All animal procedures and scoring sheets were first approved by the Animal Care Committee (Animal Use Document ID# H-08-010) at the Canadian Science Centre for Human and Animal Health, according to the guidelines set by the Canadian Council on Animal Care.
Cells and viruses
Human embryonic kidney (HEK) 293 cells, Madin-Darby canine kidney cells (MDCK), and mouse AB12 cells were maintained in Dulbecco's modified eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS), L-glutamate, sodium pyruvate (NaPyr), and antibiotics. Fetal porcine retina cells (VR1BL E1), expressing human adenovirus 5 (AdHu5) E1a and porcine adenovirus 3 (PAV3) E1b large genes, were maintained in minimum essential medium (MEM) alpha, supplemented with 10% FBS, L-glutamate, NaPyr, non-essential amino acids, HEPES buffered saline, penicillin/streptomycin, and 50 µg/ml Hygromycin B (BD Biosciences). Avian influenza H5N1 strain A/Hanoi/30408/2005 (H5N1) was generously provided by Q. Mai Le and T. Hien Nguyen, National Institute of Hygiene and Epidemiology, Hanoi, Vietnam. Virus was propagated on MDCK cells cultured with virus diluent (MEM, 0.3% bovine serum albumin, and antibiotics) containing 3.0 µg/ml TPCK-treated trypsin (TPCK-trypsin) and titered by plaque assay. All infectious recombinant adenovirus constructs were propagated on HEK 293 or VR1BL E1 cells and purified by a cesium chloride density gradient.
Vectors and construction of transfer plasmids
The full complementary DNA (cDNA) sequence from the A/Hanoi/30408/2005 H5N1 hemagglutinin (H05-HA) gene was obtained, codon optimized, and synthesized from overlapping oligonucleotide primers, as previously described 
. The H05-HA gene was first cloned into the pCAGα plasmid, containing a chicken-β-actin (CAG) promoter, to generate the pCAGα-HA construct. The pCAGα-HA expression cassette was then excised and inserted into two shuttle vector systems: pShuttle2 (Clontech) and pPAV227 (VIDO, University of Saskatchewan). Insertion of the expression cassette replaced the existing pShuttle2 cytomegalovirus (CMV) promoter with the CAG promoter, resulting in the pShuttle2-HA construct. Transfer plasmid pPAV227 was also modified to include the SV40 polyadenylation signal from pShuttle2, generating pPAV227- HA-PolyA.
The transgene cassettes from pShuttle2-HA and pPAV227-HA-PolyA were cloned into replication-deficient ΔE1ΔE3 adenoviral vectors: pAdenoX (AdHu5, Clontech) or pFPAV227 (PAV3, VIDO, University of Saskatchewan). pAdenoX-HA was generated through digestion of both pShuttle2-HA and pAdenoX by homing endonucleases I-CeuI/PI-SceI and ligation of cohesive ends using T4 DNA ligase (Invitrogen). pFPAV227-HA was generated through homologous recombination in Escherichia coli
strain BJ5183 (recBC
of linearized pPAV227-HA-PolyA (Eco47III/TthIII1) and pFPAV227 (PacI).
Production of PAV3-HA and AdHu5-HA vaccine vectors
To obtain AdHu5-HA vaccine, HEK 293 cells were transfected with 10 µg of linearized pAdenoX-HA DNA in calcium phosphate (BD Biosciences) solution and cells were cultured until the appearance of cytopathic effects (CPE). Similarly, VR1BL E1 cells were transfected with 10 ug of linearized pFPAV227-HA DNA combined with Lipofectin (Invitrogen) and cells were cultured until CPE were apparent. Amplified adenoviruses containing cell lysates were harvested, freeze-thawed three times, and purified by CsCl gradients. The integrity of the H05-HA transgene cassette was confirmed through EcoRI restriction digests and by sequencing (DNA Core, National Microbiology Laboratory) with multiple primer sets. Total virus particles (vp) was determined by OD260 and total infectious particles was determined using anti-hexon antibodies against AdHu5 (AdenoX Rapid Titer kit, Clontech) or against PAV3 (VIDO, University of Saskatchewan). Four independent vector preparations were used for each vaccine. Total infectious particles and total viral particles were determined for both adenovirus vaccines, with ratios of 1
250, and 1
300 for PAV3-HA and 1
220, and 1
250 for AdHu5-HA.
