FIG 3.

DB1-QUAD thermostability, incorporation of prefusion F protein, and F fusion activity. (A) Thermostability was analyzed at 4°C by serially titrating vials of mKate2-labeled A2, A2-line19F, DB1, and DB1-QUAD at time points ranging from 0 to 28 days. The data represent means and SD of log-transformed titers of four experimental replicates for time points 0 to 14 days and two replicates for time points 21 to 28 days, all titrated in duplicate. The asterisks denote statistical differences between A2 and A2-line19F and between DB1 and DB1-QUAD at the respective time points. There were no statistical differences between A2-line19F and DB1-QUAD titers. (B) The ratio of prefusion F to total F protein expression was measured using MPE-8 (pre-F) and motavizumab (total F) ELISAs. The data represent means and SD of at least three experimental replicates for each virus. (C) The fusion activities of line19F, BAF, and BAF-79-191-357-371 were measured using a DSP assay. 293T cells were transfected with F expression plasmids in the presence of BMS-433771 fusion inhibitor, and cell-cell fusion activity was quantified by luciferase activity at time points 0, 2, 4, 6, and 8 h post-cell mixing. Three experimental replicates were performed, and representative data from one experiment are shown. **, P < 0.005; ***, P < 0.0005; ****, P < 0.00005 by two-way ANOVA and Tukey's multiple-comparison test (A) or by Student's t test (B).