A Monomeric Uncleaved Respiratory Syncytial Virus F Antigen Retains Prefusion-Specific Neutralizing Epitopes

ABSTRACT

Respiratory syncytial virus (RSV) is the leading infectious cause of severe respiratory disease in infants and a major cause of respiratory illness in the elderly. There remains an unmet vaccine need despite decades of research. Insufficient potency, homogeneity, and stability of previous RSV fusion protein (F) subunit vaccine candidates have hampered vaccine development. RSV F and related parainfluenza virus (PIV) F proteins are cleaved by furin during intracellular maturation, producing disulfide-linked F1 and F2 fragments. During cell entry, the cleaved Fs rearrange from prefusion trimers to postfusion trimers. Using RSV F constructs with mutated furin cleavage sites, we isolated an uncleaved RSV F ectodomain that is predominantly monomeric and requires specific cleavage between F1 and F2 for self-association and rearrangement into stable postfusion trimers. The uncleaved RSV F monomer is folded and homogenous and displays at least two key RSV-neutralizing epitopes shared between the prefusion and postfusion conformations. Unlike the cleaved trimer, the uncleaved monomer binds the prefusion-specific monoclonal antibody D25 and human neutralizing immunoglobulins that do not bind to postfusion F. These observations suggest that the uncleaved RSV F monomer has a prefusion-like conformation and is a potential prefusion subunit vaccine candidate.

IMPORTANCE RSV is the leading infectious cause of severe respiratory disease in infants and a major cause of respiratory illness in the elderly. Development of an RSV vaccine was stymied when a clinical trial using a formalin-inactivated RSV virus made disease, following RSV infection, more severe. Recent studies have defined the structures that the RSV F envelope glycoprotein adopts before and after virus entry (prefusion and postfusion conformations, respectively). Key neutralization epitopes of prefusion and postfusion RSV F have been identified, and a number of current vaccine development efforts are focused on generating easily produced subunit antigens that retain these epitopes. Here we show that a simple modification in the F ectodomain results in a homogeneous protein that retains critical prefusion neutralizing epitopes. These results improve our understanding of RSV F protein folding and structure and can guide further vaccine design efforts.

INTRODUCTION

Respiratory syncytial virus (RSV) is a member of the Paramyxoviridae family of RNA viruses, which also includes human metapneumovirus, measles virus, mumps virus, Newcastle disease virus (NDV), human parainfluenzavirus 1 (PIV1) to PIV4, and PIV5. RSV is the major cause of bronchiolitis and pneumonia in infants. It is the leading cause of infant hospitalization in developed countries and is responsible for an estimated 200,000 infant deaths in developing countries each year (1, 2). RSV also causes substantial morbidity and mortality among the elderly (3, 4). There is no specific antiviral treatment recommended for RSV infection, and the only currently available prophylactic is a monoclonal antibody, palivizumab (Synagis), used to prevent disease in the highest-risk infants (5). The cost of palivizumab prevents general use, and the need for a vaccine is clear. However, despite decades of research there remains no licensed vaccine for RSV. Development of a vaccine was stymied in the 1960s when a formalin-inactivated RSV vaccine candidate made subsequent RSV disease more severe (6). Increased structural understanding of key RSV neutralization epitopes has supported a resurgence of interest in developing an RSV subunit-based vaccine.

RSV-neutralizing antibodies target the two major RSV surface antigens, the attachment protein (G) and the fusion protein (F) (7). G is variable in sequence, whereas F is highly conserved among strains, making F the more attractive vaccine antigen. RSV F is a type I viral fusion protein responsible for driving fusion of the viral envelope with host cell membranes during viral entry. Crystal structures of RSV F ectodomain trimers have documented two conformational states—prefusion and postfusion (Fig. 1C and D) (8–11). In the prefusion conformation (Fig. 1C), the heptad repeat A (HRA) region is associated with the globular head and the tip of the fusion peptide is mostly buried in the center of the protein. In the postfusion conformation (Fig. 1D), HRA and the fusion peptide (not present in published crystal structures) have extended from the globular head to attach to the target membrane and the heptad repeat B (HRB) region has rearranged to associate with the HRA region, forming a stable 6-helix bundle. This rearrangement places the host membrane bound by the fusion peptide and the viral membrane bound by the transmembrane region in close proximity to drive membrane fusion. The commercial product RespiGam (RSV immune globulin; Medimmune), made by purifying antibodies from human sera with high RSV-neutralizing titers, was shown to include antibodies specific for the prefusion F conformation (12). Indeed, depleting RSV immune globulin of antibodies that bind G and postfusion F demonstrated that the prefusion-specific F antibodies were predominantly responsible for virus neutralization by the product. The crystal structure of an RSV F ectodomain (F_ecto) stabilized by the C-terminal addition of a trimerization tag (foldon) bound to the FAb of the prefusion-specific antibody D25 identified a new antigenic site designated site Ø (9). Site Ø is formed in prefusion RSV F_ecto by the packing of the HRA region against the rest of the F globular head (a structural feature not shared by postfusion F) (Fig. 1C and D). In addition, a prefusion RSV F_ecto antigen with a trimerization tag and mutations that stabilized the HRA-globular head interactions elicited a higher neutralizing titer than did postfusion F_ecto in mice and nonhuman primates (11).

