Virus and cells
A newly identified fatal strain of Orf virus was isolated from scab specimens collected from skin lesions of a 6-week-old small-tailed Han sheep affected with Orf virus in November 2008 in the Jilin province of China . Primary ovine fetal turbinate (OFTu) cells were maintained in minimal essential medium (MEM) (Hyclone) supplemented with 10% fetal bovine serum (FBS) (Hyclone), 2 mM L-glutamine, 100 U of penicillin/ml, 100 μg of streptomycin/ml, and 20 μg of nystatin/ml. The virus was propagated in OFTu cells. For virus harvest, cell culture supernatant from infected cells were collected when approximately 90% of the culture showed cytopathic effects (CPE). After three freeze-thaw cycles, the supernatant was then cleared at 500 × g for 10 min at 4°C and stored at -80°C. The virus was purified by sucrose gradient centrifugation. Infectivity titre was assayed by the plaque method in OFTu cell culture and calculated as plaque forming units (PFU).
Construction of the expression plasmids
All expression plasmids were constructed using pcDNA3.1(+) (Invitrogen, USA) as the vector. The two primer sets used for PCR amplification are specific for either ORFV 011 (forward primer: 5'-TATAGGATCCGCCATGT GGCCGTTCTCCTCCATC-3'; reverse primer: 5'-CCGCTCGAGTTAATTTATTGGCTTGCAG-3') (restriction sites in bold) or ORFV059 (forward primer: 5'-CAAGCTTGCCACCATGGATCCACCCGAAATC-3'; reverse primer: 5'-CGAATTCTCACACGATGGCCGTGACC-3') (restriction sites in bold). The plasmids pMDT-ORFV011 and pMDT-ORFV059, containing the complete ORFV011 and ORFV059 genes of the ORFV Jilin province strain, respectively, have been described previously . The DNA vaccine constructs pcDNA3.1-ORFV011 and pcDNA3.1-ORFV059 expressing ORFV011 protein and ORFV059 protein individually were generated by subcloning the ORFV011 and ORFV059 genes into pcDNA3.1 (+), respectively. To construct the ORFV011/ORFV059 chimeric expression plasmid, the two primer sets mentioned above were modified by inserting a linker structure which encoded a (G4S)2 polypeptide in the 5' termini of the ORFV011 reverse primer and the ORFV059 forward primer. In addition, the stop codon of the ORFV011 reverse primer and the promotor of the ORFV059 forward primer were deleted. The modified primer sequences were as follows: ORFV011 (forward primer: 5'-TATAGGATCCGCCATGTGGCCGTTCTCCTCCATC-3'; reverse primer:5'-AGAGCCTCCGCCACCGGATCCACCGCCACCATTTATTGGCTTGCAGAA-3') (linker structure in italic) and ORFV059 (forward primer: 5'- GGTGGCGGTGGATCCGGTGGCGGAGGCTCTGAAATCACGGGCTACATAATC-3'; reverse primer: 5'-CGAATTCTCACACGATGGCCGTGACC-3') (linker structure in italic). The coding sequence of the ORFV011 gene without the stop codon was amplified from pMDT-ORFV011 by PCR, using ORFV011 modified primers, and the gene for ORFV059 without the promoter was amplified from pMDT-ORFV059 using ORFV059 modified primers. PCR products, including the ORFV011 and ORFV059 genes obtained by overlap PCR, were cloned into a pcDNA3.1 (+) vector, generating pcDNA3.1-ORFV 011/ORFV059 plasmid. All recombinant plasmids (data not shown) were confirmed by restriction digestion and sequence analysis.
Expression of recombinant proteins in vitro
OFTu cells were cultured in MEM containing 10% fetal bovine serum (FBS) in a 5% CO2 incubator at 37°C. Then, OFTu cells were transfected with pcDNA3.1(+) plasmid (control) or pcDNA3.1-ORFV011, pcDNA3.1-ORFV059 and pcDNA3.1-ORFV 011/ORFV059 recombinant vaccine plasmids. Briefly, OFTu cells were grown to 80% confluence in 6-well culture plates (Costar, Corning Incorporated, USA) and transfected with 8-10 μg of recombinant plasmids for each well, respectively. After 48 h post-transfection, cell lysates collected from pcDNA3.1(+) vector plasmid-transfected or pcDNA3.1-ORFV011, pcDNA3.1-ORFV059 and pcDNA3.1-ORFV059/ORFV011 recombinant vaccine plasmids-transfected OFTu cells were analyzed by SDS-PAGE and Western blot, respectively.
