Research Article: Alternative conformations of a major antigenic site on RSV F

Date Published: July 15, 2019

Publisher: Public Library of Science

Author(s): Harrison G. Jones, Michael B. Battles, Chun-Chi Lin, Siro Bianchi, Davide Corti, Jason S. McLellan, James E. Crowe.

http://doi.org/10.1371/journal.ppat.1007944

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.

Partial Text

Respiratory syncytial virus (RSV) is a ubiquitous pneumovirus which infects nearly all children in the U.S. by the age of two, with repeated infections occurring throughout life [1]. RSV is a common cause of acute lower respiratory tract infections in young children and the elderly, and in 2015 resulted in an estimated 94,000–149,000 deaths globally in children under the age of five [2]. Although few deaths of children in the United States are attributed to RSV [2, 3], severe infections requiring hospitalization are frequent and lead to estimated direct health care costs of $750 million dollars annually [4]. Currently, there is no vaccine for RSV and the only FDA-approved therapy is passive prophylaxis with the monoclonal antibody palivizumab (Synagis) [5]. However, the high cost and modest efficacy of palivizumab restricts its usage to high-risk infants [6], making the development of improved interventions a global health priority.

AM22 and RSD5 are two human antibodies that bind to antigenic site Ø and are specific for the prefusion conformation of RSV F. Although both antibodies potently neutralize RSV, similar to D25, their binding kinetics and subtype specificity have distinct differences. AM22 and D25 both preferentially bind to subtype A, whereas RSD5 preferentially binds to subtype B as a result of its fast dissociation rate constant for subtype A F proteins. In addition, previous publications using SPR and flow cytometry-based competition assays have suggested that AM22 and RSD5 do not compete with D25, but rather occupy a separate prefusion-specific epitope [37, 38]. However, comparing the crystal structures of prefusion RSV F bound to AM22, RSD5-GL, and D25, demonstrates that all three antibody epitopes overlap substantially and would prevent any two from binding simultaneously due to large steric clashes. This emphasizes the importance of structural characterization of antibody epitopes in addition to competition data, as varying antibody kinetics can mislead epitope classification when using only competition assays.

 

Source:

http://doi.org/10.1371/journal.ppat.1007944

 

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