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.IMPORTANCESARS-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.

Keywords: conformational transition; monoclonal antibody; neutralizing epitope; receptor-binding domain; severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2); spike protein.

Fig 1

Characterization of the neutralizing antibodies against authentic SARS-CoV-2. (A) Binding curves of CSW1-1805 and CSW2-1353 against RBD^B.1 were obtained by using an ELISA. Assays were performed independently three times (means ± SD). The plots were fitted with a sigmoidal function by using Igor Pro (ver 8.04, Wavemetrics). (B) Concentration-dependency of CSW1-1805 and CSW2-1353 for plaque reduction of the SARS-CoV-2 Wuhan strain. Assays were performed independently three times (means ± SD). The plots are expressed as a decreasing percentage of the plaque numbers relative to the control (without antibody) and fitted with a sigmoidal function by using Igor Pro (ver 8.04, Wavemetrics). (C) Overview of the animal challenge study. Mice were intranasally infected with 5 MLD₅₀ of rSARS-CoV-2MA-10 immediately after intraperitoneally administrating 500 µg of CSW1-1805 or isotype control. Two days later, antibodies were administered for a second time, and 6 days later, organs were collected for virus titration. (D) Body weight changes and (E) viral titers of nasal turbinate (NT) and lung samples from mice administrated CSW1-1805 (red), CSW2-1353 (blue), or isotype control (black). The dotted line in (E) indicates the detection limit.

Fig 2

Reactivity of the neutralizing antibodies against SARS-CoV-2 variants. (A) Summary of the obtained escape mutant viruses. (B) The positions of the mutated residues are shown as sticks in the structure of the RBD-ACE2 complex (PDB ID: 6m0j). Substituted amino acids in rSARS-CoV-2MA-10 and variants located near the antibody-binding interface are also shown as sticks. (C, D) Concentration-dependency of (C) CSW1-1805 and (D) CSW2-1353 for plaque reduction of SARS-CoV-2 variants. Assays were performed independently three times (means ± SD). The plots are expressed as a decreasing percentage of the plaque numbers relative to the control (without antibody) and fitted with a sigmoidal function by using Igor Pro (ver 8.04, Wavemetrics). The results for the SARS-CoV-2 Wuhan strain described in Fig. 1C are also presented for comparison.

Fig 3

Structural analysis of spike–Fab complexes. (A–C) Final sharpened maps of (A) Spike^B.1 + CSW1-1805, (B) Spike ^B.1 + CSW2-1353 (1-up RBD), and (C) Spike ^B.1 + CSW2-1353 (2-up RBD) in two orthogonal views: side view, left panels; and top view, right panels. The map regions corresponding to each protomer in the spike protein are colored in blue, red, and green, respectively. The map regions corresponding to the light and heavy chains of each Fab are colored in light green/cyan/pink and deep green/cyan/pink, respectively. (D) Superimposed 3D structures around one up-RBD. ACE2 molecules (from PDB: 7KMS) are also shown. The right panel shows approximately 90^o-rotated views of the left panel. Neighboring CSW2-1353 and ACE2 molecules are shown only in the right panel. (E, F) Close-up views of the interface between (E) the up-RBD and CSW1-1805 Fab or (F) the down-RBD and CSW2-1353 Fab. The RBD is colored in blue; the heavy and light chains of CSW1-1805 are in dark green and light green, respectively; and the heavy and light chains of CSW1-1353 are in cyan and magenta, respectively. Residues substituted in the Alpha, Beta, Gamma, Delta, and escape mutants found in this study are shown as sticks.

Fig 4

Binding analysis of the neutralizing antibodies to the down-RBD conformation. Binding curves of (A) hACE2-His-Strep, (B) CSW1-1805, and (C) CSW2-1353 to Spike^B.1 (open circle) and Spike^closed (closed circle) were obtained by using an ELISA. Assays were performed independently three times (means ± SD). The plots were fitted with a sigmoidal function by using Igor Pro (ver 8.04, Wavemetrics).

Fig 5

Comparison of neutralizing antibodies that bind to the RBD ridge. (A) Superimposed structures of CSW1-1805, 2G1, and Ab159 on the RBD in the left panel and CSW1-1805, AZD8895, COVOX-253H55L, XGv347, and S2E12 on the RBD in the right panel are shown in two orthogonal views. (B) Alignment of the amino acid sequences of the CDRs within the heavy (upper panel) and light (lower panel) chain variable domains are shown. Amino acid are colored according to the Clustal color scheme (Hydrophobic amino acids: A, I, L, M, F, W, V in blue; positively charged amino acids: K, R in red; negatively charged amino acids: E, D in magenta; polar amino acids: N, Q, S, T in green; C in pink; G in orange; P in yellow; aromatic amino acids: H, Y in cyan).