Construction of expression plasmids
Mouse-derived granulocyte macrophage-colony stimulating factor (mGM-CSF) and interleukin 2 and 4 (mIL-2, mIL-4) were fused to a short stalk, transmembrane, and cytoplasmic tail domain of influenza A/WSN/33 hemagglutinin (HA) using standard PCR methodologies as described previously . Briefly, primers, amplifying the carboxyl terminal 71 amino acids of WSN HA and the coding sequence of the cytokines, were designed to introduce the appropriate restriction sites. Nucleotides 1521–1730 coding for the 26 amino acid stalk region, the transmembrane domain, and cytoplasmic tail domain of the hemagglutinin were amplified using the forward primer 5'-CCGGATCCAATGGGACTTATGATTATCC-3' and the reverse primer 5'-CCGAATTCTCAGATGCATATTCTGCACTGC-3' to introduce restriction sites Bam HI and Eco RI (underlined), respectively. Primers specific for mGM-CSF (forward 5'-CCAAGCTTGGAGGATGTGGCTGCAGAA-3'; reverse 5'-GGGGATCCTTTTTGGACTGGTTTTTTGC-3'), mIL-2 (forward 5'-CCGGTACCAGCATGCAGCTCGCATCCTGTGTC-3'; reverse 5'-GGGGATCCTTGAGGGCTTGTTGAGATGA-3'), and mIL-4 (forward 5'-CCGGTACCGCACCATGGGTCTCAACCCCCA-3'; reverse 5'-CCGGATCCCGAGTAATCCATTTGCATGATG-3') were designed to remove stop codons and introduce Hind III (mGM-CSF) or Kpn I (mIL-2 and mIL-4) and BamHI endonuclease restriction sites on the 5' and 3' ends respectively. PCR products were generated using Platinum Pfx (Invitrogen) and GeneAmp PCR System 2400 (Applied Biosystems). Purified PCR products were subsequently digested and inserted into the respective restriction sites of pcDNA3.1 using T4 DNA Ligase (Invitrogen) according to the manufacturers protocol. Plasmid constructs, harboring the respective fusion constructs, were sequenced by the Wayne State University Sequencing Core (Applied Genomics Technology Center) to verify sequence and integrity of the constructs.
Generation of CYT-IVAC producer cell lines
Madin-Darby canine kidney (MDCK) cells were maintained in complete growth media (DMEM/10% FBS) consisting of Dulbecco's Modified Eagles Media supplemented with 10% fetal bovine serum (Atlanta Biologicals) and the antibiotics penicillin/streptomycin (100 U/100 μg). Cells were transfected with expression plasmids using Lipofectamine2000 (Invitrogen) as described previously . Stable transfectants were selected by growth in DMEM/10%FBS supplemented with Geneticin (1.5 mg/ml; Gibco). Geneticin-resistant cells were subcloned by limiting dilution plating in 96-well plates in the presence of Geneticin (G418™ Invitrogen, 1 mg/ml). Individual MDCK subclones were screened for cell surface expression and bioactivity of the respective membrane-bound cytokines.
Viral infection, purification, and inactivation
Wild-type and CYT-IVAC producer MDCK cells (90% confluent) were infected at an MOI of 1 with either influenza virus A/PR/8/34 (H1N1) or A/Udorn/72 (H3N2). Following virus adsorption (1 hr, 37°C), the inoculum was removed and DMEM/2% FBS was added. Supernatants from infected monolayers were harvested 24–36 hours post infection and cellular debris was pre-cleared at 400 × g for 15 minutes at 4°C. Virions were purified by centrifugation through two sequential 10–26% iodixanol continuous gradients (OptiPrep™, Axis-Schield) (SW41 rotor, 55,000 × g, 45 min at 4°C). Banded virus was collected and concentrated by centrifugation at 88,000 × g for 45 minutes at 4°C and subsequently re-suspended in phosphate-buffered saline, PBS. Purified virus was inactivated by treatment with 15 mM β-propiolactone for 15 minutes at 25°C. The reaction was neutralized by the addition of sodium thiosulfate (40 mM final concentration, 30 min, 25°C). Inactivated virus was diluted with PBS, pelleted by centrifugation as described and resuspended in sterile PBS. Total viral protein concentration was determined using a bicinchoninic acid protein assay kit (Pierce Biotechnology). Inactivation was confirmed by monitoring cytopathic effect in MDCK cells treated with 5 μg of inactivated virus vaccine for a period of 3–5 days at 37°C in the presence of 1.5 μg/ml TPCK-treated trypsin (Sigma).
