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.

Since the RSV F stalk comprises 55% of the total surface area of the RSV F trimer, we reasoned that expression of the head-only region of RSV F might help to focus the immune response to antigenic site Ø as well as to antigenic site II. We therefore designed 70 variants of head-only RSV F constructs using three different approaches (Fig 1C and S1 Table) . For the first approach, domain III was expressed as a simple monomer (30 designs); in the second approach, domain III was expressed as a tandemly linked dimer (18 designs); in the third approach, a trimerization domain was fused to domain III to enable the expression of a trimeric protein resembling the native pre-fusion RSV F head (22 designs). The topology of RSV F is such that removal of the RSV F stalk from the RSV F head results in two separate F 1 and F 2 polypeptides within each head protomer. Reconnection of both polypeptides to make a single polypeptide afforded an opportunity to permute circularly the domain topology, by linking either F 2 to F 1 , or F 1 to F 2 (S1 Fig). Most designs also incorporated the disulfide bond from DS-Cav1 (S155C-S290C) as well as the DS-Cav1 cavity filling mutations (S190F and V207L) [16] to stabilize the pre-fusion conformation. Numerous additional mutations included repacking of the protein interior, the addition of disulfide bonds, and the alteration of surface mutations to replace hydrophobic residues with hydrophilic residues or glycans. Moreover, various lengths of glycine rich linkers were used to link the two polypeptides of domain III and to connect them to trimerization domains. Finally, a signal peptide for secretion and a cleavable His-tag and Strep-tag to aid in purification were added to each construct. The signal peptide is colored yellow, F 2 is colored blue, F 1 is colored green, the transmembrane region is colored cyan and the cytoplasmic domain is colored gray. (B) Pre-F form of the RSV F trimer. One protomer of RSV F is depicted as a ribbon diagram and colored as in A. The other two protomers are gray surface representations. Antigenic site Ø and site II are red and orange respectively. Domains I, II and III are labeled as defined previously [9, 35] . (C) Models of head-only immunogens i-273, i-693 (upper panels), i-210 and i-447 (lower panels). For each immunogen a cartoon of the genetic construct is depicted (top) and a ribbon diagram (bottom), color-coded as in A and B with brown coloring for purification tags. PDB entries 4JHW [9] , 1RFO [36] and 1GCM [37] were used to depict RSV F, the foldon and the coiled coil respectively. RSV F residue numbering follows the numbering in PDB entry 4JHW [9] .