Article Figures & Data
Figures
- FIGURE 1.
Design, expression and characterization of recombinant RBD variants. A, Schematic diagram of SARS-CoV S protein and the fusion protein RBD-Fc. The RBD of S protein contains residues 318–510 in the S1 domain. RBD-Fc consists of RBD and a human IgG1-Fc fragment. B, Amino acid differences among the RBDs of the S proteins of the indicated isolates (Tor2, GD03T0013, and SZ3). C, Characterization of purified RBD-Fc fusion proteins by SDS-PAGE with 10% gel and Western blot with anti-RBD mAb 17H9. D, Binding of RBD variants to human or palm civet ACE2 analyzed by flow cytometry.
- FIGURE 2.
Ab responses in mice and rabbits immunized with RBD-Fc fusion proteins. A, Reactivity of mouse sera with RBD-Fc variants in ELISA. B, Binding of serially diluted mouse antisera collected after the third boost to S1-C9. C, Reactivity of rabbit sera with RBD-Fc variants in ELISA. D, Binding of serially diluted rabbit antisera collected after the third boost to S1-C9.
- FIGURE 3.
Infection of SARS pseudoviruses expressing S protein of the Tor2, GD03, or SZ3 isolate in HEK293 cells expressing human and palm civet ACE2, respectively. Infection, measured as luciferase activity of cell lysates, was assayed 2 days postinfection.
- FIGURE 4.
Potent neutralization of SARS pseudovirus infection by mouse anti-RBD Abs. Infection of HEK293 cells expressing human ACE2 or palm civet ACE2 by SARS pseudoviruses (Tor2, GD03, or SZ3) was determined in the presence of mouse antisera at a series of 3-fold dilutions. Percentage of neutralization was calculated for each sample, and the average values were plotted.
- FIGURE 5.
Cross-neutralization of SARS pseudoviruses (Tor2, GD03, or SZ3) by rabbit anti-RBD Abs. The neutralizing activity of the Abs against SARS pseudovirus infection in HEK293 cells expressing human or palm civet ACE2 was determined as described above.
Tables
Group mAb Inhibition of ACE2 Binding ND50 of Pseudovirus (μg/ml) Tor2 GD03 SZ3 Conf I 9F7 − 6.22 0.15 >50 12B11 − 4.56 0.7 >50 29G2 − 3.24 0.16 >50 Conf II 26A4 − 2.85 0.49 >50 27C1 − 1.47 0.06 >50 31H12 − 0.21 0.02 >50 Conf III 11E 12 + 1.28 3.91 >50 18D9 + 0.03 5.29 >50 Conf IV 28D6 + 0.21 0.16 0.64 30F9 + 0.02 0.08 0.21 35B5 + 0.09 0.06 0.35 Conf V 24F4 + 0.05 3.61 0.14 33G4 + 0.01 0.02 0.01 38D4 + 0.35 35.14 0.36 Conf VI 13B6 − 1.32 1.5 1.25 19B2 − 0.76 0.62 0.41 Linear 4D5 − >50 >50 >50 17H9 − >50 >50 >50 mAb RBD Variant Tor2 to SZ3 Point Mutation Tor2 GD03 SZ3 K344R F360S N479K T487S Conf I 9F7 2.72 2.97 3.13 2.34 2.07 2.01 1.91 10E 7 2.83 3.23 3.29 3.00 2.08 2.19 1.93 12B11 2.96 3.53 3.10 2.71 1.80 2.04 2.13 18C2 2.75 3.35 3.32 2.75 1.93 2.13 1.75 24H8 2.70 2.89 2.96 2.91 2.03 2.11 2.07 29G2 2.56 2.88 2.63 2.27 1.84 1.98 1.96 32H5 2.59 2.86 2.86 2.66 2.10 2.16 2.03 Conf II 20E 7 2.72 2.54 2.58 2.43 1.95 1.90 