A Strategy to Detect Emerging Non-Delta SARS-CoV-2 Variants with a Monoclonal Antibody Specific for the N501 Spike Residue

Abstract

Efforts to control SARS-CoV-2 have been challenged by the emergence of variant strains that have important implications for clinical and epidemiological decision making. Four variants of concern (VOCs) have been designated by the Centers for Disease Control and Prevention (CDC), namely, B.1.617.2 (delta), B.1.1.7 (alpha), B.1.351 (beta), and P.1 (gamma), although the last three have been downgraded to variants being monitored (VBMs). VOCs and VBMs have shown increased transmissibility and/or disease severity, resistance to convalescent SARS-CoV-2 immunity and antibody therapeutics, and the potential to evade diagnostic detection. Methods are needed for point-of-care (POC) testing to rapidly identify these variants, protect vulnerable populations, and improve surveillance. Antigen-detection rapid diagnostic tests (Ag-RDTs) are ideal for POC use, but Ag-RDTs that recognize specific variants have not yet been implemented. Here, we describe a mAb (2E8) that is specific for the SARS-CoV-2 spike protein N501 residue. The 2E8 mAb can distinguish the delta VOC from variants with the N501Y meta-signature, which is characterized by convergent mutations that contribute to increased virulence and evasion of host immunity. Among the N501Y-containing mutants formerly designated as VOCs (alpha, beta, and gamma), a previously described mAb, CB6, can distinguish beta from alpha and gamma. When used in a sandwich ELISA, these mAbs sort these important SARS-CoV-2 variants into three diagnostic categories, namely, (1) delta, (2) alpha or gamma, and (3) beta. As delta is currently the predominant variant globally, they will be useful for POC testing to identify N501Y meta-signature variants, protect individuals in high-risk settings, and help detect epidemiological shifts among SARS-CoV-2 variants.

Keywords: COVID-19; OCMS™; SARS-CoV-2; clinical diagnostic test; delta variant; monoclonal antibody; variants of concern.

Figure 1

SARS-CoV-2 L strain (Wuhan-Hu-1) spike binding by the 2E8 human mAb. (a) The 2E8 mAb binding in a direct ELISA to SARS-CoV-2 antigens: S1, S1 D614G, and nucleocapsid (Sino Biologicals); spike-pseudotyped VSV particles; and a recombinant S1 trimer. Samples were tested in triplicate. Error bars = S.E.M. (not visible due to minimal differences). (b) S1 binding to 293T-hsACE2 cells in the presence of 2E8, 4G1 (isotype control IgG), CR3022, CB6, and an ACE2-Fc fusion protein was assessed by flow cytometry. S1 cell binding, x-axis; IgG binding, y-axis. (c) Pseudovirus neutralization assay. 293T-hsACE2 cells were transduced with SARS-CoV-2 luciferase (Wuhan-Hu-1 strain) reporter viral particles (RVPs) in the presence of the mAbs, 6A (isotype control IgG), CR3022, 2E8, and CB6, and a polyclonal IgG isolated from the 2E8 B cell donor (P24). Normalized percent infection is shown; samples were tested in triplicate. Error bars = S.E.M. (d) SPR analysis of 2E8 binding to the spike S1 domain, performed on the Nicoya OpenSPR™; KD = 7.38 ± 0.58 nM.

Figure 2

Epitope binning the 2E8 on the SARS-CoV-2 S1 and RBD. We performed competition binding assays to test 2E8 binding in the presence of the CR3022, CB6, and the murine anti-S1 mAb. L-type spike (a,c) or RBD (b,d) was captured on the plate and binding of biotinylated 2E8, or the anti-spike murine mAb was tested in the presence of non-biotinylated competitor mAbs. Blue, no competitor; orange, competitor present.

Figure 3

Differential recognition of SARS-CoV-2 variant spike antigen by direct ELISA. The 2E8 (a), CB6 (b), and the mouse anti-S1 mAb (c) were tested for binding to spike antigens adhered to an ELISA plate: L RBD (Wuhan-Hu-1), α S1 (B.1.1.7), β S1 (B.1.351), γ S1 (P.1), δ RBD (B.1.617.2), K417N RBD, κ RBD (B.1.617.1), ε S1 (B.1.429). Error bars = S.E.M.

Figure 4

Differential recognition of SARS-CoV-2 variant spike antigens using a sandwich ELISA with human mAbs. The mAbs were adhered to the ELISA plate and tested for capture of soluble spike antigen. Bound antigen was detected with the mouse anti-S1 mAb. (a) Both 2E8 and CB6 are compared. (b) An anti-His tag capture antibody was used as a positive control. Antigens used (same as Figure 3): L RBD (Wuhan-Hu-1), α S1 (B.1.1.7), β S1 (B.1.351), γ S1 (P.1), δ RBD (B.1.617.2), K417N RBD, κ RBD (B.1.617.1), ε S1 (B.1.429).