Viruses, cells and synthetic genes
PRRSV IAF-Klop  and FMV09-1155278 (C.A. Gagnon, unpublished) strains were propagated in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 8% fetal bovine serum (FBS; PAA Laboratories, Inc., Etobicoke, Ontario, USA) at 37°C in a humidified atmosphere of 5% CO2 and titrated in MARC-145 cells using the Kärber method .
rAdV were propagated and titrated in the 293-PS-CymR cell line which is a cell clone derived from the HEK 293 cell line expressing the protease (PS) gene . Cells were grown in DMEM supplemented with 8% FBS and 50 μg/mL cumate (Sigma-Aldrich, St. Louis, MO, USA) at 37°C in a 5% CO2 humidified atmosphere as described .
A549 cells were propagated in DMEM supplemented with 8% of FBS and maintained at 37°C in a humidified atmosphere of 5% CO2. Unlike the HEK 293 cells, these do not express proteins encoded by the adenovirus E1 gene and were used to confirm protein expression from the rAdV .
The codons most frequently used in Sus scrofa cells and demonstrated for their ability to increase the immune response in swine  were used to generate the GP5 (ORF5)- and M (ORF6)-encoding genes (synthesized through GeneArt services; Invitrogen) on the basis of the PRRSV IAF-Klop genome sequence [Genbank accession number: U64928].
Cloning of GP5 wild-type or mutated sequences in fusion with the M sequence
Three mutations to increase the Ab and T cell immune responses specific to GP5 [22, 25] were introduced by PCR in the GP5-encoding gene. The first mutation consisted in the replacement of the GCCCTGGTGAAC nucleotide (nt) sequence (aa 27–30: ALVN) by the TCTGGGTCTGGC nt sequence (aa 27–30: SGSG) to abolish the decoy ALVN epitope . The second mutation introduced the 39 nt PADRE sequence (AKFVAAWTLKAAA) between residues 32 and 33 such that this sequence was localized between the abrogated decoy sequence and neutralizing epitope . The third mutation replaced the AAC triplet (N) by the GCC triplet (A) to abolish the N51 glycosylation site . The resultant protein produced from the mutated encoding gene was designated hereafter GP5m. GP5 wild-type- or GP5m-encoding sequences were fused by PCR to the 3’ terminal of the M gene with the insertion of the GTTACCACC (GTT) linker sequence between the M and GP5-encoding nt sequences . The sequences were validated by DNA sequencing through the McGill University Sequencing Services (Montréal, QC, Canada).
Construction of recombinant adenoviruses (rAdV)
The M-GP5- and M-GP5m-encoding sequences were inserted into the Bgl II site of the adenovirus transfer vector pAdenoVator-CMV5(CuO)-IRES-E1A . The recombinant plasmids were rescued into the genome of the pAdeasyΔPS by homologous recombination in E. coli BJ5183 cells (MP Biomedicals, Irvine, CA, USA) through electroporation (2.5 kV, 200 Ohms and 25 μF). The recombinant rAdV genome was confirmed by PCR and restriction enzyme analysis. To produce the rAdV, plasmids were linearized by Pac I digestion and 293-PS-CymR cells at 60% confluency were transfected with 2 μg of each plasmid (one well per plasmid of a 6-well tissue culture plate) using PolyFect transfection reagent (Qiagen, Valencia, CA, USA). The transfected cells were overlaid 24 h later with agarose (Invitrogen) (0.45% in DMEM supplemented with 5% FBS) and monitored daily until the appearance of viral plaques. After the confirmation of transgene expression from amplified viral clones, rAdV were produced at a large scale and purified by double cesium chloride gradient . The infectious dose of rAdV was determined in 293-PS-CymR infected cells using the Kärber method and titers were expressed in tissue culture infectious dose 50 per mL (TCID50/mL).
Western blot assay
A549 cells were seeded in 6-well tissue culture plates and infected with rAdV with a multiplicity of infection (MOI) of one. At 24 h post-infection, cells were lysed with lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% triton X-100 and EDTA-free protease-inhibitor cocktail (Roche, Indianapolis, IN, USA)) and total cell protein concentration was quantified with the DC protein assay kit (Bio-Rad, Mississauga, ON, CA, USA). For each sample, 20 μg of total cell extract was electrophoretically separated onto 12% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). The membranes were blocked in phosphate buffered saline (PBS) solution, pH 7.3, containing 0.05% Tween-20 (PBS-T) in the presence of 5% nonfat dry milk powder for 1 h at room temperature. The membranes were incubated overnight at 4°C with convalescent homologous PRRSV-specific pig antiserum obtained from a previous study  used at a 1/5000 dilution, or mouse monoclonal Abs specific to either adenovirus E1A (1/5000) (Millipore, Bedford, MA, USA), GFP (1/5000) (Roche) or GAPDH (1/10 000) (Sigma-Aldrich, St. Louis, MO, USA) proteins. Following incubation, membranes were washed three times with PBS-T and then incubated for 1 h at room temperature with anti-pig horseradish peroxidase (HRP)-conjugated IgG (Sigma-Aldrich) or anti-mouse-HRP-conjugated IgG (Bethyl, Montgomery, TX, USA). The signal was detected by enhanced chemiluminescence (ECL) (Perkin Elmer, Boston, MA, USA). The membranes were then exposed to Kodak Biomax Light-1 films.
