Cells, virus, and plasmids.
ELL-0 cells (avian fibroblasts) were obtained from the American Type Culture Collection, as were Vero cells, COS-7 cells, and HEp-2 cells. ELL-0 cells and HEp-2 cells were maintained in Eagle's minimal essential medium (MEM) (Gibco) supplemented with 10% fetal calf serum (FCS) and 2 mM glutamine. COS-7 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with nonessential amino acids, vitamins, penicillin, streptomycin, and 10% fetal calf serum. Vero cells were grown in DMEM supplemented with penicillin, streptomycin, and 5% fetal calf serum. RSV strain A2 was obtained from Ralph Tripp.
The RSV G protein cDNA was a synthetic gene obtained from Novavax, Inc. NDV NP, M, F, and HN protein genes, as well as the RSV G protein gene, were inserted into the pCAGGS expression vector as previously described (46
An NDV HN protein-RSV G protein chimeric gene was constructed by ligation of PCR-derived DNAs derived from pCAGGS-HN and pCAGGS-G. The HN sequence encoded amino acids 1 through 47, and the RSV G sequence encoded amino acids 65 through 298. The primers used to generate a DNA encoding the HN CT and TM domains were GGTTATTGTGCTGTCGACTCATTTTGGC (forward primer) and CATACTATATGCCAGGGCGGCCGCAGAGATGGCTAAG (reverse primer). This product was digested with XhoI and NotI (a site introduced without changing the amino acid sequence). The primers used to generate DNA encoding the G protein ectodomain were CTTCCCTCATCATTGCAGCGGCCGCTCTTGCCTACTCTGCGAATCATAAGGTC (forward primer which introduced a NotI site without changing the amino acid sequence) and GCCAGAAGTCAGATGGCCAAGG (reverse primer). The product was digested with NotI and MscI. The two DNA fragments were ligated into an XhoI-MscI-digested pGAGGS vector. The resulting plasmid containing the chimeric protein gene was sequenced in its entirety to verify the gene junctions (Fig. ) and to ensure that no additional changes were introduced during the PCRs.
FIG. 1. Construction and expression of NDV HN-RSV G protein chimera. (A) The diagram shows the locations of the cytoplasmic domain (CT), transmembrane domain (TM), and ectodomain of the NDV HN protein and the RSV G protein and the domains present in the NDV HN-RSV (more ...) Antibodies.
Polyclonal rabbit anti-NDV antibody was raised against UV-inactivated, purified NDV as previously described (48
). Polyclonal goat anti-RSV antibody (Biodesign) and mouse monoclonal anti-G antibody (MyBiosource) were used in Western blots. Anti-RSV F monoclonal antibody (clone 131-2A; Chemicon) was used in plaque assays. Secondary antibodies utilized were anti-goat antibody (Sigma), anti-mouse antibody (Sigma), and anti-rabbit antibody (Sigma).
Transfections were accomplished using Lipofectamine (Invitrogen) as recommended by the manufacturer. For small-scale transfections, a mixture of plasmid DNA (0.5 μg/35-mm plate) and Lipofectamine (5 μl/35-mm plate) in OptiMEM medium (Gibco) was incubated at room temperature for 45 min and then added to cells grown in 35-mm plates and previously washed with OptiMEM. Cells were incubated for 5 h at 37°C, OptiMEM was removed, and 2 ml of supplemented DMEM was added.
For quantitative preparations of VLPs, large-scale transfections of cells growing in T-150 flasks were utilized. For each T-150 flask, plasmid DNA (8 μg of each plasmid) in 1.6 ml of OptiMEM and Lipofectamine (80 μl) in 3.2 ml of OptiMEM were each incubated for 15 min at room temperature, mixed, and further incubated for 45 min at room temperature. OptiMEM (11.2 ml) was mixed with the DNA-Lipofectamine complexes and added to cells in a T-150 flask that had been washed twice with OptiMEM. Cells and DNA-Lipofectamine complexes were incubated for 5 h at 37°C, the complexes were removed, and 15 ml of complete medium was added.
