Journal: Immunology and Cell Biology

Article Title: Humoral epitope dominance and immune imprinting by SARS ‐ CoV ‐1 and SARS ‐ CoV ‐2 vaccines

doi: 10.1111/imcb.70072

Figure Lengend Snippet: Humoral epitope dominance from immunization of C57BL/6 or Ig‐humanized mice with CoV‐1 or CoV‐2 Spike MVA. C57BL/6 and Ig‐humanized mice were immunized with 2 × 10 8 PFU IV of CoV‐1, CoV‐2 or control MVA on days 0 and 14 and the immune response examined on day 28. (a) Composite model of the CoV‐2 RBD (gray, PDB 7msq) showing the co‐binding of ACE2 to the Class 1/2 epitope (blue, PDB 6m0j), S309 antibody Fab to the Class 3 epitope (orange, PDB 7r6w), EY6A antibody Fab to the Class 4 epitope (purple, PDB 6zcz), and S2H97 antibody Fab to the Class 5 epitope (green, PDB 7m7w). (b) Representative flow cytometric plots of spleen B cells from a CR3022 immunoglobulin transgenic mouse expressing membrane IgM encoding the Class 4 antibody on most B cells, showing staining with CoV‐2 RBD followed by a cocktail of each of the fluorescent antibodies and ACE2‐Fc indicated on the Y and Y axis. The gating strategy applied to identify B cells that bind CoV‐2 RBD (red box) through epitopes competed by the indicated fluorescent conjugates (dashed boxes) is shown. (c) Percentage of CoV‐2 RBD‐binding B cells from the GC (left) or PB (right) compartment which specifically block staining with fluorescent conjugates against the Class 1/2, Class 3, Class 4, or Class 5 epitope, in individual C57BL/6 mice immunized with CoV‐2 MVA or CoV‐1 MVA. (d) Percentage of total B cells from the GC (left) or PB (right) compartment which specifically block staining with fluorescent conjugates against the Class 1/2, Class 3, Class 4, or Class 5 epitope, in individual C57BL/6 mice immunized with CoV‐2 MVA or CoV‐1 MVA. (e) Percentage of CoV‐2 RBD‐binding B cells from the GC (left) or PB (right) compartment which specifically block staining with fluorescent conjugates against the Class 1/2, Class 3, Class 4, or Class 5 epitope, in individual Ig‐humanized mice immunized with CoV‐2 MVA or CoV‐1 MVA. (f) Percentage of total B cells from the GC (left) or PB (right) compartment which specifically block staining with fluorescent conjugates against the Class 1/2, Class 3, Class 4, or Class 5 epitope, in individual Ig‐humanized mice immunized with CoV‐2 MVA or CoV‐1 MVA. (g) Mean Fluorescence Intensity (MFI) of serum antibody response blocking the binding of fluorescent ACE2 (Class1/2) or canonical antibodies to the Class 3, 4, or 5 epitopes to CoV‐2 conjugated to the surface of sheep red blood cells. Data points represent individual mice. Columns show mean ± SEM. A two‐way ANOVA was applied to figures A‐K and a one‐way ANOVA to figure H. If ANOVA revealed significant differences, unpaired t ‐tests were applied to compare between different groups. Welch's correction was applied if variances between groups were statistically different. ns = not significant * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data pooled from two independent experiments.

Article Snippet: CoV‐2 variant and CoV‐1 RBD proteins and ACE2‐Fc were ordered from Sinobiological.

Techniques: Control, Binding Assay, Transgenic Assay, Expressing, Membrane, Staining, Blocking Assay, Fluorescence