Fig 4. Identification of critical residues for mAbs 2E1 and 3B1 binding with 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) Reactivity results from a representative assay are shown with four positive (wildtype RSV F) and four negative (mock-transfected) control wells included on the plate. (B) Human HEK293T cells expressing the RSV F mutation library were tested for immunoreactivity with mAb 2E1, measured on an Intellicyt high-throughput flow cytometer. Clones with reactivity of <20% relative to that of wildtype RSV F (horizontal line) yet >70% reactivity for a control mAb (vertical line) were initially identified to be critical for mAb 2E1 binding (red dots), and were verified using algorithms described elsewhere [25]. (C) Mutation of four individual residues reduced 2E1 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. (D) Mutation of three individual residues reduced 3B1 binding (red bars) but not the binding of D25 and palivizumab (gray bars). Error bars represent range of at least two replicate data points.