Replication-competent recombinant SV was rescued from the full genome SV cDNA containing an RSV F gene (for expression of a membrane-anchored form of the F protein) by a reverse genetics system using a modification of previously described methods [27
]. To create a recombinant SV that produced the membrane-anchored form of RSV F protein, the full-length cDNA of SV (Z strain) [28
] was modified to create a unique NotI
site in the non-coding region between the F and HN genes (pSV(+)N [29
], ). Viral RNA was extracted from RSV strain A-2 (American Type Culture Collection, Rockville, MD)-infected HEp-2 cells and the F gene was amplified by reverse-transcription (RT)-PCR (Titan One Tube System; Roche). The PCR forward primer included a NotI
site and the reverse primer included an SV transcription termination signal, an intergenic (IG) sequence CTT, a transcription initiation signal and a second NotI
site (see ). Both RSV F cDNA and pSV(+)N were cleaved with NotI
Design and characterization of recombinant Sendai virus expressing the RSV F protein
To rescue the recombinant virus, we infected 293T cells with a UV-inactivated, T7 RNA polymerase-expressing recombinant vaccinia virus (vTF7.3) for 1 hr at 37 °C at a multiplicity of infection (MOI, defined prior to UV treatment) of 3. Cells were co-transfected with the F-containing SV cDNA plasmid (1 μg) with three supporting T7-driven plasmids expressing the NP, P, or L genes of SV (1 μg pTF1SVNP, 1 μg pTF1SVP, and 0.1 μg pTF1SVL) in the presence of Lipofectamine (8 μl; Life Technologies, Grand Island, NY). Cells were then incubated for 40 hr. Cell lysates were subsequently prepared and inoculated into 10-day-old embryonated hens eggs. Allantoic fluids were harvested after 72 hr and virus was cloned by plaque purification on LLC-MK2 cells. Cloned recombinant SV was amplified once more in embryonated eggs to prepare vaccine stocks. Recovered virus was designated rSV-RSV-F.
Immunoprecipitation of F protein from infected HEp-2 cells
To check for RSV F protein expression by rSV-RSV-F infected cells, HEp-2 cells in 6 well plates were infected with the recombinant virus or with RSV-A2 for 1 hr at room temperature. Infected cells were then cultured for 2 days at 34°C and labeled with 35S-Trans (100 μCi/ml) overnight at 34°C. Cells were lysed with 1 ml of TNE buffer (10 mM Tris, pH 7.4; 150 mM NaCl; 0.5% NP-40). Supernatants were clarified by a high-speed spin (15,000 × g, 15 min). Labeled RSV F proteins in the clarified supernatants were captured by mixing with a murine RSV-F-specific monoclonal antibody (Clone 631, Cat#C65063, Biodesign Intl. Saco, ME) bound by sheep anti-mouse IgG Dynal Beads (M-280, cat#112.01, Dynal Biotech, Lake Success, NY). The immunocomplexes were resolved by SDS-PAGE (10% gels) and analyzed with Kodak BioMax MR film.
Purification and characterization of recombinant SV particles
HEp-2 cells were infected with recombinant SV at an MOI of 5 and harvested after incubation for 3 days at 34°C. Recombinant SV particles were purified from the cell supernatants by centrifugation on a sucrose gradient. Purified virus (quantified for protein content using a Micro BCA™ Protein Assay Kit, Pierce, Rockford, IL) was processed on an SDS gel and stained with GelCode Blue Stain Reagent. Additional gels were processed for Western blotting as described below. RSV-infected cell lysates were used as positive controls.
To obtain the control, wild-type RSV-infected cell lysate, HEp-2 cells were infected with RSV-A2 in PBS at room temperature for 1 hour. The inoculate was removed and cells were cultured in Dulbecco’s modified eagle’s medium (Cambrex Bio Science Walkersville, Inc, Walkersville, MD) supplemented with glutamine, gentamicin and 10% fetal calf serum. Cells were harvested on day 3 and lysed with 0.2 ml of TNE buffer (10 mM Tris [pH 7.4], 150 mM NaCl, 0.5% NP-40, and 1 mM EDTA). Supernatants were then clarified by centrifugation (15,000 × g, 10 min).
