Minimally mutated IGHV3-30/3-33 genes dominate the IgG anti-NANP ₆ plasma repertoire following three doses of R21/Matrix-M vaccine. (A) Schematic of the VAC053 and VAC065 clinical trials. Plasma and cells (PBMCs) were collected at the times indicated. Volunteers (n = 10) were vaccinated at three time points (days 0, 28, and 56). Ig-Seq analyses were performed at day 63 (VAC053) or day 84 (VAC065). (B) Relative abundance of NANP₆-reactive IgG lineages in a representative volunteer. Each bar represents a single IgG lineage identified by its respective CDR-H3. Red indicates the unique peptides for each lineage that were detected by LC-MS/MS. (C) Relative plasma antibody IGHV gene segment usage for each volunteer. IGHV gene identity by color is shown in D. (D) Average IGHV gene abundance in NANP₆-reactive IgG. Bar represents mean. (E) IGHV gene frequency, by lineage, in total peripheral B cells averaged across all volunteers. (F) IGHV gene frequency, by lineage, in NANP₆-nonreactive IgG averaged across all volunteers. (G) IGHJ gene usage in the same volunteer as B. (H) IGHV gene SHM levels for NANP₆-reactive and nonreactive plasma IgG antibodies and BCR sequences grouped by lineage. ****P < 0.0001 (two-tailed Kruskal–Wallis test). (I and J) Amino acid usage in CDR-H2 (I) position 50 and (J) position 52 of IGHV3-33 plasma antibodies separated by NANP₆ reactivity. I = isoleucine; L = leucine; V = valine; W = tryptophan. For each Ig-Seq analysis of polyclonal anti-NANP₆ IgG, the affinity purification was performed once, and the purified material was analyzed in triplicate by mass spectrometry.