JVI Accepts, published online ahead of print on 30 July 2014 J. Virol. doi:10.1128/JVI.01225-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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A monomeric uncleaved RSV F antigen retains pre-fusion-specific
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neutralizing epitopes
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Kurt A. Swanson,a,* Kara Balabanis,a Yuhong Xie,a Yukti Aggarwal,a Concepción Palomo,b
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Vicente Mas,b Claire Metrick,a,§ Hui Yang,a,& Christine A. Shaw,a José A. Melero,b Philip R.
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Dormitzer,a Andrea Carfia,#
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a
Novartis Vaccines, 45 Sidney Street, Cambridge, MA 02139, USA
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b
Centro Nacional de Microbiología and CIBER de Enfermedades Respiratorias, Instituto de
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Salud Carlos III, Majadahonda, 28220 Madrid, Spain
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#
Address correspondence to: [email protected]
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*Present address: Sanofi, 270 Albany Street, Cambridge, MA 02139-4234, USA
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§
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Molecular Microbiology, Sackler School of Graduate Studies, Tufts University School of
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Medicine, Boston, MA 02111, USA
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&
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Cambridge, MA 02139, USA
Present address: Department of Molecular Biology and Microbiology and Graduate Program in
Present address: Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue,
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Running Head: A monomeric RSV F adopts a pre-fusion-like conformation
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Words Text: 3647
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Words Abstract: 193
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1
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Abstract
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Respiratory syncytial virus (RSV) is the leading cause of severe respiratory disease in
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infants and a major cause of respiratory illness in the elderly. It remains an unmet vaccine need
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despite decades of research. Insufficient potency, homogeneity and stability of previous RSV
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fusion protein (F) subunit vaccine candidates have hampered vaccine development. RSV F and
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related parainfluenza virus (PIV) F proteins are cleaved by furin during intracellular maturation,
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producing disulfide-linked F1 and F2 fragments. During cell entry, the cleaved Fs rearrange from
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pre-fusion trimers to post-fusion trimers. Using RSV F constructs with mutated furin cleavage
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sites, we isolated an uncleaved RSV F ectodomain that is predominantly monomeric and requires
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specific cleavage between F1 and F2 for self-association and rearrangement into stable post-
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fusion trimers. The uncleaved RSV F monomer is folded, homogenous and displays at least two
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key RSV neutralizing epitopes shared between the pre-fusion and post-fusion conformations.
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Unlike the cleaved trimer, the uncleaved monomer binds the pre-fusion-specific monoclonal
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antibody D25 and human neutralizing immunoglobulins that do not bind to post-fusion F. These
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observations suggest that the uncleaved RSV F monomer has a prefusion-like conformation and
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is a potential pre-fusion subunit vaccine candidate.
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Importance
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RSV is the leading cause of severe respiratory disease in infants and a major cause of
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respiratory illness in the elderly. Development of an RSV vaccine was stymied when a clinical
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trial using a formalin inactivated RSV virus made disease, following RSV infection, more severe.
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Recent studies have defined the structures that the RSV F envelope glycoprotein adopts before
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and after virus entry (pre-fusion and post-fusion conformation, respectively). Key neutralization 2
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epitopes of pre-fusion and post-fusion RSV F have been identified, and a number of current
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vaccine development efforts are focused on generating easily produced subunit antigens that
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retain these epitopes. Here we show that a simple modification in the F ectodomain results in a
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homogeneous protein that retains critical pre-fusion neutralizing epitopes. These results improve
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our understanding of RSV F protein folding and structure and can guide further vaccine design
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efforts.
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Introduction
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RSV is a member of the Paramyxoviridae family of RNA viruses, which also includes
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human metapneumovirus, measles virus, mumps virus, Newcastle disease virus (NDV), human
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parainfluenzavirus (PIV) 1-4, and PIV5. RSV is the major cause of bronchiolitis and pneumonia
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in infants. It is the leading cause of infant hospitalization in developed countries and is
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responsible for an estimated 200,000 infant deaths in developing countries each year (1, 2). RSV
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also causes substantial morbidity and mortality among the elderly (3, 4). There is no specific
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antiviral treatment recommended for RSV infection, and the only currently available
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prophylactic is a monoclonal antibody, Palivizumab (Synagis™), used to prevent disease in the
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highest risk infants (5). The cost of Synagis prevents general use, and the need for a vaccine is
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clear. However, despite decades of research there remains no licensed vaccine for RSV.
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Development of a vaccine was stymied in the 1960’s when a formalin-inactivated RSV vaccine
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candidate made subsequent RSV disease more severe (6). Increased structural understanding of
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key RSV neutralization epitopes has supported a resurgence of interest in developing an RSV
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subunit-based vaccine.
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RSV-neutralizing antibodies target the two major RSV surface antigens, the attachment
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protein (G) and the fusion protein (F) (7). G is variable in sequence, whereas F is highly
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conserved between strains, making F the more attractive vaccine antigen. RSV F is a type I viral
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fusion protein, responsible for driving fusion of the viral envelope with host cell membranes
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during viral entry. Crystal structures of RSV F ectodomain trimers have documented two
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conformational states – pre-fusion and post-fusion (Fig. 1C and D) (8-11). In the pre-fusion
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conformation (Fig. 1C), the heptad repeat A (HRA) region is associated with the globular head,
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and the tip of the fusion peptide is mostly buried in the center of the protein. In the post-fusion 4
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conformation (Fig. 1D), HRA and the fusion peptide (not present in the published crystal
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structures (14,18)) have extended from the globular head to attach to the target membrane, and
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the heptad repeat B (HRB) region has rearranged to associate with the HRA region, forming a
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stable 6-helix bundle. This rearrangement places the host membrane bound by the fusion peptide
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and the viral membrane bound by the transmembrane region in close proximity to drive
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membrane fusion. The commercial product Respigam™ (Medimmune), made by purifying
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antibodies from human sera with high RSV-neutralizing titers, was shown to include antibodies
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specific for the pre-fusion F conformation (12). Indeed, depleting Respigam of antibodies that
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bind G and post-fusion F demonstrated that the pre-fusion-specific F antibodies were
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predominantly responsible for virus neutralization by the product. The crystal structure of a RSV
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F ectodomain (Fecto) stabilized by C-terminal addition of a trimerization tag (foldon) bound to the
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FAb of the pre-fusion specific antibody D25 identified a new antigenic site designated site Ø (9).
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Site Ø is formed in pre-fusion RSV Fecto by the packing of the HRA region against the rest of the
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F globular head (a structural feature not shared by post-fusion F; Fig. 1C and D). In addition, a
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pre-fusion RSV Fecto antigen with a trimerization tag and mutations that stabilized the HRA-
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globular head interactions elicited a higher neutralizing titer than did post-fusion Fecto in mice
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and non-human primates (11).
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Fusion proteins are expressed as uncleaved F0 precursors (Fig. 1A and B). Crystal structures
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of uncleaved PIV3, PIV5 and NDV Fecto revealed that furin cleavage is not required for these
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proteins to trimerize (13-15). PIV3 Fecto was crystalized in the post-fusion conformation whereas
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PIV5 Fecto was trapped in the pre-fusion conformation by the addition of a C-terminal GCN
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trimerization tag (14). Unlike PIV Fs, which each contain a single furin cleavage site, RSV F has
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two sites separated by a 27-amino acid fragment, p27 (Fig. 1B). Activation of RSV F for 5
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membrane fusion requires cleavage by furin at the two sites (16). Our initial attempts to generate
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a pre-fusion RSV Fecto trimer relied on mutating the furin cleavage sites and adding a GCN
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trimerization tag, similar to the strategy used to generate the pre-fusion PIV5 Fecto (14). However,
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addition of the GCN trimerization domain to the uncleaved RSV Fecto greatly reduced the amount
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of secreted protein. In the course of these manipulations we observed that uncleaved RSV Fecto
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was predominantly monomeric in solution. We demonstrate that this monomeric species is a
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folded, soluble protein which harbors key neutralizing epitopes including the site Ø pre-fusion
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epitope. This uncleaved species might represent a potent pre-fusion Fecto vaccine candidate.
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Materials and Methods
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Fusion protein constructs design, expression and purification
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RSV Fecto constructs with mutations to the furin cleavage sites and fusion peptide region
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(residues 109-148) were designed (Fig. 1A and B). ∆p27 furinmut Fecto contains a deletion of 23
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residues from the p27 fragment and point mutations to the furin cleavage sites. These point
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mutations prevent cleavage by intercellular furin but permit in vitro cleavage by trypsin. ∆FP
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furinwt Fecto retains the wild-type furin cleavage sites but has a fusion peptide deletion. Unlike
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∆p27 furinmut Fecto, which is purified as an uncleaved F0 species, ∆FP furinwt Fecto is cleaved by
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the cell into the F1/F2 species. DNA constructs encoding Fecto with these mutations and a C-
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terminal histidine tag were synthesized (Geneart) and cloned into the pFastBac baculovirus
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system (Invitrogen). RSV Fecto constructs were expressed in 1 L cultures for three days and
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purified by nickel affinity followed by size exclusion chromatography (GE Healthcare, Superdex
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P200). For limited proteolysis studies, Δp27 furinmut RSV Fecto was digested with trypsin (Sigma)
6
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at a weight ratio of 1:1000 trypsin to Δp27 furinmut RSV Fecto, and the resulting cleaved protein
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was purified from a size exclusion column void volume.
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Size exclusion chromatography and multi-angle light scattering (SEC-MALS)
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Size exclusion high pressure liquid chromatography (HPLC) was performed using a Waters
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2685 Separations Module HPLC with an isocratic flow rate of 0.75 mL/min coupled to a multi-
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angle light scattering (MALS) system. The mobile phase consisted of 2x PBS. The size exclusion
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column (Wyatt Technologies WTC-030S5) was used at ambient temperature with a run time of
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30 min. UV was detected at 280 nm (Waters PDA 2996), and a light scattering detector (Wyatt
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Technologies miniDawn TREOS) using a laser wavelength of 690.0 nm and a refractive index
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detector (Wyatt Technologies Optilab rEX) were added in series. A gel filtration standard
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solution (Bio-Rad) was used to generate a Stokes radius standard curve. Immediately prior to the
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measurement of the samples, the system was calibrated using a known BSA (bovine serum
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albumin) solution.
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A 100 μL injection for a target column load of 100 μg protein (RSV Fecto alone or 1:1 molar
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ratio RSV Fecto:FAb) was used. Peak retention times were used to estimate the molecular mass of
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RSV Fecto or RSV Fecto:FAb complexes based on the Stokes radius standard curve. Protein
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concentrations for identified peaks were calculated using calculated UV extinction coefficients,
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and the molecular masses for RSV Fecto or RSV Fecto:FAb complexes were determined from
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multi-angles light scattering data using Astra 5.3.4 software (Wyatt Technologies).