Western blot analyses
Proteins were separated by SDS-polyacrylamide gel electrophoresis under non-reducing conditions (to preserve antigenic determinants) and transferred to an Immobilon membrane (Millipore, Danvers, Mass.). Membranes were treated with 5% milk/TBST (Tris buffered saline plus 0.5% Tween), incubated with an RSV F-specific monoclonal antibody (antibody 1269, kindly provided by Dr. Coelingh, NIAID [30
]), washed again, and incubated with a goat anti-mouse IgG (H+L) horse radish peroxidase (HRP) conjugate (BioRad, Hercules, CA). A final reaction was with a SuperSignal West Pico Chemiluminescent (HRP) Substrate (Pierce, Rockford, Il) used as recommended by the manufacturer.
Animals and Immunizations
Groups of 5 adult female cotton rats (Sigmodon hispidus
; Harlan Sprague Dawley, Indianapolis, IN) were intranasally inoculated with the rSV-RSV-F vaccine, or wild-type SV. In some experiments, animals also received a previously-described rSV-RSV-G vaccine [29
] or an equal mixture of the rSV-RSV-F and rSV-RSV-G constructs. In each experiment, the total inoculum was 2 × 106
PFU/animal. Animals were rested for at least 9–10 days, after which mediastinal lymph nodes were collected for T-cell assays. Animals were rested for at least one month post-vaccination prior to serum sample collections and challenges.
Enzyme-linked immunosorbent assay (ELISA)
For detection of anti-RSV F-specific antibodies, ELISA plates (96-well) were coated with 1 ug/ml purified F protein (see below) and incubated overnight. Plates were blocked with PBS/3% bovine serum albumin (BSA, Sigma, St Louis, MO), after which serially diluted serum samples from the test and control cotton rats were added and incubated for at least 2 hrs at 37 °C. Plates were then washed and incubated with rabbit anti–cotton rat IgG (Virion Systems, Rockville, MD) for 30 min at room temperature. Plates were then washed, incubated with an anti-rabbit IgG-horseradish peroxidase conjugate (diluted 1:3,000 in PBS/1% BSA, Bio-Rad, Hercules, CA, Cat# 170–6515) for 30 min at room temperature, washed again, and incubated with 2,2′-azino-bis-(3-ethylbenzthiazolinesulfonic acid) (ABTS, Southern Biotechnology Associates, Inc, Birmingham, AL). Optical readings were at 405 nm.
To isolate purified RSV F protein, we utilized a recombinant SV construct that was designed to express a truncated, rather than a full-length F molecule. This SV was grown in embryonated hens eggs. Allantoic fluids were harvested three days later and fluids were centrifuged (approximately 300 × g, 10 min, to remove debris) and filtered (0.45 μM filter). Samples were passed over a PBS-equilibrated Sepharose column bound by the anti-F monoclonal antibody Palivizumab (Synagis; MedImmune Inc., Gaithersburg, MD). The column was washed with PBS and eluted with 0.2 M glycine (pH 2.8), after which pooled protein fractions were dialyzed overnight against PBS. RSV F from RSV-infected cells could also be used as the target antigen in ELISAs.
Neutralization assays and challenges
Neutralization tests were with viruses from RSV A and B isolates. Isolates were A2 (subtype A), B1 (subtype B) and VR1580 (subtype B, from the American Type Culture Collection, ATCC, Rockville, MD) and K1013 (subtype A), K1014 (subtype A), K1015 (subtype A), and K1036 (subtype B, kindly provided by Dr. J. Devincenzo, LeBonheur Children’s Hospital, Memphis, TN). Each virus was grown on HEp-2 cells except for RSV B1, which was grown on Vero cells. To conduct neutralization assays, serially diluted sera were mixed with RSV (100 to 150 PFU/well) in EMEM (Cambrex Bio Science Walkersville, Inc, Walkersville, MD) for 1 hr at 37°C. The virus-serum mixtures were inoculated onto cell monolayers in 12-well plates. Plates were incubated for 1 hr (37°C, 5% CO2) and then overlaid with medium supplemented with glutamine, antibiotics, 10% fetal calf serum and 0.75% methylcellulose (Fisher Scientific, Fair Lawn, NJ). After incubation for 4 to 6 days (37°C, 5% CO2), the methylcellulose was removed, cells were fixed with 1 ml of formalin phosphate (1:10 dilution; PROTOCOL® from Fisher Scientific) and stained with hematoxylin and eosin. Plaques were counted and the plaque numbers were compared to those in wells with normal cotton rat sera.
