A single somatic mutation acquired by antivaccinia antibody 589 reduces self- and foreign reactivity, whereas other mutations increase binding to foreign antigens. (A and B) Variable region amino acid sequence of the p589 H chain (A) and L chain (B) and sequence substitutions acquired in the immune 589 antibody (shown below in red). (C) Key depicting the number of amino acids substitutions (#aa subs) in each antibody and its binding to mature naive B cells. Data points are the mean and standard deviation of three separate experiments using PBMCs from three individual donors. Statistical significance was assessed by two-way ANOVA (**, P ≤ 0.01 for 589 vs. 589 E6Q; ****, P ≤ 0.0001 for p589 vs. 589, p589 Q6E, and 589 E6Q). SHM, somatic hypermutation. (D) Binding of each antibody to vaccinia virus measured by ELISA. Data are representative of two separate experiments with each point the mean of duplicates. Statistical significance was determined by two-way ANOVA (*, P ≤ 0.05 for p589 vs. p589 Q6E; **, P ≤ 0.01 for 589 vs. 589 E6Q). (E) Binding of p589 at 4°C (continuous line) and 37°C (dashed line). Data are representative of two independent experiments with differences evaluated by Student’s paired t test (P = 0.02). (F) Structural modeling of a hydrophobic patch of p589 (left) and 589 (right). Preimmune residues are depicted in blue with immune substitutions in dark red and oxygen in light red.