Alternative conformations of a major antigenic site on RSV F

Abstract

The respiratory syncytial virus (RSV) fusion (F) glycoprotein is a major target of neutralizing antibodies arising from natural infection, and antibodies that specifically bind to the prefusion conformation of RSV F generally demonstrate the greatest neutralization potency. Prefusion-stabilized RSV F variants have been engineered as vaccine antigens, but crystal structures of these variants have revealed conformational differences in a key antigenic site located at the apex of the trimer, referred to as antigenic site Ø. Currently, it is unclear if flexibility in this region is an inherent property of prefusion RSV F or if it is related to inadequate stabilization of site Ø in the engineered variants. Therefore, we set out to investigate the conformational flexibility of antigenic site Ø, as well as the ability of the human immune system to recognize alternative conformations of this site, by determining crystal structures of prefusion RSV F bound to neutralizing human-derived antibodies AM22 and RSD5. Both antibodies bound with high affinity and were specific for the prefusion conformation of RSV F. Crystal structures of the complexes revealed that the antibodies recognized distinct conformations of antigenic site Ø, each diverging at a conserved proline residue located in the middle of an α-helix. These data suggest that antigenic site Ø exists as an ensemble of conformations, with individual antibodies recognizing discrete states. Collectively, these results have implications for the refolding of pneumovirus and paramyxovirus fusion proteins and should inform development of prefusion-stabilized RSV F vaccine candidates.

Characterization of potent RSV neutralizing antibodies isolated from human memory B cells and identification of diverse RSV/hMPV cross-neutralizing epitopes

“…54 A recent report of two new site Ø mAbs with different RSV A and B specificities also highlighted the structural variations of this antigenic site. 45 Among the isolated highly potent mAbs in this study, we indeed found that mAbs targeting site Ø showed the most variations in neutralization potencies between the two different RSV subtypes, including a strain A-specific mAb 139B8. Similar mAbs were also reported previously from immunized mice and from human adults.…”

“…Critical residues identified for antibody binding were labeled and color-coded according to the corresponding antigenic sites. and IV binding mAbs 36,45,47 and extends to additional antigenic sites (III and V). Our data exemplified the diversity of anti-RSV mAbs and provided novel understanding into immune recognition mechanisms of RSV.…”

“…24,27,[31][32][33][34][35][36][37][38][39][40][41][42][43][44][45] Furthermore, the crystal structures of RSV and hMPV F proteins in both PreF and PostF conformations, as well as complex structures with mAbs targeting various antigenic sites, have been reported. 20,21,[28][29][30][31][32][33][34][35]37,[45][46][47][48][49] The structures of PreF and PostF antigens between RSV and hMPV are remarkably conserved, albeit only sharing about 35% sequence identity. 21,[29][30][31]46,48 MAbs that cross-neutralize both RSV and hMPV have been reported.…”

Conformational Flexibility in Respiratory Syncytial Virus G Neutralizing Epitopes

“…Likewise, residues after K187 in the C-terminal region of the CCD also appear to be flexible. Thus, RSV G is part of a growing list of antigens with flexible or intrinsically disordered regions (IDRs) that are targeted by antibodies (48)(49)(50)(51)(52)(53)(54)(55)(56). The observation of different conformations of the RSV G CCD raises several important questions.…”

Structural Insight into Paramyxovirus and Pneumovirus Entry Inhibition

“…Structures of F proteins complexed with neutralizing antibodies have been solved for a number of pneumo-and paramyxovirus family members including RSV (Figure 3), HPIV3, and NiV. Distinct antigenic sites were identified on RSV F (Figure 4): conformation-dependent site Ø that is located at the apex of prefusion F and a dominant target for nAbs [36,[76][77][78]; sites II and IV that are present in both the pre-and postfusion F conformations [67,79]; site III that likewise exists in both F conformations, but undergoes rearrangement of secondary structure elements forming the epitope [80,81]; site V located between sites Ø and III on prefusion F [82,83]; and post-fusion F site I [84,85]. A cryo-EM structure of HPIV3 F complexed with nAb PIA174 likewise locates the binding site to the apex of the prefusion F trimer, establishing contact with residues of all three F protomers [37].…”