Fig. 5: Mechanism of action of Nb70 and 3-2A2-4. | Nature Communications

Fig. 5: Mechanism of action of Nb70 and 3-2A2-4.

From: Broadly neutralizing and protective nanobodies against SARS-CoV-2 Omicron subvariants BA.1, BA.2, and BA.4/5 and diverse sarbecoviruses

Fig. 5

a The effect of monomeric Nb70 and 3-2A2-4 nanobodies on the conformational change of the SARS-CoV-2 S trimer probed by western blotting using an anti-SARS-CoV-2 S2 polyclonal antibody. Refolding to the post-fusion conformation was detected by the appearance of a proteinase K-resistant core. Digestion experiments and western blots were performed in triplicates, and one representative result is shown. b The integrated density of proteinase K-resistant core in each lane was calculated by ImageJ. c Competitive binding of monomeric Nb70 and 3-2A2-4 nanobodies with soluble ACE2 to prototype SARS-CoV-2 spike measured by cell surface staining. Spike were expressed on the surface on HEK293T, incubated with the Nb70 or 3-2A2-4 nanobody and then ACE2, followed by staining with anti-His tag-PE (nanobodies) and Streptavidin-APC (ACE2) and analyzed by FACS. A human monoclonal antibody P1A-1D1, previously isolated by our group from SARS-CoV-2 convalescent individual and showed minimal competition with ACE2, was used as the antibody control and detected by staining with anti-human IgG Fc-PE. The cell staining was repeated and one representative result is shown. d Cryo-EM density maps of the 3-2A2-4 bound to SARS-CoV-2 Omicron BA.1 spike. 3-2A2-4 is shown in purple while the SARS-CoV-2 RBD in cyan and the rest of spike in grey. Source data a, b are provided as a Source Data file.

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