Discrete somatic mutations acquired by antivaccinia antibody 166 independently remove self-reactivity and increase virus reactivity. (A and B) Variable domain amino acid sequence of the p166 H chain (A) and L chain (B) and sequence substitutions acquired in the immune 166 antibody (shown below in red). (C) Number of amino acid substitutions (#aa subs) in each antibody and binding to mature naive B cells. Data points are the mean and standard deviation of four independent experiments using PBMCs from four individual donors. Statistical significance was assessed using two-way ANOVA (****, P ≤ 0.0001 for p166 vs. 166, p166 Y26S, and 166 S26Y). SHM, somatic hypermutation. (D) Binding of each antibody to vaccinia virus measured by ELISA. The data are representative of three independent experiments with each data point showing the mean of duplicates. Statistical significance was determined by two-way ANOVA (*, P ≤ 0.05 for p166 vs. p166 Y26S; ***, P ≤ 0.001 for 166 and 166 S26Y vs. p166 and p166 Y26S). (E) Binding of p166 to mature naive B cells at 4°C (continuous line) and 37°C (dashed line). Data are representative of two independent experiments, and statistical significance was determined by Student’s paired t test (P = 0.05). (F) Three-dimensional structure of a hydrophobic patch for p166 (left) and 166 (right). Preimmune residues are depicted in blue with immune substitutions in dark red and oxygen in light red.