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.

Germline sequences of RSD5 framework regions were determined with reference to the IMGT database [63] . RSD5 and RSD5-GL (fully germlined in VH and VL framework regions, as defined by IMGT) were produced by gene synthesis (GenScript) and confirmed by sequencing. Synthesized VH and VL sequences were cloned into human Igγ1 and Igκ expression vectors (kindly provided by Michel Nussenzweig, Rockefeller University, New York, NY, USA), essentially as described [64] . Plasmids encoding antibody heavy and light chains for AM22, RSD5-WT, RSD5-GL, or for D25 were co-transfected into Expi293 cells or FreeStyle 293-F cells (Invitrogen). AM22 and D25 IgGs and Fabs were purified using Protein A agarose (Fisher) or CaptureSelect IgG-CH1 affinity matrix (Life Technologies), respectively. All IgG antibodies were eluted off the Protein A column using 0.1 M glycine pH 3.0 into a buffered solution containing 1/10 (v/v) of 1 M Tris pH 8.0. All Fabs were eluted off the CaptureSelect IgG-CH1 column using 50 mM acetic acid pH 4.0 into a buffered solution containing 1/10 (v/ v) of 1 M Tris pH 8.0. To produce RSD5-WT Fab, RSD5-WT was expressed and purified as an IgG with an HRV 3C protease site in the hinge of the heavy chain. RSD5-WT Fab was produced by digesting the IgG with HRV 3C for 2 hours at room temperature, followed by passing the solution over protein A resin to remove the Fc, and subsequently purified by SEC using a Superdex 200 column (GE). Production of RSD5-GL Fab was done by incubating RSD5-GL IgG with papain beads (Pierce). All IgGs and Fabs were buffer exchanged using a desalting column, followed by final purification by SEC using a Superdex 200 column (GE) prior to long term storage at -80˚C.