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Binding studies by surface plasmon resonance (SPR)
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The affinity of RSV Fecto constructs for Motavizumab or 101F FAbs was measured by SPR with a Biacore T100 instrument. FAbs were directly immobilized on CM5 sensor chips using 7
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amine coupling at very low levels (75 response units) and RSV Fecto construct preparations at
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various concentrations were injected at a high flow rate (50 μL/min) to avoid avidity effects and
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higher than 1:1 binding interaction. The data were processed using Biacore T100 evaluation
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software and double referenced by subtraction of the blank surface and buffer-only injection
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before global fitting of the data to a 1:1 binding model.
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Depletion of Respigam antibodies
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Antibody depletion was carried out by a method similar to that previously described (17).
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Briefly, the Δp27 furinmut Fecto and ΔFP furinwt Fecto preparations were bound covalently to
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CNBr-activated Sepharose 4g (GE Healthcare) following the manufacturer’s instructions (500
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µg of protein/160 mg Sepharose). The resins were packed into columns that were equilibrated in
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PBS. Three mg of Respigam were loaded on each of these columns. The unbound material was
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collected (referred to as Fecto-depleted Respigam). After washing the column with PBS, bound
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antibodies were eluted with glycine-HCl pH 2.5 and immediately neutralized with saturated Tris,
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concentrated and buffer exchanged to phosphate-buffered saline (PBS). The eluted antibodies
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are referred to as Fecto-captured Respigam.
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Enzyme-linked immunosorbent assay (ELISA) with depleted Respigam antibodies
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ELISA of depleted antibodies was performed as previously described (12). Briefly, purified
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101F antibody was used to coat 96 well microtiter plates overnight at 4ºC. Nonspecific antibody
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binding was blocked with 2% porcine serum in PBS with 0.05% Tween 20. An excess of
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purified proteins (either Δp27 furinmut Fecto or ΔFP furinwt Fecto, 2 µg/well) was added to each
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well, and incubation continued for 1 hour at 37ºC followed by addition of serially diluted
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antibodies and incubation for 1 hour at 37ºC. The plates were extensively washed with water 8
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after each step. Finally, bound antibodies were detected with peroxidase-labelled rabbit anti-
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human immunoglobulins and o-phenylenediamine (OPD) as substrate (GE Healthcare). The
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reaction was stopped with 2N sulfuric acid, and absorbance was read at 490 nm.
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Neutralization assay using depleted Respigam antibodies
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RSV neutralization by depleted Respigam antibodies was performed using a
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microneutralization assay described originally by Anderson et al.,1988 and modified by Martínez
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and Melero, 1998 (18, 19). Briefly, immunoglobulin dilutions were incubated with 1x105 plaque
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forming units (pfu) of the RSV Long strain for 30 minutes at 37ºC in a total volume of 50 µl.
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These mixtures were used to infect 1x105 HEp-2 cells growing in 96 well plates with Dulbecco’s
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modified Eagle’s medium (DMEM) supplemented with 2.5% fetal calf serum (FCS) that had
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been inactivated 30 minutes at 60ºC. After one hour adsorption, DMEM with 2.5% FCS was
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added, and the cultures were incubated 72 hours at 37ºC with 5% CO2. The plates were washed
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three times with 0.05% Tween 20 in PBS and fixed with 80% cold acetone in PBS. After air
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drying, viral antigen production in the fixed monolayers was measured by ELISA with a pool of
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anti-G and anti-F murine antibodies and a horseradish peroxidase (HRP)-labelled anti-mouse
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immunoglobulin preparation (Sigma).
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Electron microscopy
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RSV Fecto preparations (30 μg/mL in 25 mM Tris, 300 mM NaCl) were absorbed onto a 400-
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mesh carbon-coated grid (Electron Microscopy Sciences) and stained with 0.75% uranyl formate.
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A JEOL 1200EX microscope, operated at 80 kV, was used to analyze the samples. Micrographs
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were taken at 65,000× magnification.
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Results
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∆P27 furinmut and ∆FP furinwt Fecto oligomerization states by SEC-MALS
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PIV F ectodomains with furin cleavage site mutations form trimers in the post-fusion F
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trimer conformation (13-15, 20). To determine if ∆P27 furinmut Fecto similarly formed trimers, we
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analyzed its oligomerization state with analytical SEC coupled with MALS (Fig. 2). ∆P27
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furinmut Fecto principally eluted from the analytical column at a retention volume indicating a
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monomer, as estimated by Stokes radius. In addition, MALS analysis of the principal ∆P27
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furinmut Fecto peak gave a mass consistent with a monomer. A minor peak in the ∆P27 furinmut
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Fecto analysis ran consistent with the retention time of a trimer, suggesting some protein in this
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preparation was competent to for trimers in solution (red arrow in Fig. 2). For comparison, we
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performed SEC-MALS analysis of ∆FP furinwt Fecto, which is known by its crystal structure and
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EM analysis to be a post-fusion trimer (8). ∆FP furinwt Fecto eluted at a retention time consistent
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with a trimer, and analysis of the protein peak by MALS gave a mass consistent with a trimer
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(Fig. 2).
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Motavizumab and 101F FAb binding to ΔP27 furinmut Fecto by SPR and SEC
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We tested whether ∆P27 furinmut Fecto binds FAbs from two structurally-characterized
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neutralizing antibodies (site II (defined as Site A in (5)) binding Motavizumab and site IV
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(defined as Site C in (5)) binding 101F) using SPR (Fig. 3). Both ∆P27 furinmut and ∆FP furinwt
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Fecto bound tightly to Motavizumab and 101F FAbs. The KDs for the ∆P27 furinmut Fecto-
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Motavizumab FAb interaction (3.23 x 10-11 M) and the ∆FP furinwt Fecto-Motavizumab FAb
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interaction (2.05 x 10-11 M) were similar, as were the KDs for the ∆P27 furinmut Fecto-101F FAb
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(4.19 x 10-10 M) and ∆FP furinwt Fecto-101F FAb (1.54 x 10-10 M) interactions. 10
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To determine if FAb binding could stabilize trimerization of ∆P27 furinmut Fecto, we
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incubated the ∆P27 furinmut Fecto with a molar equivalent of either Motavizumab or 101F FAb
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and analyzed the resulting complex by SEC-MALS (Fig. 4). The retention times of the principal
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peaks for the ∆P27 furinmut Fecto-Motavizumab FAb complex and of the ∆P27 furinmut Fecto-101F
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FAb complex were consistent with 1:1 F monomer:FAb species as determined by Stokes radius
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(Fig. 4A). MALS analysis also gave masses consistent with a 1:1 F monomer:FAb complexes.
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For comparison, Stokes radius and MALS analysis for the peaks from ∆FP furinwt Fecto-FAb
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complexes were consistent with 3:3 F-FAb complexes (or an F trimer bound to three FAbs) (Fig.
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4B). As previously discussed, a shoulder peak with a retention time consistent with an F trimer
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was observed in ∆P27 furinmut Fecto preparations and corresponding shoulder peaks (indicated by
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a red arrow) were observed in the ∆P27 furinmut Fecto-FAb complex preparations with a retention
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time consistent with F trimers bound by three FAbs.
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∆P27 furinmut Fecto binding to D25 FAb by SPR and SEC
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D25 FAb binds the pre-fusion Fecto but does not bind post-fusion Fecto (9). To determine if
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∆P27 furinmut Fecto is competent to bind the pre-fusion site Ø-specific D25 FAb, we tested
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binding by SPR and SEC (Fig. 5). The KD for the ∆P27 furinmut Fecto-D25 FAb interaction was
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2.38 x 10-11 M. In the RSV Fecto -D25 crystal structure, the D25 FAb has principal contacts with
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a single protomer and modest contacts with an adjacent protomer, suggesting that binding of D25
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might occur with F trimers rather than monomers (9). To determine if binding to D25 FAb
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induced trimerization of ∆P27 furinmut Fecto, we incubated the F protein with a molar equivalent
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of D25 FAb and analyzed the complex by SEC-MALS (Fig. 5B). The retention time and Stokes
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radius of the D25-bound ∆P27 furinmut Fecto was consistent with a 1:1 F monomer:FAb species.
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Similarly, MALS analysis of the D25-bound ∆P27 furinmut Fecto gave a mass consistent with a 1:1 11
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F monomer:FAb species. We conclude that binding to D25 FAb does not induce trimerization of
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∆P27 furinmut Fecto.
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Respigam antibodies binding to ∆P27 Furinmut Fecto
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To determine if ∆P27 furinmut Fecto is competent to bind pre-fusion-specific human
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antibodies we performed binding experiments using Respigam-depletion assays similar to those
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published in Magro et al. 2012 (12). ∆P27 furinmut Fecto or ∆FP furinwt Fecto (post-fusion F trimer)
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was immobilized on resin, and the protein-bound resin was incubated with Respigam. Antibodies
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that did not bind the F protein were collected in the column flow-though (referred to as ∆P27
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furinmut Fecto-depleted Respigam or ∆FP furinwt Fecto -depleted Respigam, respectively).
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Antibodies that bound ∆P27 furinmut Fecto and were eluted are referred to as ∆P27 furinmut Fecto-
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captured Respigam. Untreated Respigam is able to bind both ∆P27 furinmut Fecto and ∆FP furinwt
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Fecto (Fig. 6A). To demonstrate that antibodies that specifically bind ∆P27 furinmut exist in
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Respigam, ∆FP furinwt Fecto–depleted Respigam was tested in ELISA for its ability to bind ∆P27
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furinmut Fecto (Fig. 6B). Although ∆FP furinwt Fecto–depleted Respigam had no significant binding
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to ∆FP furinwt Fecto (demonstrating sufficient depletion of the sera) ∆FP furinwt Fecto–depleted
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Respigam retained binding for ∆P27 furinmut Fecto (i.e. monomeric F). For comparison, ∆P27
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furinmut Fecto–depleted Respigam showed no significant binding to either ∆FP furinwt Fecto or
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∆P27 furinmut Fecto (data not shown).
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It has been previously shown that post-fusion F-depleted Respigam retained the majority of
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its neutralizing titer, and pre-fusion F-depleted Respigam had greatly diminished neutralizing
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titer (12). Furthermore, the antibodies that bind pre-fusion F represent a greater fraction of
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Respigam neutralizing titer (12). To test whether the antibodies that bind ∆P27 furinmut Fecto
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represent the majority of the neutralizing titer of Respigam, we tested the neutralizing titer of 12
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∆P27 furinmut Fecto-depleted versus ∆P27 furinmut Fecto-captured Respigam (Fig 6C). Untreated
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Respigam neutralized RSV infectivity well relative to the poorly neutralizing polyclonal
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antibodies purified from a non-immunized rabbit (negative control). ∆P27 furinmut Fecto-depleted
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Respigam has a dramatically lower neutralizing titer than Respigam, similar to the observations
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for pre-fusion F-depleted Respigam (12). ∆P27 furinmut Fecto -captured Respigam had a higher
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neutralizing titer per mass IgG than untreated Respigam. Taken together, the data indicate that
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most of the neutralizing activity of Respigam is from antibodies that bind to ∆P27 furinmut Fecto
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(i.e. monomeric F) but not to the post-fusion ∆FP furinwt Fectoand that this monomeric RSV F
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ecto-domain retains pre-fusion F specific epitopes.
