Screening and Characterization of Shark-Derived VNARs against SARS-CoV-2 Spike RBD Protein

Figures

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  Figure 1. Isolation of anti-SARS-CoV-2 RBD VNARs from RBD-immunized C. plagiosum by phage display. (A) Amplification of VNAR genes (~340 bp) and determination of the insertion rate of VNAR in the phage library. (B) Identification of RBD-specific phages from 95 clones using monoclonal phage ELISA. (C) The amino acid sequence of the four identified unique RBD-specific VNARs. (D) Reduced SDS-PAGE analysis of the purified VNAR-Fc fusions and Fc-free VNARs.

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  Figure 2. Characterization of the interaction between VNAR fusions and RBD. (A) RBD, VNAR-Fc fusions, and their molar mixture (1:1) loaded over a Superdex 200 column. The elution peak from the mixtures appeared earlier than those from RBD and VNAR-Fc fusions. (B,C) Characterization of binding affinity of isolated VNARs (B) or VNAR-Fc fusions (C) for RBD using BLI. The actual responses (colored lines) and the data fitted to a 1:1 binding model (black dotted lines) are shown. ka and kd represent the association and dissociation constants, respectively. The equilibrium dissociation constant KD was calculated as kd/ka.

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  Figure 3. Binding characterization of identified VNARs and ACE2 to various RBD by ELISA. (A–D) VNAR-Fc fusions binding to RBD of WT (A), Delta (B), Omicron (C), and SARS-CoV (D) were characterized using ELISA. Error bars indicate means ± the SD from three independent experiments. The EC50 was calculated by fitting the OD450 values from serially diluted VNAR-Fc fusions or ACE2-Fc to a sigmoidal dose–response curve.

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  Figure 4. RBD-ACE2 blocking activities of isolated VNARs characterized by BLI assay. (A–D) Blocking of ACE2 binding to RBD of WT (A), Delta (B), Omicron (C), and SARS-CoV (D) by VNARs. Biotinylated RBD was loaded on SA biosensors to a load threshold above 1.0 nm, then incubated with VNAR-Fc fusion and ACE2 sequentially. VNAR was replaced with PBST and used as a control. The inhibition rates of VNARs were calculated against the control group.

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  Figure 5. Epitope competition results of the four identified VNAR-Fc fusions by BLI. (A) The epitope competition determined by the binding signal transition of two VNAR-Fc fusions. (B) The summarized epitope competition results of VNAR-Fc fusions. The red and gray colors indicate competitive and noncompetitive relationships, respectively.

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  Figure 6. Epitope competition results between the four VNAR-Fc fusions and alpaca-derived VHHs by BLI. (A) The competition determined by binding signal transition of shark-derived VNAR-Fc fusions and alpaca-derived VHH-Fc fusions. (B) The summarized table for epitope competition of nanobodies from shark and alpaca. The red and gray colors indicate competitive and noncompetitive relationships, respectively.

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  Figure 7. In silico molecular docking of VNARs binding to RBD. (A–D) Binding mode of JM-2 (A), JM-5 (B), JM-17 (C), and JM-18 (D) to RBD. (E) The docking structures alignment of JM-2 (cyan), JM-5 (yellow), JM-17 (magenta), and JM-18 (orange) in complex with RBD (gray).

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  Figure 8. Binding sites of VNARs to WT SARS-CoV-2 RBD. (A–D) The binding sites of JM-2 (A), JM-5 (B), JM-17 (C), and JM-18 (D) to RBD. The binding residues on RBD are shown in gray. The binding residues on JM-2, JM-5, JM-17, and JM-18 are shown in cyan, yellow, magenta, and orange, respectively.

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  Figure 9. Preparation and characterization of bi-paratopic VNARs. (A) Scheme of bi-paratopic VNAR-Fc fusions. (B) SDS-PAGE analysis of purified bi-paratopic VNAR-Fc fusions. (C,D) Binding affinity of the bi-paratopic VNARs (C) or VNAR-Fc fusions (D) for WT SARS-CoV-2 RBD using BLI. The actual responses (colored lines) and the data fitted to a 1:1 binding model (black dotted lines) are shown. ka and kd represent the association constant and dissociation constant, respectively. The equilibrium dissociation constant KD was calculated as kd/ka.

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  Figure 10. Binding characterization of bi-paratopic VNARs to various RBD by ELISA. (A–D) The binding of bi-paratopic VNAR-Fc fusions to the RBD of WT (A), Delta (B), Omicron (C), and SARS-CoV (D). Error bars indicate means ± SD of three independent experiments. The EC50 was calculated by fitting the OD450 values from serially diluted VNAR-Fc fusions or ACE2-Fc to a sigmoid dose–response curve.

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  Figure 11. RBD-ACE2 blocking activities of bi-paratopic VNARs characterized by BLI assay. (A–D) Blocking of ACE2 binding to the RBDs of WT, Delta, Omicron, and SARS-CoV by bi-paratopic VNARs. SA biosensors were loaded with biotinylated RBD, then incubated with bi-paratopic VNAR-Fc fusion and ACE2 sequentially. Bi-paratopic VNAR was replaced with PBST and used as a control. The inhibition rates of bi-paratopic VNARs were calculated against the control group.