FIG 1.

RSV F ectodomain structure. (A) Linear diagram listing residue numbers corresponding to the N terminus of each segment. The furin cleavage sites (arrowheads) divide the protein into F1 and F2. DI to DIII, domains I to III; p27, excised peptide; FP, fusion peptide; HRA, -B, and -C, heptad repeats A, B, and C. (B) Sequences showing the mutations introduced into p27 and the FP region of F_ecto constructs. (Top) Wild-type RSV F sequence with p27 and fusion peptide unaltered. (Middle) ΔP27 furin_mut F_ecto sequence with mutations to the furin sites and deletion of p27 residues. (Bottom) ΔFP furin_wt F_ecto sequence with fusion peptide residues deleted. Deleted residues are shown as hyphens. (C) Surface representation of the prefusion RSV F_ecto (9). One subunit shows domains and heptad repeat regions colored as in panel A; the other two subunits are white and gray. (D) Surface representation of the postfusion RSV F_ecto (8), colored as in panel C to highlight the rearrangement of domains and heptad repeat regions after conformational change. Note the HRA (red) largely buried by the HRB (blue) upon formation of the 6-helix bundle (6HB).

Fusion proteins are expressed as uncleaved F0 precursors (Fig. 1A and B). Crystal structures of uncleaved PIV3, PIV5, and NDV F_ecto revealed that furin cleavage is not required for these proteins to trimerize (13–15). PIV3 F_ecto was crystalized in the postfusion conformation, whereas PIV5 F_ecto was trapped in the prefusion conformation by the addition of a C-terminal GCN trimerization tag (14). Unlike PIV Fs, each of which contains a single furin cleavage site, RSV F has two sites separated by a 27-amino-acid fragment, p27 (Fig. 1B). Activation of RSV F for membrane fusion requires cleavage by furin at the two sites (16). Our initial attempts to generate a prefusion RSV F_ecto trimer relied on mutating the furin cleavage sites and adding a GCN trimerization tag, similar to the strategy used to generate the prefusion PIV5 F_ecto (14). However, addition of the GCN trimerization domain to the uncleaved RSV F_ecto greatly reduced the amount of secreted protein. In the course of these manipulations, we observed that uncleaved RSV F_ecto was predominantly monomeric in solution. We demonstrate that this monomeric species is a folded, soluble protein which harbors key neutralizing epitopes, including the site Ø prefusion epitope. This uncleaved species might represent a prefusion F_ecto vaccine candidate.

MATERIALS AND METHODS

Fusion protein construct design, expression, and purification.

RSV F_ecto constructs with mutations to the furin cleavage sites and fusion peptide region (residues 109 to 148) were designed (Fig. 1A and B). Δp27 furin_mut F_ecto contains a deletion of 23 residues from the p27 fragment and point mutations to the furin cleavage sites. These point mutations prevent cleavage by intercellular furin but permit in vitro cleavage by trypsin. ΔFP furin_wt F_ecto retains the wild-type furin cleavage sites but has a fusion peptide deletion. Unlike Δp27 furin_mut F_ecto, which is purified as an uncleaved F0 species, ΔFP furin_wt F_ecto is cleaved by the cell into the F1/F2 species. DNA constructs encoding F_ecto with these mutations and a C-terminal histidine tag were synthesized (GeneArt) and cloned into the pFastBac baculovirus system (Invitrogen). RSV F_ecto constructs were expressed in 1-liter cultures for 3 days and purified by nickel affinity followed by size exclusion chromatography (Superdex P200; GE Healthcare). For limited proteolysis studies, Δp27 furin_mut RSV F_ecto was digested with trypsin (Sigma) at a weight ratio of 1:1,000 trypsin to Δp27 furin_mut RSV F_ecto, and the resulting cleaved protein was purified from a size exclusion column void volume.

