A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2

Abstract

Developing therapeutics against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could be guided by the distribution of epitopes, not only on the receptor binding domain (RBD) of the Spike (S) protein but also across the full Spike (S) protein. We isolated and characterized monoclonal antibodies (mAbs) from 10 convalescent COVID-19 patients. Three mAbs showed neutralizing activities against authentic SARS-CoV-2. One mAb, named 4A8, exhibits high neutralization potency against both authentic and pseudotyped SARS-CoV-2 but does not bind the RBD. We defined the epitope of 4A8 as the N-terminal domain (NTD) of the S protein by determining with cryo-eletron microscopy its structure in complex with the S protein to an overall resolution of 3.1 angstroms and local resolution of 3.3 angstroms for the 4A8-NTD interface. This points to the NTD as a promising target for therapeutic mAbs against COVID-19.

Fig. 1. Isolation of antigen-specific mAbs from convalescent patients of SARS-CoV-2.

(A) Reactions of plasma to SARS-CoV-2 proteins. S-ECD (extracellular domain of S protein), S1, S2, RBD (receptor binding domain), and N protein were used in ELISA to test the binding of plasma. Plasma of heathy donors were used as control, and cut-off values were calculated as optical density (OD) 450 of control × 2.1. Data were shown with mean and SD of a representative experiment. (B) The correlations between the authentic SARS-CoV-2 neutralizing antibody (NAb) titers and the pseudotyped SARS-CoV-2 NAb titers in plasma. Neutralizing assays of plasma against authentic SARS-CoV-2 were performed by using Vero E6 cells, and neutralization against pseudotyped SARS-CoV-2 were determined by using ACE2-293T cells. The correlations were calculated by means of Pearson correlation test in Graphpad 7.0. (C) Flow cytometry sorting from PBMCs of 10 convalescent patients. (D) Distribution of V gene families in heavy and light chains of all distinct clones (the total number is shown in the center of the pie charts) for each donor. (E) The number of amino acid (AA) and total nucleotide (Nt) mutations from the germline of all clonal sequences identified in (D) is shown. (F) CDR3 amino acid lengths of VH and VL of all clonal sequences identified in (D).

Fig. 2. Binding profiles of Spike protein–specific mAbs.

(A) Heatmap showing the binding of mAbs to different types of spike proteins determined by using ELISA. The EC₅₀ value for each S-mAb combination is shown, with dark red, orange, yellow, or white shading indicating high, intermediate, low, or no detectable binding, respectively. EC₅₀ values greater than 10,000 ng/ml are indicated (>). (B) Binding curves of representative mAbs. CR3022 is a control that was reported to bind SARS-CoV and SARS-CoV-2 RBD. Data were shown with mean and SD of a representative experiment. (C) Heatmap showing the competing binding of some representative S-reactive mAbs assayed in ELISA. Numbers in the box indicate the percentage binding of detecting mAb in the presence of the blocking antibody compared with the binding of detecting mAb in the absence of the blocking antibody. The mAbs were considered competing if the inhibiting percentage is <30% (black boxes with white numbers). The mAbs were judged to noncompete for the same site if the percentage is >70% (white boxes with red numbers). Gray boxes with black numbers indicate an intermediate phenotype (30 to ~70%). (D) Phylogenetic trees of all the S-specific mAbs.

Fig. 3. Neutralizing capacities of S-reactive mAbs.

(A) Neutralization of S-reactive mAbs to authentic SARS-CoV-2 in Vero-E6 cells. (B) The authentic SARS-CoV-2 virus RNA load was determined in Vero-E6 cells treated with S-reactive mAbs by using quantitative PCR. Percent infection was calculated as the ratio of RNA load in mAb-treated wells to that in wells containing virus only. (C) Neutralization of S-reactive mAbs against HIV-vectored pseudotyped SARS-CoV-2 in ACE2-293T cells. Data were shown as mean ± SD of a representative experiment.

Fig. 4. 4A8 did not block the binding of Spike protein to ACE2 receptor.

(A) BLI sensorgrams and kinetics of mAbs binding to S proteins. Global fitting curves are shown as black lines. The K_d were calculated by using a 1:1 binding model in Data Analysis Software 9.0, except for 2M-10B11, which used a heterogeneous ligand model owing to avidity effect. (B) The binding of S protein to human ACE2-overexpressing 293T cells were determined by means of flow cytometry. After the preincubation of S protein with each indicated mAb, the mAb-S mixtures were added to the ACE2-expressing cells. Cells were stained with anti-human IgG fluorescein isothiocyanate (mAb binding, x axis) and anti-His (S binding, y axis). Percentages of double-positive cells are shown. Control mAb CR3022 and 1A8 were previously reported to bind SARS-CoV RBD and Marburg glycoprotein, respectively, and ACE2-Fc protein was a human ACE2 protein conjugated with human Fc.

Fig. 5. Cryo-EM structure of the 4A8 and S-ECD complex.

The domain-colored cryo-EM map of the complex is shown on the left, and two perpendicular views of the overall structure are shown on the right. The heavy and light chains of 4A8 are colored blue and magenta, respectively. The NTDs of the trimeric S protein are colored orange. The one “up” RBD and two “down” RBDs of trimeric S protein are colored green and cyan, respectively.

Fig. 6. Interactions between the NTD and 4A8.

(A) Extensive hydrophilic interactions on the interface between NTD and 4A8. Only one NTD-4A8 is shown. (B to D) Detailed analysis of the interface between NTD and 4A8. Polar interactions are indicated by red dashed lines. The residues involved in hydrophobic interactions are presented as spheres.