Fig 2. Identification of critical residues for monoclonal antibody 4D7 binding using shotgun mutagenesis epitope mapping.

A shotgun mutagenesis alanine scanning library was constructed for the RSV F protein. The library contains 368 individual mutations at residues identified as surface exposed on the prefusion and postfusion forms of RSV F proteins. Each well of the mutation array plate contained one mutant with a defined substitution. (A) Human HEK293T cells expressing the RSV F mutation library were tested for immunoreactivity with 4D7, measured on an Intellicyt high-throughput flow cytometer. Clones with reactivity of <15% relative to that of wildtype RSV F (horizontal line) yet >70% reactivity for a control monoclonal antibody were initially identified to be critical for 4D7 binding (red dots), and were verified using algorithms described elsewhere [23] (U.S. patent application 61/938,894). (B) Mutation of three individual residues reduced 4D7 binding (red bars) but not the binding of D25 and palivizumab (gray bars). Error bars represent range (half of the maximum minus minimum values) of at least two replicate data points. (C) Comparison of 4D7 binding epitope on prefusion and postfusion RSV F structures. Prefusion RSV F structure is shown in magenta and postfusion F structure shown in green. Residues 384 to 392 are shown in stick representation highlighting both main chain and side chain atoms, and the rest of the structure is shown in line representation with only main chain bonds depicted.