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.

Antigenic screening of head-only RSV F immunogens All constructs were assessed using a 96-well microplate format for high throughput expression followed by an ELISA-based antigenic evaluation as described previously [16] . Briefly, 24 h prior to transfection HEK 293T cells (Thermo Fisher Scientific, MA) were seeded in each well of a 96-well microplate at a density of 2.5x10 5 cells/ml in expression medium (high glucose DMEM supplemented with 10% ultra-low IgG fetal bovine serum and 1x-non-essential amino acids), and incubated at 37°C, 5% CO 2 for 20 h. Plasmid DNA and TrueFect-Max (United Bio-Systems, MD) were mixed and added to the growing cells, and the 96-well plate incubated at 37°C, 5% CO 2 . One day post transfection, enriched medium (high glucose DMEM plus 25% ultra-low IgG fetal bovine serum, 2x nonessential amino acids, 1x glutamine) was added to each well, and the 96-well plate was returned to the incubator for continuous culture. On day five post transfection, supernatants with the expressed RSV F variants were harvested and tested by ELISA for binding to D25 and motavizumab antibodies using Ni 2+ -NTA microplates. After incubating the harvested supernatants at 4°C for one week, two weeks and five weeks, ELISAs were repeated for recognition by antibodies D25, AM22 and 5C4 respectively.