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. 2021 Oct 8;218(12):e20211003. doi: 10.1084/jem.20211003

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© 2021 Shinnakasu et al.

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Figure 3. — Glycan engineering of the CoV-2 head-RBD elicited cross-neutralizing activities against CoV-1 and WIV1. (A) BALB/c mice were prime-boost-immunized with CoV-2 RBD WT, GM9, GM14, or CoV-1 RBD WT as shown in Fig. 2. Sera were collected 7 d after boost and preincubated with CoV-2 S, PaGX S, SHC014 S, WIV1 S, or CoV-1 S–pseudotyped VSVΔG-luc for 1 h. The mixture was incubated with VeroE6 TMPRESS2 cells overnight. CoV-2 RBD WT (n = 11); GM9 (n = 11); GM14 (n = 11); CoV-1 RBD WT (n = 8). (B) Neutralization activities of plasma samples from prepandemic healthy donors (HC, n = 8) or convalescent COVID-19 patients (n = 24) against CoV-2, CoV-1, and WIV1 S–pseudotyped VSVΔG-luc. (C) Donor information. (D) ADE assay. Schematic representation of ADE assay (left). ADE of infection of Raji cells by MW05 mouse IgG1 mAb (middle). CoV-2 S–pseudotyped VSVΔG-luc was preincubated with different concentrations of MW05 mouse IgG1 mAb, or control mouse IgG1, and then added onto Raji cells. The luciferase activity was measured at 16 h after infection. Serially diluted sera from immunized mice showed no significant ADE activity (right). MW05 mouse IgG1 and irrelevant mouse IgG1 (0.1 µg/ml) were used for positive and negative control, respectively. CoV-2 WT (n = 4); GM9 (n = 5); GM14 (n = 5); CoV-1 WT (n = 4); preimmune sera (n = 3). Representative results from two or three independent experiments are shown (A–D). Data are mean ± SEM (D). Dotted lines in the graphs (NT50 = 20) represent the lower limit of detection (A and B). *, P < 0.05; **, P < 0.01; ***, P < 0.001; unpaired Student’s t test (A and B).