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Limited-proteolysis of ∆P27 furinmut Fecto
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Uncleaved post-fusion PIV trimers (F0) form rosettes, self-associated by aggregation of
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their fusion peptides, after in vitro cleavage into the F1/F2 species (20). To determine if
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uncleaved ∆P27 furinmut Fecto was competent to form rosettes of post-fusion trimers upon in vitro
267
cleavage, ∆P27 furinmut Fecto was digested with trypsin (Fig. 7A). Upon treatment with trypsin
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uncleaved ∆P27 furinmut Fecto was cleaved into the native F1/F2 species of wild-type Fecto, further
269
suggesting that the protein retains a mostly globular fold. Cleaved ∆P27 furinmut Fecto was further
270
purified from the SEC void volume and analyzed by electron microscopy (Fig. 7B). ∆FP furinwt
271
Fecto was previously described as mono-dispersed elongated crutches (8); however, cleaved ∆P27
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furinmut Fecto forms rosettes of elongated crutches similar to the post-fusion rosettes described for
273
RSV and paramyxoviruses (20, 21). The observations that ∆P27 furinmut Fecto is resistant to
274
degradation by limited proteolysis and competent to forms rosettes of post-fusion F trimers
275
suggest that the uncleaved ∆P27 furinmut Fecto (i.e.. monomeric F) is a well-folded pre-fusion-like
276
molecule structurally competent to rearrange into the post-fusion conformation (Fig. 7C). We 13
277
also attempted to observe the monomeric RSV F by electron microscopy, but we were
278
unsuccessful, which is likely due to the protein’s relatively small size (i.e. 65 KDa).
279
Discussion
280
RSV remains one of the most important, unmet vaccine needs. Renewed interest in
281
developing an RSV F subunit-based vaccine was triggered first by information from the
282
structurally-characterized PIV F proteins and now from structural studies of RSV F itself. Recent
283
data have demonstrated that RSV Fecto stabilized in a pre-fusion conformation elicits higher
284
neutralizing titers than post-fusion RSV Fecto (11, 12), and there is high interest in developing a
285
pre-fusion RSV F subunit vaccine. The novel, pre-fusion-specific site Ø epitope, recognized by
286
the D25 FAb and formed by the HRA region folding against the globular head, appears to be
287
largely responsible for the improved immunogenicity (9). For this reason, antigens presenting the
288
site Ø epitope will likely become components of next generation RSV vaccine candidates.
289
Our initial attempts to generate a stable pre-fusion RSV Fecto antigen focused on the strategy
290
used to stabilize PIV5 F in its metastable, pre-fusion conformation (data not shown) (14). We
291
attempted to trimerize an uncleaved (furin sites mutated) or cleaved (wild type furin sites) RSV
292
Fecto using a C-terminal GCN trimerization domain, but found the protein secretion from the cell
293
was greatly reduced. We similarly tried expressing a cleaved RSV Fecto using a C-terminal foldon
294
tag but this protein formed rosettes similar to a post-fusion Fecto. For this reason, we examined
295
the oligomerization states of RSV F proteins without trimerization tags. SEC-MALS
296
demonstrated that uncleaved ∆P27 furinmut Fecto is predominantly a monomer in solution (Fig. 2).
297
This observed difference in the cleavage-dependence of PIV F and RSV F trimerization may be
298
linked to the presence of a single furin cleavage site in PIV F but two cleavage sites in RSV F, 14
299
flanking a p27 insertion (Fig. 1). The uncleaved, monomeric Fecto (∆P27 furinmut Fecto) has some
300
features consistent with the post-fusion trimer (∆FP furinwt Fecto). Specifically, two neutralizing
301
epitopes present on both pre-fusion and post-fusion Fecto, sites II and IV, are available for binding
302
by their respective FAbs, from Motavizumab and 101F, as demonstrated by SPR (Fig. 3) and
303
SEC-MALS (Fig. 4). A linear peptide harboring the Motavizumab epitope is capable of
304
transiently adopting its native conformation and being stabilized by FAb binding; however, the
305
affinity of the FAb-peptide interaction is ~6,000-fold less than the folded post-fusion protein-
306
FAb affinity (10). Thus, the tight binding of uncleaved, monomeric Fecto to the Motavizumab
307
FAb suggests the epitope is pre-formed on the protein surface, as it is on the trimer (Fig. 3).
308
SEC-MALS indicates that, upon binding either the Motavizumab FAb or the 101F FAb, the
309
monomer does not self-assemble into a trimer (Fig. 4). This finding suggests that the constraints
310
preventing trimerization of the uncleaved monomer are not relieved by FAb binding.
311
Further evidence suggests that the RSV Fecto monomer is a folded species. When the RSV Fecto
312
monomer is treated with trypsin, it is cleaved into its biologically relevant F1/F2 species. This
313
limited and specific proteolysis suggests that the RSV Fecto monomer has a globular fold,
314
protecting it from non-specific trypsin digestion. Upon cleavage into F1/F2, RSV Fecto
315
spontaneously self-associates into rosettes of post-fusion trimers (Fig. 7B). This behavior is
316
analogous to the rosette formation observed for the PIV Fs (20).
317
If the RSV Fecto monomer is in a pre-fusion-like conformation, the HRA region would be
318
packed against the globular head, presenting novel HRA epitopes lost in the post-fusion
319
conformation (Fig. 7C). Unlike the post-fusion Fecto trimer, the Fecto monomer is capable of
320
binding the site Ø, pre-fusion-specific, FAb D25 (Fig. 5). Furthermore, the Fecto monomer binds
321
antibodies in the human sera-derived Respigam commercial product that do not bind the post15
322
fusion Fecto antigen (Fig. 6B). Finally, previous studies have shown that post-fusion Fecto antigens
323
are unable to bind and deplete the majority of neutralizing activity from Respigam (12).
324
However, the Fecto monomer is able to bind and deplete the majority of neutralizing antibodies
325
from Respigam, suggesting that the Fecto monomer retains the important site Ø epitopes present
326
in other pre-fusion antigens and presumably on the RSV F surface on the virion.
327
Retention of site Ø is a desired characteristic of next generation RSV F antigens, and
328
therefore the uncleaved RSV F monomer here described represents a potential pre-fusion subunit
329
vaccine candidate. However, the RSV F monomer described here readily refolds in the post-
330
fusion conformation in presence of trace amounts of trypsin or contaminant trypsin-like
331
proteases and, therefore, is not stable enough for evaluation in immunization studies. Future
332
work will be aimed at stabilizing the RSV F monomer by introducing additional mutations that
333
lock jt more stably in a pre-fusion conformation amenable to in vivo studies.
334 335
Acknowledgements
336
Work in Madrid was supported in part by grant SAF2012-31217 from Plan Nacional I+D+I
337
(Ministerio de Economía y Competitividad).
16
338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383
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Nair H, Nokes DJ, Gessner BD, Dherani M, Madhi SA, Singleton RJ, O'Brien KL, Roca A, Wright PF, Bruce N, Chandran A, Theodoratou E, Sutanto A, Sedyaningsih ER, Ngama M, Munywoki PK, Kartasasmita C, Simoes EA, Rudan I, Weber MW, Campbell H. 2010. Global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis. Lancet 375:1545-1555. Hall CB. 2010. Respiratory syncytial virus, p. 2207-2221. In Mandell GL BJ, Dolin R (ed.), Mandell, Douglas, and Bennett's Principles and Practice of Infectious Disease, vol. 7th edition, Churchill Livingstone, New York. Thompson WW, Shay DK, Weintraub E, Brammer L, Cox N, Anderson LJ, Fukuda K. 2003. Mortality associated with influenza and respiratory syncytial virus in the United States. JAMA : the journal of the American Medical Association 289:179-186. Han LL, Alexander JP, Anderson LJ. 1999. Respiratory syncytial virus pneumonia among the elderly: an assessment of disease burden. The Journal of infectious diseases 179:25-30. Johnson S, Oliver C, Prince GA, Hemming VG, Pfarr DS, Wang SC, Dormitzer M, O'Grady J, Koenig S, Tamura JK, Woods R, Bansal G, Couchenour D, Tsao E, Hall WC, Young JF. 1997. Development of a humanized monoclonal antibody (MEDI-493) with potent in vitro and in vivo activity against respiratory syncytial virus. The Journal of infectious diseases 176:1215-1224. Kim HW, Canchola JG, Brandt CD, Pyles G, Chanock RM, Jensen K, Parrott RH. 1969. Respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine. American journal of epidemiology 89:422-434. Anderson R, Huang Y, Langley JM. 2010. Prospects for defined epitope vaccines for respiratory syncytial virus. Future microbiology 5:585-602. Swanson KA, Settembre EC, Shaw CA, Dey AK, Rappuoli R, Mandl CW, Dormitzer PR, Carfi A. 2011. Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers. Proceedings of the National Academy of Sciences of the United States of America 108:9619-9624. McLellan JS, Chen M, Leung S, Graepel KW, Du X, Yang Y, Zhou T, Baxa U, Yasuda E, Beaumont T, Kumar A, Modjarrad K, Zheng Z, Zhao M, Xia N, Kwong PD, Graham BS. 2013. Structure of RSV Fusion Glycoprotein Trimer Bound to a Prefusion-Specific Neutralizing Antibody. Science. McLellan JS, Yang Y, Graham BS, Kwong PD. 2011. Structure of respiratory syncytial virus fusion glycoprotein in the postfusion conformation reveals preservation of neutralizing epitopes. Journal of virology 85:7788-7796. McLellan JS, Chen M, Joyce MG, Sastry M, Stewart-Jones GB, Yang Y, Zhang B, Chen L, Srivatsan S, Zheng A, Zhou T, Graepel KW, Kumar A, Moin S, Boyington JC, Chuang GY, Soto C, Baxa U, Bakker AQ, Spits H, Beaumont T, Zheng Z, Xia N, Ko SY, Todd JP, Rao S, Graham BS, Kwong PD. 2013. Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus. Science 342:592-598. Magro M, Mas V, Chappell K, Vazquez M, Cano O, Luque D, Terron MC, Melero JA, Palomo C. 2012. Neutralizing antibodies against the preactive form of respiratory syncytial virus fusion protein offer unique possibilities for clinical intervention. Proceedings of the National Academy of Sciences of the United States of America 109:3089-3094. Yin HS, Paterson RG, Wen X, Lamb RA, Jardetzky TS. 2005. Structure of the uncleaved ectodomain of the paramyxovirus (hPIV3) fusion protein. Proceedings of the National Academy of Sciences of the United States of America 102:9288-9293. 17
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Yin HS, Wen X, Paterson RG, Lamb RA, Jardetzky TS. 2006. Structure of the parainfluenza virus 5 F protein in its metastable, prefusion conformation. Nature 439:38-44. Swanson K, Wen X, Leser GP, Paterson RG, Lamb RA, Jardetzky TS. 2010. Structure of the Newcastle disease virus F protein in the post-fusion conformation. Virology 402:372-379. Gonzalez-Reyes L, Ruiz-Arguello MB, Garcia-Barreno B, Calder L, Lopez JA, Albar JP, Skehel JJ, Wiley DC, Melero JA. 2001. Cleavage of the human respiratory syncytial virus fusion protein at two distinct sites is required for activation of membrane fusion. Proceedings of the National Academy of Sciences of the United States of America 98:9859-9864. Sastre P, Melero JA, Garcia-Barreno B, Palomo C. 2005. Comparison of affinity chromatography and adsorption to vaccinia virus recombinant infected cells for depletion of antibodies directed against respiratory syncytial virus glycoproteins present in a human immunoglobulin preparation. Journal of medical virology 76:248-255. Anderson LJ, Bingham P, Hierholzer JC. 1988. Neutralization of respiratory syncytial virus by individual and mixtures of F and G protein monoclonal antibodies. Journal of virology 62:42324238. Martinez I, Melero JA. 1998. Enhanced neutralization of human respiratory syncytial virus by mixtures of monoclonal antibodies to the attachment (G) glycoprotein. The Journal of general virology 79 ( Pt 9):2215-2220. Connolly SA, Leser GP, Yin HS, Jardetzky TS, Lamb RA. 2006. Refolding of a paramyxovirus F protein from prefusion to postfusion conformations observed by liposome binding and electron microscopy. Proceedings of the National Academy of Sciences of the United States of America 103:17903-17908. Calder LJ, Gonzalez-Reyes L, Garcia-Barreno B, Wharton SA, Skehel JJ, Wiley DC, Melero JA. 2000. Electron microscopy of the human respiratory syncytial virus fusion protein and complexes that it forms with monoclonal antibodies. Virology 271:122-131.