Expression of PAV3-HA and AdHu5-HA vaccines
Protein expression of H05-HA by both vectors was confirmed in HEK 293, VRIBL E1, and AB12 cells using standard Western blotting techniques. To evaluate in vivo expression, groups of 6 BALB/c mice were vaccinated with 1010 vp of PAV3-HA or AdHu5-HA and muscle tissue was harvested 4 days following immunization. Muscle tissues were homogenized and normalized per gram of muscle tissue in radioimmunoprecipitation (RIPA) buffer. Expression of each vaccine was detected from 75 µg of loaded muscle tissue.
Immunization and viral challenge
Groups of 10 BALB/c (Charles River Canada) were vaccinated with 108, 109, or 1010 vp of recombinant adenovirus PAV3-HA or AdHu5-HA by intramuscular (I.M.) administration. Each vaccine was diluted in 100 µl and 50 µl was administered in each of the right and left hind limbs. All mice were anesthetized and challenged after 28 days through intranasal inoculation with 100 times the dose of A/Hanoi/30408/2005 virus required to obtain 50% survival (100 Lethal Dose 50 or 100LD50) in 50 µl virus diluent. The LD50 for the H5N1-H05 virus was 1.05 plaque forming units (pfu), therefore 100LD50 was 105 pfu. Mice were monitored for 15 to 20 days following challenge and signs of disease including weight loss, labored breathing, ruffled fur, and death were observed according to an approved scoring chart. All animal procedures were approved by the Institutional Animal Care Committee at the National Microbiology Laboratory (NML) at the Public Health Agency of Canada (PHAC), according to the guidelines of the Canadian Council on Animal Care. All infectious work was performed in the high biocontainment laboratory at NML/PHAC.
Enzyme-linked immunosorbent spot assay
Groups of 4 BALB/c mice were vaccinated with 1010 vp of PAV3-HA or AdHu5-HA vaccines. The day before each experiment, ELISPOT-IFNγ (BD Biosciences) plates were coated with purified mouse IFNγ and incubated at 4°C, overnight. As well, overlapping 15mer peptides (Mimitopes, Australia) spanning the entire H05-HA protein were resuspended overnight in dimethyl sulfoxide (DMSO) and allocated in pools by matrix format. Spleens were harvested on days 8, 10, 14, and 21 post-immunization and splenocytes were plated at 5×105 cells/well in RPMI 1640 (supplemented with 10% FBS, L-glutamine, NaPyr, HEPES, non-essential amino acids, 5×10−3 M 2-β-mercaptoethanol, and antibiotics) and restimulated with each of the peptide pools (2.5 µg/ml per well). An individual 9mer peptide representing the immunodominant H5N1 H05-HA (IYSTVASSL, conserved influenza A viruses) epitope was also evaluated, along with negative controls PR8-NP (TYQRTRALV, A/PuertoRico/8/34 (H1N1) nucleoprotein) and ZGP (TELRTFSI, Zaire Ebolavirus glycoprotein). ELISPOT-IFNγ plates were incubated at 37°C overnight (18–20 hours) and washed the following day according to the manufacturer's instructions. AEC Substrate Set (BD Biosciences) was used to develop spots formed by interferon gamma secreting cells. Spots were visualized and counted using an ELISPOT plate reader (AID ELISPOT reader, Cell Technology, Colombia, Maryland).
Flow cytometric analysis
Splenocytes obtained on day 10 post-vaccination, plated at 2×106 cells/well, were restimulated with 9mer H05-HA, PR8-NP, or 8mer ZGP individual peptides in DMEM (supplemented with 10% FBS, L-glutamine, NaPyr, HEPES, non-essential amino acids, 5×10−3 M β-mercaptoethanol, and antibiotics), IL2, and GolgiStop (Brefeldin A, BD Biosciences). Cells were stimulated for 5 hours and stained with anti-mouse CD8-FITC (fluorescein isothiocyanate) at 4°C for 30 minutes. BD Cytofix and permwash protocol was used to fix and permeabilized cells according to the manufacturer instructions. The following day, cells were stained for interferon gamma using anti-mouse IFNγ-PE (phycoerythrin). All samples were read on the LSRII Flow cytometer (BD Biosciences). Data was analyzed using BD FACSDiva 6.0.1 software (BD Biosciences).