Recombinant GP5 production
Plasmid pGEX4T1 (Pharmacia Biotech, Piscataway, NJ, USA) encoding wild-type GP5 of the PRRSV IAF-Klop strain was obtained from a previous study . Recombinant GP5 in fusion with Glutathione S-transferase (GST), designated hereafter rGP5, was produced in BL21 (DE3)pLysS competent E. coli cells (Promega, Madison, WI, USA) upon induction at OD(600 nm) of 1.2 with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 4 h at 37°C. Bacterial cells were lysed by sonication in buffer (PBS, 0.5% Tween-20, 0.5% triton X-100, 0.5% NP40) and the whole bacteria protein extract was separated through 12% SDS-PAGE. The band corresponding to the rGP5 expected molecular weight was excised from the gel and electroeluted. The purified protein was dialyzed against PBS. The protein concentration was quantified with the DC protein assay kit as described above. The identity of the eluted protein was confirmed by Western blot using convalescent PRRSV-specific pig antiserum as above. The protein stock was then stored at −80°C for further use.
Immunization of piglets and experimental challenge
Animal protocols were approved by the University’s and the Dairy and Swine Research and Development Centre’s (DSRDC) Animal Protection Institutional Committees according to the regulations of the Canadian Council for Animal Care. Seventy, three-week-old, Yorkshire-Landrace x Duroc pigs from the herd located at the DSRDC, Agriculture and Agri-Food Canada (Sherbrooke, QC, Canada) were used. At weaning, pigs at 21 days of age were divided into five groups of 14 pigs according to a randomized complete block design. The pigs were housed in the nursery rooms of the DSRDC swine complex. For each group, six pigs were kept separately for subsequent experimental challenge. Pigs were fed ad libitum with commercial non-medicated feed and had access to water. The swine herd was seronegative for PRRSV, transmissible gastroenteritis virus, Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae prior to the experiment.
Pigs of three groups were immunized by both the intramuscular (IM) and intranasal (IN) routes at days 0 and 21 with 108 TCID50 of rAdV expressing GFP (negative control), M-GP5 or M-GP5m. Pigs of group four were primed at day 0 IM and IN with 108 TCID50 of rAdV expressing M-GP5m, and boosted at day 21 IM with 250 μg of rGP5 supplemented with 500 μg of QuilA (Brenntag Biosector, Frederikssund, Denmark), and mixed in equal volume with Freund’s incomplete adjuvant (Sigma-Aldrich). This group was designated M-GP5m/rGP5 hereafter. Pigs of group five were inoculated at days 0 and 21 IM with 2 mL of the commercial Ingelvac PRRSV MLV vaccine (Boehringer Ingelheim, St Joseph, MO, USA) inactivated 1 h at 56°C prior to the inoculation (designated hereafter as the inactivated vaccine). The inactivation process was conducted to avoid unwanted propagation of live PRRSV in the PRRSV-free swine herd of the research center. For IN immunization, pigs were sedated with stresnil (6–8 mg/kg of weight) given by the IM route. Blood samples were collected at 0, 21, 28, 35 and 49 days post-immunization (dpi). At 49 dpi, 8 pigs of each group were sedated and anesthetized (10 mg/kg of ketamine and 5 mg/kg xylazine) prior to being euthanized by exsanguination. The animal lungs were collected to perform bronchoalveolar lavage with 30 mL of PBS, pH 7.3.
The remaining pigs (n = 6) of each group were transferred at 42 dpi to the Faculty of Veterinary Medicine of the University of Montréal for experimental challenge. After an adaptation time of 7 days, the pigs were challenged (e.g. at 49 dpi) IN with 2 × 105 TCID50 of the PRRSV FMV09-1155278 strain. The FMV09-1155278 strain was selected for experimental challenge on the basis of preliminary experiments in two pigs showing viremia from day 3 following infection with the virus. Animals were monitored daily for the presence of clinical signs of cough, dyspnea, diarrhea and inappetence. Blood samples were collected and rectal temperature was monitored at 0, 3, 5, 7, 10, 14 and 21 days post-challenge (dpc). Animals were weighed at days 0, 7, 14 and 21 dpc to determine the average daily body weight gain. At 21 dpc, pigs were euthanized to perform bronchoalveolar lavage with 30 mL of PBS as described above and for pathological examination. Determination of the percentage of macroscopic lung lesions, based on the consolidation of lung tissues, was determined according to a scoring system described elsewhere .
