Fig 2: The SARS-CoV-2 Spike D614G variant displays similar ACE2 binding kinetics but altered proteolytic cleavage.(a) Schematic diagram of SARS-CoV-2 Spike protein structure with the added C9 affinity tag on the C-terminus. Spike cleavage fragments S1, S2, and S2’ are also indicated. (b, c) Association and dissociation binding curves of Spike D614 (b) and Spike G614 (c) with hACE2. Blue curves represent Spike protein binding profiles at 200 nM, 100 nM, 50 nM, 25 nM, and 6.25 nM. Red curves represent the best global fit using a 2:1 heterogeneous ligand model. (d) Western blot of total protein lysate from HEK293FT cells after transfection with D614 Spike, G614 Spike, or mock transfection. (Upper) Detection of full-length Spike and cleavage fragments using an anti-C9 (rhodopsin) antibody. (Lower) Detection of GAPDH via anti-GAPDH antibody. (e) Fraction of cleaved (S2 + S2’) to uncleaved (full-length) fragments for Spike D614 and G614 (n = 4 replicates, error bars are s.e.m.). (f) Fold-change in cleavage between Spike variants (D614/G614) (n = 4 replicates, error bars are s.e.m.). (g) Western blot of boiled S-virus pseudotyped with D614 Spike or G614 Spike. (Upper) Detection of full-length Spike and cleavage fragments using an anti-C9 (rhodopsin) antibody. (Lower) Detection of p24 capsid protein via anti-24 antibody. (h) Fraction of cleaved (S2 + S2’) to uncleaved (full-length) fragments for Spike D614 and G614 on lentiviral particles (n = 4 replicates, error bars are s.e.m.). (i) Fold-change in cleavage between Spike variants on lentiviral particles (D614/G614) (n = 4 replicates, error bars are s.e.m.). *p=0.05, ***p=0.001. Significance testing was done with an unpaired two-tailed t-test.

Fig 3: Correlation of anti-RBD IgG levels and ACE2 binding inhibition with SARS-CoV-2 disease severity. Bar charts showing mean ACE2 binding inhibitions (%) against wild-type and delta RBD are correlated with WHO grades for disease severity for samples 7–49 days post PCR (a,b) and = 50 days post PCR (c,d). Mean anti-RBD WT IgG and anti-RBD delta IgG levels are shown for samples 7–49 days post PCR (e,f) and = 50 days post PCR (g,h). Individual samples are displayed as colored dots, bars indicate the mean of the dataset with error bars representing standard deviation. Number of samples is given below the columns (n). If no samples for a group were available, the column is labeled with “n/a”. WHO grade 1—ambulatory/no limitations of activities, 2—ambulatory/limitation of activities, 3—hospitalized, mild disease/no oxygen therapy, 4—hospitalized, mild disease/mask or nasal prongs, 6—hospitalized, severe disease/intubation + mechanical ventilation, 7—hospitalized, severe disease/ventilation + additional organ support (pressors, RRT, ECMO), 8—Death. The study did not contain samples of WHO grade 5.

Fig 4: Characterization of the stable formation of binding module-virus complex(A and B) The binding curve and dissociation constant of TriApTDF against SARS-CoV-2 RBD-beads of (A) wild-type and (B) L452R/T478K mutant in binding buffer.(C) Cy5-labeled binding module from substrate rupture induced by the interaction of ACE2-expressing HEK293T cells and SARS-CoV-2 at the low tensile force of TGT; almost invisible cell adherence on the substrate.(D) Significant cell adhesion occurs as the TGT failed to rupture at high tensile force. Scale bar, 100 µm.

Fig 5: Correlation between anti-RBD IgG MFI signals and ACE2 binding inhibition (%) of serum samples from COVID-19 patients for wild-type and 11 RBD mutants. Regression analysis comparing ACE2 binding inhibition (%) and IgG responses (MFI) for wild-type and all RBD mutants included in the study. Each circle represents one sample (n = 168). For longitudinal donors with more than one sample available, the sample closest to 20 days post positive PCR diagnosis was selected. The percentage next to the bracket indicates the proportion of samples with ACE2 binding inhibition = 20% (in orange). Spearman’s correlation coefficient (r) is specified for every correlation.