409 410
18
411
Figure legends
412
Figure 1. RSV F ectodomain structure. (A) Linear diagram listing residue numbers
413
corresponding to the N terminus of each segment. The furin cleavage sites (arrowheads) divide
414
the protein into F1 and F2. DI-III, domains I-III; p27, excised peptide; FP, fusion peptide; HRA,
415
-B, and –C heptad repeats A, B, and C are indicated. (B) Sequences showing the mutations
416
introduced into p27 and FP region of Fecto constructs. Top, wild-type RSV F sequence with p27
417
and fusion peptide unaltered. Middle, ∆P27 furinmut Fecto sequence with mutations to the furin
418
sites and deletion of p27 residues. Bottom, ∆FP furinwt Fecto sequence with fusion peptide
419
residues deleted. Deleted residues shown as hyphens. (C) Surface representation of the pre-
420
fusion RSV Fecto (9). One subunit colored by domains and heptad repeat regions as in A; the
421
other two are white and gray. (D) Surface representation of the post-fusion RSV Fecto (8), colored
422
as in C to highlight the rearrangement of domains and heptad repeat regions after conformational
423
change. Notice the HRA (red) largely buried by the HRB (blue) upon forming the six helix
424
bundle (6HB).
425
Figure 2. Analytical size exclusion chromatography analysis of RSV Fecto constructs. Top,
426
open circles represent the chromatogram of ∆P27 furinmut Fecto in which closed circles represent
427
the chromatogram of ∆FP furinwt Fecto. The principal peak of the cleaved ∆FP furinwt Fecto is
428
approximately 10.5 minutes. The principal peak of the uncleaved ∆P27 furinmut Fecto is
429
approximately 11.2 minutes, with a minor shoulder peak at approximately 10.5 minutes (arrow).
430
Bottom, masses of Fecto constructs measured by SEC-MALS. The molecular masses estimated by
431
the protein sequence for a monomer ∆P27 furinmut Fecto and trimer ∆FP furinwt Fecto are shown in
432
the first column. The masses for the RSV Fecto constructs as measured by Stokes radius and
433
MALS analysis are shown in second and third columns, respectively. 19
434
Figure 3. Binding studies by SPR of Fecto and FAbs. 101F and Motavizumab FAbs were
435
immobilized on a sensor chip, and varying concentrations of Fecto were injected with mass
436
absorption response units (RU) monitored as a function of time (sensorgrams are labeled
437
according to Fecto construct, FAb chip, and concentration as indicated). Fitting the resulting
438
curves to a 1:1 binding stoichiometry resulted in ka and kd and fit values as indicated in the
439
table.
440
Figure 4. Analytical size exclusion chromatography analysis of RSV Fecto constructs bound
441
to FAbs. (A) Black circles represent the chromatogram of ∆P27 furinmut Fecto where blue and red
442
circles represent the chromatogram of 101F FAb-bound and Motavizumab FAb-bound ∆P27
443
furinmut Fecto, respectively. Upon FAb binding the principal peak of ∆P27 furinmut Fecto shifts from
444
approximately 11.2 minutes to 10.7 minutes. An arrow shows a shoulder peak shifting to a
445
retention time of approximately 9.5 minutes. (B) Chromatogram of ∆FP furinwt Fecto with or
446
without FAb binding, colored as in A. Upon FAb binding the principal peak of ∆FP furinwt Fecto
447
shifts from approximately 10.5 minutes to approximately 9.5 minutes. C) Masses of Fecto
448
constructs predicted by the protein sequence for a monomer ∆FP furinwt Fecto and trimer ∆P27
449
furinmut Fecto bound to a single or three FAbs, respectively, are shown in the first column. The
450
masses for the RSV Fecto constructs bound to FAbs as measured by Stokes radius and MALS
451
analysis are shown in columns two and three, respectively.
452
Figure 5. Pre-fusion-specific D25 FAb binding to ∆P27 furinmut Fecto by SPR and SEC-
453
MALS. (A) D25 FAb was immobilized on a sensor chip, and varying concentrations of ∆P27
454
furinmut Fecto were injected with mass absorption response units (RU) monitored as a function of
455
time (Fecto concentrations are indicated). Fitting the resulting curves to a 1:1 binding 20
456
stoichiometry resulted in ka and kd and fit values as indicated in the table below. The resulting
457
KD for D25 FAb binding to ∆P27 furinmut Fecto is 2.38 x 10-11 M. (B) Top, SEC chromatogram of
458
∆P27 furinmut Fecto. Bottom, chromatogram of D25 FAb-bound ∆P27 furinmut Fecto. Arrows
459
indicate the expected retention time of RSV Fecto monomer (FM), unbound FAb (FAb), and RSV
460
Fecto monomer bound to FAb (FM+FAb). Below, masses of unbound ∆P27 furinmut Fecto or FAb-
461
bound ∆P27 furinmut Fecto predicted by sequence are shown in the first column. The measured
462
masses for ∆P27 furinmut Fecto and D25 FAb-bound ∆P27 furinmut Fecto by Stokes radius and
463
MALS analysis are shown in the second and third columns, respectively.
464
Figure 6. ∆P27 furinmut Fecto is recognized by unique neutralizing antibodies of Respigam.
465
(A) ∆FP furinwt Fecto or ∆P27 furinmut Fecto was adsorbed on an ELISA plate, and different
466
concentrations of Respigam were analyzed for binding. Respigam bound both ∆FP furinwt Fecto
467
(circles) and ∆P27 furinmut Fecto (squares) similarly. (B) ∆FP furinwt Fecto or ∆P27 furinmut Fecto
468
was adsorbed on an ELISA plate and ∆FP furinwt Fecto-depleted Respigam was analyzed at
469
different concentrations for binding. ∆FP furinwt Fecto-depleted-Respigam did not significantly
470
bind ∆FP furinwt Fecto (circles) but retained significant binding to ∆P27 furinmut Fecto (squares). (C)
471
∆P27 furinmut Fecto-depleted and –daptured Respigam were tested for neutralizing titer. Relative
472
to the strongly neutralizing, untreated Respigam (circles), antibodies from ∆P27 furinmut Fecto–
473
depleted Respigam (squares) retain a poorer neutralizing titer whereas antibodies from ∆P27
474
furinmut Fecto–captured Respigam (diamonds) retain a higher neutralizing titer per mass IgG
475
(plotted on the x-axis).
476
Figure 7. Limited proteolysis of ∆P27 furinmut Fecto induces protein trimerization and
477
association by the fusion peptide. (A) SDS-PAGE gel showing limited proteolysis of RSV F 21
478
constructs. RSV ∆P27 furinmut Fecto prior to digestion with trypsin is labeled as “Monomer” and
479
produces the F0 band. Two lots of RSV ∆FP furinwt Fecto are labeled as “Trimer” and produce F1
480
and F2 bands in the absence of trypsin. RSV ∆P27 furinmut Fecto produces F1 and F2 bands after
481
digestion with trypsin. (B) Electron microscopy of the RSV Fecto rosettes formed spontaneously
482
by trypsin treatment of ∆P27 furinmut Fecto. The rosettes have the elongated crutch shape of post-
483
fusion RSV Fecto and proteins are associated at the end of the stalk where the hydrophobic fusion
484
peptide is located. (C) Hypothetical model of ∆P27 furinmut Fecto as it undergoes conformational
485
rearrangement after cleavage into the F1/F2 species. Top, model of uncleaved ∆P27 furinmut Fecto
486
based on pre-fusion structure with Site A (sites II), Site C (site IV) and site Ø formed on the
487
protein surface. HRB in blue is likely unfolded and represented as a dashed line. A black arrow
488
represents trypsin digestion of the monomer into F1/F2 leading to a conformational change of the
489
pre-fusion-like monomer into the post-fusion trimer. Bottom, a model of RSV Fecto rosettes
490
formed by post-fusion trimers associated by interactions between their fusion peptides (green).
491
Two neutralizing epitopes, Sites A and C, remain on the protein surface while the pre-fusion site
492
Ø epitope is lost as the HRA (red) is largely buried by the HRB (blue).
493
22
1
A monomeric uncleaved RSV F antigen retains pre-fusion-specific
2
neutralizing epitopes
3 4
Kurt A. Swanson,a,* Kara Balabanis,a Yuhong Xie,a Yukti Aggarwal,a Concepción Palomo,b
5
Vicente Mas,b Claire Metrick,a,§ Hui Yang,a,& Christine A. Shaw,a José A. Melero,b Philip R.
