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 major goal for RSV-vaccine development is to identify immunogens that can boost antibody responses to neutralization-sensitive epitopes in the setting of pre-existing immunity. Increasing serum-neutralizing activity in pregnant women would help to increase their protective response and to extend the period of maternally-acquired protection from RSV. Likewise, in elderly people who have experienced repeated RSV infections throughout life, but still have modest levels of serum-neutralizing activity, boosting the response to neutralization-sensitive epitopes could achieve greater than threshold levels of protective immunity. Therefore, we took a structure-based design approach to produce immunogens that focused immune recognition on the apical surfaces of the pre-F trimer. We removed the stalk portion from the RSV F trimeric glycoprotein to create 70 RSV F head-only immunogens. Fifty of these designs were recognized by antibodies to the metastable antigenic site Ø (D25, AM22 and 5C4) and were stable for at least one hour at 70°C, a temperature at which DS-Cav1 rapidly inactivates (Table 1) . The thermostable head-only immunogens included 26 monomers, 6 dimers, and 18 trimers. Four of these, a monomer (i-273), a dimer (i-693) and two trimers (i-210 and i-447), were expressed, purified, and characterized for antigenicity, overall stability, structural integrity, and immunogenicity. Although all four of these head-only immunogens displayed nanomolar affinity to antigenic site Ø-directed antibodies and had physical stability that was similar or exceeded that of DS-Cav1, only one of the immunogens (the i-447 trimer) was able to elicit RSV-neutralizing titers in mice that were comparable to those elicited by DS-Cav1 (Fig 4A) . The lower overall immunogenicity of these head-only immunogens may relate to increased flexibility between protomers, as we recently found that the reduction of interprotomer flexibility in pre-F trimers yielded higher immunogenicity [42] .