Polyacrylamide gel electrophoresis, silver staining, and Western analysis.
Proteins in extracts, virus, or VLPs were resolved in 8% polyacrylamide gels as previously described (60
). Silver staining of proteins in the polyacrylamide gels was accomplished as recommended by the manufacturer (Pierce). For quantification of individual proteins in the polyacrylamide gels, different concentrations of bovine serum albumin (BSA) were electrophoresed in the same gel. A standard curve based on the stain of the BSA (using a Bio-Rad densitometer to measure the intensity of staining) was used to determine the concentration of each of the proteins in the purified VLPs or virus. For Western analysis, proteins in the polyacrylamide gels were transferred to polyvinylidene difluoride (PVDF) membranes (PerkinElmer) by dry transfer (iblot; Invitrogen). Proteins were detected in the blots as previously described (39
At 24 h posttransfection, heparin was added to the cells at a final concentration of 10 μg/ml. At 72 h posttransfection and again at 96 h posttransfection, cell supernatants were collected and cell debris was removed by centrifugation at 5,000 × g (Sorvall GSA SLA-1500 rotor). VLPs in the supernatant were pelleted by centrifugation in a type 19 rotor (Beckman) at 28,000 × g for 12 h. The resulting pellet was resuspended in TNE buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 5 mM EDTA), Dounce homogenized, and layered on top of a discontinuous sucrose gradient composed of 2 ml 65% sucrose and 4 ml 20% sucrose. The gradients were centrifuged in an SW28 rotor (Beckman) at 80,000 × g for 6 h. The fluffy layer at the 20 to 65% sucrose interface, containing the VLPs, was collected, mixed with 2 volumes of 80% sucrose, placed on top of a 1-ml layer of 80% sucrose in an SW41 Beckman centrifuge tube, and then overlaid with 3.5 ml of 50% sucrose and 2 ml of 10% sucrose. The gradients were centrifuged for 18 h at 175,000 × g. The VLPs, which floated to the interface of the 50% and 10% sucrose layers, were collected and concentrated by centrifugation in an SW50.1 rotor for 16 h at 140,000 × g. All sucrose solution concentrations are given as weights per volume, all sucrose solutions were dissolved in TNE buffer, and all centrifugations were done at 4°C.
RSV (multiplicity of infection [MOI] of 0.1) in 5 ml of DMEM without serum was added to confluent Vero cells growing in T-150 flasks. Cells were incubated with virus for 2 h at 37°C, and then 15 ml of DMEM with 5% fetal calf serum was added. Infected cells were incubated for 3 to 4 days at 37°C. The cells were then scraped into the cell supernatant, and the suspended cells were frozen at −80°C and then thawed. The resulting cell lysates were clarified by centrifugation at 1,200 × g (Sorvall) for 20 min. Virus in the supernatant was precipitated using polyethylene glycol 8000 (PEG 8000; 50% [wt/vol]), which was added to the supernatant to a final concentration of 10% and incubated for 90 min with stirring at 4°C. The precipitated virus was pelleted by centrifugation at 3,000 × g for 20 min at 4°C, snap-frozen, and stored at −80°C. The thawed virus pellet was resuspended in 10% sucrose in TNE, homogenized, and layered on top of a discontinuous sucrose gradient composed of 1 ml of 60%, 3 ml of 45%, and 4 ml of 30% sucrose (all dissolved in TNE buffer), and the gradient was centrifuged at 160,000 × g in an SW41 rotor for 90 min. The visible virus band between the 30% and 45% sucrose layers was collected. The viral protein content (M, NP, F, and G) of purified RSV was determined as described above, using known amounts of BSA included in the same gel.
RSV UV inactivation.
Purified virus was diluted in 2 ml of phosphate-buffered saline (PBS) in a 60-mm tissue culture dish and placed on a rotating platform 10 cm from a germicidal lamp (G15T8; Sylvania) for 20 min, a time previously determined to inactivate 100% of the virus, as measured by plaque assay. The efficacy of UV inactivation was determined in a plaque assay.