6
Dormitzer,a Andrea Carfia,#
7 8
a
Novartis Vaccines, 45 Sidney Street, Cambridge, MA 02139, USA
9
b
Centro Nacional de Microbiología and CIBER de Enfermedades Respiratorias, Instituto de
10
Salud Carlos III, Majadahonda, 28220 Madrid, Spain
11 12
#
Address correspondence to: [email protected]
13 14
*Present address: Sanofi, 270 Albany Street, Cambridge, MA 02139-4234, USA
15
§
16
Molecular Microbiology, Sackler School of Graduate Studies, Tufts University School of
17
Medicine, Boston, MA 02111, USA
18
&
19
Cambridge, MA 02139, USA
Present address: Department of Molecular Biology and Microbiology and Graduate Program in
Present address: Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue,
20 21
Running Head: A monomeric RSV F adopts a pre-fusion-like conformation
22 23
Words Text: 3647
24
Words Abstract: 193
25
1
26
Abstract
27
Respiratory syncytial virus (RSV) is the leading cause of severe respiratory disease in
28
infants and a major cause of respiratory illness in the elderly. It remains an unmet vaccine need
29
despite decades of research. Insufficient potency, homogeneity and stability of previous RSV
30
fusion protein (F) subunit vaccine candidates have hampered vaccine development. RSV F and
31
related parainfluenza virus (PIV) F proteins are cleaved by furin during intracellular maturation,
32
producing disulfide-linked F1 and F2 fragments. During cell entry, the cleaved Fs rearrange from
33
pre-fusion trimers to post-fusion trimers. Using RSV F constructs with mutated furin cleavage
34
sites, we isolated an uncleaved RSV F ectodomain that is predominantly monomeric and requires
35
specific cleavage between F1 and F2 for self-association and rearrangement into stable post-
36
fusion trimers. The uncleaved RSV F monomer is folded, homogenous and displays at least two
37
key RSV neutralizing epitopes shared between the pre-fusion and post-fusion conformations.
38
Unlike the cleaved trimer, the uncleaved monomer binds the pre-fusion-specific monoclonal
39
antibody D25 and human neutralizing immunoglobulins that do not bind to post-fusion F. These
40
observations suggest that the uncleaved RSV F monomer has a prefusion-like conformation and
41
is a potential pre-fusion subunit vaccine candidate.
42
Importance
43
RSV is the leading cause of severe respiratory disease in infants and a major cause of
44
respiratory illness in the elderly. Development of an RSV vaccine was stymied when a clinical
45
trial using a formalin inactivated RSV virus made disease, following RSV infection, more severe.
46
Recent studies have defined the structures that the RSV F envelope glycoprotein adopts before
47
and after virus entry (pre-fusion and post-fusion conformation, respectively). Key neutralization 2
48
epitopes of pre-fusion and post-fusion RSV F have been identified, and a number of current
49
vaccine development efforts are focused on generating easily produced subunit antigens that
50
retain these epitopes. Here we show that a simple modification in the F ectodomain results in a
51
homogeneous protein that retains critical pre-fusion neutralizing epitopes. These results improve
52
our understanding of RSV F protein folding and structure and can guide further vaccine design
53
efforts.
3
54
Introduction
55
RSV is a member of the Paramyxoviridae family of RNA viruses, which also includes
56
human metapneumovirus, measles virus, mumps virus, Newcastle disease virus (NDV), human
57
parainfluenzavirus (PIV) 1-4, and PIV5. RSV is the major cause of bronchiolitis and pneumonia
58
in infants. It is the leading cause of infant hospitalization in developed countries and is
59
responsible for an estimated 200,000 infant deaths in developing countries each year (1, 2). RSV
60
also causes substantial morbidity and mortality among the elderly (3, 4). There is no specific
61
antiviral treatment recommended for RSV infection, and the only currently available
62
prophylactic is a monoclonal antibody, Palivizumab (Synagis™), used to prevent disease in the
63
highest risk infants (5). The cost of Synagis prevents general use, and the need for a vaccine is
64
clear. However, despite decades of research there remains no licensed vaccine for RSV.
65
Development of a vaccine was stymied in the 1960’s when a formalin-inactivated RSV vaccine
66
candidate made subsequent RSV disease more severe (6). Increased structural understanding of
67
key RSV neutralization epitopes has supported a resurgence of interest in developing an RSV
68
subunit-based vaccine.
69
RSV-neutralizing antibodies target the two major RSV surface antigens, the attachment
70
protein (G) and the fusion protein (F) (7). G is variable in sequence, whereas F is highly
71
conserved between strains, making F the more attractive vaccine antigen. RSV F is a type I viral
72
fusion protein, responsible for driving fusion of the viral envelope with host cell membranes
73
during viral entry. Crystal structures of RSV F ectodomain trimers have documented two
74
conformational states – pre-fusion and post-fusion (Fig. 1C and D) (8-11). In the pre-fusion
75
conformation (Fig. 1C), the heptad repeat A (HRA) region is associated with the globular head,
76
and the tip of the fusion peptide is mostly buried in the center of the protein. In the post-fusion 4
77
conformation (Fig. 1D), HRA and the fusion peptide (not present in the published crystal
78
structures (14,18)) have extended from the globular head to attach to the target membrane, and
79
the heptad repeat B (HRB) region has rearranged to associate with the HRA region, forming a
80
stable 6-helix bundle. This rearrangement places the host membrane bound by the fusion peptide
81
and the viral membrane bound by the transmembrane region in close proximity to drive
82
membrane fusion. The commercial product Respigam™ (Medimmune), made by purifying
83
antibodies from human sera with high RSV-neutralizing titers, was shown to include antibodies
84
specific for the pre-fusion F conformation (12). Indeed, depleting Respigam of antibodies that
85
bind G and post-fusion F demonstrated that the pre-fusion-specific F antibodies were
86
predominantly responsible for virus neutralization by the product. The crystal structure of a RSV
87
F ectodomain (Fecto) stabilized by C-terminal addition of a trimerization tag (foldon) bound to the
88
FAb of the pre-fusion specific antibody D25 identified a new antigenic site designated site Ø (9).
89
Site Ø is formed in pre-fusion RSV Fecto by the packing of the HRA region against the rest of the
90
F globular head (a structural feature not shared by post-fusion F; Fig. 1C and D). In addition, a
91
pre-fusion RSV Fecto antigen with a trimerization tag and mutations that stabilized the HRA-
92
globular head interactions elicited a higher neutralizing titer than did post-fusion Fecto in mice
93
and non-human primates (11).
94
Fusion proteins are expressed as uncleaved F0 precursors (Fig. 1A and B). Crystal structures
95
of uncleaved PIV3, PIV5 and NDV Fecto revealed that furin cleavage is not required for these
96
proteins to trimerize (13-15). PIV3 Fecto was crystalized in the post-fusion conformation whereas
97
PIV5 Fecto was trapped in the pre-fusion conformation by the addition of a C-terminal GCN
98
trimerization tag (14). Unlike PIV Fs, which each contain a single furin cleavage site, RSV F has
99
two sites separated by a 27-amino acid fragment, p27 (Fig. 1B). Activation of RSV F for 5
100
membrane fusion requires cleavage by furin at the two sites (16). Our initial attempts to generate
101
a pre-fusion RSV Fecto trimer relied on mutating the furin cleavage sites and adding a GCN
102
trimerization tag, similar to the strategy used to generate the pre-fusion PIV5 Fecto (14). However,
103
addition of the GCN trimerization domain to the uncleaved RSV Fecto greatly reduced the amount
104
of secreted protein. In the course of these manipulations we observed that uncleaved RSV Fecto
105
was predominantly monomeric in solution. We demonstrate that this monomeric species is a
106
folded, soluble protein which harbors key neutralizing epitopes including the site Ø pre-fusion
107
epitope. This uncleaved species might represent a potent pre-fusion Fecto vaccine candidate.
108
Materials and Methods
109
Fusion protein constructs design, expression and purification
110
RSV Fecto constructs with mutations to the furin cleavage sites and fusion peptide region
111
(residues 109-148) were designed (Fig. 1A and B). ∆p27 furinmut Fecto contains a deletion of 23
112
residues from the p27 fragment and point mutations to the furin cleavage sites. These point
113
mutations prevent cleavage by intercellular furin but permit in vitro cleavage by trypsin. ∆FP
114
furinwt Fecto retains the wild-type furin cleavage sites but has a fusion peptide deletion. Unlike
115
∆p27 furinmut Fecto, which is purified as an uncleaved F0 species, ∆FP furinwt Fecto is cleaved by
116
the cell into the F1/F2 species. DNA constructs encoding Fecto with these mutations and a C-
117
terminal histidine tag were synthesized (Geneart) and cloned into the pFastBac baculovirus
118
system (Invitrogen). RSV Fecto constructs were expressed in 1 L cultures for three days and
119
purified by nickel affinity followed by size exclusion chromatography (GE Healthcare, Superdex
120
P200). For limited proteolysis studies, Δp27 furinmut RSV Fecto was digested with trypsin (Sigma)
6
121
at a weight ratio of 1:1000 trypsin to Δp27 furinmut RSV Fecto, and the resulting cleaved protein
122
was purified from a size exclusion column void volume.
123
Size exclusion chromatography and multi-angle light scattering (SEC-MALS)
124
Size exclusion high pressure liquid chromatography (HPLC) was performed using a Waters
125
2685 Separations Module HPLC with an isocratic flow rate of 0.75 mL/min coupled to a multi-
126
angle light scattering (MALS) system. The mobile phase consisted of 2x PBS. The size exclusion
127
column (Wyatt Technologies WTC-030S5) was used at ambient temperature with a run time of
128
30 min. UV was detected at 280 nm (Waters PDA 2996), and a light scattering detector (Wyatt
129
Technologies miniDawn TREOS) using a laser wavelength of 690.0 nm and a refractive index
130
detector (Wyatt Technologies Optilab rEX) were added in series. A gel filtration standard
131
solution (Bio-Rad) was used to generate a Stokes radius standard curve. Immediately prior to the
132
measurement of the samples, the system was calibrated using a known BSA (bovine serum
133
albumin) solution.
134
A 100 μL injection for a target column load of 100 μg protein (RSV Fecto alone or 1:1 molar
135
ratio RSV Fecto:FAb) was used. Peak retention times were used to estimate the molecular mass of
136
RSV Fecto or RSV Fecto:FAb complexes based on the Stokes radius standard curve. Protein
137
concentrations for identified peaks were calculated using calculated UV extinction coefficients,
138
and the molecular masses for RSV Fecto or RSV Fecto:FAb complexes were determined from
139
multi-angles light scattering data using Astra 5.3.4 software (Wyatt Technologies).