Size exclusion chromatography and multiangle light scattering (SEC-MALS).

Size exclusion high pressure liquid chromatography (HPLC) was performed using a Waters 2685 separation module HPLC instrument with an isocratic flow rate of 0.75 ml/min coupled to a multiangle light scattering (MALS) system. The mobile phase consisted of 2× phosphate-buffered saline (PBS). The size exclusion column (WTC-030S5; Wyatt Technology) was used at ambient temperature with a run time of 30 min. UV was detected at 280 nm (PDA 2996; Waters), and a light-scattering detector (miniDawn TREOS; Wyatt Technology) using a laser wavelength of 690.0 nm and a refractive-index detector (Optilab rEX; Wyatt Technology) were added in series. A gel filtration standard solution (Bio-Rad) was used to generate a Stokes radius standard curve. Immediately prior to the measurement of the samples, the system was calibrated using a known bovine serum albumin (BSA) solution.

A 100-μl injection for a target column load of 100 μg protein (RSV F_ecto alone or a 1:1 molar ratio of RSV F_ecto:FAb) was used. Peak retention times were used to estimate the molecular mass of RSV F_ecto or RSV F_ecto:FAb complexes based on the Stokes radius standard curve. Protein concentrations for identified peaks were calculated using calculated UV extinction coefficients, and the molecular masses for RSV F_ecto or RSV F_ecto:FAb complexes were determined from multiangle light scattering data using Astra 5.3.4 software (Wyatt Technologies).

Binding studies by surface plasmon resonance.

The affinity of RSV F_ecto constructs for motavizumab or 101F FAbs was measured by surface plasmon resonance (SPR) with a Biacore T100 instrument. FAbs were directly immobilized on CM5 sensor chips using amine coupling at very low levels (75 response units), and RSV F_ecto construct preparations at various concentrations were injected at a high flow rate (50 μl/min) to avoid avidity effects and higher than 1:1 binding interactions. The data were processed using Biacore T100 evaluation software and double referenced by subtraction of the blank surface and buffer-only injection values before global fitting of the data to a 1:1 binding model.

Depletion of RSV immune globulin antibodies.

Antibody depletion was carried out by a method similar to that previously described (17). Briefly, the Δp27 furin_mut F_ecto and ΔFP furin_wt F_ecto preparations were bound covalently to CNBr-activated Sepharose 4B (GE Healthcare) according to the manufacturer's instructions (500 μg of protein/160 mg Sepharose). The resins were packed into columns that were equilibrated in PBS. Three milligrams of RSV immune globulin was loaded on each of these columns. The unbound material (referred to as F_ecto-depleted RSV immune globulin) was collected. After the column was washed with PBS, bound antibodies were eluted with glycine-HCl (pH 2.5), immediately neutralized with saturated Tris, and concentrated, and the buffer was exchanged for PBS. The eluted antibodies are referred to as F_ecto-captured RSV immune globulin.

Enzyme-linked immunosorbent assay with depleted RSV immune globulin antibodies.

Enzyme-linked immunosorbent assay (ELISA) of depleted antibodies was performed as previously described (12). Briefly, purified 101F antibody was used to coat 96-well microtiter plates overnight at 4°C. Nonspecific antibody binding was blocked with 2% porcine serum in PBS with 0.05% Tween 20. An excess of purified proteins (either Δp27 furin_mut F_ecto or ΔFP furin_wt F_ecto, 2 μg/well) was added to each well, and incubation continued for 1 h at 37°C followed by the addition of serially diluted antibodies and incubation for 1 h at 37°C. The plates were extensively washed with water after each step. Finally, bound antibodies were detected with peroxidase-labeled rabbit anti-human immunoglobulins and o-phenylenediamine (OPD) as the substrate (GE Healthcare). The reaction was stopped with 2 N sulfuric acid, and absorbance was read at 490 nm.

Neutralization assay using depleted RSV immune globulin antibodies.

