Characterization of a neutralizing antibody that recognizes a loop region adjacent to the receptor-binding interface of the SARS-CoV-2 spike receptor-binding domain

ABSTRACT Although the global crisis caused by the coronavirus disease 2019 (COVID-19) pandemic is over, the global epidemic of the disease continues. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the cause of COVID-19, initiates infection via the binding of the receptor-binding domain (RBD) of its spike protein to the human angiotensin-converting enzyme II (ACE2) receptor, and this interaction has been the primary target for the development of COVID-19 therapeutics. Here, we identified neutralizing antibodies against SARS-CoV-2 by screening mouse monoclonal antibodies and characterized an antibody, CSW1-1805, that targets a narrow region at the RBD ridge of the spike protein. CSW1-1805 neutralized several variants in vitro and completely protected mice from SARS-CoV-2 infection. Cryo-EM and biochemical analyses revealed that this antibody recognizes the loop region adjacent to the ACE2-binding interface with the RBD in both a receptor-inaccessible “down” state and a receptor-accessible “up” state and could stabilize the RBD conformation in the up-state. CSW1-1805 also showed different binding orientations and complementarity determining region properties compared to other RBD ridge-targeting antibodies with similar binding epitopes. It is important to continuously characterize neutralizing antibodies to address new variants that continue to emerge. Our characterization of this antibody that recognizes the RBD ridge of the spike protein will aid in the development of future neutralizing antibodies. IMPORTANCE SARS-CoV-2 cell entry is initiated by the interaction of the viral spike protein with the host cell receptor. Therefore, mechanistic findings regarding receptor recognition by the spike protein help uncover the molecular mechanism of SARS-CoV-2 infection and guide neutralizing antibody development. Here, we characterized a SARS-CoV-2 neutralizing antibody that recognizes an epitope, a loop region adjacent to the receptor-binding interface, that may be involved in the conformational transition of the receptor-binding domain (RBD) of the spike protein from a receptor-inaccessible “down” state into a receptor-accessible “up” state, and also stabilizes the RBD in the up-state. Our mechanistic findings provide new insights into SARS-CoV-2 receptor recognition and guidance for neutralizing antibody development.

The RBD is colored in blue, the heavy and light chains of CSW1-1805 in dark green and light green, respectively, and the heavy and light chains of CSW1-1353 in cyan and magenta, respectively.

Fig
Fig. S1.Preparation of recombinant SARS-CoV-2 Spike protein.(A) Schematic view of the SARS-CoV-2 spike ectodomain (residues 1-1208) with the GSAS substitution of the furin cleavage site (RRAR; residues 682-685) and the K986P/V987P mutations, followed by the C-terminal, the foldon trimerization motif, and an 8 x His-tag.SP, signal peptide; NTD, N-terminal domain; RBD, receptor-binding domain.(B) A representative elution chromatogram of the recombinant spike protein used in this study.The area around the elution peak is shown in the inset with the fraction numbers.(C) SDS-PAGE of each fraction represented in (B)

Fig. S4 .
Fig. S4.Sequence analysis of the SARS-CoV-2 spike gene of escape mutants.Parts of the sequence electrogram of the SARS-CoV-2 S gene in escape mutants that emerged in the presence of (A) CSW1-1805 or (B) CSW2-1353.The letter of the amino acid at the substituted position is colored in red.

Fig. S6 .
Fig. S6.Cryo-EM analysis of the spike-CSW1-1805 complex.(A) Typical motion-corrected micrographs (x 600,000).(B) Selected 2D class averages aligned in descending order of particle number from left to right and top to bottom.(C) Final sharpened maps in two orthogonal views: top view, left panel; and side view, right panel.The local resolution distributions are colored as shown in the color bar.(D) FSC curve for the final map.The dashed blue line indicates the FSC = 0.143 criterion.

Fig. S7 .
Fig. S7.Cryo-EM analysis of the spike-CSW2-1353 complex.(A) Typical motion-corrected micrograph (x 600,000).(B) Selected 2D class averages aligned in descending order of particle number from left to right and top to bottom.(C, D) Final sharpened maps of (C) 1-up RBD and (D) 2-up RBD datasets in two orthogonal views: top view, left panel; and side view, right panel.The local resolution distributions are colored as shown in the color bar.(E, F) FSC curves for the final maps of (E) 1-up RBD and (F) 2-up RBD datasets.The dashed blue lines indicate the FSC = 0.143 criterion.

Fig. S8 .
Fig. S8.Amino acid sequences of variable regions of CSW1-1805 and CSW2-1353.Amino acid sequences of the heavy (VH) and light (VL) chains of CSW1-1805 and CSW2-1353.Residues in the complementary determinant regions are defined using the AbM definition and highlighted in yellow.

Fig. S9 .
Fig. S9.Close-up views of the spike-Fab complexes.(A, B) Final sharpened maps of (A) Spike+CSW1-1805 and (B) Spike+CSW2-1353 (1-up RBD) datasets around the Fab molecules.The map regions corresponding to each chain are colored differently (in the same color as Figure 3A, B).Model structures of Fabs were generated by using SWISS-MODEL and fitted into the maps.(C) Structural comparison of the up-RBDs bound to CSW2-1353 and CSW1-1805.The coloring is the same as in (A) and (B).(D-J) The interface between (D-F) the up-RBD and CSW1-1805 Fab or (G-J) the down-RBD and CSW2-1353 Fab.

Fig. S10 .
Fig. S10.Cryo-EM analysis of the spike C480A mutant.(A) Typical motion-corrected micrograph (x 600,000).(B) Selected 2D class averages aligned in descending order of particle number from left to right and top to bottom.(C) Final sharpened maps