140
Binding studies by surface plasmon resonance (SPR)
141 142
The affinity of RSV Fecto constructs for Motavizumab or 101F FAbs was measured by SPR with a Biacore T100 instrument. FAbs were directly immobilized on CM5 sensor chips using 7
143
amine coupling at very low levels (75 response units) and RSV Fecto construct preparations at
144
various concentrations were injected at a high flow rate (50 μL/min) to avoid avidity effects and
145
higher than 1:1 binding interaction. The data were processed using Biacore T100 evaluation
146
software and double referenced by subtraction of the blank surface and buffer-only injection
147
before global fitting of the data to a 1:1 binding model.
148
Depletion of Respigam antibodies
149
Antibody depletion was carried out by a method similar to that previously described (17).
150
Briefly, the Δp27 furinmut Fecto and ΔFP furinwt Fecto preparations were bound covalently to
151
CNBr-activated Sepharose 4g (GE Healthcare) following the manufacturer’s instructions (500
152
µg of protein/160 mg Sepharose). The resins were packed into columns that were equilibrated in
153
PBS. Three mg of Respigam were loaded on each of these columns. The unbound material was
154
collected (referred to as Fecto-depleted Respigam). After washing the column with PBS, bound
155
antibodies were eluted with glycine-HCl pH 2.5 and immediately neutralized with saturated Tris,
156
concentrated and buffer exchanged to phosphate-buffered saline (PBS). The eluted antibodies
157
are referred to as Fecto-captured Respigam.
158
Enzyme-linked immunosorbent assay (ELISA) with depleted Respigam antibodies
159
ELISA of depleted antibodies was performed as previously described (12). Briefly, purified
160
101F antibody was used to coat 96 well microtiter plates overnight at 4ºC. Nonspecific antibody
161
binding was blocked with 2% porcine serum in PBS with 0.05% Tween 20. An excess of
162
purified proteins (either Δp27 furinmut Fecto or ΔFP furinwt Fecto, 2 µg/well) was added to each
163
well, and incubation continued for 1 hour at 37ºC followed by addition of serially diluted
164
antibodies and incubation for 1 hour at 37ºC. The plates were extensively washed with water 8
165
after each step. Finally, bound antibodies were detected with peroxidase-labelled rabbit anti-
166
human immunoglobulins and o-phenylenediamine (OPD) as substrate (GE Healthcare). The
167
reaction was stopped with 2N sulfuric acid, and absorbance was read at 490 nm.
168
Neutralization assay using depleted Respigam antibodies
169
RSV neutralization by depleted Respigam antibodies was performed using a
170
microneutralization assay described originally by Anderson et al.,1988 and modified by Martínez
171
and Melero, 1998 (18, 19). Briefly, immunoglobulin dilutions were incubated with 1x105 plaque
172
forming units (pfu) of the RSV Long strain for 30 minutes at 37ºC in a total volume of 50 µl.
173
These mixtures were used to infect 1x105 HEp-2 cells growing in 96 well plates with Dulbecco’s
174
modified Eagle’s medium (DMEM) supplemented with 2.5% fetal calf serum (FCS) that had
175
been inactivated 30 minutes at 60ºC. After one hour adsorption, DMEM with 2.5% FCS was
176
added, and the cultures were incubated 72 hours at 37ºC with 5% CO2. The plates were washed
177
three times with 0.05% Tween 20 in PBS and fixed with 80% cold acetone in PBS. After air
178
drying, viral antigen production in the fixed monolayers was measured by ELISA with a pool of
179
anti-G and anti-F murine antibodies and a horseradish peroxidase (HRP)-labelled anti-mouse
180
immunoglobulin preparation (Sigma).
181
Electron microscopy
182
RSV Fecto preparations (30 μg/mL in 25 mM Tris, 300 mM NaCl) were absorbed onto a 400-
183
mesh carbon-coated grid (Electron Microscopy Sciences) and stained with 0.75% uranyl formate.
184
A JEOL 1200EX microscope, operated at 80 kV, was used to analyze the samples. Micrographs
185
were taken at 65,000× magnification.
9
186
Results
187
∆P27 furinmut and ∆FP furinwt Fecto oligomerization states by SEC-MALS
188
PIV F ectodomains with furin cleavage site mutations form trimers in the post-fusion F
189
trimer conformation (13-15, 20). To determine if ∆P27 furinmut Fecto similarly formed trimers, we
190
analyzed its oligomerization state with analytical SEC coupled with MALS (Fig. 2). ∆P27
191
furinmut Fecto principally eluted from the analytical column at a retention volume indicating a
192
monomer, as estimated by Stokes radius. In addition, MALS analysis of the principal ∆P27
193
furinmut Fecto peak gave a mass consistent with a monomer. A minor peak in the ∆P27 furinmut
194
Fecto analysis ran consistent with the retention time of a trimer, suggesting some protein in this
195
preparation was competent to for trimers in solution (red arrow in Fig. 2). For comparison, we
196
performed SEC-MALS analysis of ∆FP furinwt Fecto, which is known by its crystal structure and
197
EM analysis to be a post-fusion trimer (8). ∆FP furinwt Fecto eluted at a retention time consistent
198
with a trimer, and analysis of the protein peak by MALS gave a mass consistent with a trimer
199
(Fig. 2).
200
Motavizumab and 101F FAb binding to ΔP27 furinmut Fecto by SPR and SEC
201
We tested whether ∆P27 furinmut Fecto binds FAbs from two structurally-characterized
202
neutralizing antibodies (site II (defined as Site A in (5)) binding Motavizumab and site IV
203
(defined as Site C in (5)) binding 101F) using SPR (Fig. 3). Both ∆P27 furinmut and ∆FP furinwt
204
Fecto bound tightly to Motavizumab and 101F FAbs. The KDs for the ∆P27 furinmut Fecto-
205
Motavizumab FAb interaction (3.23 x 10-11 M) and the ∆FP furinwt Fecto-Motavizumab FAb
206
interaction (2.05 x 10-11 M) were similar, as were the KDs for the ∆P27 furinmut Fecto-101F FAb
207
(4.19 x 10-10 M) and ∆FP furinwt Fecto-101F FAb (1.54 x 10-10 M) interactions. 10
208
To determine if FAb binding could stabilize trimerization of ∆P27 furinmut Fecto, we
209
incubated the ∆P27 furinmut Fecto with a molar equivalent of either Motavizumab or 101F FAb
210
and analyzed the resulting complex by SEC-MALS (Fig. 4). The retention times of the principal
211
peaks for the ∆P27 furinmut Fecto-Motavizumab FAb complex and of the ∆P27 furinmut Fecto-101F
212
FAb complex were consistent with 1:1 F monomer:FAb species as determined by Stokes radius
213
(Fig. 4A). MALS analysis also gave masses consistent with a 1:1 F monomer:FAb complexes.
214
For comparison, Stokes radius and MALS analysis for the peaks from ∆FP furinwt Fecto-FAb
215
complexes were consistent with 3:3 F-FAb complexes (or an F trimer bound to three FAbs) (Fig.
216
4B). As previously discussed, a shoulder peak with a retention time consistent with an F trimer
217
was observed in ∆P27 furinmut Fecto preparations and corresponding shoulder peaks (indicated by
218
a red arrow) were observed in the ∆P27 furinmut Fecto-FAb complex preparations with a retention
219
time consistent with F trimers bound by three FAbs.
220
∆P27 furinmut Fecto binding to D25 FAb by SPR and SEC
221
D25 FAb binds the pre-fusion Fecto but does not bind post-fusion Fecto (9). To determine if
222
∆P27 furinmut Fecto is competent to bind the pre-fusion site Ø-specific D25 FAb, we tested
223
binding by SPR and SEC (Fig. 5). The KD for the ∆P27 furinmut Fecto-D25 FAb interaction was
224
2.38 x 10-11 M. In the RSV Fecto -D25 crystal structure, the D25 FAb has principal contacts with
225
a single protomer and modest contacts with an adjacent protomer, suggesting that binding of D25
226
might occur with F trimers rather than monomers (9). To determine if binding to D25 FAb
227
induced trimerization of ∆P27 furinmut Fecto, we incubated the F protein with a molar equivalent
228
of D25 FAb and analyzed the complex by SEC-MALS (Fig. 5B). The retention time and Stokes
229
radius of the D25-bound ∆P27 furinmut Fecto was consistent with a 1:1 F monomer:FAb species.
230
Similarly, MALS analysis of the D25-bound ∆P27 furinmut Fecto gave a mass consistent with a 1:1 11
231
F monomer:FAb species. We conclude that binding to D25 FAb does not induce trimerization of
232
∆P27 furinmut Fecto.
233
Respigam antibodies binding to ∆P27 Furinmut Fecto
234
To determine if ∆P27 furinmut Fecto is competent to bind pre-fusion-specific human
235
antibodies we performed binding experiments using Respigam-depletion assays similar to those
236
published in Magro et al. 2012 (12). ∆P27 furinmut Fecto or ∆FP furinwt Fecto (post-fusion F trimer)
237
was immobilized on resin, and the protein-bound resin was incubated with Respigam. Antibodies
238
that did not bind the F protein were collected in the column flow-though (referred to as ∆P27
239
furinmut Fecto-depleted Respigam or ∆FP furinwt Fecto -depleted Respigam, respectively).
240
Antibodies that bound ∆P27 furinmut Fecto and were eluted are referred to as ∆P27 furinmut Fecto-
241
captured Respigam. Untreated Respigam is able to bind both ∆P27 furinmut Fecto and ∆FP furinwt
242
Fecto (Fig. 6A). To demonstrate that antibodies that specifically bind ∆P27 furinmut exist in
243
Respigam, ∆FP furinwt Fecto–depleted Respigam was tested in ELISA for its ability to bind ∆P27
244
furinmut Fecto (Fig. 6B). Although ∆FP furinwt Fecto–depleted Respigam had no significant binding
245
to ∆FP furinwt Fecto (demonstrating sufficient depletion of the sera) ∆FP furinwt Fecto–depleted
246
Respigam retained binding for ∆P27 furinmut Fecto (i.e. monomeric F). For comparison, ∆P27
247
furinmut Fecto–depleted Respigam showed no significant binding to either ∆FP furinwt Fecto or
248
∆P27 furinmut Fecto (data not shown).