RSV neutralization by depleted RSV immune globulin antibodies was performed using a microneutralization assay described originally by Anderson et al. in 1988 and modified by Martínez and Melero in 1998 (18, 19). Briefly, immunoglobulin dilutions were incubated with 1 × 10⁵ PFU of the RSV Long strain for 30 min at 37°C in a total volume of 50 μl. These mixtures were used to infect 1 × 10⁵ HEp-2 cells growing in 96-well plates with Dulbecco's modified Eagle's medium (DMEM) supplemented with 2.5% fetal calf serum (FCS) that had been inactivated 30 min at 60°C. After 1 h of adsorption, DMEM with 2.5% FCS was added, and the cultures were incubated 72 h at 37°C with 5% CO₂. The plates were washed three times with 0.05% Tween 20 in PBS and fixed with 80% cold acetone in PBS. After air drying, viral antigen production in the fixed monolayers was measured by ELISA with a pool of anti-G and anti-F murine antibodies and a horseradish peroxidase (HRP)-labeled anti-mouse immunoglobulin preparation (Sigma).

Electron microscopy (EM).

RSV F_ecto preparations (30 μg/ml in 25 mM Tris, 300 mM NaCl) were absorbed onto a 400-mesh-sized carbon-coated grid (Electron Microscopy Sciences) and stained with 0.75% uranyl formate. A JEOL 1200EX microscope operated at 80 kV was used to analyze the samples. Micrographs were taken at a magnification of ×65,000.

RESULTS

ΔP27 furin_mut and ΔFP furin_wt F_ecto oligomerization states by SEC-MALS.

PIV F ectodomains with furin cleavage site mutations form trimers in the postfusion F trimer conformation (13, 15, 20). To determine if ΔP27 furin_mut F_ecto similarly formed trimers, we analyzed its oligomerization state with analytical SEC coupled with MALS (Fig. 2). ΔP27 furin_mut F_ecto principally eluted from the analytical column at a retention volume indicating a monomer, as estimated by Stokes radius. In addition, MALS analysis of the principal ΔP27 furin_mut F_ecto peak gave a mass consistent with a monomer. A minor peak in the ΔP27 furin_mut F_ecto analysis ran consistent with the retention time of a trimer, suggesting some protein in this preparation was competent for trimerization in solution (Fig. 2, red arrow). For comparison, we performed SEC-MALS analysis of ΔFP furin_wt F_ecto, which is known by its crystal structure and EM analysis to be a postfusion trimer (8). ΔFP furin_wt F_ecto eluted at a retention time consistent with a trimer, and analysis of the protein peak by MALS gave a mass consistent with a trimer (Fig. 2).

FIG 2.

Analytical size exclusion chromatography analysis of RSV F_ecto constructs. (Top) Open circles represent the chromatogram of ΔP27 furin_mut F_ecto, and closed circles represent the chromatogram of ΔFP furin_wt F_ecto. The principal peak of the cleaved ΔFP furin_wt F_ecto is at approximately 10.5 min. The principal peak of the uncleaved ΔP27 furin_mut F_ecto is at approximately 11.2 min, with a minor shoulder peak at approximately 10.5 min (arrow). (Bottom) Masses of F_ecto constructs measured by SEC-MALS. The molecular masses estimated according to the protein sequence for a monomer ΔP27 furin_mut F_ecto and trimer ΔFP furin_wt F_ecto are shown in the second column. The masses for the RSV F_ecto constructs as measured by Stokes radius and MALS analysis are shown in the third and fourth columns, respectively.

Motavizumab and 101F FAb binding to ΔP27 furin_mut F_ecto by SPR and SEC.

Using SPR, we tested whether ΔP27 furin_mut F_ecto binds FAbs from two structurally characterized neutralizing antibodies, motavizumab, which binds site II (defined as site A in reference 5), and 101F, which binds site IV (defined as site C in reference 5) (Fig. 3). Both ΔP27 furin_mut and ΔFP furin_wt F_ecto bound tightly to motavizumab and 101F FAbs. The K_D values (equilibrium dissociation constants) for the ΔP27 furin_mut F_ecto-motavizumab FAb interaction (3.23 × 10⁻¹¹ M) and the ΔFP furin_wt F_ecto-motavizumab FAb interaction (2.05 × 10⁻¹¹ M) were similar, as were the K_D values for the ΔP27 furin_mut F_ecto-101F FAb (4.19 × 10⁻¹⁰ M) and ΔFP furin_wt F_ecto-101F FAb (1.54 × 10⁻¹⁰ M) interactions.

FIG 3.

