Characterization of SARS-CoV-2 Escape Mutants to a Pair of Neutralizing Antibodies Targeting the RBD and the NTD

Abstract

Mutations in the spike protein of SARS-CoV-2 can lead to evasion from neutralizing antibodies and affect the efficacy of passive and active immunization strategies. Immunization of mice harboring an entire set of human immunoglobulin variable region gene segments allowed to identify nine neutralizing monoclonal antibodies, which either belong to a cluster of clonally related RBD or NTD binding antibodies. To better understand the genetic barrier to emergence of SARS-CoV-2 variants resistant to these antibodies, escape mutants were selected in cell culture to one antibody from each cluster and a combination of the two antibodies. Three independently derived escape mutants to the RBD antibody harbored mutations in the RBD at the position T478 or S477. These mutations impaired the binding of the RBD antibodies to the spike protein and conferred resistance in a pseudotype neutralization assay. Although the binding of the NTD cluster antibodies were not affected by the RBD mutations, the RBD mutations also reduced the neutralization efficacy of the NTD cluster antibodies. The mutations found in the escape variants to the NTD antibody conferred resistance to the NTD, but not to the RBD cluster antibodies. A variant resistant to both antibodies was more difficult to select and only emerged after longer passages and higher inoculation volumes. VOC carrying the same mutations as the ones identified in the escape variants were also resistant to neutralization. This study further underlines the rapid emergence of escape mutants to neutralizing monoclonal antibodies in cell culture and indicates the need for thorough investigation of escape mutations to select the most potent combination of monoclonal antibodies for clinical use.

Keywords: NTD; RBD; SARS-CoV-2; SARS-CoV-2 escape mutations; neutralizing monoclonal antibodies; spike protein; variants of concern.

Figure 1

Neutralization of escape variants and identification of mutations. (A) IC50s of the antibodies indicated for the TRES 6 escape variants selected in three independent experiments (Exp) and the wild type virus. IC50s were taken from [27]; (B) IC50s of the antibodies indicated for TRES328 escape variants selected in two independent experiments and the wild type virus; (C) IC50s of the antibodies indicated for an escape mutant selected in the presence of TRES6 and TRES328 and without antibody and the wild type viral isolate; (D) viral load (C_T) in cell culture supernatants of the indicated passages and antibodies used for selection. Double the inoculation volume was needed to maintain sufficient viral load levels during passaging in the presence of TRES6 and TRES328. (E–G) Mutations identified through amplicon sequencing of the TRES6 (E), TRES328 (F), and TRES6+TRES328 (G) viral escape variants. Mutations identified in the first isolate are displayed in yellow, in the second isolate in green, and the third isolate in orange. Map not drawn to scale. FP: fusion peptide; HR: heptad repeat; TM: transmembrane domain; CP: cytoplasmic domain.

Figure 2

Characterization of S of TRES6 escape variants. (A,B,D,E,G,H,J,K,M,N,P,Q) Percent inhibition of lentiviral vector particles pseudotyped with SARS-CoV-2 S carrying the indicated RBD and/or NTD mutations. IC50s of the indicated RBD (red) and NTD (blue) cluster antibodies are displayed in ng/mL in the brackets. (C,F,I,L,O,R) Binding of the RBD (red) and NTD (blue) cluster antibodies and an S2-binding antibody (grey) to S with the indicated mutations. The binding capacity of cluster 1 and cluster 2 antibodies to HEK-293T cells expressing SARS-CoV-2 S proteins carrying a T478I (C), T478I+N74K (F), and T478K (I); or T478K+I68R (L), S477N (O), and S477N+G261R (R). Neutralization and binding of the control D614G variant is given in Figure 5A–C.

Figure 3

Characterization of S of TRES328 escape variants. (A,B,D,E) Percent inhibition of lentiviral vector particles pseudotyped with SARS-CoV-2 S carrying the indicated RBD and/or NTD mutations. IC50s of the indicated RBD (red) and NTD (blue) cluster antibodies are shown in brackets in ng/mL. (C,F) Binding of the RBD (red) and NTD (blue) cluster antibodies and a S2 binding antibody (grey) to S with the indicated mutations. Neutralization and binding of the D614G wild-type spike protein is given in Figure 5A–C. (G) Structural modeling of TRES328 (yellow) in complex with the NTD of wild-type spike protein (pink). NTD residues are represented as blue sticks and TRES328 residues as yellow sticks in order to highlight the position of Y145 and H245 or to illustrate the stabilizing electrostatic interactions (black dashed lines) between K147 and E72 of TRES328 and K150 and E54 of TRES328, respectively.

Figure 4

Characterization of S of the TRES6+TRES328 double escape variant. (A,B) Percent inhibition of lentiviral vector particles pseudotyped with SARS-CoV-2 S carrying the D215G and S477N mutations. IC50s of RBD (red) and NTD (blue) cluster antibodies are shown in ng/mL in the brackets. Neutralization and binding of the D614G wild-type spike protein is given in Figure 5A–C. (C) Binding of the RBD (red) and NTD (blue) cluster antibodies and an S2 binding antibody (grey) to S with the D215G and S477N mutations. (D) Structural modeling of TRES328 (yellow) in complex with the NTD of wild-type spike protein (pink). The NTD residues R214 and D215 are shown as sticks to illustrate that D215 is not located within the TRES328 interface and to highlight the close proximity of the R214 and D215 side chains, which form in some structures a salt bridge as strong electrostatic and stabilizing interaction (black dashed lines). The mutation to glycine (D215G) destroys this salt bridge interaction with R214, probably resulting in an increased flexibility of the arginine side chain, which could thereby possibly lead to a conformational change of the NTD.

Figure 5

Characterization of SARS-CoV-2 S of the indicated VOCs. (A,B,D,E,G,H,J,K) Percent inhibition of lentiviral vector particles pseudotyped with S of the indicated VOCs. The relevant mutations within the NTD and RBD of the respective mutation are given in brackets. IC50s of the indicated RBD (red) and NTD (blue) cluster antibodies are shown in ng/mL in the brackets. (C,F,I,L) Binding of the RBD (red) and NTD (blue) cluster antibodies and an S2-binding antibody (grey) to S of the VOCs indicated.