Table 3.

Mapping of VRC-01 3-2 αIM residues required for antibody binding by site-directed mutagenesis. Site-directed mutagenesis was used to create a “mini-library” in which each position in the BC and FG loops of VRC01 3-2 αIM was permitted to be either the wild-type sequence or a serine residue (except in the case of methionine and histidine, which were replaced by either serine, asparagine, arginine, and isoleucine). The resulting library was then selected for binding to either the VRC-01 Mab (left) or the irrelevant M2 anti-FLAG antibody (right). A selection of clones, 23 panned on VRC-01 and 24 panned on anti-FLAG, were sequenced; results are presented. Wild-type VRC-01 sequences are unshaded, while substitutions are color coded by amino acid (e.g., red denotes serine). Sequences of the clones selected against the VRC-01 3-2 αIM demonstrate the critically important role of the complete FG loop and 4 of 7 residues within the BC loop in contributing to the binding of the VRC-01 3-2 αIM to its cognate antibody. In contrast, sequences of the clones selected against the irrelevant M2 anti-FLAG antibody reveal the diversity of the parental “mini-library”.