Shared N417-Dependent Epitope on the SARS-CoV-2 Omicron, Beta, and Delta Plus Variants

Abstract

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, several variants of concern (VOCs) have arisen which are defined by multiple mutations in their spike proteins. These VOCs have shown variable escape from antibody responses and have been shown to trigger qualitatively different antibody responses during infection. By studying plasma from individuals infected with either the original D614G, Beta, or Delta variants, we showed that the Beta and Delta variants elicit antibody responses that are overall more cross-reactive than those triggered by D614G. Patterns of cross-reactivity varied, and the Beta and Delta variants did not elicit cross-reactive responses to each other. However, Beta-elicited plasma was highly cross-reactive against Delta Plus (Delta+), which differs from Delta by a single K417N mutation in the receptor binding domain, suggesting that the plasma response targets the N417 residue. To probe this further, we isolated monoclonal antibodies from a Beta-infected individual with plasma responses against Beta, Delta+, and Omicron, which all possess the N417 residue. We isolated an N417-dependent antibody, 084-7D, which showed similar neutralization breadth to the plasma. The 084-7D MAb utilized the IGHV3-23*01 germ line gene and had somatic hypermutations similar to those of previously described public antibodies which target the 417 residue. Thus, we have identified a novel antibody which targets a shared epitope found on three distinct VOCs, enabling their cross-neutralization. Understanding antibodies targeting escape mutations, such as K417N, which repeatedly emerge through convergent evolution in SARS-CoV-2 variants, may aid in the development of next-generation antibody therapeutics and vaccines. IMPORTANCE The evolution of SARS-CoV-2 has resulted in variants of concern (VOCs) with distinct spike mutations conferring various immune escape profiles. These variable mutations also influence the cross-reactivity of the antibody response mounted by individuals infected with each of these variants. This study sought to understand the antibody responses elicited by different SARS-CoV-2 variants and to define shared epitopes. We show that Beta and Delta infections resulted in antibody responses that were more cross-reactive than the original D614G variant, but they had differing patterns of cross-reactivity. We further isolated an antibody from Beta infection which targeted the N417 site, enabling cross-neutralization of Beta, Delta+, and Omicron, all of which possess this residue. The discovery of antibodies which target escape mutations common to multiple variants highlights conserved epitopes to target in future vaccines and therapeutics.

Keywords: SARS-CoV-2; antibody cross-reactivity; antibody isolation; variants.

FIG 1

Comparison of plasma cross-reactivities elicited by three distinct SARS-CoV-2 variants. (A) Plasma from D614G, Beta, and Delta infections during three distinct SARS-CoV-2 waves were tested for neutralization breadth against a range of VOCs using a pseudovirus-based neutralization assay. Fold changes in neutralization are shown above each variant. Plasma neutralization titer is measured as an ID₅₀ (reciprocal plasma dilution causing 50% reduction of infection). Black horizontal bars represent geometric means. The threshold of detection for the neutralization assay is an ID₅₀ of >20. (B) Spider plots were derived from GMTs for plasma triggered by D614G, Beta, or Delta against multiple VOCs. The GMTs for each plasma set were normalized against titers to the autologous virus, and breadth was expressed as area under the curve. (C) Plasma from Beta-infected individuals was tested against the Delta and Delta+ variants. Fold changes in neutralization are shown above each variant. Plasma neutralization titer is measured as an ID₅₀. Black horizontal bars represent geometric means. The threshold of detection for the neutralization assay is an ID₅₀ of >20.

FIG 2

Characterization of plasma and an isolated monoclonal antibody from a Beta-infected individual. (A) A sequence-confirmed Beta-infected hospitalized individual, SA-01-0084 (084), was selected for this study. Plasma and peripheral blood mononuclear cells were collected 2 days post-positive PCR for further analysis. (B) Plasma from SA-01-0084 was tested for neutralization activity in a pseudovirus-based neutralization assay against a range of VOC/VOIs. Plasma neutralization titer is measured as an ID₅₀. The threshold of detection for the neutralization assay is an ID₅₀ of >20. (C) The gating strategy for the isolation of SARS-CoV-2 spike-specific B cells via single-cell sorting is shown. Each dot represents a cell, and the dots in the sorted cell red gate represent Beta spike-positive B cells that had CD3⁻, CD14⁻, CD16⁻, and CD19⁺ markers. These cells were single-cell sorted into 96-well plates and amplified through antibody gene-specific PCR. (D) MAb 084-7D was tested for neutralization activity in a pseudovirus-based neutralization assay against the same VOCs/VOIs as tested with the plasma. MAb neutralization was measured as a monoclonal antibody concentration causing 50% reduction of infection (IC₅₀, in micrograms per milliliter). The threshold of detection for the neutralization assay is an IC₅₀ of >20 μg/mL. (E) A SARS-CoV-2 ELISA was used to map the specificity of the 084-7D MAb. The MAb was tested against the D614G and Beta N-terminal domain, spike and RBD antigens, as well as SBD (subdomain 1) proteins mutated to include Beta-specific SBD single mutations, K417N, E484K, and N501Y. The starting concentration of the MAb was 5 μg/mL. The threshold of detection for the binding assay is an optical density at 450 nm (OD₄₅₀) of >0.04. Lines for D614G SBD, E484K, and N501Y are all at negative and superimposed.

FIG 3

MAb 084-7D displayed antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC) with cross-reactivity toward Beta and Omicron. (A) ADCP activity of MAb 084-7D was measured using THP-1 phagocytosis assay and the phagocytosis score is shown as an area under the curve (AUC) measure. The threshold of detection for this assay is an AUC of  >0. (B) ADCC activity of MAb 094-7D was measured using an infectious ADCC assay. The percent killing activity is shown as an AUC measure. The threshold of detection for the ADCC assay is an AUC of >8,000. In both ADCP and ADCC assays, P2B-2F6 was used a positive control for D614G activity, CR3022 was used as a positive control for all variants, and palivizumab, a respiratory syncytial virus (RSV)-specific MAb, was used as a negative control. Experiments were conducted in duplicate, and bars represent the means with standard deviations of two experiments.

FIG 4

Genetic analysis of MAb 084-7D compared to a similar N417-dependent MAb. Heavy chain (A) and light chain (B) analysis of MAb 087-7D was conducted using IMGT/V-QUEST (https://www.imgt.org/IMGT_vquest/analysis). A multiple-sequence alignment of the germ line gene and MAb sequences for 084-7D and CAB-A17 was generated using ClustalW (https://www.genome.jp/tools-bin/clustalw). Key positions implicated in the development of breadth in the CAB-A17 MAb are shown in bold, and mutations from germ line gene residues in these regions are shown in red.