Binding studies by SPR of F_ecto and FAbs. 101F and motavizumab FAbs were immobilized on a sensor chip, various concentrations of F_ecto were injected, and mass absorption response units (RU) were monitored as a function of time (sensorgrams are labeled according to F_ecto construct, FAb chip, and concentration, as indicated). Fitting the resulting curves to a 1:1 binding stoichiometry resulted in k_a (association rate constant), k_d (dissociation rate constant), and fit values as indicated in the table. R_max, maximum analyte binding capacity of the surface in RU. Ms, mol × seconds.

To determine if FAb binding could stabilize trimerization of ΔP27 furin_mut F_ecto, we incubated the ΔP27 furin_mut F_ecto with a molar equivalent of either motavizumab or 101F FAb and analyzed the resulting complex by SEC-MALS (Fig. 4). The retention times of the principal peaks for the ΔP27 furin_mut F_ecto-motavizumab FAb complex and for the ΔP27 furin_mut F_ecto-101F FAb complex were consistent with 1:1 F monomer:FAb species as determined by Stokes radius (Fig. 4A). MALS analysis also gave masses consistent with 1:1 F monomer:FAb complexes. In comparison, Stokes radius and MALS analysis results for the peaks from ΔFP furin_wt F_ecto-FAb complexes were consistent with those for 3:3 F-FAb complexes (or an F trimer bound to three FAbs) (Fig. 4B). As previously discussed, a shoulder peak with a retention time consistent with an F trimer was observed in ΔP27 furin_mut F_ecto preparations and corresponding shoulder peaks (Fig. 4B, red arrow) were observed in the ΔP27 furin_mut F_ecto-FAb complex preparations with a retention time consistent with F trimers bound by three FAbs.

FIG 4.

Analytical size exclusion chromatography analysis of RSV F_ecto constructs bound to FAbs. (A) Black circles represent the chromatogram of ΔP27 furin_mut F_ecto, and blue and red circles represent the chromatogram of 101F FAb-bound and motavizumab FAb-bound ΔP27 furin_mut F_ecto, respectively. Upon FAb binding, the principal peak of ΔP27 furin_mut F_ecto shifts from approximately 11.2 min to 10.7 min. An arrow shows a shoulder peak shifting to a retention time of approximately 9.5 min. (B) Chromatogram of ΔFP furin_wt F_ecto with or without FAb binding, colored as in panel A. Upon FAb binding, the principal peak of ΔFP furin_wt F_ecto shifts from approximately 10.5 min to approximately 9.5 min. (C) Masses of F_ecto constructs predicted by the protein sequence for a monomer ΔFP furin_wt F_ecto and trimer ΔP27 furin_mut F_ecto bound to a single Fab or to three FAbs, respectively, are shown in the second column. Masses for the RSV F_ecto constructs bound to FAbs as measured by Stokes radius and MALS analysis are shown in columns three and four, respectively.

ΔP27 furin_mut F_ecto binding to D25 FAb by SPR and SEC.

D25 FAb binds the prefusion F_ecto but does not bind postfusion F_ecto (9). To determine if ΔP27 furin_mut F_ecto is competent to bind the prefusion site Ø-specific D25 FAb, we tested binding by SPR and SEC (Fig. 5). The K_D for the ΔP27 furin_mut F_ecto-D25 FAb interaction was 2.38 × 10⁻¹¹ M. In the RSV F_ecto -D25 crystal structure, the D25 FAb has principal contacts with a single protomer and modest contacts with an adjacent protomer, suggesting that binding of D25 might occur with F trimers rather than monomers (9). To determine if binding to D25 FAb induced trimerization of ΔP27 furin_mut F_ecto, we incubated the F protein with a molar equivalent of D25 FAb and analyzed the complex by SEC-MALS (Fig. 5B). The retention time and Stokes radius of the D25-bound ΔP27 furin_mut F_ecto was consistent with 1:1 F monomer:FAb species. Similarly, MALS analysis of the D25-bound ΔP27 furin_mut F_ecto gave a mass consistent with 1:1 F monomer:FAb species. We conclude that binding to D25 FAb does not induce trimerization of ΔP27 furin_mut F_ecto.

FIG 5.

