Fig 2: Apparent activities of two distinct ACE2 derivatives produced in plants to RBDs plotted against half maximal inhibitory concentration (IC50) of authentic SARS-CoV-2 neutralization. The IC50 values of the gACE2 (glycosylated) and dACE2 (deglycosylated) were calculated using normalized optical density (OD) data obtained from quadruplicated test dilutions in GraphPad Prism v8.2 software (GraphPad). The OD values from untreated (cell control) wells were used as normalization standards. A non-linear regression analysis was performed using log (inhibitor) versus normalized response-variable slope. The R square values were recorded as 0.86 and 0.89 for dACE2 and ACE2, respectively. ACE2, gACE2, glycosylated ACE2; dACE2, plant produced deglycosylated ACE2.

Fig 3: Binding affinity of gRBD, dRBD, and commercial RBDs of SARS-CoV-2 to ACE2, the receptor of SARS-CoV-2. The samples of 100 ng of plant-produced gRBD, dRBD, and commercially available (A) recombinant SARS-CoV-2 S protein RBD (amino acids Arg319–Phe541, with a C-terminal 8×His tag, expressed in 293E cells, cat. no. 793606, BioLegend, USA), or (B) SARS-CoV-2 spike protein (RBD) coronavirus active protein (amino acids Arg319–Phe541, produced in baculovirus–insect cells, cat. no. MBS2563882, MyBioSource, San Diego, CA, USA) were incubated on plates coated with ACE2. After incubation, anti-SARS-CoV-2 spike RBD polyclonal antibodies were added into each well and detected with rabbit IgG conjugated with HRP. Each point on the graph was derived from three replica for each dilution. Data are shown as mean ± standard error of the mean (SEM) of triplicates in each sample dilution. Statistical significance (p < 0.05) was calculated using the one-way ANOVA test with Tukey’s multiple comparisons. p value for each group is shown in parentheses. ** p < 0.01, *** p < 0.001; OD—optical density; Kd—dissociation constant.

Fig 4: A Western blot analysis of human ACE2s, produced in Nicotiana benthamiana plants. dACE2: angiotensin-converting enzyme 2 (ACE2) co-expressed with bacterial Endo H, produced in N. benthamiana, different concentration (dilutions) of crude extract were loaded into wells; gACE2: western blot analysis of human ACE2, produced in N. benthamiana plants; different concentration (dilutions) of crude extract were loaded into wells; C-undiluted crude extract from non-infiltrated N. benthamiana, was loaded into well; gPA83: 25, 50, and 100 ng of purified plant produced dPA83 of Bacillus anthracis, loaded as a control protein to quantify the expression levels of ACE2 and ACE2 proteins. Purified anti-His Tag antibody (Cat. No. 652502, BioLegend) was used as primary and mouse IgG used as secondary antibodies to detect ACE2 proteins.

Fig 5: Binding activity of plant produced glycosylated or deglycosylated variants of ACE2 with commercial or plant produced, glycosylated or deglycosylated forms of spike proteins (Flag tagged). Commercial or plant-produced spike protein was coated with an ELISA plate at a concentration of 200 ng/well. Different concentration of plant produced, Sephacryl® S-200 HR column purified, gACE2 or dACE2 proteins (His-tagged) were added. Purified anti-human monoclonal ACE2 antibody (Cat. No. 375801, BioLegend) was used as a primary and rat IgG used as secondary antibodies. Com S: commercial Spike protein, active Recombinant 2019-nCoV Spike Protein, RBD, His Tag, produced in Baculovirus-Insect Cells, Cat. no. MBS2563882); pp-gRBD: plant produced glycosylated Receptor Binding Domain of Spike protein (Mamedov et al., 2021); pp-dRBD: plant produced deglycosylated RBD (Mamedov et al., 2021); pp-gACE2: plant produced gACE2; pp-dACE2: plant produced Endo H in vivo dACE2; Endo H, plant produced Flag-tagged protein was used as negative control. (A,B) Graph for binding affinity between pp-gACE2 and pp-dACE2 to spike protein variants. (A) A graph was plotted with non-linear regression analysis in GraphPad software. Points refer to absorbance for each sample dilutions and lines were plotted according to Kd value. (B) Column bar graph of Kd values determined with non-linear regression analysis in GraphPad software.