1.90 26A4 2.78 3.60 3.38 2.63 2.23 2.09 1.91 27C1 2.59 2.75 2.57 2.83 2.08 2.13 2.12 31H12 2.69 3.15 2.86 2.69 2.17 2.32 2.08 34E 10 2.65 3.22 3.06 3.07 2.31 2.46 1.97 Conf III 11E 12 2.51 1.80 2.66 1.99 1.39 1.64 1.57 18D9 2.49 2.28 2.77 2.28 1.48 1.30 1.78 Conf IV 28D6 2.68 3.21 3.03 3.10 2.23 2.18 2.05 30F9 2.73 3.15 3.35 3.28 2.24 2.26 2.17 35B5 2.59 3.42 3.22 3.21 2.29 2.38 2.20 Conf V 24F4 2.75 2.53 3.31 3.58 2.41 2.39 1.95 33G4 2.68 2.85 3.05 3.11 2.50 2.48 2.34 38D4 2.56 2.02 2.98 2.85 2.09 2.40 1.96 Conf VI 13B6 2.73 2.75 2.80 3.14 1.67 2.06 2.02 19B2 2.72 2.72 2.67 2.77 1.94 1.78 2.00 44B5 2.61 3.11 2.76 2.77 2.07 2.12 2.02 45F6 2.56 3.10 2.78 2.66 2.06 2.17 2.08 Linear 4D5 2.72 3.19 3.56 1.42 1.04 1.08 1.22 17H9 2.75 3.51 3.38 3.77 2.32 2.62 2.16 Control 3A3b 0.03 0.05 0.02 0.03 0.04 0.03 0.02 -
a Ags were coated to ELISA plates at 1 μg/ml and mAbs were tested at 10 μg/ml. OD450 value >0.2 was considered positive reaction and highlighted in boldface.
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b 3A3 is a control mAb targeting the S2 domain.
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- Table III.
Characterization of neutralization epitopes with non-natural RBD mutants by ELISAa
Group MAb Deletion of Single-Point Mutation aa 318–326 aa 491–510 K390A D429A R441A E452A D454A D463A Conf I 9F7 0.08 0.00 2.15 0.18 0.02 1.36 0.01 1.47 10E 7 0.11 0.00 2.39 0.19 0.01 1.44 0.03 1.71 12B11 0.03 0.00 2.40 0.09 0.03 0.19 0.00 0.45 18C2 0.03 0.01 2.45 0.12 0.01 0.03 0.00 0.34 24H8 0.07 −0.01 2.32 0.19 0.02 0.29 0.00 1.35 29G2 0.06 0.00 2.41 0.17 0.01 0.71 0.02 1.37 32H5 0.08 0.00 2.51 0.20 0.01 0.82 0.01 1.46 Conf II 20E 7 0.02 −0.01 2.36 0.12 0.02 0.65 0.00 1.24 26A4 0.02 0.00 2.42 0.14 0.02 0.65 0.00 1.08 27C1 0.07 0.03 2.36 0.19 0.12 0.79 0.01 1.32 31H12 0.08 0.00 2.48 0.20 0.01 1.03 0.00 1.87 34E 10 0.08 −0.01 2.74 0.20 0.01 1.02 0.01 1.72 Conf III 11E 12 0.01 0.00 2.05 0.01 0.02 0.25 0.01 0.05 18D9 0.01 0.00 2.18 0.01 0.02 0.39 0.00 0.44 Conf IV 28D6 0.07 0.00 2.37 0.07 0.02 1.69 0.00 1.49 30F9 0.06 0.00 2.43 0.05 0.02 1.59 0.00 1.37 35B5 0.08 −0.01 2.48 0.11 0.02 1.65 0.01 1.57 Conf V 24F4 1.73 1.22 2.75 1.45 0.73 2.59 1.95 0.86 33G4 2.03 1.56 2.50 1.71 1.05 2.56 2.06 2.02 38D4 1.27 1.06 2.49 0.77 0.50 2.41 1.30 0.77 Conf VI 13B6 0.00 −0.01 2.32 0.15 0.02 1.40 0.00 1.28 19B2 0.04 −0.01 2.36 0.00 0.01 1.15 0.00 0.86 44B5 0.13 0.00 2.41 0.20 0.03 1.70 0.04 1.99 45F6 0.08 0.00 2.43 0.20 0.02 1.78 0.03 1.62 Linear 4D5 2.70 2.20 1.75 0.89 0.83 3.03 2.44 0.82 17H9 2.95 3.62 2.63 2.78 3.59 3.23 0.19 4.18 Control 3A3 0.01 0.02 0.02 0.00 0.03 0.02 0.01 0.03 -
a Ags were coated to ELISA plates at 1 μg/ml, and mAbs were tested at 10 μg/ml. OD450 value >0.2 was considered positive reaction and highlighted in boldface.
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