Serum for the detection of HA specific antibodies by neutralization or hemagglutination inhibition assays was collected from each mouse following immunization, as previously described 
. Briefly, all samples were treated with receptor-destroying enzyme (RDE, 1
3 ratio, Accurate Chemical) overnight at 37°C and inactivated the following day for 45 minutes at 56°C. Starting with a 1
10 dilution, two-fold serial dilutions of serum were done in a 96-well round-bottom microtiter plate in virus diluent (MEM, 0.3% BSA, and antibiotics), mixed with 100 pfu/well of homologous A/Hanoi/30408/2005 virus and incubated at 37°C for 60 minutes. For hemagglutination inhibition (HI) assays, two-fold serial dilutions of each sample were performed in phosphate buffered saline (PBS) and 50 µl/well was added to a 96-well V-bottom microtiter plate. Four hemagglutinating doses of homologous A/Hanoi/30408/2005 were added to each well and the plates were incubated at room temperature for one hour. Horse red blood cells (0.5% erythrocytes in 0.85% saline) were added to each well and incubated for 60 minutes. Hemagglutination titer was determined as the reciprocal of the highest dilution where red blood cells did not agglutinate. For neutralization assays, the serum/virus mixture was added onto subconfluent MDCK cells in a 96-well flat-bottom microtiter plate and incubated for 5–10 minutes at room temperature. Virus diluent supplemented with 3.0 µg/ml TPCK-trypsin was added into each well (100 µl/well) and incubated at 37°C, 5% CO2
, for 48 hours. Cells were monitored for the presence or absence of CPE using a light microscope. The highest serum dilution which did not exhibit CPE was scored as positive for neutralizing antibody and titers were reported as the reciprocal of the dilution. All infectious in vitro
work was performed in the high containment laboratory of NML/PHAC.
An enzyme-linked immunosorbant assay (ELISA) was performed to evaluate total IgG antibodies present in serum at 25 days and 1 year following vaccination. 96-well plates (NUNC) were coated with 1 ug/ml H5 HA antigen, overnight at 4°C. The following day, serial tenfold dilutions of serum were added to the plates and incubated for 60 minutes at room temperature. Following three washes, a goat anti-mouse conjugated to HRP was applied as the secondary antibody for 45 minutes. After an additional three washes, a 3,3′,5,5′-tetramethylbenzidine (TMB) solution was added to react with the HRP conjugate and incubated in the dark for 20 minutes. The reaction was stopped using sulphuric acid solution. The absorbance was detected at 450 nm (OD450). The endpoint cut-off (A450
0.100) was determined as the last serum dilution which was greater than two times the optical density of the negative control. Endpoint titres were reported as the reciprocal of the first serum dilution that was higher than the cut-off.
Rapid vaccination regimen and detection of viral load
Groups of 15 BALB/c mice were vaccinated with a 1010
vp/mouse dose of PAV3-HA or AdHu5-HA 5, 8, or 10 days prior to challenge with 100LD50 of H5N1-H05 virus. Nine animals were monitored for survival and lung tissue and blood were harvested from the remaining 6 mice three days post-infection. Virus load was determined by the tissue culture infectious dose which will result in cytopathic effects (CPE) of fifty percent of cell culture (TCID50). TCID50 was performed by added serial dilutions of homogenized lung tissue onto MDCK cells and monitoring the presence of CPE after 48 hours. The TCID50 titre was calculated using the Reed & Muench method 
and normalized per gram of lung tissue.
Graphs and Statistical analysis
All graphs were generated using GraphPad Prism 5.0 and data were analyzed for statistical difference by performing unpaired t test, or one-way analysis of variance (ANOVA) when appropriate. The differences in the mean or raw values among treatment groups were considered significant when p<0.05.