Selection and characterization of human respiratory syncytial virus escape mutants resistant to a polyclonal antiserum raised against the F protein

2012 ◽  
Vol 157 (6) ◽  
pp. 1071-1080 ◽  
Author(s):  
Lorena Tomé ◽  
Sandra Frabasile ◽  
Claudia Candia ◽  
Alvaro Pittini ◽  
Natalia Farina ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Polyclonal Antiserum ◽  
Human Respiratory Syncytial Virus ◽  
F Protein ◽  
Virus Escape ◽  
Escape Mutants ◽  
Syncytial Virus

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

2017 ◽  
Vol 91 (13) ◽  
Author(s):  
Normand Blais ◽  
Martin Gagné ◽  
Yoshitomo Hamuro ◽  
Patrick Rheault ◽  
Martine Boyer ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Antibody Response ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Human Respiratory Syncytial Virus ◽  
Vaccine Antigen ◽  
Significant Advance ◽  
F Protein ◽  
Syncytial Virus

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. 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.

scholarly journals Characterization of the epitope for anti-human respiratory syncytial virus F protein monoclonal antibody 101F using synthetic peptides and genetic approaches

2007 ◽  
Vol 88 (10) ◽  
pp. 2719-2723 ◽  
Author(s):  
Sheng-Jiun Wu ◽  
Albert Schmidt ◽  
Eric J. Beil ◽  
Nicole D. Day ◽  
Patrick J. Branigan ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Antigenic Site ◽  
Neutralizing Antibody ◽  
Human Respiratory Syncytial Virus ◽  
Peptide Sequence ◽  
F Protein ◽  
Syncytial Virus ◽  
A Minor ◽  
Key Residues

Chimeric 101F (ch101F) is a mouse–human chimeric anti-human respiratory syncytial virus (HRSV) neutralizing antibody that recognizes residues within antigenic site IV, V, VI of the fusion (F) glycoprotein. The binding of ch101F to a series of peptides overlapping aa 422–438 spanning antigenic site IV, V, VI was analysed. Residues 423–436 comprise the minimal peptide sequence for ch101F binding. Substitution analysis revealed that R429 and K433 are critical for ch101F binding, whilst K427 makes a minor contribution. Binding of ch101F to a series of single mutations at positions 427, 429 and 433 in the F protein expressed recombinantly on the cell surface confirmed the peptide results. Sequence analysis of viruses selected for resistance to neutralization by ch101F indicated that a single change (K433T) in the F protein allowed ch101F escape. The results confirm that ch101F and palivizumab have different epitope specificity and define key residues for ch101F recognition.

scholarly journals Distinct patterns of within-host virus populations between two subgroups of human respiratory syncytial virus

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Gu-Lung Lin ◽  
Simon B. Drysdale ◽  
Matthew D. Snape ◽  
Daniel O’Connor ◽  
Anthony Brown ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Respiratory Syncytial Virus ◽  
Population Level ◽  
Human Respiratory Syncytial Virus ◽  
Sequencing Data ◽  
F Protein ◽  
Consensus Sequences ◽  
Escape Mutants ◽  
Syncytial Virus ◽  
Acid Alteration

AbstractHuman respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection in young children globally, but little is known about within-host RSV diversity. Here, we characterised within-host RSV populations using deep-sequencing data from 319 nasopharyngeal swabs collected during 2017–2020. RSV-B had lower consensus diversity than RSV-A at the population level, while exhibiting greater within-host diversity. Two RSV-B consensus sequences had an amino acid alteration (K68N) in the fusion (F) protein, which has been associated with reduced susceptibility to nirsevimab (MEDI8897), a novel RSV monoclonal antibody under development. In addition, several minor variants were identified in the antigenic sites of the F protein, one of which may confer resistance to palivizumab, the only licensed RSV monoclonal antibody. The differences in within-host virus populations emphasise the importance of monitoring for vaccine efficacy and may help to explain the different prevalences of monoclonal antibody-escape mutants between the two subgroups.