Antibody response to PRRSV
The presence of serum or bronchoalveolar lavage fluid (BALF) GP5-specific Abs was evaluated by an indirect ELISA using Immulon 2HB 96-well microtiter plates (Thermo Labsystems, Franklin, MA, USA). The plates were coated with 0.1 μg of rGP5 per well diluted in 0.05 M sodium carbonate buffered solution (pH 9.6) to a final volume of 100 μL. Following an overnight incubation at 4°C, the plates were washed 4 times with PBS-T and then saturated with 150 μL of PBS-T containing 1% bovine serum albumin (BSA) overnight at 4°C. One hundred μL of pig serum (used at a 1/200 dilution) or BALF diluted two-fold in PBS-T with 1% BSA were added into wells (in duplicate) and incubated for 2 h at 37°C. Plates were washed as described above and anti-pig HRP-conjugated IgG (used at a 1/10 000 dilution) or anti-pig HRP-conjugated IgA (used at a 1/25 000 dilution) (AbD Serotec, Raleigh, NC, USA) in PBS-T with 1% BSA were added for 1 h at 37°C. Plates were washed and the HRP signal was detected by adding 100 μL of tetramethylbenzidine (TMB, Sigma-Aldrich) per well. After an incubation of 20 min at room temperature, the reaction was stopped by adding 50 μL 1M H2SO4 to each well. Optical density (OD) was determined at 450 nm (using Tecan Infinite M1000 reader, Tecan Group Ltd, Männedorf, Switzerland). For each serum sample, the average OD was corrected by subtracting the OD of the uncoated well from the OD obtained with the antigen-coated well.
The IDEXX PRRS X3 HerdChek ELISA (IDEXX Laboratories, Westbrook, ME, USA) was used for detection of PRRSV-specific Abs in serum of animals. Serum samples with a calculated S/P ratio greater than 0.4 were considered positive as recommended by the manufacturer.
Serum neutralization assay
Serum or BALF samples were heat inactivated at 56°C for 30 min. Serial two-fold dilutions (starting at 1/2) of each sample were done in DMEM, and the neutralization test was performed by a viral cytopathic effect inhibition method using the IAF-Klop or FMV09-1155278 strain, 105 cells/well of MARC-145 cells plated the day before the assay and four wells per specimen dilution . The NAb titer was calculated at 96 h post cell infection and expressed as the reciprocal of the highest sample dilution neutralizing 100 TCID50 of the virus.
Cellular immune response to PRRSV
Swine peripheral blood mononuclear cells (PBMC) from pigs to be challenged afterwards with PRRSV were isolated at 28, 35 and 49 dpi by density gradient centrifugation using Ficoll-Paque Plus (specific density of 1.077 g/mL; GE Healthcare, Piscataway, NJ, USA). PBMC were suspended in Roswell Park Memorial Institute 1640 medium (RPMI; Invitrogen) supplemented with penicillin (100 U/mL)/streptomycin (100 μg/mL) (Invitrogen), 10% FBS, 0.05 mM β-mercaptoethanol (Fisher Scientific, Nepean, Ontario, USA) and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Invitrogen). PBMC were seeded at a concentration of 4 × 105 cells per well in 96-well plates. Quadruplicate wells were exposed to 25 μL of either 200 TCID50 of heat-inactivated PRRSV IAF-Klop strain, cell culture medium (control cell cultures), or 1 μg/mL of Concanavalin A (Con A; Sigma-Aldrich) used as a positive control for lymphocyte functional activity. All cell cultures were incubated for 3 days at 37°C, and pulsed with 0.5 μCi of tritiated thymidine (specific activity, 6.7 Ci/mmol, Perkin Elmer) 18 h before harvesting cells onto a Wallac silica membrane (Perkin Elmer). The bound radioactivity was measured with Wallac Microbeta 1450 Trilux liquid scintillation counter (Perkin Elmer). The cell blastogenic responses were expressed by calculating the stimulation index (SI) which represents the ratio of the mean counts per minute (CPM) incorporated by the virus or mitogen-stimulated containing cell cultures to the mean CPM incorporated by the control cell cultures.
RNA extraction and PRRSV real-time PCR
The QIAamp Viral RNA kit (Qiagen) was used to isolate viral RNA from the serum samples at 0, 5, 7 and 10 dpc as described in the manufacturer’s instructions. A commercial PRRSV real-time PCR diagnostic kit (NextGen, Tetracore Inc., Gaithersburg, MD, USA) was used for PRRSV quantification as recommended by the manufacturer. The quantification of PRRSV was determined by comparing the sample results with a standard curve based on the amount of serially diluted IAF-Klop strain produced in MARC-145 cells and titrated as TCID50/mL of viral particles in the MARC-145-infected cell culture supernatant . The PRRSV qRT-PCR results were expressed in TCID50/mL of serum.
Data were analyzed with a Proc MIX procedure of SAS (SAS Institute, Inc., Cary,NC, USA). Analysis of differences in Ab responses to GP5 in sera before and after challenge was performed with a Kruskal-Wallis test with Bonferroni adjustment. The other data were analyzed by ANOVA with Tukey adjustment.