Structure-Based Design of Head-Only Fusion Glycoprotein Immunogens for Respiratory Syncytial Virus

Boyington, Jeffrey C.; Joyce, M. Gordon; Sastry, Mallika; Stewart-Jones, Guillaume B. E.; Chen, Man; Kong, Wing-Pui; Ngwuta, Joan O.; Thomas, Paul V.; Tsybovsky, Yaroslav; Yang, Yongping; Zhang, Baoshan; Chen, Lei; Druz, Aliaksandr; Georgiev, Ivelin S.; Ko, Kiyoon; Zhou, Tongqing; Mascola, John R.; Graham, Barney S.; Kwong, Peter D.

Head-only RSV F variants were expressed by transient transfection of Expi293F cells (Thermo Fisher Scientific, MA) using 293fectin (Thermo Fisher Scientific, MA). The culture supernatants were harvested 5 days post transfection and centrifuged at 10,000g to remove cell debris. The supernatants were sterile-filtered and the head only RSV F variants were purified by nickel and Strep-Tactin (iba, MO) affinity chromatography followed by size-exclusion chromatography (SEC) using a Superdex 200 16/60 column (GE Healthcare, PA). The purification tags were removed from the RSV F proteins by digestion with 1.0 U restriction-grade biotinylated thrombin (Novagen, WI) per mg of immunogen overnight at room temperature. Cleaved immunogens were subsequently passed over a streptavidin column (GE Healthcare, PA) to capture the thrombin and further purified by a second round of SEC in phosphate-buffered saline (PBS) prior to analysis or immunization. The molecular weights of proteins eluted from SEC were estimated by fitting elution volumes to a regression line in a plot of Log molecular weight versus (Ve-Vo)/(Vt-Vo) for several standard proteins where Ve is elution volume, Vo is the void volume of the column, and Vt is the total volume of the column.