Extended Data Fig. 4: Fluorescence-activated cell sorting (FACS) of human anti-RBD memory B cells and subsequent BCR sequencing.

a-b, Panels showing IgG (a) and IgM (b) surface expression of anti-RBD memory cells exactly as in Fig. 2b and c, but with the 5 representative individuals from whom cells were subsequently sorted highlighted in yellow. c, Gating strategy for single-cell sorting of RBD-specific memory B cells. Dual-labelled (RBD-PE⁺/-AF647⁺) CD20⁺ CD3⁻ CD8⁻ CD16⁻ Ova⁻ cells were sorted. d, Representative flow cytometry plots show RBD-binding cells that were sorted from the 5 mAb recipients. e, Pie charts show the distribution of antibody sequences derived from cells isolated from 10 vaccinated control individual after vax2. The upper panel shows IgM, and the lower panel depicts IgG sequences^9,11. The number in the inner circle indicates the number of sequences analysed for the individual denoted above the circle. Slices coloured in shades of blue indicate cells that are clonally expanded (same IGHV and IGLV genes, with highly similar CDR3s). Pie slice size is proportional to the number of clonally related sequences. The black outline and % value indicate the frequency of clonally expanded sequences detected within an individual. White pie areas indicate the proportion of sequences isolated only once. For C005, there were no IgM transcripts amplified at the timepoint assayed. f-h, Comparison of the frequency distribution of V gene usage for the IgH and IgL among antibodies isolated from mRNA-vaccinated mAb recipients (this study) and controls^9,11, after vax2, and from database of shared clonotypes of human antibodies from Soto et al.⁴³. Graphs show relative abundance of human IGHV (f), IGKV (g), and IGLV (h) genes within the human V gene database (in grey, Sequence Read Archive accession SRP010970), antibodies isolated from mAb recipients (in green) or vaccinated controls (in blue). Colours of stars indicate levels of statistical significance for the following frequency comparisons: black – vaccinated controls vs. database; red – mAb recipients vs. database; blue – mAb recipients vs. vaccinated controls.