Prefusion-specific D25 FAb binding to ΔP27 furin_mut F_ecto by SPR and SEC-MALS. (A) D25 FAb was immobilized on a sensor chip, various concentrations of ΔP27 furin_mut F_ecto were injected, and mass absorption response units (RU) were monitored as a function of time (F_ecto concentrations are indicated). Fitting the resulting curves to 1:1 binding stoichiometry resulted in k_a, k_d, and fit values as indicated in the table below. The resulting K_D for D25 FAb binding to ΔP27 furin_mut F_ecto is 2.38 × 10⁻¹¹ M. (B) (Top) SEC chromatogram of ΔP27 furin_mut F_ecto. (Bottom) Chromatogram of D25 FAb-bound ΔP27 furin_mut F_ecto. Arrows indicate the expected retention times of RSV F_ecto monomer (F_M), unbound FAb (FAb), and RSV F_ecto monomer bound to FAb (F_M+FAb). Below, the masses of unbound ΔP27 furin_mut F_ecto or FAb-bound ΔP27 furin_mut F_ecto predicted by sequence are shown in the second column. The measured masses for ΔP27 furin_mut F_ecto and D25 FAb-bound ΔP27 furin_mut F_ecto by Stokes radius and MALS analysis are shown in the third and fourth columns, respectively.

RSV immune globulin antibodies binding to ΔP27 furin_mut F_ecto.

To determine if ΔP27 furin_mut F_ecto is competent to bind prefusion-specific human antibodies, we performed binding experiments using RSV immune globulin-depletion assays similar to those published in Magro et al. (12). ΔP27 furin_mut F_ecto or ΔFP furin_wt F_ecto (postfusion F trimer) was immobilized on resin, and the protein-bound resin was incubated with RSV immune globulin. Antibodies that did not bind the F protein were collected in the column flow-through (referred to as ΔP27 furin_mut F_ecto-depleted RSV immune globulin or ΔFP furin_wt F_ecto-depleted RSV immune globulin, respectively). Antibodies that bound ΔP27 furin_mut F_ecto and were eluted are referred to as ΔP27 furin_mut F_ecto-captured RSV immune globulin. Untreated RSV immune globulin is able to bind both ΔP27 furin_mut F_ecto and ΔFP furin_wt F_ecto (Fig. 6A). To demonstrate that antibodies that specifically bind ΔP27 furin_mut exist in RSV immune globulin, we tested ΔFP furin_wt F_ecto-depleted RSV immune globulin in ELISA for its ability to bind ΔP27 furin_mut F_ecto (Fig. 6B). Although ΔFP furin_wt F_ecto-depleted RSV immune globulin had no significant binding to ΔFP furin_wt F_ecto (demonstrating sufficient depletion of the sera), ΔFP furin_wt F_ecto-depleted RSV immune globulin retained binding for ΔP27 furin_mut F_ecto (i.e., monomeric F). In comparison, ΔP27 furin_mut F_ecto-depleted RSV immune globulin showed no significant binding to either ΔFP furin_wt F_ecto or ΔP27 furin_mut F_ecto (data not shown).

FIG 6.

ΔP27 furin_mut F_ecto is recognized by unique neutralizing antibodies of RSV immune globulin (Respigam). (A) ΔFP furin_wt F_ecto or ΔP27 furin_mut F_ecto was adsorbed on an ELISA plate, and different concentrations of RSV immune globulin were analyzed for binding. RSV immune globulin bound both ΔFP furin_wt F_ecto (circles) and ΔP27 furin_mut F_ecto (squares) similarly. (B) ΔFP furin_wt F_ecto or ΔP27 furin_mut F_ecto was adsorbed on an ELISA plate, and ΔFP furin_wt F_ecto-depleted RSV immune globulin was analyzed at different concentrations for binding. ΔFP furin_wt F_ecto-depleted RSV immune globulin did not significantly bind ΔFP furin_wt F_ecto (circles) but retained significant binding to ΔP27 furin_mut F_ecto (squares). (C) ΔP27 furin_mut F_ecto-depleted and -captured RSV immune globulins were tested for neutralizing titers. Relative to those from the strongly neutralizing, untreated RSV immune globulin (circles), antibodies from ΔP27 furin_mut F_ecto-depleted RSV immune globulin (squares) retain a lower neutralization titer, whereas antibodies from ΔP27 furin_mut F_ecto-captured RSV immune globulin (diamonds) retain a higher neutralizing titer per mass of IgG (plotted on the x axis).

