Neutralizing antibody 5-7 defines a distinct site of vulnerability in SARS-CoV-2 spike N-terminal domain

Abstract

Antibodies that potently neutralize SARS-CoV-2 target mainly the receptor-binding domain or the N-terminal domain (NTD). Over a dozen potently neutralizing NTD-directed antibodies have been studied structurally, and all target a single antigenic supersite in NTD (site 1). Here, we report the cryo-EM structure of a potent NTD-directed neutralizing antibody 5-7, which recognizes a site distinct from other potently neutralizing antibodies, inserting a binding loop into an exposed hydrophobic pocket between the two sheets of the NTD β sandwich. Interestingly, this pocket was previously identified as the binding site for hydrophobic molecules, including heme metabolites, but we observe that their presence does not substantially impede 5-7 recognition. Mirroring its distinctive binding, antibody 5-7 retains neutralization potency with many variants of concern (VOCs). Overall, we reveal that a hydrophobic pocket in NTD proposed for immune evasion can be used by the immune system for recognition.

Keywords: COVID-19; N-terminal domain; NTD; SARS-CoV-2; antigenic supersite; cryo-EM; neutralizing antibody; variants of concern.

Graphical abstract

Figure 1

Antibody 5-7 targets a hydrophobic pocket in NTD (A) Cryo-EM reconstruction for spike complex with antibody 5-7 from 2 orthogonal views. The map shown is a composite map obtained combining the overall map (3.50 Å resolution) and the locally refined map for the 5-7:NTD interface (3.82 Å resolution). NTD is shown in orange, RBD in green, glycans in red, antibody heavy chain in blue, and light chain in gray. (B) Details of antibody 5-7 recognition of NTD showing the overall interface (left), recognition by CDR H3 (center), and recognition by CDR H1 and H2 (right). CDR H1, H2, and H3 are colored in shades of blue; CDR L1, L2, and L3 are colored in shades of gray. Nitrogen atoms are colored in blue, oxygen atoms in red, and hydrogen bonds (distance <3.2 Å) are represented as dashed lines. See also Figures S1–S4 and Table S1.

Figure 2

Antibody 5-7 adopts a different binding mode compared to antibodies targeting the NTD supersite (A) Structural superposition of all NTD-directed antibodies deposited in the PDB on NTD in the context of SARS-CoV-2 spike trimer. All neutralizing antibodies target the NTD supersite (magenta) except for 5-7 (blue) and P008_056 (green). The non-neutralizing antibody DH1052 (yellow) binds to a different region at the “bottom” of NTD. (B) Comparison between NTD supersite (magenta) and 5-7 epitope (blue) on NTD. The overlap region only includes 3 residues (150–152) located in the N3 loop. (C) The hydrophobic pocket that accommodates 5-7 CDR H3 (left) has been previously reported to bind polysorbate 80 (right). See also Figures S1, S4, S6, and S7.

Figure 3

SARS-CoV-2 neutralization profiles for 5-7 and two NTD supersite targeting antibodies (A) Neutralization of wild-type (USA/WA1), B.1.1.7, B.1.351, P.1, B.1.526, and B.1.429 authentic viruses by NTD-directed mAbs 5-7, 5-24, and 4-8. The horizontal dotted lines on each graph indicate 50% and 90% neutralization. Data are means ± SEMs of technical triplicates and represent 1 of 2 independent experiments. (B) Fold increase or decrease in IC₅₀ of the mAbs against pseudoviruses containing all of the combined mutations or the NTD single mutation of B.1.1.7, B.1.351, P.1, B.1.526. and B.1.427/9, relative to the wild-type (D614G) virus, presented as a heatmap in which darker colors indicate a greater change. Red, resistance >5-fold; green, sensitization > 5-fold. See also Figures S5 and S7.

Figure 4

Antibody 5-7 binds apart from most of the mutations in emerging SARS-CoV-2 variants (A) The majority of NTD mutations in different SARS-CoV-2 variants are not within the 5-7 binding region. Mutations in B.1.1.7 are colored magenta, B.1.351 orange, P.1 blue, B.1.526 green, and B.1.427/9 red. The NTD supersite is outlined in yellow. (B) Comparison of residue entropy between NTD supersite, 5-7 epitope, and other residues on NTD. Larger entropy means more diversification. Left and center panels show the footprint and residue entropy for the supersite and 5-7 epitope on the NTD surface. Right: residues are represented as dots, and the geometric mean and 95% confidence interval (CI) of the 3 groups of residues are shown in blue, red, and green, respectively. Antibody 5-7 epitope residues are the most conserved among the 3 groups. The p values between different groups were calculated using the Kolmogorov-Smirnov test. See also Figure S4.