To further study the subcellular localization of target protein, the pcDNA3.1(+) vector plasmid-transfected or pcDNA3.1-ORFV011, pcDNA3.1-ORFV059 and pcDNA3.1-ORFV011/ORFV059 recombinant vaccine plasmids-transfected OFTu cells were subjected to an IFA test employing rabbit anti-ORFV polyclonal antibody (kindly provided by the Jilin Institute for Veterinary Research, diluted at 1:100). The secondary antibody used was FITC-conjugated goat anti-rabbit IgG (H + L) (1:500 dilution in PBS). After immunostaining, the normal control and transfected cells were observed under a fluorescent microscope.
Large-scale preparations of DNA vaccine plasmids were obtained using alkaline lysis. Sixty 6-8-week-old female Balb/c mice (purchased from the Laboratory Animal Center of Jilin University in China) were used for experiments in accordance with the guidelines for animal experimentation of Jilin University. The animals were maintained under pathogen-free conditions, randomly divided into six groups (ten mice per group), and immunized by skin scarification  with one of the following formulations: (π) the A group was immunized with PBS, (θ) the B group with 100 μg pcDNA3.1(+) plasmids, (ρ) the C group with 100 μg pcDNA3.1-ORFV011 plasmids, (σ) the D group with 100 μg pcDNA3.1-ORFV059 plasmids, (τ) the E group with 50 μg pcDNA3.1-ORFV011 plasmids plus 50 μg pcDNA3.1-ORFV059 plasmids, and (φ) the F group with 100 μg pcDNA3.1-ORFV011/ORFV059 plasmids. The mice were boosted in the same manner on days 14 and 28. Blood samples were collected from the tail artery at weeks 0, 1, 2, 3, 4, 5, 6, 7 and 8 post immunization. The sera were inactivated at 56°C for 30 min and stored at -80°C until assayed by ELISA (IgG at each time-points, IgG1/IgG2a isotype at 1 week post the final boost immunization, virus-neutralizing antibody at 2 weeks post immunization). On day 10 after the final boost immunization, 5 mice of each group were sacrificed by cervical dislocation, and their splenocytes were isolated for a lymphocyte proliferation assay and flow cytometry assay.
ORFV-specific antibody titer assay
ORFV-specific antibody responses were determined using an indirect ELISA, with the purified ORFV as the antigen. Briefly, Costar High Binding 96-well plates (eBioscience, San Diego, CA) were coated overnight at 4°C with 0.1 μg of purified ORFV diluted in 100 μl of 0.1 M NaHCO3 buffer (pH 9.6). The plates were blocked with PBS/0.1% Tween-20/2% BSA (150 μl/well) for 1 h at 37°C. After washing three times with PBS/0.1% Tween-20, sera were added in a dilution of 1:20 in PBS/0.1% Tween-20/2% BSA. The plates were incubated for 90 min at 37°C, washed three times, and then incubated with 100 μl of horseradish-peroxidase (HRP)-conjugated goat anti-mouse IgG (Zhongshan Biotechnology Company, Beijing, China) at a dilution of 1:5000 in PBS/0.1% Tween-20/2% BSA for 1 h at 37°C. After three washes, the presence of IgG was detected with 100 μl of TMB according to the manufacturer's instructions. The reaction was stopped by adding 50 μl of 2 M H2SO4. The OD value was read at 450 nm. Results were expressed as an antibody endpoint titer, determined when the OD value is 3-fold greater than the background value obtained with a 1:20 dilution of serum from PBS-injected mice. The purified ORFV antigen was replaced with OFTu cell supernanant as a negative control.
Immunoglobulin isotyping ELISA
In order to determine the IgG1 and IgG2a subtypes in mice immunized with ORFV vaccine plasmids, purified ORFV was diluted to optimal concentrations in 0.1 M NaHCO3 buffer (pH 9.6), which was used as coating antigen. Costar High Binding 96-well plates (eBioscience, San Diego, CA) were coated with 0.1 μg of purified ORFV diluted in 100 μl of 0.1 M NaHCO3 buffer (pH 9.6). The plates were blocked with PBS/0.1% Tween-20/2% BSA (150 μl/well) for 1 h at 37°C. After washing three times with PBS/0.1% Tween-20, the sera were diluted at 1:20 in PBS/0.1% Tween-20. After a 1 h incubation, the plates were washed with PBS/0.1% Tween-20 and goat anti mouse IgG1, IgG2a (Sigma Chemical Co., SA Locus, Mo.) diluted to 1:1000 were used to detect IgG1 and IgG2a subtypes, respectively. After incubating for 60 min, the plates were washed with PBS/0.1% Tween-20, and horseradish-peroxidase (HRP)-conjugated rabbit anti-goat IgG (Zhongshan Biotechnology Company, Beijing, China) diluted to 1:4000 in PBS/0.1% Tween-20 was added to each well. The reaction was visualized after 1 h of incubation with TMB. The reaction was stopped by adding 50 μl of 2 M H2SO4. The OD value was read at 450 nm. Results were expressed as an antibody endpoint titer, determined when the OD value is 3-fold greater than the background value obtained with a 1:20 dilution of serum from PBS-injected mice.
