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.

All fortéBio assays were performed at 30°C in tilted black 384-well plates (Greiner Bio-One, NC) with agitation set to 1,000 rpm in and a well volume of 50 μl. Trimeric and dimeric RSV F head proteins i-210, i-447, and i-693 were immobilized using anti-Strep-tag IgG. Due to reduced avidity of the interaction between the anti-Strep-tag antibody and the monomeric RSV F head proteins, i-273 was immobilized using the higher affinity antigenic site II-directed motavizumab IgG. Pre-immobilized anti-mouse Fc biosensor tips were loaded with anti-Streptag (100 μg/ml in PBS + 0.2% BSA) for 600 s and pre-immobilized anti-human Fc biosensor tips were loaded with motavizumab IgG (50 μg/ml in PBS + 0.2% BSA) for 300 s. Variability in loading within a row of eight tips did not exceed 0.1 nm for each of these steps. Biosensor tips were equilibrated for 120 s in PBS + 0.2% BSA prior to loading RSV F variants for 300 s. Biosensor tips were then equilibrated for 120 s in PBS + 0.2% BSA prior to measuring association of D25 Fab (50 μg/ml in PBS + 0.2% BSA) for 300 s. Fabs were allowed to dissociate for 300 s in PBS + 0.2% BSA. Parallel correction to subtract systematic baseline drift was carried out by subtracting the measurements recorded for a loaded sensor incubated in PBS + 1% BSA. For trimeric RSV F head proteins i-210 and i-447, as well as monomeric RSV F head protein i-273, physical stability is reported as the ratio of steady state D25-binding level before and after stress treatment. Due to reduced avidity of the interaction between the anti-Strep-tag antibody and the dimeric RSV F head protein i-693, some dissociation of the antigen from the biosensor was observed over the 420 seconds of equilibration and D25 binding. To account for this, the physical stability of i-693 is reported as the ratio of maximum D25-binding level before and after stress treatment.