Animals were challenged intranasally with RSV isolates A2, B1, K1015 or VR1580 at doses of 1.5–7.5 × 106
PFU/cotton rat. Lungs were harvested 3 days later for virus measurements and 5 days later for immunopathology studies (as described by Prince et. al. [5
Peptide synthesis and IFN-gamma ELISPOT assays
For analyses of RSV-specific T-cells, overlapping peptides (derived from the RSV A2 F sequence, Genbank Accession # AAB86664) were prepared by the Hartwell Center for Bioinformatics and Biotechnology at St. Jude Children’s Research Hospital. Peptides were generally 15 amino acids in length and were initiated at intervals of 5 amino acids to cover the entire length of the RSV F protein. They were then combined to form eleven pools (see detail below).
Multiscreen-hemagglutinin filtration plates (Millipore, Bedford, MA) were incubated overnight at 4°C with 3.3 μg/ml goat anti-cotton rat IFN-gamma antibody (R & D Systems, Minneapolis, MN), washed, and incubated for at least 1 hr at 37°C with complete tumor medium (CTM [32
], a modified Eagle’s medium [Invitrogen, Grand Island, NY] supplemented with 10% fetal calf serum, dextrose [500 μg/m], glutamine [2 mM], 2-mercaptoethanol [3 × 10−5
M], essential and non-essential amino acids, sodium pyruvate, sodium bicarbonate, and antibiotics). Mediastinal lymph node cells were harvested from cotton rats 10 days after vaccination. Fresh cells were suspended in CTM and added to wells (2.5-1 × 106
cells/well) containing the peptide pools (final concentration of each peptide, approximately 10 μM). Positive control wells received 4 μg/ml Con A (Sigma-Aldrich, St. Louis, MO) rather than peptide. The plates were incubated for 48 hr at 37 °C, washed four times with PBS, and washed four times with PBS wash buffer (PBS with 0.05% Tween 20). Biotinylated goat anti-cotton rat IFN-gamma antibody (100 μl of 0.5 μg/ml; R & D Systems, Minneapolis, MN) in PBS containing 0.05% Tween 20 and 1% FCS was then added to each well, and plates were incubated at 37 °C for at least 2 hr. After additional washing, streptavidin-conjugated alkaline phosphatase (Cat# D0396, DAKO, Copenhagen, Denmark) diluted 1:500 in PBS wash buffer was added. One hour later, plates were rinsed with wash buffer and water and the IFN-gamma spots were developed by adding 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium alkaline phosphatase substrate (Sigma-Aldrich). Spots were counted with an Axioplan 2 microscope and software (Carl Zeiss, Munich-Hallbergmoos, Germany).
Measurement of virus replication
Three days after intranasal RSV challenge, virus was quantified in the lungs of vaccinated animals. Briefly, cotton rats were sacrificed and the lungs were harvested and homogenized on ice with a mechanical Dounce homogenizer (PowerGen125 PCR Tissue Homogenizing kit; Fisher Scientific) to yield a final homogenate of 5 ml in PBS. Homogenates were centrifuged (approximately 1500 × g, 10 min) and supernatants were collected for cryopreservation prior to testing. For all viruses except the B1 isolate, serially diluted supernatants were inoculated into multiple wells of HEp-2 cell monolayers in 6–12 well plates in EMEM; after 1 hr at 37 °C, 5% CO2, the wells were overlaid with medium supplemented with glutamine, antibiotics, 10% fetal calf serum and 0.75% methylcellulose. After incubation for 4 to 6 days (37°C, 5% CO2), the methylcellulose was removed, the cells were fixed with formalin phosphate, and the plates were stained with hematoxylin and eosin for plaque counting. For RSV B1, the same procedure was followed except that samples were applied to Vero cell monolayers (ATCC, Rockville, MD). Plaque forming units within cotton rat lungs were calculated.
Lungs isolated 5 days after challenge with RSV were prepared for histological analyses as previously described [29
]. Briefly, lungs were inflated via the trachea with 10% neutral buffered formalin and submerged in formalin for overnight fixation. The fixed tissue was embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin. Lung pathology was scored on the basis of three parameters: peribronchiolitis (inflammatory cells around small airways); alveolitis (inflammatory cells within alveolar spaces); and interstitial pneumonitis (inflammatory cell infiltrates and thickening of alveolar walls). Scoring was performed as described by Prince et. al. [5
] in a blinded manner by a veterinary pathologist (KB) using a scale of 0 (no inflammation) to 100 (maximum inflammation within the experiment). Relative comparisons between test and control animals were evaluated independently for each parameter and for each experiment.