Fig 2: The AlphaLISA assay monitors ACE2-RBD interactions. A) ACE2-Avi and RBD-Fc were titrated against each other from 300 – 0.1 nM in matrix format to determine the optimal protein concentrations for the AlphaLISA assay. ACE2-Avi and RBD-Fc were mixed at the concentrations indicated and allowed to equilibrate at 25°C for 30 minutes. Streptavidin Donor beads and Protein A Acceptor beads were then introduced to the solutions to a final concentration of 5 µg/mL of each bead. After 40 minutes of incubation at 25°C the signal intensity was read using a PheraStar plate reader. B) Assay suitability to 1536-well format was determined by combining 4 nM ACE2-Avi with 4 nM RBD-Fc in PBS + 0.05 mg/mL BSA. The mixture was incubated at 25 °C for 30 minutes. Streptavidin donor beads (10 µg/mL) were added to columns 1–8 of a 1536-well plate. Protein A acceptor beads (10 µg/mL) were added to columns 5–8. The entire mixture was incubated at 25°C for 40 minutes before reading the low-signal control (columns 1–4) and high-signal control (columns 5–8) using a PheraSTAR plate reader with an AlphaLISA module. Z` and signal-to-background (S:B) were calculated from these sample measurements. C, D) To confirm the AlphaLISA signal related to ACE2-RBD interactions, we tested the ability of His-S1 (another form of the SARS-CoV-2 Spike protein) and untagged ACE2 to lower the AlphaLISA signal in a dose-dependent manner. C) His-S1 was mixed in dose-response with 4 nM ACE2-Avi and allowed to incubate at 25°C for 30 minutes before adding 4 nM RBD-Fc. D) Untagged ACE2 was pre-incubated in dose-response with 4 nM RBD-Fc and allowed to incubate at 25°C for 30 minutes before adding 4 nM ACE2-Avi. Both His-S1 and untagged ACE2 showed dose-dependent signal loss, indicating the AlphaLISA signal is mediated by RBD-Fc binding to ACE2-Avi.

Fig 3: Logo Plot Representation of Mutation Effects on Binding and Expression, Related to Figure 3Letter height indicates preference of each site for individual amino acids with respect to ACE2 binding (height above the center line) or RBD expression (height below the center line). Blue letters indicate the unmutated SARS-CoV-2 amino acid, and, where applicable, green letters indicate differences found in SARS-CoV-1. Yellow highlights mark residues that contact ACE2 in the SARS-CoV-2 or SARS-CoV-1 crystal structures. See the STAR Methods for details of how the amino acid preferences are calculated from the experimental measurements.

Fig 4: Decoy vesicles confer widespread inhibition of emerging SARS-CoV-2 Spike variants. Left: Dose-response curves depicting the inhibition of various strains of SARS-CoV-2 Spike pseudotyped lentivirus by WT-ACE2 UC-EVs. The parental strain is the D614G Spike-lenti. Curves are normalized to the percent of cells transduced at the lowest EV dose in a particular curve. Symbols represent the mean of three biological replicates; error bars are standard error of the mean. Data are representative of two independent experiments. Right: Log(ID50) values calculated from the dose response curves. Numbers above each point report the relative resistance for a given strain relative to the parental strain (D614G). Error bars represent 99% confidence intervals for the parameter (ID50) estimation.

Fig 5: Neutralization of SARS-CoV-2 by RBD-specific sdAbs.a Neutralization of 5 sdAbs against SARS-CoV-2pp. SARS-CoV-2pp was pre-incubated with 5-fold serially diluted sdAbs before inoculation of human ACE2 transfected 293T cells. At 48 h post infection, luciferase activities were measured, and percent neutralization was calculated. The experiments were performed independently at least twice and similar results were obtained. One representative data of one experiment were shown and data were average values of three replicates (n = 3). b Determination of neutralization activities of 5 sdAbs against live SARS-CoV-2. Absolute quantification of SARS-CoV-2 RNA copy number in culture supernatants was performed using real time RT-PCR method, and percent neutralization was calculated. The experiments were performed independently at least twice and similar results were obtained. One representative data of one experiment were shown and data were average values of two replicates (n = 2). c Summary of the half maximal neutralization concentration (EC50) values of the 5 sdAbs against both SARS-CoV-2pp and live virus.