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.

Designed constructs were codon optimized for expression in human cells, and transfected and expressed in HEK 293T cells by using a 96-well format, which coupled expression with evaluation of antigenic recognition [16] . As several of the most potently neutralizing RSV F antibodies target antigenic site Ø, three monoclonal antibodies (mAbs) specific for this site were used as readout: antibodies D25, 5C4 and AM22. Additionally, because soluble RSV F trimers are metastable and spontaneously convert to a post-fusion conformation, D25 binding was also evaluated after one week at 4°C, and AM22 and 5C4 binding were evaluated after two and five weeks, respectively, at 4°C. Out of 70 designs, 51 (73%) had D25 ELISA readouts of 1.5 OD 450 or higher after one week at 4°C (S2-S4 Tables). To further gauge the stability of these immunogens, D25 recognition was also evaluated after one hour at 60, 70, 80, 90 and 100°C. Fifty of the head-only designs retained the majority of their D25 antigenicity after one hour at 70°C, conditions under which the parent DS-Cav1 RSV F trimer completely lost D25 recognition [16] . At 80°C and above, we observed a substantial decrease in D25 binding for most of the head-only immunogens (S2-S4 Tables).