Characterization of Pre-F-GCN4t, a Modified Human Respiratory Syncytial Virus Fusion Protein Stabilized in a Noncleaved Prefusion Conformation

J Virol. 2017 Jun 9;91(13):e02437-16. doi: 10.1128/JVI.02437-16. Print 2017 Jul 1.


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.

Keywords: hydrogen/deuterium exchange mass spectrometry; pre-F conformation; respiratory syncytial virus F protein; vaccine antigen.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Monoclonal / metabolism
  • Antibodies, Neutralizing / metabolism
  • Antibodies, Viral / metabolism
  • Humans
  • Macromolecular Substances / ultrastructure
  • Mass Spectrometry
  • Microscopy, Electron
  • Models, Molecular
  • Protein Binding
  • Protein Folding
  • Protein Multimerization
  • Protein Stability
  • Recombinant Proteins / chemistry*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology
  • Recombinant Proteins / metabolism*
  • Viral Fusion Proteins / chemistry*
  • Viral Fusion Proteins / genetics
  • Viral Fusion Proteins / immunology
  • Viral Fusion Proteins / metabolism*


  • Antibodies, Monoclonal
  • Antibodies, Neutralizing
  • Antibodies, Viral
  • F protein, human respiratory syncytial virus
  • Macromolecular Substances
  • Recombinant Proteins
  • Viral Fusion Proteins