249
It has been previously shown that post-fusion F-depleted Respigam retained the majority of
250
its neutralizing titer, and pre-fusion F-depleted Respigam had greatly diminished neutralizing
251
titer (12). Furthermore, the antibodies that bind pre-fusion F represent a greater fraction of
252
Respigam neutralizing titer (12). To test whether the antibodies that bind ∆P27 furinmut Fecto
253
represent the majority of the neutralizing titer of Respigam, we tested the neutralizing titer of 12
254
∆P27 furinmut Fecto-depleted versus ∆P27 furinmut Fecto-captured Respigam (Fig 6C). Untreated
255
Respigam neutralized RSV infectivity well relative to the poorly neutralizing polyclonal
256
antibodies purified from a non-immunized rabbit (negative control). ∆P27 furinmut Fecto-depleted
257
Respigam has a dramatically lower neutralizing titer than Respigam, similar to the observations
258
for pre-fusion F-depleted Respigam (12). ∆P27 furinmut Fecto -captured Respigam had a higher
259
neutralizing titer per mass IgG than untreated Respigam. Taken together, the data indicate that
260
most of the neutralizing activity of Respigam is from antibodies that bind to ∆P27 furinmut Fecto
261
(i.e. monomeric F) but not to the post-fusion ∆FP furinwt Fectoand that this monomeric RSV F
262
ecto-domain retains pre-fusion F specific epitopes.
263
Limited-proteolysis of ∆P27 furinmut Fecto
264
Uncleaved post-fusion PIV trimers (F0) form rosettes, self-associated by aggregation of
265
their fusion peptides, after in vitro cleavage into the F1/F2 species (20). To determine if
266
uncleaved ∆P27 furinmut Fecto was competent to form rosettes of post-fusion trimers upon in vitro
267
cleavage, ∆P27 furinmut Fecto was digested with trypsin (Fig. 7A). Upon treatment with trypsin
268
uncleaved ∆P27 furinmut Fecto was cleaved into the native F1/F2 species of wild-type Fecto, further
269
suggesting that the protein retains a mostly globular fold. Cleaved ∆P27 furinmut Fecto was further
270
purified from the SEC void volume and analyzed by electron microscopy (Fig. 7B). ∆FP furinwt
271
Fecto was previously described as mono-dispersed elongated crutches (8); however, cleaved ∆P27
272
furinmut Fecto forms rosettes of elongated crutches similar to the post-fusion rosettes described for
273
RSV and paramyxoviruses (20, 21). The observations that ∆P27 furinmut Fecto is resistant to
274
degradation by limited proteolysis and competent to forms rosettes of post-fusion F trimers
275
suggest that the uncleaved ∆P27 furinmut Fecto (i.e.. monomeric F) is a well-folded pre-fusion-like
276
molecule structurally competent to rearrange into the post-fusion conformation (Fig. 7C). We 13
277
also attempted to observe the monomeric RSV F by electron microscopy, but we were
278
unsuccessful, which is likely due to the protein’s relatively small size (i.e. 65 KDa).
279
Discussion
280
RSV remains one of the most important, unmet vaccine needs. Renewed interest in
281
developing an RSV F subunit-based vaccine was triggered first by information from the
282
structurally-characterized PIV F proteins and now from structural studies of RSV F itself. Recent
283
data have demonstrated that RSV Fecto stabilized in a pre-fusion conformation elicits higher
284
neutralizing titers than post-fusion RSV Fecto (11, 12), and there is high interest in developing a
285
pre-fusion RSV F subunit vaccine. The novel, pre-fusion-specific site Ø epitope, recognized by
286
the D25 FAb and formed by the HRA region folding against the globular head, appears to be
287
largely responsible for the improved immunogenicity (9). For this reason, antigens presenting the
288
site Ø epitope will likely become components of next generation RSV vaccine candidates.
289
Our initial attempts to generate a stable pre-fusion RSV Fecto antigen focused on the strategy
290
used to stabilize PIV5 F in its metastable, pre-fusion conformation (data not shown) (14). We
291
attempted to trimerize an uncleaved (furin sites mutated) or cleaved (wild type furin sites) RSV
292
Fecto using a C-terminal GCN trimerization domain, but found the protein secretion from the cell
293
was greatly reduced. We similarly tried expressing a cleaved RSV Fecto using a C-terminal foldon
294
tag but this protein formed rosettes similar to a post-fusion Fecto. For this reason, we examined
295
the oligomerization states of RSV F proteins without trimerization tags. SEC-MALS
296
demonstrated that uncleaved ∆P27 furinmut Fecto is predominantly a monomer in solution (Fig. 2).
297
This observed difference in the cleavage-dependence of PIV F and RSV F trimerization may be
298
linked to the presence of a single furin cleavage site in PIV F but two cleavage sites in RSV F, 14
299
flanking a p27 insertion (Fig. 1). The uncleaved, monomeric Fecto (∆P27 furinmut Fecto) has some
300
features consistent with the post-fusion trimer (∆FP furinwt Fecto). Specifically, two neutralizing
301
epitopes present on both pre-fusion and post-fusion Fecto, sites II and IV, are available for binding
302
by their respective FAbs, from Motavizumab and 101F, as demonstrated by SPR (Fig. 3) and
303
SEC-MALS (Fig. 4). A linear peptide harboring the Motavizumab epitope is capable of
304
transiently adopting its native conformation and being stabilized by FAb binding; however, the
305
affinity of the FAb-peptide interaction is ~6,000-fold less than the folded post-fusion protein-
306
FAb affinity (10). Thus, the tight binding of uncleaved, monomeric Fecto to the Motavizumab
307
FAb suggests the epitope is pre-formed on the protein surface, as it is on the trimer (Fig. 3).
308
SEC-MALS indicates that, upon binding either the Motavizumab FAb or the 101F FAb, the
309
monomer does not self-assemble into a trimer (Fig. 4). This finding suggests that the constraints
310
preventing trimerization of the uncleaved monomer are not relieved by FAb binding.
311
Further evidence suggests that the RSV Fecto monomer is a folded species. When the RSV Fecto
312
monomer is treated with trypsin, it is cleaved into its biologically relevant F1/F2 species. This
313
limited and specific proteolysis suggests that the RSV Fecto monomer has a globular fold,
314
protecting it from non-specific trypsin digestion. Upon cleavage into F1/F2, RSV Fecto
315
spontaneously self-associates into rosettes of post-fusion trimers (Fig. 7B). This behavior is
316
analogous to the rosette formation observed for the PIV Fs (20).
317
If the RSV Fecto monomer is in a pre-fusion-like conformation, the HRA region would be
318
packed against the globular head, presenting novel HRA epitopes lost in the post-fusion
319
conformation (Fig. 7C). Unlike the post-fusion Fecto trimer, the Fecto monomer is capable of
320
binding the site Ø, pre-fusion-specific, FAb D25 (Fig. 5). Furthermore, the Fecto monomer binds
321
antibodies in the human sera-derived Respigam commercial product that do not bind the post15
322
fusion Fecto antigen (Fig. 6B). Finally, previous studies have shown that post-fusion Fecto antigens
323
are unable to bind and deplete the majority of neutralizing activity from Respigam (12).
324
However, the Fecto monomer is able to bind and deplete the majority of neutralizing antibodies
325
from Respigam, suggesting that the Fecto monomer retains the important site Ø epitopes present
326
in other pre-fusion antigens and presumably on the RSV F surface on the virion.
327
Retention of site Ø is a desired characteristic of next generation RSV F antigens, and
328
therefore the uncleaved RSV F monomer here described represents a potential pre-fusion subunit
329
vaccine candidate. However, the RSV F monomer described here readily refolds in the post-
330
fusion conformation in presence of trace amounts of trypsin or contaminant trypsin-like
331
proteases and, therefore, is not stable enough for evaluation in immunization studies. Future
332
work will be aimed at stabilizing the RSV F monomer by introducing additional mutations that
333
lock jt more stably in a pre-fusion conformation amenable to in vivo studies.
334 335
Acknowledgements
336
Work in Madrid was supported in part by grant SAF2012-31217 from Plan Nacional I+D+I
337
(Ministerio de Economía y Competitividad).
16
338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383
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Nair H, Nokes DJ, Gessner BD, Dherani M, Madhi SA, Singleton RJ, O'Brien KL, Roca A, Wright PF, Bruce N, Chandran A, Theodoratou E, Sutanto A, Sedyaningsih ER, Ngama M, Munywoki PK, Kartasasmita C, Simoes EA, Rudan I, Weber MW, Campbell H. 2010. Global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis. Lancet 375:1545-1555. Hall CB. 2010. Respiratory syncytial virus, p. 2207-2221. In Mandell GL BJ, Dolin R (ed.), Mandell, Douglas, and Bennett's Principles and Practice of Infectious Disease, vol. 7th edition, Churchill Livingstone, New York. Thompson WW, Shay DK, Weintraub E, Brammer L, Cox N, Anderson LJ, Fukuda K. 2003. Mortality associated with influenza and respiratory syncytial virus in the United States. JAMA : the journal of the American Medical Association 289:179-186. Han LL, Alexander JP, Anderson LJ. 1999. Respiratory syncytial virus pneumonia among the elderly: an assessment of disease burden. The Journal of infectious diseases 179:25-30. Johnson S, Oliver C, Prince GA, Hemming VG, Pfarr DS, Wang SC, Dormitzer M, O'Grady J, Koenig S, Tamura JK, Woods R, Bansal G, Couchenour D, Tsao E, Hall WC, Young JF. 1997. Development of a humanized monoclonal antibody (MEDI-493) with potent in vitro and in vivo activity against respiratory syncytial virus. The Journal of infectious diseases 176:1215-1224. Kim HW, Canchola JG, Brandt CD, Pyles G, Chanock RM, Jensen K, Parrott RH. 1969. Respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine. American journal of epidemiology 89:422-434. Anderson R, Huang Y, Langley JM. 2010. Prospects for defined epitope vaccines for respiratory syncytial virus. Future microbiology 5:585-602. Swanson KA, Settembre EC, Shaw CA, Dey AK, Rappuoli R, Mandl CW, Dormitzer PR, Carfi A. 2011. Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers. Proceedings of the National Academy of Sciences of the United States of America 108:9619-9624. McLellan JS, Chen M, Leung S, Graepel KW, Du X, Yang Y, Zhou T, Baxa U, Yasuda E, Beaumont T, Kumar A, Modjarrad K, Zheng Z, Zhao M, Xia N, Kwong PD, Graham BS. 2013. Structure of RSV Fusion Glycoprotein Trimer Bound to a Prefusion-Specific Neutralizing Antibody. Science. McLellan JS, Yang Y, Graham BS, Kwong PD. 2011. Structure of respiratory syncytial virus fusion glycoprotein in the postfusion conformation reveals preservation of neutralizing epitopes. Journal of virology 85:7788-7796. McLellan JS, Chen M, Joyce MG, Sastry M, Stewart-Jones GB, Yang Y, Zhang B, Chen L, Srivatsan S, Zheng A, Zhou T, Graepel KW, Kumar A, Moin S, Boyington JC, Chuang GY, Soto C, Baxa U, Bakker AQ, Spits H, Beaumont T, Zheng Z, Xia N, Ko SY, Todd JP, Rao S, Graham BS, Kwong PD. 2013. Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus. Science 342:592-598. Magro M, Mas V, Chappell K, Vazquez M, Cano O, Luque D, Terron MC, Melero JA, Palomo C. 2012. Neutralizing antibodies against the preactive form of respiratory syncytial virus fusion protein offer unique possibilities for clinical intervention. Proceedings of the National Academy of Sciences of the United States of America 109:3089-3094. Yin HS, Paterson RG, Wen X, Lamb RA, Jardetzky TS. 2005. Structure of the uncleaved ectodomain of the paramyxovirus (hPIV3) fusion protein. Proceedings of the National Academy of Sciences of the United States of America 102:9288-9293. 17
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Yin HS, Wen X, Paterson RG, Lamb RA, Jardetzky TS. 2006. Structure of the parainfluenza virus 5 F protein in its metastable, prefusion conformation. Nature 439:38-44. Swanson K, Wen X, Leser GP, Paterson RG, Lamb RA, Jardetzky TS. 2010. Structure of the Newcastle disease virus F protein in the post-fusion conformation. Virology 402:372-379. Gonzalez-Reyes L, Ruiz-Arguello MB, Garcia-Barreno B, Calder L, Lopez JA, Albar JP, Skehel JJ, Wiley DC, Melero JA. 2001. Cleavage of the human respiratory syncytial virus fusion protein at two distinct sites is required for activation of membrane fusion. Proceedings of the National Academy of Sciences of the United States of America 98:9859-9864. Sastre P, Melero JA, Garcia-Barreno B, Palomo C. 2005. Comparison of affinity chromatography and adsorption to vaccinia virus recombinant infected cells for depletion of antibodies directed against respiratory syncytial virus glycoproteins present in a human immunoglobulin preparation. Journal of medical virology 76:248-255. Anderson LJ, Bingham P, Hierholzer JC. 1988. Neutralization of respiratory syncytial virus by individual and mixtures of F and G protein monoclonal antibodies. Journal of virology 62:42324238. Martinez I, Melero JA. 1998. Enhanced neutralization of human respiratory syncytial virus by mixtures of monoclonal antibodies to the attachment (G) glycoprotein. The Journal of general virology 79 ( Pt 9):2215-2220. Connolly SA, Leser GP, Yin HS, Jardetzky TS, Lamb RA. 2006. Refolding of a paramyxovirus F protein from prefusion to postfusion conformations observed by liposome binding and electron microscopy. Proceedings of the National Academy of Sciences of the United States of America 103:17903-17908. Calder LJ, Gonzalez-Reyes L, Garcia-Barreno B, Wharton SA, Skehel JJ, Wiley DC, Melero JA. 2000. Electron microscopy of the human respiratory syncytial virus fusion protein and complexes that it forms with monoclonal antibodies. Virology 271:122-131.
