Impact of SHM on the neutralizing capacity of GM9_TH8 lineage. (A) The alignment of 11 HC somatic variants of GM9_TH8 lineage selected from multiple immune compartments, in addition to the HC of cloned GM9_TH8 mAb, with levels of SHM ranging from 0 to 24% at the amino acid level. The top row shows the inferred germline sequence of HC, where the VD and DJ junctions were determined based on the least-mutated variant found in the blood Rep-seq data. (B) Maximum likelihood phylogenetic tree showing HC sequences from the GM9_TH8 lineage with the 12 expressed GM9_TH8 variants (representative data from two independent experiments) indicated with arrows. (C) Binding curves for each of the 12 GM9_TH8 variants analyzed in conventional ELISA (PBS) or in the presence of high salt (NaSCN; representative data from duplicate wells from two independent experiments). (D) Autologous tier 2 neutralizing activity of the 12 GM9_TH8 variants as IgG or Fab shown in the order of increasing SHM (data from one independent experiment). (E) Scatter plots of SHM versus Fab neutralizing activity (IC₅₀ or IC₈₀) were generated, and the Spearman rank correlation coefficient (ρ) was computed using Prism software (GraphPad, v7; ρ = 0.837). Asterisk indicates that the non-neutralizing PB_seq681430_post-1 Ab was assigned IC₅₀ and IC₈₀ values of 100 for the purpose of the calculation. Data points are color-coded according to compartment. The analyses of Ab function were performed twice. (F) Representation of the GM9_TH8seq732127 Fab structure. LC is shown in light blue/gray and HC in dark blue, with HCDR1 in pink, HCDR2 in magenta, HCDR3 in orange, and FR3 in green. Mutated residues are shown in spheres and labeled. Left: Shared SHM between the GM9_TH8 HC and the GM9_TH8seq732127 HC. Middle: SHM at the same position but mutated to a different aa residue for GM9_TH8 (first letter) compared to GM9_TH8seq732127 (second letter). Right: All SHM in the GM9_TH8seq732127 HC.