Fig 1: Detection of neutralizing antibodies in sera against SARS-CoV-2 pseudovirus infection. (A) Inhibition of SARS-CoV-2 pseudovirus entry by ACE2-Ig. S-C19del or VSV-G pseudovirions were preincubated with ACE2-Ig and added to 293T-ACE2 cells. (B) Sera from convalescent COVID-19 patients neutralized the SARS-CoV-2 pseudovirus. Serum sample from healthy individual was tested as a negative control. RLU were detected at 72 h post-pseudovirus inoculation. The data are presented as the mean percentages of inhibition ± SDs of duplicate wells.
Fig 2: 3D-Structural modeling and MD simulation of interactions of SARS-CoV-2 S1 proteins with ACE2 receptor(A–H) 3D models of non-glycosylated (A) and differently N-glycosylated S1 proteins: (B) Man 5, (C) NA2F, (D) NA3F, (E) A2F, and (F) A3F; (G) the relationship between required binding energy and the glycan molecular volumes; (H) the relationship between glycan molecular volumes and glycan molecular mass.
Fig 3: Depicting results of CC- and S-plate using the calculation sheet, fitting of calibration curve replicates to sigmoidal function (A), fitting of individual mice serum neutralization N% (of RBD-ACE2 binding) as a function of serum dilution (B), and the resulting mean group nAb titer column graph (C).
Fig 4: Dalbavancin inhibits binding of SARS-CoV-2 spike protein to ACE2 in vitro.a ACE2 (0.5 μg) and SARS-CoV-2 spike protein (0.5 μg) were mixed and treated with various candidate peptide drugs (10 μM). Co-precipitated proteins were identified by western blot analysis using anti-ACE2 antibody. Analyzed proteins are indicated on the right. For positive control (bottom), ACE2-His (0.5 μg) and SARS-CoV-2 spike protein (0.5 μg) were mixed and treated with various concentration of ACE2-hFc, and then co-precipitated proteins were identified by western blot analysis using anti-His-tag antibody. b ACE2 (2 μg/ml) was crosslinked to microplates by N-oxysuccinimide esters for ELISA. SARS-CoV-2 spike protein (10 ng/mL) and tested candidate drugs (1 μM) were incubated with ACE2, and extra un-crosslinked ACE2 protein (100 ng/mL) was used as a positive control. SARS-CoV-2 spike protein antibodies were used for chromogenic reaction. c Binding curves of immobilized human ACE2 with SARS-CoV-2 spike protein (left, positive control) and dalbavancin (right). Concentration-response SPR experiment showing binding of dalbavancin to ACE2 with an equilibrium dissociation constant (KD) of ~147 nM.
Fig 5: SPR binding curves of immobilized human ACE2 with de-N-glycosylated S1 proteins of SARS-CoV-2(A and B) Column (A): deglycosylated S1 proteins by PNGase F, and column (B): the same S1 proteins pretreated with inactive PNGase F under identical conditions as controls. The best fit of the data to a 1:1 binding model is shown in the dash line, and the KD values are the mean ± SD of two independent experiments.
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