409 410
18
411
Figure legends
412
Figure 1. RSV F ectodomain structure. (A) Linear diagram listing residue numbers
413
corresponding to the N terminus of each segment. The furin cleavage sites (arrowheads) divide
414
the protein into F1 and F2. DI-III, domains I-III; p27, excised peptide; FP, fusion peptide; HRA,
415
-B, and –C heptad repeats A, B, and C are indicated. (B) Sequences showing the mutations
416
introduced into p27 and FP region of Fecto constructs. Top, wild-type RSV F sequence with p27
417
and fusion peptide unaltered. Middle, ∆P27 furinmut Fecto sequence with mutations to the furin
418
sites and deletion of p27 residues. Bottom, ∆FP furinwt Fecto sequence with fusion peptide
419
residues deleted. Deleted residues shown as hyphens. (C) Surface representation of the pre-
420
fusion RSV Fecto (9). One subunit colored by domains and heptad repeat regions as in A; the
421
other two are white and gray. (D) Surface representation of the post-fusion RSV Fecto (8), colored
422
as in C to highlight the rearrangement of domains and heptad repeat regions after conformational
423
change. Notice the HRA (red) largely buried by the HRB (blue) upon forming the six helix
424
bundle (6HB).
425
Figure 2. Analytical size exclusion chromatography analysis of RSV Fecto constructs. Top,
426
open circles represent the chromatogram of ∆P27 furinmut Fecto in which closed circles represent
427
the chromatogram of ∆FP furinwt Fecto. The principal peak of the cleaved ∆FP furinwt Fecto is
428
approximately 10.5 minutes. The principal peak of the uncleaved ∆P27 furinmut Fecto is
429
approximately 11.2 minutes, with a minor shoulder peak at approximately 10.5 minutes (arrow).
430
Bottom, masses of Fecto constructs measured by SEC-MALS. The molecular masses estimated by
431
the protein sequence for a monomer ∆P27 furinmut Fecto and trimer ∆FP furinwt Fecto are shown in
432
the first column. The masses for the RSV Fecto constructs as measured by Stokes radius and
433
MALS analysis are shown in second and third columns, respectively. 19
434
Figure 3. Binding studies by SPR of Fecto and FAbs. 101F and Motavizumab FAbs were
435
immobilized on a sensor chip, and varying concentrations of Fecto were injected with mass
436
absorption response units (RU) monitored as a function of time (sensorgrams are labeled
437
according to Fecto construct, FAb chip, and concentration as indicated). Fitting the resulting
438
curves to a 1:1 binding stoichiometry resulted in ka and kd and fit values as indicated in the
439
table.
440
Figure 4. Analytical size exclusion chromatography analysis of RSV Fecto constructs bound
441
to FAbs. (A) Black circles represent the chromatogram of ∆P27 furinmut Fecto where blue and red
442
circles represent the chromatogram of 101F FAb-bound and Motavizumab FAb-bound ∆P27
443
furinmut Fecto, respectively. Upon FAb binding the principal peak of ∆P27 furinmut Fecto shifts from
444
approximately 11.2 minutes to 10.7 minutes. An arrow shows a shoulder peak shifting to a
445
retention time of approximately 9.5 minutes. (B) Chromatogram of ∆FP furinwt Fecto with or
446
without FAb binding, colored as in A. Upon FAb binding the principal peak of ∆FP furinwt Fecto
447
shifts from approximately 10.5 minutes to approximately 9.5 minutes. C) Masses of Fecto
448
constructs predicted by the protein sequence for a monomer ∆FP furinwt Fecto and trimer ∆P27
449
furinmut Fecto bound to a single or three FAbs, respectively, are shown in the first column. The
450
masses for the RSV Fecto constructs bound to FAbs as measured by Stokes radius and MALS
451
analysis are shown in columns two and three, respectively.
452
Figure 5. Pre-fusion-specific D25 FAb binding to ∆P27 furinmut Fecto by SPR and SEC-
453
MALS. (A) D25 FAb was immobilized on a sensor chip, and varying concentrations of ∆P27
454
furinmut Fecto were injected with mass absorption response units (RU) monitored as a function of
455
time (Fecto concentrations are indicated). Fitting the resulting curves to a 1:1 binding 20
456
stoichiometry resulted in ka and kd and fit values as indicated in the table below. The resulting
457
KD for D25 FAb binding to ∆P27 furinmut Fecto is 2.38 x 10-11 M. (B) Top, SEC chromatogram of
458
∆P27 furinmut Fecto. Bottom, chromatogram of D25 FAb-bound ∆P27 furinmut Fecto. Arrows
459
indicate the expected retention time of RSV Fecto monomer (FM), unbound FAb (FAb), and RSV
460
Fecto monomer bound to FAb (FM+FAb). Below, masses of unbound ∆P27 furinmut Fecto or FAb-
461
bound ∆P27 furinmut Fecto predicted by sequence are shown in the first column. The measured
462
masses for ∆P27 furinmut Fecto and D25 FAb-bound ∆P27 furinmut Fecto by Stokes radius and
463
MALS analysis are shown in the second and third columns, respectively.
464
Figure 6. ∆P27 furinmut Fecto is recognized by unique neutralizing antibodies of Respigam.
465
(A) ∆FP furinwt Fecto or ∆P27 furinmut Fecto was adsorbed on an ELISA plate, and different
466
concentrations of Respigam were analyzed for binding. Respigam bound both ∆FP furinwt Fecto
467
(circles) and ∆P27 furinmut Fecto (squares) similarly. (B) ∆FP furinwt Fecto or ∆P27 furinmut Fecto
468
was adsorbed on an ELISA plate and ∆FP furinwt Fecto-depleted Respigam was analyzed at
469
different concentrations for binding. ∆FP furinwt Fecto-depleted-Respigam did not significantly
470
bind ∆FP furinwt Fecto (circles) but retained significant binding to ∆P27 furinmut Fecto (squares). (C)
471
∆P27 furinmut Fecto-depleted and –daptured Respigam were tested for neutralizing titer. Relative
472
to the strongly neutralizing, untreated Respigam (circles), antibodies from ∆P27 furinmut Fecto–
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depleted Respigam (squares) retain a poorer neutralizing titer whereas antibodies from ∆P27
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furinmut Fecto–captured Respigam (diamonds) retain a higher neutralizing titer per mass IgG
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(plotted on the x-axis).
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Figure 7. Limited proteolysis of ∆P27 furinmut Fecto induces protein trimerization and
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association by the fusion peptide. (A) SDS-PAGE gel showing limited proteolysis of RSV F 21
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constructs. RSV ∆P27 furinmut Fecto prior to digestion with trypsin is labeled as “Monomer” and
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produces the F0 band. Two lots of RSV ∆FP furinwt Fecto are labeled as “Trimer” and produce F1
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and F2 bands in the absence of trypsin. RSV ∆P27 furinmut Fecto produces F1 and F2 bands after
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digestion with trypsin. (B) Electron microscopy of the RSV Fecto rosettes formed spontaneously
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by trypsin treatment of ∆P27 furinmut Fecto. The rosettes have the elongated crutch shape of post-
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fusion RSV Fecto and proteins are associated at the end of the stalk where the hydrophobic fusion
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peptide is located. (C) Hypothetical model of ∆P27 furinmut Fecto as it undergoes conformational
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rearrangement after cleavage into the F1/F2 species. Top, model of uncleaved ∆P27 furinmut Fecto
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based on pre-fusion structure with Site A (sites II), Site C (site IV) and site Ø formed on the
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protein surface. HRB in blue is likely unfolded and represented as a dashed line. A black arrow
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represents trypsin digestion of the monomer into F1/F2 leading to a conformational change of the
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pre-fusion-like monomer into the post-fusion trimer. Bottom, a model of RSV Fecto rosettes
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formed by post-fusion trimers associated by interactions between their fusion peptides (green).
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Two neutralizing epitopes, Sites A and C, remain on the protein surface while the pre-fusion site
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Ø epitope is lost as the HRA (red) is largely buried by the HRB (blue).
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