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.

To assess the physical stability of the pre-fusion conformation of RSV F head region proteins under various stress conditions, we treated the proteins with a variety of pharmaceutically relevant stresses such as extreme pH, high temperature, low and high osmolarity, and repeated freeze/thaw cycles [16, 17, 27] . The physical stability of treated RSV F head-only proteins was evaluated by retention of binding to the antigen-binding fragment (Fab) of the site Ø-specific antibody D25. All treatments were carried out at a protein concentration of 100 μg/ml. For pH treatments, the RSV F glycoprotein solution was adjusted to either pH 3.5 with citrate buffer or pH 10 with N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) buffer, incubated at room temperature for 60 minutes and subsequently neutralized to pH 7.5 with Tris buffer. Temperature treatments were carried out by incubating the RSV F glycoprotein solutions at 50°C, 70°C or 90°C for 60 minutes in a PCR cycler with heated lid. For osmolarity treatments, RSV F glycoprotein solutions originally containing 150 mM NaCl were either diluted with 2.5 mM Tris buffer (pH 7.5) to an osmolarity of 10 mM NaCl or adjusted with 4.5 M MgCl 2 to a final concentration of 3.0 M MgCl 2 . Protein solutions were incubated for 60 minutes at room temperature and then returned to 150 mM salt by adding 5 M NaCl or dilution with 2.5 mM Tris buffer, respectively, and concentrated to 50 μg/ml. The freeze/thaw treatment was carried out by repeatedly freezing RSV F glycoprotein solutions in liquid nitrogen and thawing at 37°C ten times. All RSV F glycoproteins were diluted to 80 μg/ml with PBS + 1% BSA to reduce non-specific binding, and their ability to bind D25 Fab was measured with a fortéBio Octet instrument using the protocol described below.