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.

Head-only boosting can focus the RSV-neutralizing response to antigenic sites Ø and II We next investigated whether the increased RSV-neutralization titers from the heterologous boosts of i-693 and i-447 were directed towards antigenic sites Ø or II. We previously developed DS-Cav1 probes with knock-out (KO) mutations in antigenic site Ø or antigenic site II [15] , and we used these site-specific KO probes to quantify the site-specific immune response. First, we measured the relative binding affinity of immune sera to DS-Cav1, DS-Cav1 site Ø-KO, and DS-Cav1 site II-KO probes. With the homologous DS-Cav1 prime/boost sera, no significant difference was observed in binding to the three probes ( Fig 5A) . This was not entirely surprising given that the individual knock-out mutations for antigenic sites Ø and II cover only a small percentage (1-3%) of the surface area of their respective probes. In contrast, the site Ø-KO probe bound significantly less to both i-447 and i-693 boosted sera (P = 0.0015 and 0.0021 respectively) than to the DS-Cav1 probe (Fig 5A) . The site II-KO probe also bound significantly less to i-447 boosted sera than to the DS-Cav1 probe (P = 0.0015), but its binding was statistically comparable to DS-Cav1 with i-693 boosted sera. We next measured the competitive effect of DS-Cav1 antigenic site Ø and site II KO probes on neutralization. DS-Cav1 added to sera from each of the three groups of immunized mice (3×DS-Cav1, 2×DS-Cav1 + 1×i-693 and 2×DS-Cav1 + 1×i-447) competed for sera very efficiently as expected, resulting in significantly reduced neutralization (P = <0.0001) (Fig 5B) . However, the site Ø KO and site II KO probes competed much less effectively than DS-Cav1, highlighting the importance of these sites (Fig 5B) . In the DS-Cav1 and i-693-boosted group, antigenic site II KO probe competed more strongly than the antigenic site Ø KO probe (P = 0.0354 and 0.0431 respectively), indicating antigenic site Ø to be the dominant target of neutralization by these sera. In the i-447-boosted group, however, there was no significant difference in neutralization competition between antigenic site Ø and site II KO probes. Thus, with regards to site-specific induced responses, our results indicate the RSV-neutralizing response boosted by head-only immunogens i-693 and i-447 to indeed be more focused on head-specific sites Ø and II, especially antigenic site Ø.