Fig 1: Structure of the NT-193 Fab-RBD complex reveals a distinct binding mode(A) Overviews of the NT-193 complex at two angles are shown on the left (magenta, NT-193 heavy chain; pink, NT-193 light chain; orange, RBD; numbers 1–3 represent CDR-H1, CDR-H2, and CDR-H3, respectively). The structure of the ACE2-RBD complex is shown on the right (PDB: 6m0j). ACE2 is shown in cyan.(B) Comparison of the NT-193 complex (colored the same as in A) with the representative class 1/RBS-A antibody complex, B38 (dark and light blue), and class 2/RBS-B antibody complex, COVA2-39 (dark and light green).(C) CDR binding sites (magenta and pink, stick representations) are shown on the surface model of RBD (gray).(D) Comparison between ACE2 and NT-193 binding sites on RBS. The binding areas for ACE2 are shown in cyan. The regions surrounded by dotted lines indicate the binding areas for light chains (red, LC) and heavy chains (black, HC).(E) Amino acid sequence conservation of RBS between CoV1 and CoV2. Yellow, conserved; Light orange, similar; gray, non-conserved. The dotted lines are the same as in (D).(F) Mapping of antibody binding residues on RBD. The residues that contact ACE2, antibody, or RBD within a distance of 4 Å are defined as binding residues. The RBD residues conserved with CoV1 are colored similarly to those in (E). The binding residues of RBD for ACE2 and antibody are mapped with color. The mutations in VOCs are shown (red).(G) Recognition mode of NT-193 toward RBD. Left, schematic of the NT-193-RBD complex. The light chain of NT-193 uses CDR-L1/L3 (blue) and DE loop (cyan) for the recognition of RBD. CDR-H3 (magenta) and CDR-L3 dominantly bind to RBD. Right, cartoon models of CDR-Hs (magenta and pink), CDR-Ls (blue), and the DE loop (blue and cyan) of NT-193 are shown with a meshed surface.See also Figures S3–S5 and Tables S1 and S2.
Fig 2: 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.
Fig 3: 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 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 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.
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