Alternative conformations of a major antigenic site on RSV F

Jones, Harrison G.; Battles, Michael B.; Lin, Chun-Chi; Bianchi, Siro; Corti, Davide; McLellan, Jason S.

DS-Cav1 or postfusion RSV F from subtype A (strain A2) or subtype B (strain B9320), as well as a subtype A mutant with a K209Q substitution, with a C-terminal 6x-His or 8x-His tag was immobilized on a Ni-NTA sensor chip to a total of 80-150 response units using a Biacore X100 (GE). A buffer-only sample was injected over the DS-Cav1 or postfusion RSV F and reference flow cells for reference subtraction, followed by serial 3-fold dilutions of Fab (AM22, RSD5-GL, RSD5-WT, or D25) from 300 nM to 46.5 pM in HBS-P+, with a duplication of the 11.1 nM concentration. For the DS-Cav1 subtype A mutant with a K209Q substitution, only AM22 Fab was evaluated. For the assay evaluating AM22 Fab binding to strain B9320, the highest AM22 Fab concentration used was 1 uM in HBS-P+ buffer, followed by serial 3-fold dilutions to the lowest concentration of 152 pM. For the assay evaluating binding to postfusion RSV F of subtype A or B, a concentration of 300 nM of each Fab in HBS-P+ buffer was used. The data were double-reference subtracted and fit to a 1:1 binding model using the Biacore X100 or Scrubber2 analysis software. Final binding curves were displayed using GraphPad Prism Version 7.03 for Windows.