It has been previously shown that postfusion F-depleted RSV immune globulin retained the majority of its neutralizing titer and that prefusion F-depleted RSV immune globulin had a greatly diminished neutralizing titer (12). Furthermore, the antibodies that bind prefusion F represent a greater fraction of RSV immune globulin neutralizing titer (12). To test whether the antibodies that bind ΔP27 furin_mut F_ecto represent the majority of the neutralizing titer of RSV immune globulin, we tested the neutralizing titer of ΔP27 furin_mut F_ecto-depleted versus that of ΔP27 furin_mut F_ecto-captured RSV immune globulin (Fig. 6C). Untreated RSV immune globulin neutralized RSV infectivity well relative to the poorly neutralizing polyclonal antibodies purified from a nonimmunized rabbit (negative control). ΔP27 furin_mut F_ecto-depleted RSV immune globulin has a dramatically lower neutralizing titer than RSV immune globulin, similar to observations for prefusion F-depleted RSV immune globulin (12). ΔP27 furin_mut F_ecto-captured RSV immune globulin had a higher neutralizing titer per mass of IgG than untreated RSV immune globulin. Taken together, the data indicate that most of the neutralizing activity of RSV immune globulin is from antibodies that bind to ΔP27 furin_mut F_ecto (i.e., monomeric F) but not to the postfusion ΔFP furin_wt F_ecto and that this monomeric RSV F ectodomain retains prefusion F-specific epitopes.

Limited proteolysis of ΔP27 furin_mut F_ecto.

Uncleaved postfusion PIV trimers (F0) form rosettes, self-associated by aggregation of their fusion peptides, after in vitro cleavage into the F1/F2 species (20). To determine if uncleaved ΔP27 furin_mut F_ecto was competent to form rosettes of postfusion trimers upon in vitro cleavage, we digested ΔP27 furin_mut F_ecto with trypsin (Fig. 7A). Upon treatment with trypsin, uncleaved ΔP27 furin_mut F_ecto was cleaved into the native F1/F2 species of wild-type F_ecto, further suggesting that the protein retains a mostly globular fold. Cleaved ΔP27 furin_mut F_ecto was further purified from the SEC void volume and analyzed by electron microscopy (Fig. 7B). ΔFP furin_wt F_ecto was previously described as mono-dispersed elongated crutches (8); however, cleaved ΔP27 furin_mut F_ecto forms rosettes of elongated crutches similar to the postfusion rosettes described for RSV and paramyxoviruses (20, 21). The observations that ΔP27 furin_mut F_ecto is resistant to degradation by limited proteolysis and is competent to form rosettes of postfusion F trimers suggest that the uncleaved ΔP27 furin_mut F_ecto (i.e., monomeric F) is a well-folded prefusion-like molecule structurally competent to rearrange into the postfusion conformation (Fig. 7C). We also attempted to observe the monomeric RSV F by electron microscopy, but we were unsuccessful, which is likely due to the protein's relatively small size (i.e., 65 kDa).

FIG 7.

Limited proteolysis of ΔP27 furin_mut F_ecto induces protein trimerization and association by the fusion peptide. (A) Sodium dodecyl sulfate-polyacrylamide gel showing limited proteolysis of RSV F constructs. RSV ΔP27 furin_mut F_ecto prior to digestion with trypsin is labeled “Monomer” and produces the F0 band. Two lots of RSV ΔFP furin_wt F_ecto are labeled “Trimer” and produce F1 and F2 bands in the absence of trypsin. RSV ΔP27 furin_mut F_ecto is labeled “Rosettes” and produces F1 and F2 bands after digestion with trypsin. (B) Electron microscopy image of the RSV F_ecto rosettes formed spontaneously after trypsin treatment of ΔP27 furin_mut F_ecto. The rosettes have the elongated crutch shape of postfusion RSV F_ecto, and proteins are associated at the end of the stalk where the hydrophobic fusion peptide is located. (C) Hypothetical model of ΔP27 furin_mut F_ecto as it undergoes conformational rearrangement after cleavage into the F1/F2 species. (Top) Model of uncleaved ΔP27 furin_mut F_ecto based on prefusion structure with Site A (sites II), Site C (site IV) and site Ø formed on the protein surface. HRB in blue is likely unfolded and is represented as a dashed line. A black arrow represents trypsin digestion of the monomer into F1/F2, leading to a conformational change of the prefusion-like monomer into the postfusion trimer. (Bottom) A model of RSV F_ecto rosettes formed by postfusion trimers associated by interactions between their fusion peptides (green). Two neutralizing epitopes, Sites A and C, remain on the protein surface, while the prefusion site Ø epitope is lost as the HRA (red) is largely buried by the HRB (blue).

