Impact of the N501Y substitution of SARS-CoV-2 Spike on neutralizing monoclonal antibodies targeting diverse epitopes

Gene sequences of published nAbs downloaded from the National Center of Biotechnology Information (NCBI) were synthesized and cloned into the human full-length IgG1 expression vectors (Sangon Biotech, Shanghai). Paired heavy and light chains were co-transfected into 293 F cells, and antibodies were purified from cell supernatants using protein A columns according to the manufacturer’s instructions (National Engineering Research Center for Biotechnology, Beijing) after five days. Purified nAbs were quantified using a NanoDrop spectrophotometer and stored at 4 °C.

SARS-CoV-2 pseudotyped-virus was generated by co-transfection of HEK-293T cells in T75 flask with 10 μg of SARS-CoV-2 spike-expressing plasmid and 20 μg of an env-deficient HIV-1 backbone vector (pNL4-3.Luc.R-E-). Two days post-transfection, pseudovirus-containing culture supernatant was harvested, clarified by centrifugation, filtered and then stored at −80 °C in 1.5-ml aliquots. To determine the neutralizing activity, serially diluted monoclonal antibodies were incubated with equal volume of diluted pseudovirus at 37 °C for 1 h. The antibody-virus mixtures were subsequently added into pre-seeded HEK-293T-hACE2 cells in duplicate. After a 48-h incubation, culture medium was removed and 100 μl of the Bright-Lite Luciferase reagent (Vazyme Biotech, Nanjing, China) was added to the cells. After a 2-min incubation at room temperature, 90 μl of cell lysate was transferred to 96-well white solid plates for measurements of luminescence using the Varioskan™ LUX multimode microplate reader (Thermo Fisher Scientific). The 50% inhibitory concentrations (IC₅₀) was calculated using GraphPad Prism software by log (inhibitor) vs. normalized response—Variable slope (four parameters) model and fourfold of IC₅₀ was set as the cutoff of significant change [11].

The binding assays of monoclonal antibodies to the wild type and N501Y mutant SARS-CoV-2 RBDs were performed using the Biacore 8 K system (GE Healthcare). Specifically, one flow cell of the CM5 sensor chips were covalently coated with the wild type or N501Y mutant RBDs (Sino Biological, Beijing) in 10 mM sodium acetate buffer (pH 5.0) for a final RU (response units) around 250, whereas the other flow cell was left uncoated and blocked as a control. All the assays were run at a flow rate of 30 µl/min in HBS-EP buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.05% Tween-20). Serially diluted antibodies were injected for 60 s respectively and the resulting data were fit in a 1:1 binding model with Biacore Evaluation software (GE Healthcare). Every measurement was performed three times and the individual values were used to produce the mean affinity constant and standard deviation.

According to the competition with ACE2 and the accessibility of neutralizing epitopes on the RBD in ‘up’ or ‘down’ conformations, RBD-specific nAbs were classified into four classes[12]. As shown in Additional file 1: Fig. S1, nAbs of Class 1 and Class 2 bind to the receptor-binding motif (RBM) directly and near-RBM region on the RBD of spike, respectively, both of which could disturb the interaction between the RBD of virus and the cell receptor-ACE2. The nAbs of Class 3 and Class 4 do not compete with ACE2 directly, bind to the two sides of the base of RBD away from RBM, and usually cross-react with SARS-CoV. In this study, the twelve-nAb panel contains 3 nAbs in Class 1 (CB6, P2C-1F11, and REGN10933), 4 nAbs in Class 2 (BD-23, BD-368-2, P2B-2F6, and P2C-1A3), 2 nAbs in Class 3 (S309 and REGN10987), 1 nAb in Class 4 (EY6A), and 2 unclassified nAbs (COV2-2196 and CV07-287). To assess the effect of the N501Y substitution on the recognition with nAbs, we measured the neutralizations and binding affinities of these nAbs against the Wuhan-Hu-1 strain (WT) and N501Y mutant by pseudovirus neutralizing assay and surface plasmon resonance (SPR).

Class 1 nAbs directly bound to the ACE2-binding site on the RBD, which shared the largest number of residues between their epitopes and ACE2-binding site. As shown in Table 1, the neutralizing activity and binding affinity of CB6 against N501Y were decreased distinctly compared to WT (7.38- and 6.67-fold, respectively), with another two Class 1 nAbs (P2C-1F11 and REGN10933) slightly affected (Figs. 1, 2, Table 1, Additional file 1: Fig. S2, Fig. S3). Reasonably, previous structural analysis of the CB6/RBD complex revealed that the epitope residue N501 contacted directly with CB6 but not with P2C-1F11 and REGN10933 [1, 3, 13]. Additionally, the free energy perturbation method also predicted that N501Y weaken the binding affinity of CB6 to RBD [14].

