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.

A fortéBio Octet Red384 instrument (Pall ForteBio LLC, CA) was used to measure binding kinetics of RSV F head-only variants to antigenic site Ø (D25, AM22) and site II-targeting motavizumab antibodies. All assays were performed with agitation set to 1,000 rpm in PBS supplemented with 1% bovine serum albumin (BSA) in order to minimize nonspecific interactions. The final volume for all solutions was 50-80 μl/well. Assays were performed at 30°C in tilted black 384-well plates (Greiner Bio-One, NC). RSV F head-only variants (50 μg/ml) in PBS buffer was used to load anti-His-tag probes (HIS1K) for 300 s. Typical capture levels for each loading step were between 0.6 and 1.2 nm, and variability within a row of eight tips did not exceed 0.1 nm for each of these steps. The nm unit is a measure of the change in the interference pattern of white light reflected from the surface of the biosensor tip compared to an internal reference. This was measured in real-time and correlates with a change in the number of bound molecules on the biosensor tip surface. This can also be defined as a change in response units measured in nm [25, 26] . Biosensor tips were then equilibrated for 90 s in PBS + 1% BSA prior to measuring association with antigen binding fragments (Fabs) in solution (0.016 μM to 0.5 μM) for 300 s; Fabs were then allowed to dissociate for 300-1200 s. Parallel correction to subtract systematic baseline drift was carried out by subtracting the measurements recorded for a loaded sensor incubated in PBS + 1% BSA. Data analysis and curve fitting were carried out using Octet analysis software, version 8.0 (Pall ForteBio LLC, CA). Experimental data were fitted with a binding equation describing a 1:1 interaction. Global analyses of the complete data sets assuming reversible binding (full dissociation) were carried out using nonlinear least-squares fitting allowing a single set of binding parameters to be obtained simultaneously for all concentrations measured in a single experiment.