title: Recombinant Soluble Respiratory Syncytial Virus F Protein That Lacks Heptad Repeat B, Contains a GCN4 Trimerization Motif and Is Not Cleaved Displays Prefusion-Like Characteristics.
abstract: The 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.
authors.affiliation: Virology Division, Department of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht University, 3894 CL, Utrecht, The Netherlands; Mucosis B.V., Meditech Center, L.J. Zielstraweg 1, 9713 GX Groningen, The Netherlands.
authors.forename: Ivy
authors.initials: I
authors.lastname: Widjaja
authors.affiliation: Virology Division, Department of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht University, 3894 CL, Utrecht, The Netherlands; Mucosis B.V., Meditech Center, L.J. Zielstraweg 1, 9713 GX Groningen, The Netherlands.
authors.forename: Alan
authors.initials: A
authors.lastname: Rigter
authors.affiliation: Department of Immunology, University Medical Center Utrecht, 3508 AB, Utrecht, The Netherlands.
authors.forename: Shamir
authors.initials: S
authors.lastname: Jacobino
authors.affiliation: Virology Division, Department of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht University, 3894 CL, Utrecht, The Netherlands.
authors.forename: Frank J M
authors.initials: FJ
authors.lastname: van Kuppeveld
authors.affiliation: Mucosis B.V., Meditech Center, L.J. Zielstraweg 1, 9713 GX Groningen, The Netherlands.
authors.forename: Kees
authors.initials: K
authors.lastname: Leenhouts
authors.affiliation: Centro Nacional de Microbiología and CIBERES, Instituto de Salud Carlos III, Majadahonda, 28220, Madrid, Spain.
authors.forename: Concepción
authors.initials: C
authors.lastname: Palomo
authors.affiliation: Centro Nacional de Microbiología and CIBERES, Instituto de Salud Carlos III, Majadahonda, 28220, Madrid, Spain.
authors.forename: Jose A
authors.initials: JA
authors.lastname: Melero
authors.affiliation: Department of Immunology, University Medical Center Utrecht, 3508 AB, Utrecht, The Netherlands.
authors.forename: Jeanette H W
authors.initials: JH
authors.lastname: Leusen
authors.affiliation: Mucosis B.V., Meditech Center, L.J. Zielstraweg 1, 9713 GX Groningen, The Netherlands.
authors.forename: Bert Jan
authors.initials: BJ
authors.lastname: Haijema
authors.affiliation: Virology Division, Department of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht University, 3894 CL, Utrecht, The Netherlands.
authors.forename: Peter J M
authors.initials: PJ
authors.lastname: Rottier
authors.affiliation: Virology Division, Department of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht University, 3894 CL, Utrecht, The Netherlands.
authors.forename: Cornelis A M
authors.initials: CA
authors.lastname: de Haan
mesh_terms: D057134:Antibodies, Neutralizing / Q000494:pharmacology; D000914:Antibodies, Viral / Q000494:pharmacology*; D001665:Binding Sites; D045744:Cell Line, Tumor; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology / Q000821:virology; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020545:Respiratory Mucosa / Q000378:metabolism* / Q000473:pathology / Q000821:virology; D018113:Respiratory Syncytial Virus, Human / Q000235:genetics* / Q000378:metabolism; D014760:Viral Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D011994:Recombinant Proteins; D014760:Viral Fusion Proteins