Widjaja, IvyRigter, AlanJacobino, Shamirvan Kuppeveld, Frank J MLeenhouts, KeesPalomo-Sanz, ConcepcionMelero, Jose AntonioLeusen, Jeanette H WHaijema, Bert JanRottier, Peter J Mde Haan, Cornelis A M2022-03-282022-03-282015-06PLoS One. 2015 Jun 24;10(6):e0130829.http://hdl.handle.net/20.500.12105/13868The respiratory syncytial virus (RSV) fusion protein F is considered an attractive vaccine candidate especially in its prefusion conformation. We studied whether recombinant soluble RSV F proteins could be stabilized in a prefusion-like conformation by mutation of heptad repeat B (HRB). The results show that soluble, trimeric, non-cleaved RSV F protein, produced by expression of the furin cleavage site-mutated F ectodomain extended with a GCN4 trimerization sequence, is efficiently recognized by pre- as well as postfusion-specific antibodies. In contrast, a similar F protein completely lacking HRB displayed high reactivity with prefusion-specific antibodies recognizing antigenic site Ø, but did not expose postfusion-specific antigenic site I, in agreement with this protein maintaining a prefusion-like conformation. These features were dependent on the presence of the GCN4 trimerization domain. Absence of cleavage also contributed to binding of prefusion-specific antibodies. Similar antibody reactivity profiles were observed when the prefusion form of F was stabilized by the introduction of cysteine pairs in HRB. To study whether the inability to form the 6HB was responsible for the prefusion-like antibody reactivity profile, alanine mutations were introduced in HRB. Although introduction of alanine residues in HRB inhibited the formation of the 6HB, the exposure of postfusion-specific antigenic site I was not prevented. In conclusion, proteins that are not able to form the 6HB, due to mutation of HRB, may still display postfusion-specific antigenic site I. Replacement of HRB by the GCN4 trimerization domain in a non-cleaved soluble F protein resulted, however, in a protein with prefusion-like characteristics, suggesting that this HRB-lacking protein may represent a potential prefusion F protein subunit vaccine candidate.engVoRhttp://creativecommons.org/licenses/by/4.0/Antibodies, NeutralizingAntibodies, ViralBinding SitesCell Line, TumorEpithelial CellsGene ExpressionHEK293 CellsHumansModels, MolecularProtein BindingProtein MultimerizationProtein Structure, TertiaryRecombinant ProteinsRespiratory MucosaRespiratory Syncytial Virus, HumanViral Fusion ProteinsAtribución 4.0 Internacional26107504106e013082910.1371/journal.pone.01308291932-6203PloS Oneopen access