DISCUSSION

RSV remains one of the most important pathogens for which a vaccine is needed. Renewed interest in developing an RSV F subunit-based vaccine was triggered first by information from the structurally characterized PIV F proteins and now from structural studies of RSV F itself. Recent data have demonstrated that RSV F_ecto stabilized in a prefusion conformation elicits higher neutralizing titers than postfusion RSV F_ecto (11, 12), and there is high interest in developing a prefusion RSV F subunit vaccine. The novel prefusion-specific site Ø epitope, recognized by the D25 FAb and formed by the HRA region folding against the globular head, appears to be largely responsible for the improved immunogenicity (9). For this reason, antigens presenting the site Ø epitope will likely become components of next-generation RSV vaccine candidates.

Our initial attempts to generate a stable prefusion RSV F_ecto antigen focused on the strategy used to stabilize PIV5 F in its metastable, prefusion conformation (data not shown) (14). We attempted to trimerize an uncleaved (with mutated furin sites) or cleaved (with wild-type furin sites) RSV F_ecto using a C-terminal GCN trimerization domain but found the protein secretion from the cell was greatly reduced. We similarly tried expressing a cleaved RSV F_ecto using a C-terminal foldon tag, but this protein formed rosettes similar to those of a postfusion F_ecto. For this reason, we examined the oligomerization states of RSV F proteins without trimerization tags. SEC-MALS demonstrated that uncleaved ΔP27 furin_mut F_ecto is predominantly a monomer in solution (Fig. 2). This observed difference in the cleavage dependence of PIV F and RSV F trimerization may be linked to the presence of a single furin cleavage site in PIV F but two cleavage sites in RSV F flanking a p27 insertion (Fig. 1). The uncleaved, monomeric F_ecto (ΔP27 furin_mut F_ecto) has some features consistent with the postfusion trimer (ΔFP furin_wt F_ecto). Specifically, two neutralizing epitopes present on both prefusion and postfusion F_ecto, sites II and IV, are available for binding by their respective FAbs from motavizumab and 101F, as demonstrated by SPR (Fig. 3) and SEC-MALS (Fig. 4). A linear peptide harboring the motavizumab epitope is capable of transiently adopting its native conformation and being stabilized by FAb binding; however, the affinity of the FAb-peptide interaction is ∼6,000-fold less than folded postfusion protein-FAb affinity (10). Thus, the tight binding of uncleaved, monomeric F_ecto to the motavizumab FAb suggests the epitope is preformed on the protein surface, as it is on the trimer (Fig. 3). SEC-MALS indicates that, upon binding either the motavizumab FAb or the 101F FAb, the monomer does not self-assemble into a trimer (Fig. 4). This finding suggests that the constraints preventing trimerization of the uncleaved monomer are not relieved by FAb binding.

Further evidence suggests that the RSV F_ecto monomer is a folded species. When the RSV F_ecto monomer is treated with trypsin, it is cleaved into its biologically relevant F1/F2 species. This limited and specific proteolysis suggests that the RSV F_ecto monomer has a tight fold that protects it from nonspecific trypsin digestion. Upon cleavage into F1/F2, RSV F_ecto spontaneously self-associates into rosettes of postfusion trimers (Fig. 7B). This behavior is analogous to the rosette formation observed for the PIV Fs (20).

If the RSV F_ecto monomer is in a prefusion-like conformation, the HRA region would be packed against the globular head, presenting novel HRA epitopes lost in the postfusion conformation (Fig. 7C). Unlike the postfusion F_ecto trimer, the F_ecto monomer is capable of binding the site Ø, prefusion-specific FAb D25 (Fig. 5). Furthermore, the F_ecto monomer binds antibodies in the human serum-derived RSV immune globulin commercial product that do not bind the postfusion F_ecto antigen (Fig. 6B). Finally, previous studies have shown that postfusion F_ecto antigens are unable to bind and deplete the majority of neutralizing activity from RSV immune globulin (12). However, the F_ecto monomer is able to bind and deplete the majority of neutralizing antibodies from RSV immune globulin, suggesting that the F_ecto monomer retains the important site Ø epitopes present in other prefusion antigens and presumably on the RSV F that is present on the surface of the virion.

Retention of site Ø is a desired characteristic of next-generation RSV F antigens, and therefore the uncleaved RSV F monomer here described represents a potential prefusion subunit vaccine candidate. However, the RSV F monomer described here readily refolds into the postfusion conformation in the presence of trace amounts of trypsin or contaminant trypsin-like proteases and therefore is not stable enough for evaluation in immunization studies. Future work will be aimed at stabilizing the RSV F monomer by introducing additional mutations that lock it more stably in a prefusion conformation amenable to in vivo studies.