A Naïve Phage Display Library-Derived Nanobody Neutralizes SARS-CoV-2 and Three Variants of Concern

Figures & data

Figure 1 Panning of specific nanobodies against the SARS-CoV-2 S1 protein by naïve nanobody library. (a) Schematic diagram of nanobody screening. (b) The anti-SARS-CoV-2 spike S1 nanobodies were enriched 149-fold after three rounds of screening. (c) Phage ELISA was used to identify an anti-SARS-CoV-2 S1 individual clone after three rounds of bio-panning. (d) SARS-CoV-2 S1 specific nanobodies were identified from the top 20 clones that were specifically bound to SARS-CoV-2 S1. (e) Alignment of the amino acid sequence of three screened SARS-CoV-2 S1 nanobodies.

Figure 2 Purification and identification of the SARS-CoV-2 S1-specific nanobody. The top three nanobodies with the highest affinity for SARS-CoV-2 S1 were identified and named Nb-H6, Nb-G7, and Nb-B7. (a) SDS-PAGE analysis of purified nanobodies Nb-H6, Nb-G7, and Nb-B7. (M) Marker. The molecular weight (kDa) is indicated on the left-hand side. (b) The affinity of Nb-H6, Nb-G7, and Nb-B7 to SARS-CoV-2 S1 (left panel) and SARS-CoV-2 RBD (right panel) at various concentrations (200, 50, 12.5, 3.12, 0.78, 0.19, 0.049, and 0.012 nM). The results are presented as mean absorbance values at OD₄₅₀ nm ± SD (n = 3).

Figure 3 Characterization of the SARS-CoV-2 nanobody Nb-H6. (a) The binding kinetics of Nb-H6 with the immobilized SARS-CoV-2 S1 and RBD were measured using biolayer interferometry (BLI). (b) Competitive binding assays by ELISA. SARS-CoV-2 S1 (left panel) and RBD (right panel) bind to ACE2 after competitive blocking with serially diluted Nb-H6. IC₅₀ values were calculated by nonlinear regression fitting to a variable slope, a four-parameter dose-response model. Data are presented as mean ± SD of three technical replicates. (c) Table summarizing the affinity K_D, association (K_on), and dissociation constants (K_off) determined by BLI as well as the competition assay IC₅₀ values for Nb-H6.

Figure 4 Evaluation of the thermal and pH stability of Nb-H6. Nb-H6 was incubated at different temperatures or pH levels, and the affinity of Nb-H6 binding to SARS-CoV-2 S1 was determined by ELISA. (a) Nb-H6 was incubated at 4, 30, 60, and 90 °C for 5 minutes; (b) Nb-H6 was incubated at 90 °C for 5, 15, 30, 45, 60, 75, 90, and 105 minutes; and (c) Nb-H6 was incubated at different pHs for 2 hours and the binding capacity was measured using ELISA. Data are shown as mean ± SD, n=3.

Figure 5 Nb-H6 suppresses SARS-CoV-2 infection in vitro. The SARS-CoV-2 spike pseudotyped GFP-luciferase lentivirus and authentic SARS-CoV-2 were mixed with serially diluted Nb-H6 and then infected host cells (HEK293-hACE2 and Vero E6, respectively) for infection. (a) Green fluorescence images were captured to visualize Nb-H6 neutralization against the SARS-CoV-2 pseudovirus (left panel), and the luciferase reaction assay was used to determine the IC₅₀ value (right panel). Data are shown as mean ± SD (n = 3). (b) The authentic virus neutralization assay was performed to verify the anti-authentic SARS-CoV-2 ability of Nb-H6. Anti-SARS-CoV-2 N protein antibody and HRP-linked secondary antibody (left panel) were used to label the infected cells (left panel). For the calculation of the IC₅₀, the foci were counted by the focus formation assay (FFA) (right panel). The scale bar in each image represents 200 μm. Data are shown as mean ± SD (n = 6). All IC₅₀ values were calculated using a four-parameter logistic curve.

Figure 6 The binding activity of Nb-H6 with SARS-CoV-2 variants S1 and RBD. (a) The binding kinetics of Nb-H6 with the immobilized S1 of variants Alpha (B.1.1.7), Delta (B.1.617.2), Lambda (C.37), and Omicron (BA.2) were detected by BLI. (b) The binding capacity of Nb-H6 with RBD of variants Lambda (C.37), Delta (B.1.617.2), and Omicron (BA.2 and BA.5) was measured by BLI. (c) Table summarizing the affinity K_D, association (K_on), and dissociation constants (K_off) determined by BLI.

Figure 7 Nb-H6 inhibits SARS-CoV-2 variant pseudovirus infection. The SARS-CoV-2 variant spike pseudotyped GFP-luciferase lentivirus was incubated with different concentrations of Nb-H6 for 1 hour and subsequently infected HEK293-hACE2 cells for 6 hours to allow virus entry. Representative fluorescence images and dose-response curve for Nb-H6 neutralization of SARS-CoV-2 variants Alpha B.1.1.7 (a), Delta B.1.617.2 (b), Omicron BA.2 (c), and Omicron (BA.5) (d) pseudovirus at 48 hours post-infection. The scale bar in each image represents 200 μm. The IC₅₀ was calculated using a four-parameter logistic curve. Data are shown as mean ± SD (n =3).

Figure 8 The amino acid residues Asn72 and Arg97 of Nb-H6 are critical for binding to SARS-CoV-2 RBD. (a) Predicted amino acids involved in the binding interactions between SARS-CoV-2 spike RBD (gray) and Nb-H6 (Orange). The Nb-H6 amino acid residues Asn72 and Arg97 are shown to form hydrogen bonds with the SARS-CoV-2 residues Tyr449 and Tyr489, respectively. (b) Amino acid sequence alignment of RBM (blue box) of wild-type and variant RBD. The conserved sites Y449 and Y489 were highlighted with a red box. (c) Affinity of Nb-H6 N72A or Nb-H6 R97A binding to SARS-CoV-2 S1 and RBD at various concentrations (800, 266.67, 88.89, 29.63, 9.88, 3.29, 1.10, and 0.37 nM), with Nb-H6 as a positive control. The results are presented as mean absorbance values at OD₄₅₀ nm ± SD (n = 3). (d) Biolayer interferometry (BLI) was used to measure the binding kinetics of Nb-H6, Nb-H6 N72A, and Nb-H6 R97A with the immobilized SARS-CoV-2 spike S1 and RBD. (e) Competitive binding assays by ELISA. SARS-CoV-2 S1 (left panel) and RBD (right panel) bind to ACE2 after competitive blocking with serially diluted Nb-H6, Nb-H6 N72A, and Nb-H6 R97A. (f) The affinity of Nb-H6 binding to RBD mutants Y449A and Y489A was assessed by ELISA (left penal) and Biolayer interferometry (right penal). Nb-H6 was used at concentrations (400, 100, 25, 6.25, 1.56, 0.39, 0.10, and 0.02 nM) in ELISA, and results are presented as mean absorbance values at OD₄₅₀ nm ± SD (n = 3). Data are presented as mean ± SD of three technical replicates.