Characterization of Pre-F-GCN4t, a Modified Human Respiratory Syncytial Virus Fusion Protein Stabilized in a Noncleaved Prefusion Conformation
Autor: | Josée Demers, Ann-Muriel Steff, Vincent Dewar, Yoshitomo Hamuro, Normand Blais, Patrick Rheault, Denis Martin, Martine Boyer, Martin Gagné, Guy Baudoux, Jean-Louis Ruelle |
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Rok vydání: | 2017 |
Předmět: |
Models
Molecular 0301 basic medicine Protein Folding Macromolecular Substances medicine.drug_class Immunology Plasma protein binding Antibodies Viral Monoclonal antibody Microbiology Mass Spectrometry 03 medical and health sciences Antigen Virology Vaccines and Antiviral Agents medicine Humans Neutralizing antibody chemistry.chemical_classification biology Protein Stability Antibodies Monoclonal Antibodies Neutralizing Fusion protein Recombinant Proteins Microscopy Electron 030104 developmental biology chemistry Insect Science biology.protein Protein folding Protein Multimerization Antibody Glycoprotein Viral Fusion Proteins Protein Binding |
Zdroj: | Journal of Virology. 91 |
ISSN: | 1098-5514 0022-538X |
Popis: | 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. IMPORTANCE Human 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. |
Databáze: | OpenAIRE |
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