The Class 2 nAbs shared less or none overlapping residues with ACE2-binding site, but still showed strong competitions with ACE2. P2B-2F6 and P2C-1A3 both recognized an overlapped epitope on RBD yet with distinct angles of approach compared to P2C-1F11 (Class 1) [13]. BD-368-2 recognized RBD with a similar angle as P2B-2F6, and both inhibited the viral entry by a clash between the light chain and ACE2 [15]. These three nAbs of Class 2 did not contact with N501 epitope directly, so kept stable neutralizing and binding activities against N501Y mutant (Table 1, Additional file 1: Fig. S2, Fig. S3). Unexpectedly, the N501Y mutant virus was more sensitive (6.58-fold) to the neutralization of BD-23, another Class 2 antibody which also contact directly with N501 (Fig. 1, Table 1). Consistently, surface plasmon resonance analysis also revealed that the binding affinity of BD-23 to N501Y variant was increased 6.65-fold as compared with the wild type RBD (Fig. 2, Table 1, Additional file 1: Fig. S3).

Another two classes of nAbs recognized two completely distinct epitopes away from the ACE2-binding site. S309 (Class 3) bound both ‘up’ and ‘down’ states of the RBD, whereas EY6A (Class 4) only recognized the RBD epitope in up conformation [12]. Targeting the relatively conserved epitopes, S309 and EY6A both cross-neutralized SARS-CoV-2 and SARS-CoV. Seventeen out of 22 epitope residues recognized by S309 were conserved between SARS-CoV-2 and SARS-CoV, and 21 of 31 residues in the interaction between EY6A and RBD were conserved with a SARS-CoV specific nAb (CR3022) [16, 17]. Not surprisingly, the N501Y mutation did not affect the neutralizing and binding activities of S309 and EY6A (Figs. 1, 2, Table 1, Additional file 1: Fig. S2, Fig. S3).

The mutation N501Y in the circulating strain in England is not the first time identified from SARS-CoV-2 live virus. A previous study reported that N501Y existed in a mouse-adapted strain, named MASCp6, which was isolated by serial passaging of SARS-CoV-2 in the respiratory tract of mice and led to interstitial pneumonia in this mouse challenge model. Structural remodeling showed that the binding capacity of RBD of viral spike to mouse ACE2 protein was increased by the substitution of N501Y [18]. Another study developed a novel strain-MASCp36 by passaging of SARS-CoV-2 from MASCp6, which caused 100% fatality in aged mice. Sequence analysis revealed that another two amino acid substitutions (K417N and Q493H) appeared in RBD of SARS-CoV-2 spike besides N501Y. These mutations contributed the enhancement of binding affinity between RBD and mouse ACE2 which has been certified in vitro by surface plasmon resonance and structure analysis [19].

Indeed, it is reported that the neutralizing activities of four nAbs (lacking analysis of neutralizing epitope) against SARS-CoV-2_N501Y, and one nAb (03-1F9) showed a decrease of six-fold in the neutralization [20]. In addition, the K417N and E484K contribute more than a single N501Y substitution in the resistance of virus to the neutralization by nAbs in a study which measured the neutralizing potencies of eight human nAbs against two SARS-CoV-2 variants both carrying N501Y mutation (N501Y.V1 and N501Y.V2) [8]. Our results were similar with that of above two studies, which indicated that the N501Y mutation may partially affect the neutralizing potencies of some nAbs. Furthermore, a study analyzed the neutralization of SARS-CoV-2 variants by several classes of nAbs based on their different epitopes [10]. Consistently, we also revealed that current mutations on the RBD mainly influenced the neutralizations of nAbs targeting directly to the RBM and near-RBM region. Like S309 and EY6A, some nAbs bind the base of RBD and recognize the epitopes away from the RBM, whose neutralizations are barely affected by the current mutations of SARS-CoV-2. These results also explained the phenomenon that though the neutralization geometric mean titers of sera samples whether from convalescent patients or vaccinated individuals indeed reduced in different levels, a part of sera samples as a mixture of polyclonal nAbs with diverse epitopes still maintained the neutralizing activities against SARS-CoV-2 variants to a certain extent [8‐10].