tested CoV S-proteins but do not neutralize, are indicated by unfilled dots. TXG-0078, which comprises its own cross-reactivity pattern, is highlighted in pink.

Figure 2.. Quantifying the development of mAb breadth of binding using antigen barcodes

(A) Ring plots showing the antigen barcode classifications of 6,305 SARS-CoV-2-specific mAbs from participants CC10, CC25, CC31, and CC66 (additional participants found in Figure S1). Individual mAbs are represented by radial vectors, with each antigen ring (WA1: blue, Alpha: green, Beta: yellow, Gamma: orange, Kappa: magenta) colored if the given B cell was classified as positive for the respective antigen. Antibodies are sorted by breadth, with breadth decreasing in the clockwise direction. (B) Isotype distribution of SARS-CoV-2-specific mAbs for each donor. (C) Isotype distribution of mAbs, grouped by cross-variant breadth. (D) VH gene use of SARS-CoV-2-specific mAbs, grouped by cross-variant breadth. (E) Number of VH nucleotide mutations in SARS-CoV-2-specific mAbs, grouped by breadth. White line represents the median, with the inter-quartile interval representing 50% of the data. (F) Number of VH nucleotide mutations in SARS-CoV-2-specific mAbs, grouped by time point (T1, T2, and T3 are post-infection but pre-vaccination and T4 is post-infection and post-vaccination). (G) Median cross-variant breadth of all mAbs isolated at each time point, grouped by donor. The dashed black line represents the median of all mAbs from participants. (H) Phylogenetic representation of a single public SARS-CoV-2-specific clonotype. All mAbs in the clonotype use the IgG1 isotype except for a single IgA1 clone (indicated by text and an unfilled marker). (I) Complementarity-determining region (CDR) alignments of sequences in the public clonotype shown in (H). Mutations (CDR1 and −2) or untemplated residues (junction) shared between multiple participants are highlighted in color (CC10: magenta, CC25: green, CC66: blue). The clone IgA1 mAb is indicated by an unfilled marker. (J) Junction alignments of a second public clonotype comprising TXG-0078 (from donor 531) and CC25.1 (from donor CC25). Untemplated positions (N- and P-additions) are indicated in bold, and identical residues in untemplated regions are highlighted in red. (K) Distribution of cross-donor alignment scores obtained by iterative pairwise alignment of heavy-chain amino acid sequences. Only sequences recovered from different doors were compared. Distributions resulting from a comparison of sequences encoding the same V-gene (green), V-genes from the same family (orange) or V-genes from different families (blue) are shown. The alignment score between TXG-0078 and CC25.1 is highlighted. (L) Ring plots showing antigen barcode classifications of two additional participants (CC62 and CC67). Antigen rings are colored if the respective mAb was classified as antigen positive (WA1: magenta, Beta: yellow, Kappa: green, SARS-CoV: blue, YN02 RBD: purple). Ring plots for two additional donors (CC24 and CC42) can be found in Figure S1. (M) Neutralization of SARS-CoV (red) and several SARS-CoV-2 variants by mAb CC24.2. Neutralization assays were performed in triplicate at each concentration. (N) Epitope binning of mAb CC24.2 with several RBD-specific mAbs. Two competitor mAbs (CC25.4 and CC25.56) bind the site 5 epitope, and the other competitor mAbs (CC25.54, CC84.24, and CC84.2) bind other RBD epitopes. Binding inhibition (dark gray) or lack of binding inhibition (light gray) is shown, with PBS used as a negative control.

Figure 3.. Structural definition of the broad, NTD-supersite antibody TXG-0078

(A) Representative 2D classes obtained by cryoelectron microscopy (cryo-EM) analysis of TXG-0078 Fab in complex with SARS-CoV-2 spike. (B) Two cryo-EM reconstructions of the TXG-0078-spike complex at 3.14 (rosy brown/gray) and 3.34 Å (red) resolutions showing conformational flexibility of the Fab-NTD region. (C) Cryo-EM reconstruction (3.9 Å) of the NTD-Fab region obtained by local refinement. The spike NTD is colored gray, and the Fab is colored rosy brown. (D) The atomic model of TXG-0078 Fab Fv bound to NTD docked in the local refinement map. (E) The atomic model reveals that TXG-0078 targets the NTD supersite, as has been described for other antibodies, though it is capable of weakly binding endemic human coronaviruses. The N3 and N5 loops comprise the epitope of TXG-0078; the CDRH3 of TXG-0078 reaches into a pocket in the N5 loop, and the light chain is only minimally engaged in binding. Epitope and paratope residues are colored in light blue and red, respectively. (F) Multiple sequence alignment of spikes from diverse TXG-0078-reactive beta-CoVs was performed using Clustal Omega. Epitope residues are displayed in (F), showing conservation of proline at position 251.

Figure 4.. TXG-0078 protects transgenic ACE2 mice from live SARS-CoV-2 challenge

(A) Mice (n = 20) were administered 300 μg of TXG-0078, CC24.2, a cocktail of both TXG-0078 and CC24.2, or a control antibody targeting Zika virus (ZIKV-1). Each group contains 5 animals. The mean percentage of change in baseline weight is shown for each group. Shading indicates standard error. Significant differences between groups (p ≤ 0.05) are indicated by asterisks in the color of each group displaying significantly different weight loss than the control (ZIKV-1) group. Significance was determined using a one-way analysis of variance (ANOVA) with Dunnet’s multiple comparisons test. (B) Percentage of baseline weight at day 5 is shown for all mice, separated by treatment group.