Formalin-inactivated RSV (FI-RSV) was prepared by a modification of the method of Prince et al. (64
). Virus was harvested from infected HEp-2 cells, clarified by centrifugation (1,000 × g
for 20 min at 4°C), filtered through a 0.8-μm filter, formalin treated by the addition of a 1/10 volume of a 1:400 dilution of 37% formaldehyde in water, and incubated for 3 days at 36°C with gentle stirring. The formalin-treated virus was pelleted at 100,000 × g
for 60 min at 10°C and resuspended in a 1/20 volume of EPES MEM and a 1/10 volume of SPG (2 M sucrose, 110 mM potassium phosphate, pH 7.1, 5 mM monosodium glutamate). The resuspended virus was centrifuged at 1,000 × g
for 15 min at 4°C, and the supernatant was incubated with 4 mg/ml aluminum hydroxide (Alhydrogel) overnight at room temperature. The virus-alum mixture was centrifuged at 1,000 × g
for 15 min, and the aluminum hydroxide pellet (adsorbed virus) was resuspended in a one-half volume of HMEM and a 1/10 volume of SPG and stored in 0.5-ml aliquots at 4°C. The amount of virus bound to the alum was estimated to be 44% by measuring the amount of total protein in the formalin-inactivated virus sample before and after alum adsorption. The amount of virus adsorbed to the alum was calculated to be equivalent to 6.1 × 108
PFU/ml. This estimate was based on the known virus titer prior to formalin treatment, the volume of the virus prior to formalin inactivation, the percent protein bound to the alum, and the concentration factor for each step in the procedure.
RSV immunoplaque assays.
Vero cells (1.5 × 105/well) were added to 24-well tissue culture plates (Costar) and incubated overnight at 37°C or until confluent. After removal of the medium, serial dilutions (in 200 μl of DMEM without serum) of RSV in virus stocks or in lung tissue clarified supernatants were adsorbed to monolayers that had been washed with DMEM without serum, in duplicate. Plates were incubated at 37°C for 2 h, the supernatant was removed, and each well received 1 ml of methylcellulose overlay (1 volume of 2× DMEM containing 10% fetal calf serum and 2% penicillin-streptomycin and 1 volume of 2% methylcellulose [Sigma]). After 3 to 6 days of incubation at 37°C, the overlay was removed, the cells were washed with PBS, and the monolayers were fixed with 1 ml of ice-cold acetone-methanol (60:40) for 10 min. After air dying, plates were blocked in 5% nonfat dry milk in PBS for 10 min at room temperature. Anti-F monoclonal antibody (1:800 dilution) was added to wells and incubated for 2 hours, followed by a 1-hour incubation with secondary anti-mouse immunoglobulin G (IgG) antibody conjugated to alkaline phosphatase. Antibodies were diluted in 5% nonfat milk, and plates were incubated at 37°C. Plates were washed twice with PBS containing 0.5% Tween 20 (Sigma) after each antibody incubation step. Individual plaques were developed using a DAB substrate kit (Vector Laboratories) as recommended by the manufacturer.
Mouse sera were complement inactivated or not and then diluted in DMEM without serum. Purified RSV was diluted to approximately 75 to 150 PFU in 100 μl. Dilutions of mouse sera in 100-μl aliquots were added to the virus and incubated for 1 h at 37°C. The mixture was then added to prewashed, confluent monolayers of Vero cells growing in 24-well tissue culture dishes, and the cells were incubated at 37°C for 1 hour. The antibody-virus mixture was removed, and 1 ml of methylcellulose overlay was added to each well as described above. Plates were incubated for 3 to 4 days, and plaques were stained as described above.
Animal immunization and challenge protocols.
