Fig 2. Surface-plasmon resonance and pseudovirus inhibition studies of SARS1-RBD, SARS2-RBD, PgCoV-RBD, LyRa11-RBD.

(A) Biacore surface plasmon resonsance (SPR) studies were performed with the indicated RBD-Fc (yellow and blue) captured on the sensor chip. The soluble monomeric forms of the indicated ACE2 variants (green, with a four-amino-acid C-terminal tag in red) were injected for analysis as represented in the figure. Biacore X100 sensorgrams representing results from studies in which the indicated RBD-Fc variants were captured by an anti-human Fcγ antibody immobilized on a CM5 chip after instantaneous background depletion. Monomeric forms of tagged soluble human ACE2 (hACE2-CT), human ACE2 lacking its active-site histidines (hACE2-NN-CT), or pangolin ACE2-CT were injected at five concentrations, serially diluted by two-fold from the highest concentration (100 nM for human ACE2 variants). The experiment is representative of two with similar results. (B) The association rate constant (k_on), dissociation rate constant (k_off), and equilibrium dissociation constant (K_d) were calculated and plotted for comparison from the study shown in (A). (C) 293T-hACE2 cells were pre-incubated with the indicated concentrations of the indicated RBD-Fc variants, and subsequently infected with retroviral pseudoviruses (PV) pseudotyped with the S proteins of SARS-CoV-1 (SARS1-S-PV), SARS-CoV-2 (SAR2-S-PV), or with the G protein of vesicular stomatitis virus (VSV-G-PV), as diagrammed. Nonlinear fit for log(inhibitor) vs. response was calculated for each RBD. In both SARS1-S-PV and SARS2-S-PV panels, SARS-RBD-Fc, SARS2-RBD-Fc, PgCoV-RBD-Fc, LyRa11-RBD-Fc bottom plateaus CI did not overlap with values from VSV-G-PV. Error bars in indicate standard error of the mean (S.E.M) and are representative of results from at least two independent experiments. Asterisks indicate lack of 95% CI overlap of RBD-Fc constructs with respect to VSV-G group.