Cell surface expression and viral incorporation of membrane-bound cytokines (Immunofluorescence Microscopy)
MDCK cells were grown to 90% confluency on glass cover slips in 24 well plates. Cells were washed with phosphate buffered saline (PBS) and fixed with 3% paraformaldehyde (PF) in 250 mM HEPES for 10 minutes at room temperature (RT). PF was removed and 50 mM glycine in PBS was added for 10 minutes at RT to quench any remaining PF. Cells were washed 2 times with PBS and blocked with 2% chicken serum in PBS for 30 minutes at RT. For immunostaining cells were incubated sequentially with rat anti-cytokine specific antibody (BD Pharmagen) and chicken anti-rat IgG conjugated Alexa Fluor® 488 antibody (Invitrogen/Molecular Probes). All antibodies were diluted in PBS/2% chicken serum. Cover slips were mounted on slides using ProLong Antifade (Invitrogen/Molecular Probes). Immunofluorescent staining was visualized using a Nikon E800 Epifluorescence Microscope. Digital images were captured using a Roper CoolSnap FX digital camera and analyzed using MetaMorph Imaging Software (Universal Imaging).
To visualize viral incorporation of membrane-bound cytokines, CYT-IVAC producer cells, grown on cover slips, were infected with filamentous influenza A/Udorn/72 at an MOI of 1. The cells were fixed at 8 hr post-infection with 3% PF and blocked as described above. Cells were incubated with rat anti-cytokine specific primary antibody and Alexa Fluor® 488 conjugated secondary antibody as described above. Additionally, cells were incubated with goat anti-H3 antibody and secondary chicken Alexa Fluor® 594 conjugated anti-goat IgG (Invitrogen/Molecular Probes). Cover slips were mounted and immunofluorescence was analyzed as described above.
Western blot analysis
Vaccines were solubilized in Laemmli Buffer (BioRad) (LB) and heated at 96°C for 10 minutes to denature proteins. Samples were separated on 12% PAGE-SDS and subsequently blotted to PVDF membrane. Membranes were probed by sequential incubation with rat anti-GM-CSF (BD Bioscience), followed by goat anti-rat IgG horseradish-peroxidase conjugated secondary antibody (Santa Cruz). Membranes were exposed to ECL or Femto solution per manufacturers (Pierce) instructions and membranes were visualized using Chemdoc XRS (BioRad).
Total Cytokine and Hemagglutinin Quantitation by Slot Blot Assay
Serial dilutions of vaccines at 1, 0.5 and 0.25 μg (cytokine quantification) or 1, 0.2 and 0.04 μg (HA quantification) of total viral protein, as well as serial diluted recombinant cytokine (2000 ng to 1.95 ng) were blotted on PVDF membranes using a slot blot apparatus. Membranes were blocked with 5% milk solution and subsequently incubated sequentially with diluted primary antibody, specific for the respective cytokine (rat anti-GM-CSF, IL-2, or IL-4, BD Bioscience) or hemagglutinin (mouse anti-HA, Meridian Life Science® Inc or rabbit anti-H1N1/Pan H1, Pierce® Inc) followed by the respective horseradish-peroxidase conjugated secondary antibody (goat anti-rat IgG (Santa Cruz), goat anti-mouse IgG (BioRad) or goat anti-rabbit IgG (Sigma). Membranes were exposed to ECL or Femto solution per manufacturers (Pierce®) instructions and chemiluminescent signals were recorded using a Chemdoc XRS (BioRad). Images were processed with ImageJ software (NIH freeware) and standard curves for each cytokine were generated using optical pixel densities. Total cytokine content for each vaccine preparation was extrapolated from standard curves and is expressed as the average of the three dilutions evaluated for each vaccine in nanograms (ng) of cytokine per microgram (μg) of total viral protein. The signal intensity of the HA specific signal for each vaccine was calculated for each dilution and the average pixel density per μg of total viral protein is given.
