Crystal Structure and Immunogenicity of the DS-Cav1-Stabilized Fusion Glycoprotein From Respiratory Syncytial Virus Subtype B

Abstract

Background:Respiratory syncytial virus (RSV) subtypes, A and B, co-circulate in annual epidemics and alternate in dominance. We have shown that a subtype A RSV fusion (F) glycoprotein, stabilized in its prefusion conformation by DS-Cav1 mutations, is a promising RSV-vaccine immunogen, capable of boosting RSV-neutralizing titers in healthy adults. In both humans and vaccine-tested animals, neutralizing titers elicited by this subtype A DS-Cav1 immunogen were ~ 2- to 3-fold higher against the homologous subtype A virus than against the heterologous subtype B virus.Methods:To understand the molecular basis for this subtype difference, we introduced DS-Cav1 mutations into RSV strain B18537 F, determined the trimeric crystal structure, and carried out immunogenicity studies.Results:The B18537 DS-Cav1 F structure at 2-Å resolution afforded a precise delineation of prefusion F characteristics, including those of antigenic site Ø, a key trimer-apex site. Structural comparison with the subtype A prefusion F indicated 11% of surface residues to be different, with an alpha-carbon root-mean-square deviation (RMSD) of 1.2 Å; antigenic site Ø, however, differed in 23% of its surface residues and had an alpha-carbon RMSD of 2.2 Å. Immunization of vaccine-tested animals with DS-Cav1-stabilized B18537 F induced neutralizing responses ~100-fold higher than with postfusion B18537 F. Notably, elicited responses neutralized RSV subtypes A and B at similar levels and were directed towards both conserved equatorial and diverse apical regions.Conclusion:We propose that structural differences in apical and equatorial sites–coupled to differently focused immune responses–provide a molecular explanation for observed differences in elicited subtype A and B neutralizing responses.

Convergent structural features of respiratory syncytial virus neutralizing antibodies and plasticity of the site V epitope on prefusion F

“…This is likely due to the conservation of a small polar, uncharged side chain for both A and B strains, thus maintaining a similar environment for recognition by RSB1. On the other hand, the Asp200Asn substitution would likely result in the loss of a salt bridge with RSB1 Arg53 LCDR2 (Fig 2D), and comparison with the RSV B structure (PDB 6Q0S) [40] indicates there would be a shift in the local surface potential from a negatively charged region into a more neutral or positive surface (S6 Fig) . This would seemingly disfavor binding to the RSB1 positively charged Arg53 LCDR2 . However, we found that the single mutant Asp200Asn, as well as the double mutant Asp200Asn-Asn276Ser, were again not sufficient to impact the binding affinity for RSB1.…”

Cross-Protective Antibodies Against Common Endemic Respiratory Viruses

“…3c ; also ref. 52 ]. This is notable because escape mutations to D25 have been identified in infants who suffered a breakthrough infection with RSV subtype B after receiving nirsevimab 53 .…”

Humoral Immunity to Hantavirus Infection

“…Understanding the ultrastructural arrangement and dynamics of Gn and Gc on the surface of the virion and how that may contribute to the exposure or occlusion of important antigenic sites is critical in development of medical countermeasures. For example, understanding the antigenic sites exposed in the respiratory syncytial virus (RSV) F protein and molecular determinants of prefusion stabilization has led to the development of a highly immunogenic RSV vaccine (63). Most recently, knowledge of the molecular-level dynamics of the receptor binding domain (RBD) in coronaviruses gave way to the rapid development of a prefusion stabilized spike protein vaccine that exposes immunogenic sites in the "up" form of the RBD (64).…”