scholarly journals Trivalency of a Nanobody Specific for the Human Respiratory Syncytial Virus Fusion Glycoprotein Drastically Enhances Virus Neutralization and Impacts Escape Mutant Selection

2016 ◽  
Vol 60 (11) ◽  
pp. 6498-6509 ◽  
Author(s):  
Concepción Palomo ◽  
Vicente Mas ◽  
Laurent Detalle ◽  
Erik Depla ◽  
Olga Cano ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Antigenic Site ◽  
Serial Passage ◽  
Human Respiratory Syncytial Virus ◽  
Escape Mutant ◽  
Comprehensive Description ◽  
Mutant Selection ◽  
Fusion Glycoprotein ◽  
Escape Mutants ◽  
Syncytial Virus

ABSTRACTALX-0171 is a trivalent Nanobody derived from monovalent Nb017 that binds to antigenic site II of the human respiratory syncytial virus (hRSV) fusion (F) glycoprotein. ALX-0171 is about 6,000 to 10,000 times more potent than Nb017 in neutralization tests with strains of hRSV antigenic groups A and B. To explore the effect of this enhanced neutralization on escape mutant selection, viruses resistant to either ALX-0171 or Nb017 were isolated after serial passage of the hRSV Long strain in the presence of suboptimal concentrations of the respective Nanobodies. Resistant viruses emerged notably faster with Nb017 than with ALX-0171 and in both cases contained amino acid changes in antigenic site II of hRSV F. Detailed binding and neutralization analyses of these escape mutants as well as previously described mutants resistant to certain monoclonal antibodies (MAbs) offered a comprehensive description of site II mutations which are relevant for neutralization by MAbs and Nanobodies. Notably, ALX-0171 showed a sizeable neutralization potency with most escape mutants, even with some of those selected with the Nanobody, and these findings make ALX-0171 an attractive antiviral for treatment of hRSV infections.

Molecular characterization of the glycoprotein and fusion protein in human respiratory syncytial virus subgroup A: Emergence of ON-1 genotype in Iran

2019 ◽  
Vol 71 ◽  
pp. 166-178 ◽  
Author(s):  
Somayeh Shatizadeh Malekshahi ◽  
Shaghayegh Razaghipour ◽  
Yazdan Samieipoor ◽  
Farhad B. Hashemi ◽  
Ali Akbar Rahbari Manesh ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Fusion Protein ◽  
Molecular Characterization ◽  
Human Respiratory Syncytial Virus ◽  
Syncytial Virus

scholarly journals RAGE inhibits human respiratory syncytial virus syncytium formation by interfering with F-protein function

2013 ◽  
Vol 94 (8) ◽  
pp. 1691-1700 ◽  
Author(s):  
Jane Tian ◽  
Kelly Huang ◽  
Subramaniam Krishnan ◽  
Catherine Svabek ◽  
Daniel C. Rowe ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Epithelial Cells ◽  
Syncytium Formation ◽  
Human Respiratory Syncytial Virus ◽  
Host Cells ◽  
Type I ◽  
F Protein ◽  
Viral Spread ◽  
Infected Cells ◽  
Syncytial Virus

Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infection. Infection is critically dependent on the RSV fusion (F) protein, which mediates fusion between the viral envelope and airway epithelial cells. The F protein is also expressed on infected cells and is responsible for fusion of infected cells with adjacent cells, resulting in the formation of multinucleate syncytia. The receptor for advanced glycation end products (RAGE) is a pattern-recognition receptor that is constitutively highly expressed by type I alveolar epithelial cells. Here, we report that RAGE protected HEK cells from RSV-induced cell death and reduced viral titres in vitro. RAGE appeared to interact directly with the F protein, but, rather than inhibiting RSV entry into host cells, virus replication and budding, membrane-expressed RAGE or soluble RAGE blocked F-protein-mediated syncytium formation and sloughing. These data indicate that RAGE may contribute to protecting the lower airways from RSV by inhibiting the formation of syncytia, viral spread, epithelial damage and airway obstruction.

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