Hemagglutination units (HAU) were determined by agglutination of chicken red blood cells as previously described . Briefly, serial diluted vaccine preparations were mixed with an equal volume of fresh 0.5% chicken red blood cells and incubated at room temperature for 30 minutes. Red blood cell agglutination was recorded and HAU per μg of total viral protein is expressed as the reciprocal of the last dilution of virus that resulted in agglutination.
Bioassays of membrane-bound cytokines
Bone marrow (BM) cells, as indicator cells for mGM-CSF bioactivity, were prepared from the femurs of female Balb/c mice. Briefly, bone marrow was flushed from the femurs with RPMI and the cell suspension passed through a 70 μm cell strainer. Red blood cells were lysed using RBC lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 0.01% EDTA). Cells were washed 2 times with RPMI and re-suspended in complete RPMI (10% FBS, 20 mM L-glutamine, 1 M HEPES, 100 mM Sodium Pyruvate, 55 μM 2β-Mercaptoethanol, Penicillin/Streptomycin (100 units/100 μg/ml)). For MDCK based bioassays, BM cells (2 × 105/well) were added to wells of a 96 well plate containing 90% confluent, mitomycin C (50 μg/ml) treated wild type or CYT-IVAC producer (mGM-CSF~HA) MDCK cells. For virus based bioassays and quantitation of viral incorporated bioactive GM-CSF, BM cells (2 × 105) or MPRO cells (5 × 103) , respectively, were added to wells of a 96 well plate containing inactivated A/PR/8/34 wild type or A/PR/8/34 mGM-CSF~HA. Recombinant GM-CSF was also used to establish a standard curve by which virus-incorporated bioactive GM-CSF could be quantitated. Plates were incubated at 37°C for 72 hours (BM) or 48 hours (MPRO cells). For the last 18 hours of incubation for the cell-based bioassay, cells were pulsed with 3H-thymidine then harvested and counted using a scintillation counter. For the viral based bioassay, Alamar Blue® (Invitrogen) was added to each well at 10% of the total volume for the last 24 hours and Alamar Blue® reduction was determined from the absorbance values recorded at 570 nm and 600 nm after 72 (BM) or 48 (MPRO) hours.
CTLL-2 cells (a gift from Dr. Robert Swanborg, Wayne State University) were used as indicator cells for the bioactivity of mIL-2. Cells were maintained in complete RPMI supplemented with recombinant mouse IL-2 (10 ng/ml). CTLL-2 cells (5 × 103) were added to 96 well plates containing mitomycin C treated cells (wild-type or mIL-2 CYT-IVAC producer cells) or inactivated virus (A/PR/8/34 wild-type or A/PR/8/34 mIL-2~HA) as described above. Recombinant IL-2 was also used to establish a standard curve by which virus-incorporated bioactive IL-2 could be quantitated. Plates were incubated at 37°C for 48 hours. For the last 18 hours of incubation for the cell-based bioassay, cells were pulsed with 3H-thymidine then harvested and counted using a scintillation counter. For the virus particle based bioassay, Alamar Blue® was added to each well for the last 24 hours and absorbance was read at 570 nm and 600 nm after 48 hours.
CT.4s cells (gift from Dr. William Paul and Dr. Jane Hu-Li, Laboratory of Immunology, National Institute of Health) were used to determine mIL-4 bioactivity . Cells were maintained in complete RPMI supplemented with recombinant mouse IL-4 (2 ng/ml). CT.4s cells (5 × 103) were added to 96 well plates containing mitomycin C treated MDCK cells (wild-type or mIL-4 CYT-IVAC producer cells) or inactivated virus (A/PR/8/34 wild-type or A/PR/8/34 mIL-4~HA) as described above. Recombinant IL-4 was also used to establish a standard curve by which virus-incorporated bioactive IL-4 could be quantitated. Plates were incubated at 37°C for 48 hours. For the last 18 hours of incubation for the cell-based bioassay, cells were pulsed with 3H-thymidine, harvested and counted using a scintillation counter. For the viral based bioassay, Alamar Blue® was added to each well for the last 24 hours and absorbance was read at 570 nm and 600 nm after 48 hours.
Standard curves for recombinant GM-CSF, IL-2 and IL-4 were deduced from the difference data of the 570 nm and 600 nm absorbance readings for each dilution of recombinant protein using Prism (GraphPad Software, Inc.). Difference data, collected from various dilutions of GM-CSF, IL-2, or IL-4-bearing CYT-IVAC preparations, was applied to their respective standard curve for quantitation of bioactive membrane-bound cytokine for each CYT-IVAC on a per microgram basis.