Three-week-old BALB/c mice, from Jackson Laboratories or Taconic Laboratories, were housed in groups of five under pathogen-free conditions in microisolator cages at the University of Massachusetts Medical Center animal quarters. All protocols requiring open cages were accomplished in biocontainment hoods. Mice were immunized by intraperitoneal (i.p.) or intramuscular (i.m.) inoculation of different concentrations of VLPs or UV-inactivated RSV in 0.5 ml (i.p.) or 0.05 ml (i.m.) of PBS containing 30% sucrose. Other groups of mice were lightly anesthetized with isoflurane and then infected by intranasal (i.n.) inoculation of RSV (1 × 106 to 3 × 106 PFU/mouse in 50 μl). Mice that received an immunization boost were injected i.p. or i.m. with 10 μg of VLPs or UV-inactivated RSV/mouse or received 1 × 106 to 3 × 106 PFU/mouse of live RSV (i.n.). (The RSV protein concentration in 1 × 106 to 3 × 106 PFU was, at most, 400-fold less than the protein concentration in the VLP or UV-RSV preparation.)
Mice challenged with live RSV were lightly anesthetized as described above and infected i.n. with 1 × 106 to 3 × 106 PFU of virus.
Detection of virus in lung tissue.
Mice were anesthetized with isoflurane and exsanguinated after severing of the right caudal artery. Lungs were removed aseptically, placed in 0.5 ml of 30% sucrose in PBS, and stored at −80°C. Upon thawing, lungs were weighed and then homogenized using a pestle (Kontes). The homogenate was centrifuged at 12,000 rpm for 15 min, and the virus titer in the supernatant was determined by plaque assay as described above.
Determination of antibody titers by ELISA.
Antigens used as targets in enzyme-linked immunosorbent assays (ELISAs) were RSV-infected Vero cell extracts or extracts from 293T cells transfected with pGAGGS-G. To prepare cell extracts, cell monolayers were washed in cold PBS and lysed in TNE buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 5 mM EDTA) containing 1% Triton X-100. All antigens were placed in carbonate buffer, pH 9.6, added to microtiter plates (Costar), and incubated overnight at 4°C. The amount of transfected extract added to each well was adjusted so that the amounts of G protein were comparable, as determined by Western blotting.
After binding of the target antigen, wells were blocked in 50 μl PBS containing 1% BSA at room temperature for 1 to 2 h, washed three times in PBS, and drained. Different dilutions of mouse sera were added to the microtiter wells in 50 μl of PBS-BSA and then incubated for 1 h at room temperature. After removal of the mouse sera and washing of the wells three times, a biotinylated anti-mouse antibody (1:4,000 dilution) (Sigma) in 50 μl of PBS-BSA was added, and the microtiter plates were incubated for 1 h at room temperature. The microtiter plates were then washed three times in PBS, and horseradish peroxidase (HRP)-conjugated neutravidin (1:4,000 dilution) (Pierce) was added in 50 μl of PBS-BSA. The microtiter plates were incubated for 1 h at room temperature and washed four times in PBS. TMB (3,3′,5,5′,tetramethylbenzidene) substrate (Sigma) in a 50-μl volume was added to each well and incubated for 15 to 20 min. The reaction was stopped with 50 μl 1 N H2SO4, and the optical density was read in a plate reader (Molecular Devices).
Pulmonary histology of RSV-infected mice.
For histological analysis of lung tissue, mice were anesthetized with isoflurane and exsanguinated after severing of the right caudal artery. The lungs were then fixed via infusion through the trachea with 4% formalin, removed, immersed in 4% formalin for 24 h, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) or periodic acid-Schiff stain (PAS) by the University of Massachusetts Core Facility. Six sections per mouse were obtained. Sections from each mouse were scored blindly for the degree of inflammation (H&E stains) of blood vessels, airways, or interstitial spaces, on a scale of 0 to 3, as previously described (52
). For sections stained with PAS, the percentage of airways positive for PAS in 25 randomly selected airways was determined.
Statistical analyses of data were accomplished using Graph Pad Prism 5 software.