Determination of virus-neutralizing titers
To perform the virus neutralization assay, serum samples were diluted 1:5 in MEM medium and heat-activated for 30 min at 56°C. This 1:5 starting dilution of inactivated sera was serially diluted two-fold in MEM medium in 96-well flat bottom plates and incubated with 0.1 μg of purified ORFV diluted in 50 μl MEM for 90 min at 37°C. All sera were run in triplicate. Subsequently, 100 μl of OFTu cell suspension were added to each well at a concentration of 45,000 cells/well. The plates were incubated for 5 days at 37°C in a 5% CO2 incubator. The neutralization titer of a serum is the dilution at which 50% of the wells are protected against virus infection. The titer is expressed as ND50 and calculated using the Reed and Muench method.
Single-cell suspensions of spleens were prepared from the immunized mice at day 10 post the final boost immunization. Briefly, spleens from freshly killed mice were disrupted by using monofilament nylon filters, and the cells were collected and centrifuged at 500 × g for 20 min. The cell pellets were suspended in 10 ml RPMI 1640 and then centrifuged at 500 × g for 10 min at room temperature. The cells were resuspended in RPMI 1640 supplemented with 10% FBS at a concentration of 1 × 107 cells/ml before the lymphocyte proliferation assay and flow cytometry assay.
Cellular proliferation assay
Untreated splenocytes were cultured in RPMI 1640 medium with 10% fetal serum. The cells were suspended in complete RPMI 1640 to achieve the concentration of 1 × 106 splenocytes/well. The primary incubation was at 37°C in a 5% CO2 incubator for 72 h, followed by addition of 10 μl MTT (5 mg/ml) per well and a secondary incubation at 37°C in a 5% CO2 incubator for another 4 h. The microplates were centrifuged, the unreacted MTT was removed, then 100 μl dimethyl sulfoxide (DMSO) was added per well to solubilize formazan. The plates were then incubated for 10 min with shaking. The absorbance of the plate was read using a 570 nm filter within minutes following shaking. The treated cells were cultured under the same conditions, but with the addition of purified ORFV antigen (at a final concentration of 5 μg/ml) in complete RPMI 1640 medium. The mitogen, Con A was added at 5 μg/ml concentration in the positive control wells. The geometric means and standard deviations for triplicate sets of samples were calculated. Lymphocyte proliferation is expressed as a stimulation index (SI), which is defined as the mean of experimental data divided by the mean of the unstimulated control [20, 21].
Flow cytometry assay
In order to determine the changes of specific T cell subsets in mice immunized with ORFV vaccine plasmids, the flow cytometry assay were performed. 1 × 106 splenocytes collected from the immunized mice at day 10 post the final boost immunization were suspended in PBS and stained with FITC-conjugated anti-mouse CD3, PE-Cy5-conjugated anti-mouse CD8, and PE-conjugated anti-mouse CD4 for 30 min on ice. After three washes with PBS, the cells were centrifuged at 500 × g for 10 min. The cells were suspended in 200 μl PBS. At least 10,000 cells were analyzed per sample. The stained cells were analyzed by a BD FACSAria flow cytometer, and the data were analyzed with Cellquest V3.3 software.
The production of ORFV-specific cytokines (IL-2, IL-4, IL-6, IFN-γ and TNF-α) was assessed by culturing splenocytes (1 × 106 cells/ml) in triplicate with purified ORFV antigen (5 μg/ml). Control stimuli included RPMI 1640 medium alone or ConA at 5 μg/ml. The supernatants were harvested after 72 h at 37°C in a 5% CO2 incubator, filtered and stored at -20°C until assayed. The presence of IL-2, IL-4, IL-6, IFN-γ and TNF-α in mouse splenocytes culture supernatants were determined by commercial cytokines immunoassay kits (Dakewa Biotech Company, Shenzhen, China) according to the manufacturer's instructions. Supplied standards were used to generate a standard curve. The detection limits of the assay were 2.5 pg/ml, 3.0 pg/ml, 3.5 pg/ml, 2.0 pg/ml, 2.0 pg/ml for IL-2, IL-4, IL-6, IFN-γ and TNF-α, respectively.
All data were analyzed using the statistical software program Systat 10 (SPSS). The distribution of data was determined using descriptive statistics. Data which were not normally distributed were transformed by ranking. Differences in ELISA titers were investigated using one-way analysis of variance (ANOVA) performed on the rank. The means of the rank-transformed variables were compared using Tukey's multiple comparison test. Values of * P < 0.05, ** P < 0.01 were considered statistically significant.