title: A monomeric uncleaved respiratory syncytial virus F antigen retains prefusion-specific neutralizing epitopes.
abstract: Respiratory syncytial virus (RSV) is the leading infectious cause of severe respiratory disease in infants and a major cause of respiratory illness in the elderly. There remains an unmet vaccine need despite decades of research. Insufficient potency, homogeneity, and stability of previous RSV fusion protein (F) subunit vaccine candidates have hampered vaccine development. RSV F and related parainfluenza virus (PIV) F proteins are cleaved by furin during intracellular maturation, producing disulfide-linked F1 and F2 fragments. During cell entry, the cleaved Fs rearrange from prefusion trimers to postfusion trimers. Using RSV F constructs with mutated furin cleavage sites, we isolated an uncleaved RSV F ectodomain that is predominantly monomeric and requires specific cleavage between F1 and F2 for self-association and rearrangement into stable postfusion trimers. The uncleaved RSV F monomer is folded and homogenous and displays at least two key RSV-neutralizing epitopes shared between the prefusion and postfusion conformations. Unlike the cleaved trimer, the uncleaved monomer binds the prefusion-specific monoclonal antibody D25 and human neutralizing immunoglobulins that do not bind to postfusion F. These observations suggest that the uncleaved RSV F monomer has a prefusion-like conformation and is a potential prefusion subunit vaccine candidate. Importance: RSV is the leading infectious cause of severe respiratory disease in infants and a major cause of respiratory illness in the elderly. Development of an RSV vaccine was stymied when a clinical trial using a formalin-inactivated RSV virus made disease, following RSV infection, more severe. Recent studies have defined the structures that the RSV F envelope glycoprotein adopts before and after virus entry (prefusion and postfusion conformations, respectively). Key neutralization epitopes of prefusion and postfusion RSV F have been identified, and a number of current vaccine development efforts are focused on generating easily produced subunit antigens that retain these epitopes. Here we show that a simple modification in the F ectodomain results in a homogeneous protein that retains critical prefusion neutralizing epitopes. These results improve our understanding of RSV F protein folding and structure and can guide further vaccine design efforts.
authors: Novartis Vaccines, Cambridge, Massachusetts, USA.
authors: Kurt A
authors: KA
authors: Swanson
authors: Novartis Vaccines, Cambridge, Massachusetts, USA.
authors: Kara
authors: K
authors: Balabanis
authors: Novartis Vaccines, Cambridge, Massachusetts, USA.
authors: Yuhong
authors: Y
authors: Xie
authors: Novartis Vaccines, Cambridge, Massachusetts, USA.
authors: Yukti
authors: Y
authors: Aggarwal
authors: Centro Nacional de Microbiología and CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.
authors: Concepción
authors: C
authors: Palomo
authors: Centro Nacional de Microbiología and CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.
authors: Vicente
authors: V
authors: Mas
authors: Novartis Vaccines, Cambridge, Massachusetts, USA.
authors: Claire
authors: C
authors: Metrick
authors: Novartis Vaccines, Cambridge, Massachusetts, USA.
authors: Hui
authors: H
authors: Yang
authors: Novartis Vaccines, Cambridge, Massachusetts, USA.
authors: Christine A
authors: CA
authors: Shaw
authors: Centro Nacional de Microbiología and CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.
authors: José A
authors: JA
authors: Melero
authors: Novartis Vaccines, Cambridge, Massachusetts, USA.
authors: Philip R
authors: PR
authors: Dormitzer
authors: Novartis Vaccines, Cambridge, Massachusetts, USA Centro Nacional de Microbiología and CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain andrea.carfi@novartis.com.
authors: Andrea
authors: A
authors: Carfi
mesh_terms: D057134:Antibodies, Neutralizing / Q000276:immunology*; D000956:Antigens, Viral / Q000276:immunology*; D000939:Epitopes / Q000276:immunology*; D006801:Humans; D059748:Proteolysis; D012136:Respiratory Syncytial Viruses / Q000276:immunology*
chemical_list: D057134:Antibodies, Neutralizing; D000956:Antigens, Viral; D000939:Epitopes