Panel A shows schematic diagram of prime boost immunization and analysis timeline without axes. Panel B shows line graph of serum anti RBD antibody levels over time with vertical axis anti RBD immunoglobulin and horizontal axis days post prime. Panel C shows scatter plot of germinal center B cell percentage with vertical axis percentage of germinal center B cells of B220 positive cells and horizontal axis mouse genotypes. Panel D shows scatter plot of RBD binding germinal center B cells with vertical axis percentage of RBD binding germinal center B cells and horizontal axis mouse genotypes. Panel E shows pie charts of antibody binding categories including non binders spike binders and RBD binders without axes. Panel F shows scatter plot of clonal sequence percentage with vertical axis percentage of clonal sequences and horizontal axis mouse genotypes. Panel G shows dot plot of mutation counts with vertical axis number of mutations and horizontal axis mouse genotypes. Panel H shows scatter plot of Shannon entropy with vertical axis entropy score and horizontal axis mouse genotypes. Panel I shows scatter plot of inverse Simpson diversity index with vertical axis diversity index and horizontal axis mouse genotypes. Panel J shows bar graphs of immunoglobulin gene usage with vertical axis frequency percentage and horizontal axis gene identities across mouse genotypes. Panel K shows schematic diagram of experimental protocol for secondary analysis without axes. Panel L shows scatter plot of germinal center B cell percentage with vertical axis percentage of germinal center B cells of B220 positive cells and horizontal axis mouse genotypes. Panel M shows scatter plot of RBD binding germinal center B cells with vertical axis percentage of RBD binding germinal center B cells and horizontal axis mouse genotypes.
Prime boost vaccination. (A) Diagram of the experimental protocol for A–J. (B) ELISA quantification of total anti-RBD antibodies in the serum measured weekly from day 6 to day 97 after prime immunization. Thin lines represent single mice; bold line indicates the group mean. Dashed red lines indicate booster immunizations. (C) Percentage of GC B cells among B220+ B cells in the draining LN at day 98. Gating as in Fig. 2 E. (D) Percentage of RBD-binding GC B cells in the draining LN at day 98. Each dot represents one mouse. Gating as in Fig. 2 K. Each dot represents one mouse. Bars indicate the mean ± SD (E) Pie charts depicting the distribution of RBD-binding, spike-binding, or nonbinding recombinant antibodies cloned from the largest expanded clones of four mice/group of GC B cells on day 98 from draining LNs. BT50 <10 µg/ml is considered binders. Inner circle numbers indicate the number of recombinant antibodies analyzed. (F) Dot plot showing the percentage of clonal sequences from GC B cells on day 98. Each dot represents one mouse. Bars indicate the median. (G) Number of mutations in GC B cells on day 98. Each dot represents one clone. Bars indicate the median. (H) Dot plot depicting Shannon’s entropy for sequences from F. (I) Dot plots depicting the inverse Simpson’s index for sequences from F. Each dot represents one mouse. Bars indicate the median. (J) Bar graphs showing the relative abundance of mouse Ighv and Igkv gene usage among sequences from F. Top 10 most frequent genes ranked by the mean frequency in mM-only mice are shown. Bars indicate the mean ± SD. (K) Diagram of the experimental protocol for L and M. (L) Percentage of GC B cells among B220+ B cells in the draining LN on day 98. Each dot represents one mouse. Gating as in Fig. 2 E. Bars indicate the mean ± SD. (M) Percentage of RBD-binding GC B cells. Each dot represents one mouse. Gating as in Fig. 2 K. Bars indicate the mean ± SD. Statistics in C, D, J, L, and M indicate nonparametric one-way ANOVA P values. Statistics in E–I indicate the two-sided Mann–Whitney U test. n.s., not significant P > 0.05. All flow cytometric experiments are from at least two independent experiments.