Fig 2: Distinguishing SARS-CoV-2 wild-type antigens from variant antigens. a) Schematic illustration showing the use of the ACE2-based biosensor to distinguish SARS-CoV-2 wild-type antigens from the variants. Two color-labeled SARS-CoV2-specific antibodies were co-deposited onto the conjugate pad. A specific antibody labeled with red CNB detects both wild-type S1 and the S1 variants; however, a second specific antibody labeled with blue CNB detects only wild-type S1. Therefore, the color of the test line depends on whether wild-type S1 (purple) or an S1 variant (red) is detected. b) The results of the tests. Performance was evaluated using SARS-CoV-2 S1 antigens (wild-type, alpha variant, and beta variant) serially diluted from 1000 ng/mL to 20 ng/mL c) Representative images of test lines and the blue-to-red ratios. At the reaction end-point (after 20 min), the test lines were analyzed using commercial software (ImageJ) to examine the RGB composition. d) Bar graph showing the blue-to-red ratio obtained from the serially diluted sample tests. The cut-off-value (C.O.V) was determined as the mean value of the blue-to-red ratio for the wild-type S1 minus three times the standard deviation.

Fig 3: Decreased binding of the A372T mutant to human ACE2(a) Functional ELISA was used to determine the binding affinity of different S protein receptor-binding domains (RBDs). Plates were coated with recombinant human ACE2 receptor (2 µg/mL at 100 µL/well) and then probed with varying concentrations (0.256–4000 ng/mL) of purified RBDs from WT SARS-CoV-2 (S A372), A372T, and N501Y (positive control). To determine EC50 values, the absorbance values (450 nM) were fit to a sigmoidal, 4PL nonlinear model using Prism 9 (GraphPad). The experiment was repeated in two independent replicates with four total technical replicates per sample. Error bars represent standard deviation of the mean.(B) The EC50 values were compared by one-way ANOVA with Dunnett’s multiple comparisons test. ****p < 0.0001 compared with WT SARS-CoV-2 (A372). Error bars represent standard deviation of the mean.

Fig 4: Evaluation of prophylactic and therapeutic efficacy of F6-b8-Fc in a mouse ACE2-adapted model(A) The overview of study design for evaluating F6-ab8-Fc efficacy in a SARS-CoV-2 mouse model.(B) Percent survival curves for each F6-ab8-Fc treatment group as indicated.(C) Lung viral titers (PFUs) in lung tissue for the F6-ab8-Fc treatment groups. The limit of detection (LoD) is 100 PFU/lobe.(D) Lung hemorrhage scores of live mice. T tests were used to evaluate statistical differences. *p < 0.05, **p < 0.01, ***p < 0.001, ns. no significance.

Fig 5: Concentration-dependent inhibition of the entry of SARS-CoV-2 pseudovirus (VSV-?G) into hACE2/Furin-expressing cells by MeBlu. Entry of VSV-?G pseudoviruses bearing the SARS-CoV-2 S protein (plus GFP reporters) in ACE2/Furin overexpressing host cells (Vero E6) in the presence of increasing concentrations of MeBlu was quantified via GFP fluorescence in a live imaging system. Normalized data are shown on a semilogarithmic scale and fitted with a classic sigmoidal curve used to calculate IC50.