Figure 6
NIH-CoVnb-112 stability and potent inhibition of SARS-CoV-2 pseudovirus following nebulization. (a) An Aerogen Solo High-Performance Vibrating Mesh nebulizer was placed in line with a custom glass bead condenser to allow for collection of the nanobody following nebulization. (Image element courtesy of Aerogen.) (b) A pre-nebulization and post-nebulization sample of purified NIH-CoVnb-112 was analyzed by SDS-PAGE gel. The dominant band for each sample remains at approximately 14 kDa indicating no detectable degradation or aggregation of NIH-CoVnb-112 following nebulization. (c) Size exclusion chromatography demonstrated a prominent peak eluting at 13.5 mL elution volume in both pre and post-nebulization samples. (d) A fluorescence reporter assay utilizing SARS-CoV-2 spike protein pseudotyped lentivirus was used to demonstrate potent inhibition of the RBD:ACE2 interaction. HEK293 cells overexpressing human ACE2 were cultured for 24 h with pseudotyped virus which was pretreated with NIH-CoVnb-112 at different concentrations. Inhibition of the spike RBD occurs when the virus is not able to transduce the HEK293-ACE2 cells and subsequently produce RFP reporter protein. e Following 48 hr incubation, HEK293-ACE2 cells were analyzed by flow cytometry to quantify the fluorescence level. NIH-CoVnb-112 potently inhibited viral transduction both pre and post-nebulization with an EC50 of 0.323 µg/mL (23.1 nM) and 0.116 µg/mL (8.3 nM) respectively. (Figure elements generated using BioRender.com).
