Fig 1.
Isolation of neutralizing antibodies from the memory B cells of a convalescent donor.
A. Identification of C-03-0020 donor with significant binding to SARS-CoV-2 RBD. B. High neutralizing activity of C-03-0020 plasma against SARS-CoV-2 in live virus neutralization assay. C. High neutralizing activity of C-03-0020 plasma against SARS-CoV-2 in pseudovirus neutralization assay. D. Neutralization potential of RBD-reactive mAbs were evaluated against SARS-CoV-2 pseudovirus. E. CDRH3 sequence, germline usage neutralization potency of isolated mAbs against SARS-CoV and SARS-CoV-2.
Fig 2.
Isolated monoclonal antibodies can neutralize all circulating variants of concern and interest.
A. Binding affinities of THSC20.HVTR04 and THSC20.HVTR26 to the SARS-CoV-2 (Wuhan) receptor binding domain (RBD) protein by BLI-Octet. Biotinylated wild type SARS-CoV-2 RBD antigen was immobilized on Streptavidin (SA) biosensors and binding affinity of monoclonal antibodies to RBD was tested using three-fold serial dilutions of mAbs starting with 33.3 nM and lowest 0.41 nM (five different concentrations were tested). Association and dissociation was assessed for 500 seconds each. Data shown is reference-subtracted and aligned using Octet Data Analysis software v11.1 (Forte Bio). Curve fitting was performed using a 1:1 binding model and Kon, Koff and KD values were determined with a global fit. B. Binding of mAbs to SARS-CoV-2 spike protein expressed on 293T cells as assessed by mean fluorescent intensity (MFI) in a flow cytometry. C. Live virus focus reduction neutralization assay. The ability of the two top mAbs (THSC20.HVTR04 and THSC20.HVTR26) was assessed by dose-dependent foci reduction neutralization (FRNT) live virus neutralization assay in Vero-E6 cells. D. Expanded pseudovirus neutralization assay of THSC20.HVTR04 and THSC20.HVTR26 against circulating VOCs (Alpha, Beta, Gamma, Delta) and VOIs (Kappa, Delta Plus). Other known mAbs (REGN10933, REGN10987, CC12.1 and CC6.36) were included in the experiment as benchmarking controls. Representative dose response curves are shown with each concentration response tested in duplicate. Values shown are mean with SEM.
Fig 3.
Neutralization potential of isolated monoclonal antibodies against Omicron.
Neutralization of pseudovirus expressing Omicron spike by all the five newly isolated mAbs was carried out as described above. Average of three independent experiments with each concentration response tested in duplicate were used to prepare the dose response curves. Values shown are mean with SEM.
Fig 4.
Neutralizing antibodies from the same donor target non-competing epitopes on RBD.
A. Monoclonal antibodies were evaluated for epitope competition using BLI. Biotinylated RBD was captured using streptavidin biosensor and indicated mAbs at a concentration of 100μg/ml first incubated for 10 min followed by incubation with 25μg/ml of competing antibodies for 5 min. B. ACE2-mAb competition for RBD. Inhibition of SARS-CoV-2 RBD binding by five mAbs to the cell surface hACE2 was assessed by flow cytometry.
Fig 5.
X-ray crystallography of THSC20.HVTR04 and THSC20.HVTR26 complexed with SARS-CoV-2 RBD.
A. Schematic of THSC20.HVTR26 Fab (PDB 7Z0X, shown in dark and light green for heavy and light chains respectively) and THSC20.HVTR04 Fab (PBD 7Z0Y, shown in dark and light blue), bound to SARS-CoV-2 spike RBD (shown in purple/pink respectively). Structures were aligned by RBD. B. Surface view of SARS-CoV-2 RBD from PDB 7Z0X (top) or 7Z0Y (bottom), viewed from the angle of ACE2 approach, with the ACE2 binding site colored gold. Complementarity determining regions are shown in cartoon view, labelled, and colored as in A. C. Surface view of THSC20.HVTR26 Fab variable domains (colored as in A). RBD residues 475–489 and F456 are shown in purple cartoon and stick view. Key amino acids E484, T478, F486, and N487 are labelled. D. Surface view of THSC20.HVTR04 Fab variable domains (colored as in A). RBD residues 439–450 and 498–502 are shown in purple cartoon and stick view. Key amino acids V445, K444, P499, N439, and N440 are labelled. E. Primary sequence of SARS-CoV-2 RBD positions 410 to 510 (purple), and mutations associated with the Beta (cyan), Delta+ (lime), or Omicron (salmon) variants. The percentage of accessible surface for each amino acid that was bound by THSC20.HVTR04 or THSC20.HVTR26 was calculated (% buried surface area) and plotted using blue or green dots respectively, where each dot represents ~10% of the total solvent accessible surface area.
Fig 6.
Negative stain EM analysis of mAbs complexed with SARS-CoV-2 spike protein.
Negative stain EM analysis of mAbs THSC20.HVTR04 (blue) and THSC20.HVTR26 (green) complexed with SARS-CoV-2 6P Mut7 spike protein. PDB 6VYB (one RBD-up) was fit into maps i-iii, and PDB 6VXX (three RBD’s down) was fit into map iv.
Fig 7.
Neutralizing mAbs are able to protect against Wuhan and Delta variants in K18-hACE2 mice model.
A. Experimental design of the efficacy assessment in K18-hACE2 transgenic mice model. K18-hACE2 mice were passively given intraperitoneal injection of the two individual mAbs or mixture of two mAbs 24 h prior to intranasal inoculation with 105 PFU of SARS-CoV-2 Wuhan or Delta isolate. Each mouse was administered with single dose of 10 mg/kg body weight (equivalent to 200 ug /animal) for testing against Wuhan isolate and four different doses of 10 mg, 2.5 mg, 0.625 mg and 0.156 mg mAb per kg body weight (equivalent to 200μg, 50μg, 12.5μg and 3.125μg per animal respectively) for testing against Delta variant isolate. B-D. The prophylactic effect of THSC20.HVTR04 and THSC20.HVTR26 alone and in combination against Wuhan isolate on preventing body weight loss at concentration of 10 mg per kg body weight (B), lung viral load assessed at day 6 (C) and correlation of percent body weight change with lung viral load at day 6 (D). E-G. The prophylactic effect of THSC20.HVTR04 and THSC20.HVTR26 in combination against SARS-CoV-2 Delta variant on preventing body weight loss at four different concentrations as 10 mg, 2.5 mg, 0.625 mg and 0.156 mg mAb per kg body weight, day-wise and on day 6, respectively (E), Lung viral load assessed at day 6 (F), and correlation of percent body weight change with lung viral load at day 6 (G).