Broad SARS-CoV-2 neutralization by monoclonal and bispecific antibodies derived from a Gamma-infected individual

Abstract

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has remained a medical threat due to the evolution of multiple variants that acquire resistance to vaccines and prior infection. Therefore, it is imperative to discover monoclonal antibodies (mAbs) that neutralize a broad range of SARS-CoV-2 variants. A stabilized spike glycoprotein was used to enrich antigen-specific B cells from an individual with a primary Gamma variant infection. Five mAbs selected from those B cells showed considerable neutralizing potency against multiple variants, with COVA309-35 being the most potent against the autologous virus, as well as Omicron BA.1 and BA.2, and COVA309-22 having binding and neutralization activity against Omicron BA.4/5, BQ.1.1, and XBB.1. When combining the COVA309 mAbs as cocktails or bispecific antibodies, the breadth and potency were improved. In addition, the mechanism of cross-neutralization of the COVA309 mAbs was elucidated by structural analysis. Altogether these data indicate that a Gamma-infected individual can develop broadly neutralizing antibodies.

Keywords: Immunology; Structural biology; Virology.

Graphical abstract

Figure 1

Selection of B cells and antibodies from a SARS-CoV-2 Gamma-infected individual (A) Sorting strategy of singlet viable CD19⁺ SARS-CoV-2 Gamma S-specific B cells. Gamma S-specific B cells were selected by double staining of Gamma S labeled with two different fluorescent dyes (Gamma S-AF647, Gamma S-BV421). In addition, Gamma S-positive B cells were stained for IgD, CD27, IgG, and IgM expression. Frequency of WT S cross-reactive B cells is also indicated. SSC-A, side scatter area; FSC-H, forward scatter height; FSC-A, forward scatter area. (B) Flow cytometry-based screening of HEK293T-produced non-purified COVA309 supernatants against Gamma, Beta, and WT S expressed on HEK293T cells, shown in the percentage of binding. 293T NT, non-transfected cells. COVA1-16 and CH3I antibodies were included as positive and negative controls, respectively. 14 mAbs (in bold) were selected for larger scale expression. (C) Heatmap showing the percentage of mean fluorescence intensity (MFI) of HEK293F-produced and purified COVA309 mAbs binding to SARS-CoV-2 variant S expressed on HEK293T cells, as assessed by flow cytometry. VOCs, variants of concern; VOIs, variants of interest. COVA1-16 and D25 (an RSV F specific mAb) are used as positive and negative controls, respectively. (D) Heatmap depicting the binding of COVA309 mAbs to SARS-CoV-2 variant RBDs, as determined by ELISA. Color scale indicates the area under the curve (AUC) for each mAb. (E) Half maximal inhibitory concentrations (IC₅₀) of SARS-CoV-2 VOC pseudoviruses neutralization for COVA309 mAbs, COVA1-16, and COVA1-18.^,, The cutoff was set at 10 μg/mL (light gray bar) for variants up to Omicron BA.2, and at 50 μg/mL (dark gray bar) for Omicron BA.4/5, BQ.1.1, and XBB.1. Each dot represents the mean value from two or three experiments in which three or four replicates per mAb were performed. (F) Neutralization of authentic SARS-CoV-2 viruses by COVA309 mAbs. The cutoff was set at 10 μg/mL (gray bar). Color code is the same as for the pseudovirus neutralization. COVA1-16 and COVA1-18 mAbs were tested against WT, Alpha, Beta, Gamma, Omicron BA.1, BA.2, and BA.5 to corroborate the pseudovirus neutralization findings. Each dot represents the mean value from one or two experiments performed in duplo.

Figure 2

COVA309 mAb epitope determination (A) Biolayer interferometry plot depicts competition between the COVA309 mAbs and ACE-2, for binding to Gamma S coated on the chip. The black curves represent the baseline. (B) Example of BLI plots showing COVA309-22 and -38 mAbs competing with COVA1-16 and CR3022 for binding to the WT S. The black curves represent the baseline, when no analyte was added. (C) Heatmap showing the percentage of residual binding of the ACE-2 receptor and other known SARS-CoV-2 mAbs after competition with COVA309 mAbs, as determined by BLI assay. For the mAbs competition, WT S was loaded on biosensors, and COVA2-14 (S2 binder), COVA1-22 (NTD binder), COVA2-04 (RBS-A), J08 (RBS-B), COVA2-15 (RBS-D), S309 (S309 site), COVA1-16 (CR3022 site), and CR3022 (CR3022 site)^,,, were included as competitors. (D) Crystal structure of COVA309-22 Fab in complex with SARS-CoV-2 WT RBD at a 3.7 Å resolution (left). The HC and LC are colored in orange and yellow, respectively. CR3022 Fab (right) is reported as a comparison (PDB: 6W41). (E) Detailed representation of the main residues involved in the COVA309-22-WT RBD interaction. (F) COVA309-22 HC (orange) and LC (yellow) bind the base and lateral face of the RBD, far from the ACE-2 receptor binding site (green; PDB: 6M0J). (G) Front view (top row) and top view (bottom row) of low-resolution NS-EM reconstructions of COVA309-03, -10, -35, and -38 in complex with either Omicron 6P S or Gamma 6P S. Composite NS-EM maps of COVA309 Fabs indicate that all COVA309 mAbs align on one RBD in the up conformation.

Figure 3

Characterization of bsAbs and antibody cocktails (A) IC₅₀ values for pseudovirus neutralization of SARS-CoV-2 VOCs by bsAbs (left) and corresponding antibody cocktails (right). For antibody cocktails, a 1:1 ratio was used. The cutoff was set at 10 μg/mL (gray bar). Each dot represents the mean value from two or three experiments in which three or four replicates per mAb were performed. (B) Neutralization of authentic SARS-CoV-2 viruses by bsAbs and antibody cocktails. The cutoff was set at 10 μg/mL (gray bar). Each dot represents the mean value from one or two experiments performed in duplo.