Pathogen-free, 8-week- and 18-month-old, female BALB/c mice were purchased from the National Institute of Aging (NIA, Bethesda, MD). All animal procedures were conducted according to the guidelines of the Institutional Animal Care and Use Committee.
The fusion subunit F protein (sequence containing amino acids 1–524) of RSV A2 was expressed in Chinese Hamster Ovary (CHO) cells (ATCC, Manassas, VA) and immuno-affinity purified with anti-RSV F monoclonal antibody (Palivizumab, MedImmune, Gaithersburg, MD) to > 95% purity. The antigenic sites of the protein were preserved as determined by an ELISA sandwich assay. Purified RSV F protein was used for both animal immunizations and coating in ELISA assays. Our data suggested that any anti-CHO host cell protein response generated in young or aged mice was not detectable in an ELISA assay using 0.5 μg/mL coating concentration of RSV F. Wild-type (wt) RSV A2 and Green Fluorescent Protein (GFP)-tagged RSV A2 were grown and titrated in Vero cells. Virus stocks were stabilized in sucrose-phosphate (1X SP) buffer (0.2 M sucrose, 0.0038 M KH2PO4, 0.0072 M KH2PO4), snap-frozen and stored at −70°C.
All mice were primed at day 0 and boosted at day 14 intramuscularly (i.m) in both quadriceps. Placebo groups were given 100 μl of phosphate buffered saline (PBS), while F vaccine groups received 0.3 μg of F protein. This dose was based on our previous observation that 0.3 μg of F without adjuvant was only partially protective upon wt RSV A2 challenge (unpublished results). For RSV F + alum group, mice were given 0.3 μg of F protein mixed (by vortexing for 30 min at RT) with 100 μg of Alhydrogel (Aluminum Hydroxide, Brenntag Biosector, Denmark). Lastly, for the live RSV group, mice were intranasally (i.n.) infected on day 0 with 106 PFU wt RSV strain A2. Protection was assessed by intranasal inoculation of all mice with 106 PFU RSV strain A2 on day 28. Body weight was measured daily following wt RSV challenge until animals were euthanized.
Peripheral blood was collected from the submandibular vein 27 days post-priming. Collected blood was left at room temperature (RT) for 20 min. The tubes were centrifuged at 7000 rpm for 10 min and sera stored at −80°C.
RSV-specific neutralizing antibody titers in mouse sera were measured using GFP-expressing RSV A2 in a micro-neutralization assay. Mouse sera from all treatment and placebo groups were heat-inactivated at 56°C for 45 min and serially diluted three-fold in growth medium. Equal volumes of the diluted sera were mixed with GFP-expressing RSV A2 virus to yield 500 PFU/well. Virus only and hyperimmune serum with a known neutralizing antibody titer were included on each plate as controls. The serum + virus (or virus only) mixture was incubated at 33°C, 5% CO2 for 1 h to allow time for the neutralizing antibodies in the serum to neutralize the virus particles. Confluent monolayers of Vero cells (ATCC, Manassas, VA) prepared in separate 96-well plates were infected with the GFP-expressing RSV A2 virus only or the serum/virus mixture from the above step. The infected Vero cell plates were incubated at 33°C, 5% CO2 for 22 h. The plates were washed and the fluorescent viral foci enumerated using an IsoCyteTM Reader (Blueshift Biotechnologies, Sunnyvale, CA). The 50% reduction in viral foci (antibody EC50 titers) was calculated using a 4-parameter curve fit algorithm.
Serum IgG, IgG1, IgG2a, and Lung IgG ELISA
RSV F-specific IgG antibody titers in serum were measured using ELISA. Three sets of high binding 96-well plates intended to measure IgG, IgG1, and IgG2a, respectively, were coated overnight at 4°C with 0.5 μg/mL of CHO-expressed recombinant RSV F diluted in PBS. Plates were washed with PBS/0.05% Tween-20 and blocked with PBS/0.05% Tween-20 containing 0.5% bovine serum albumin (BSA, EMD Biosciences, Gibbstown, NJ) for 1 h at 37°C. The samples and hyperimmune serum reference standard (MedImmune, Mountain View, CA) were serially diluted 2-fold in PBS/0.05% Tween-20/0.5% BSA following an initial pre-dilution of 1:50. After washing, the pre-diluted samples were added to the three plates and the plates incubated at 37°C for 1 hour. Horseradish peroxidase (HRP)-conjugated, goat anti-mouse IgG, IgG1, or IgG2a antibodies (Jackson ImmunoResearch, West Grove, PA) diluted 1:20,000 in PBS/0.05% Tween-20/0.5% BSA were added, respectively, to the three plates. After washing, the plates were then developed with 3,3´,5,5´-tetramethylbenzidine (TMB, Sigma, St. Louis, MO) and stopped with 1N HCl (Sigma, St. Louis, MO). The absorbance was measured at 450 nm on a SpectraMax plate reader and analyzed using SoftMax® Pro (Molecular Devices Inc., Sunnyvale, CA). Titers were reported as Log2EC50 determined using a 4-parameter curve fit for each sample curve.
Viral plaque assay
Mice were euthanized at day 4 post-challenge. A Beadbeater (Biospec Products, Bartlesville, OK) was used to homogenize the lungs as previously described. Lung homogenates were serially diluted and used to inoculate subconfluent HEp-2 cells in 24-well plates. After 1 h adsorption at RT on a rocking platform, the cells were overlaid with MEM/10% FBS/1% penicillin G/streptomycin sulfate/amphotericin B solution /0.75% methylcellulose. After six days, the overlay medium was removed and the cells fixed with methanol. Plaques were visualized by immunodetection as described.
Lung homogenates prepared for the viral plaque assay as described above were also evaluated in a Luminex-based cytokine profiling assay. Mouse cytokine multiplex kits, custom designed to include IL-5, IL-13, IFN-γ, RANTES, MCP-1, eotaxin, KC, IP-10, and MIP-1α were purchased from Millipore (Billerica, MA). The assay was performed according to the manufacturer’s instructions. The plates were analyzed on a Bio-Rad Luminex reader (Hercules, CA), and individual cytokine levels were expressed as pg/mL. IFNγ and IL-5 were used to represent TH1 and TH2 cytokines, respectively.
Heart-lung blocks were harvested 4 days post-infection and fixed overnight in 4% paraformaldehyde. Lungs were transferred to 70% ethanol and then embedded in paraffin blocks as described previously. Tissue sections (5 μm) were stained with hematoxylin and eosin (H&E) to assess histologic changes. H&E-stained slides were digitally scanned using a Zeiss MIRAX MIDI microscope. Slides were examined and scored by a pathologist who was blinded to the experimental groups. Lymphocytes, neutrophils, macrophages, and eosinophils were assessed in peribronchiolar, perivascular, interstitial, and alveolar spaces as described. For the eosinophil score, groups were assessed for severity of eosinophilic infiltrate on a scale of 0 to 5 in the peribronchiolar, perivascular, interstitial, and alveolar spaces, where 0 = no eosinophils present, 1=1-10 in area, 2=11-20, 3=21-30, 4=31-40, 5= 41–50.