Animal experiments were performed in accordance with NIH guidelines and with approval by the Institutional Animal Care and Use Committee of the Virginia State University and Polytechnic Institute. Groups of 8–10 week old female Balb/c mice (NCI, Charles, River Laboratories) were immunized subcutaneously with 0.375 μg total viral protein of β-propiolactone inactivated A/PR/8/34 wild-type, A/PR/8/34 mGM-CSF~HA, A/PR/8/34 mIL-2~HA, or A/PR/8/34 IL-4~HA diluted in PBS. PBS alone acted as the negative vehicle control. Serum was collected on day 21 post-vaccination by retro-orbital bleeding. Mice were challenged with 1000 TCID50 of mouse-adapted Influenza A/PR/8/34 (100 LD50) on day 35 post-vaccination. Weight loss and survival was monitored following challenge.
Enzyme linked immunosorbent assay (ELISA)
Antiviral antibody levels in sera of vaccinated animals were determined by a standard enzyme-linked immunosorbent assay using whole virus as the coating antigen. Briefly, Immuno Plates (Nunc) were coated with 10 hemagglutination units (HAU) of inactivated A/PR/8/34 in coating buffer (sodium bicarbonate, pH 9.6) and blocked overnight at 4°C in PBST buffer (phosphate buffered saline with 0.05% Tween 20) supplemented with 2% BSA. Plates were washed 3 times with wash buffer (PBS containing 0.05% Tween 20). Serum samples, collected on day 21 post vaccination, were added to wells of ELISA plates and plates were incubated with shaking overnight at 4°C. Plates were washed 3 times with PBST. Horseradish Peroxidase (HRP) conjugated secondary antibody (anti-mouse IgG, IgG1, or IgG2a; Southern Biotech), diluted in PBST with 2% BSA, was added and plates were incubated with shaking for 1.5 hours at RT. Plates were washed 3 times with wash buffer and wells were incubated with substrate (2,2'-Azino-Bis(3-Ethylbenzthiazoline-6-Sulfonic Acid; Sigma) for 30 minutes at RT, followed by the addition of 1% SDS to stop the reaction. Absorbance was measured at 405 nm using a plate reader (SpectraFluor Plus, Tecan) and O.D. readings were plotted against a standard curve to determine the amount of influenza specific antibody per milliliter of serum.
Microneutralization Assay for determination of virus neutralizing antibody titers
Neutralizing antibody titers were determined for serum samples collected from mice on day 21 post-vaccination as described in the WHO Manual on Animal Influenza Diagnosis and Surveillance . Briefly, two-fold serial dilutions of serum in PBS were incubated with 100 TCID50 of influenza A/PR/8/34 for 1 hour at room temperature. The serum/virus cocktail was added to MDCK cells for 1 hour at 37°C. Serum/virus cocktail was removed and cells were incubated for 3 days at 37°C in the presence of 1.5 μg/ml TPCK-treated trypsin (Sigma). Neutralizing titer was determined to be the reciprocal of the last dilution of serum that protected MDCK cells from cytopathic effect.
Quantitation of viral loads in lungs
Viral loads in the lung tissue of vaccinated mice were determined by collecting lungs on day 4 post-challenge. Lungs were weighed and flash frozen in DMEM with liquid nitrogen. Lung tissue was homogenized, pelleted and supernatants were collected. Lung homogenates were brought to equal volume with DMEM. Viral titers of lung homogenates were determined from serial 10-fold sample dilutions and incubation with MDCK cells for 1 hour at 37°C to allow for virus adsorption. Subsequently, cells were washed and incubated for 3 days at 37°C in the presence of 1.5 μg/ml TPCK-treated trypsin (Sigma) and cytopathic effects were recorded. Viral loads were reported as 50% tissue culture infectious dose units (TCID50/ml) as determined by the Reed-Muench method .
Statistical analysis using Prism software (Graphpad) was conducted with the help of Dr. Stephen Were (statistician for VA-MD Regional College of Veterinary Medicine). ELISA antibody titer data was analyzed by One-way ANOVA on normalized log transformed data using Dunnett's multiple comparison test with PR/8/34 wild-type group as the control. Comparison of survival curves was analyzed using Fisher's exact test.