title: Characterization of Pre-F-GCN4t, a Modified Human Respiratory Syncytial Virus Fusion Protein Stabilized in a Noncleaved Prefusion Conformation.
abstract: The human respiratory syncytial virus (hRSV) fusion (F) protein is considered a major target of the neutralizing antibody response to hRSV. This glycoprotein undergoes a major structural shift from the prefusion (pre-F) to the postfusion (post-F) state at the time of virus-host cell membrane fusion. Recent evidences suggest that the pre-F state is a superior target for neutralizing antibodies compared to the post-F state. Therefore, for vaccine purposes, we have designed and characterized a recombinant hRSV F protein, called Pre-F-GCN4t, stabilized in a pre-F conformation. To show that Pre-F-GCN4t does not switch to a post-F conformation, it was compared with a recombinant post-F molecule, called Post-F-XC. Pre-F-GCN4t was glycosylated and trimeric and displayed a conformational stability different from that of Post-F-XC, as shown by chemical denaturation. Electron microscopy analysis suggested that Pre-F-GCN4t adopts a lollipop-like structure. In contrast, Post-F-XC had a typical elongated conical shape. Hydrogen/deuterium exchange mass spectrometry demonstrated that the two molecules had common rigid folding core and dynamic regions and provided structural insight for their biophysical and biochemical properties and reactivity. Pre-F-GCN4t was shown to deplete hRSV-neutralizing antibodies from human serum more efficiently than Post-F-XC. Importantly, Pre-F-GCN4t was also shown to bind D25, a highly potent monoclonal antibody specific for the pre-F conformation. In conclusion, this construct presents several pre-F characteristics, does not switch to the post-F conformation, and presents antigenic features required for a protective neutralizing antibody response. Therefore, Pre-F-GCN4t can be considered a promising candidate vaccine antigen.IMPORTANCEHuman respiratory syncytial virus (RSV) is a global leading cause of infant mortality and adult morbidity. The development of a safe and efficacious RSV vaccine remains an important goal. The RSV class I fusion (F) glycoprotein is considered one of the most promising vaccine candidates, and recent evidences suggest that the prefusion (pre-F) state is a superior target for neutralizing antibodies. Our study presents the physicochemical characterization of Pre-F-GCN4t, a molecule designed to be stabilized in the pre-F conformation. To confirm its pre-F conformation, Pre-F-GCN4t was analyzed in parallel with Post-F-XC, a molecule in the post-F conformation. Our results show that Pre-F-GCN4t presents characteristics of a stabilized pre-F conformation and support its use as an RSV vaccine antigen. Such an antigen may represent a significant advance in the development of an RSV vaccine.
authors.affiliation: GSK Vaccines, Laval, Canada normand.x.blais@gsk.com.
authors.forename: Normand
authors.initials: N
authors.lastname: Blais
authors.affiliation: GSK Vaccines, Laval, Canada.
authors.forename: Martin
authors.initials: M
authors.lastname: Gagné
authors.affiliation: ExSAR Corporation, Monmouth Junction, New Jersey, USA.
authors.forename: Yoshitomo
authors.initials: Y
authors.lastname: Hamuro
authors.affiliation: GSK Vaccines, Laval, Canada.
authors.forename: Patrick
authors.initials: P
authors.lastname: Rheault
authors.affiliation: GSK Vaccines, Laval, Canada.
authors.forename: Martine
authors.initials: M
authors.lastname: Boyer
authors.affiliation: GSK Vaccines, Laval, Canada.
authors.forename: Ann-Muriel
authors.initials: AM
authors.lastname: Steff
authors.affiliation: GSK Vaccines, Rixensart, Belgium.
authors.forename: Guy
authors.initials: G
authors.lastname: Baudoux
authors.affiliation: GSK Vaccines, Rixensart, Belgium.
authors.forename: Vincent
authors.initials: V
authors.lastname: Dewar
authors.affiliation: GSK Vaccines, Laval, Canada.
authors.forename: Josée
authors.initials: J
authors.lastname: Demers
authors.affiliation: GSK Vaccines, Rixensart, Belgium.
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Ruelle
authors.affiliation: GSK Vaccines, Laval, Canada.
authors.forename: Denis
authors.initials: D
authors.lastname: Martin
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism; D057134:Antibodies, Neutralizing / Q000378:metabolism; D000914:Antibodies, Viral / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances / Q000648:ultrastructure; D013058:Mass Spectrometry; D008854:Microscopy, Electron; D008958:Models, Molecular; D011485:Protein Binding; D017510:Protein Folding; D055503:Protein Multimerization; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014760:Viral Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; C526850:F protein, human respiratory syncytial virus; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014760:Viral Fusion Proteins
keywords: hydrogen/deuterium exchange mass spectrometry; pre-F conformation; respiratory syncytial virus F protein; vaccine antigen