Transformation and expression of the PCV2 cap gene in S. cerevisiaeand production of yeast extracts for microencapsulation
PCV2 cap gene expression was optimized as previously described by Bucarey et al. , with some modifications. Briefly, the pYES2::opt-cap plasmid was transformed into the expression host, S. cerevisiae INVSc1 (genotype: MATα his3Δ1 leu2 trp1-289 ura3-52/MATα his3Δ1 leu2 trp1-289 ura3-; phenotype: His-, Leu-, Trp-, Ura-) using the lithium acetate/single-stranded carrier DNA/polyethylene glycol method . Transformed colonies were cultured in selective autotrophic yeast nitrogen base (YNB) URA ̄ medium (6.7 g of YNB (US Biological, MA, USA), 5 g of casamino acid, 20 g of glucose, 0.03 g of tryptophan (Sigma-Aldrich Co., MO, USA), and 20 g of bactoagar (US Biological, Ma, USA) in 1,000 ml of distilled water) for 48 h at 30°C. The recombinant colonies were picked and transferred into 10 ml of liquid YNB URA ̄ medium and cultured overnight at 30°C until the optical density reached 0.6–0.7 at 600 nm (OD600). The cells were then harvested, washed twice with phosphate-buffered saline (PBS), and inoculated into 50 ml of induction medium (YNB URA ̄ medium containing 2% galactose (Sigma-Aldrich Co., MO, USA) instead of glucose) to a final OD600 of 0.1–0.3. The cells were then cultured at 30°C with shaking. The cells were induced for 24 h, harvested by centrifugation at 1,500 × g for 5 min at 4°C, and then resuspended in 5 ml of 0.6 M KCl. The cell walls were then digested with liticase (0.1 mg/ml; Sigma-Aldrich Co., MO, USA) at 37°C for 1 h. The resulting protoplasts were sonicated on ice (5 × 60 s cycles with 20 s intervals) using a 102C model Branson Digital Sonifier (Branson Ultrasonics Corporation, CT, USA) operated at 40% amplitude. The raw cell extracts were clarified by centrifugation at 1,500 × g for 5 min at 4°C and analysed by SDS-PAGE followed by Western blotting with a mouse anti-Cap PCV2-specific monoclonal antibody (isotype IgG2a; 1:100 dilution; Jeno Biotech Inc., Republic of Korea), as described by Bucarey et al. . The raw extracts (without clarification) were lyophilized and ground to produce the yeast powder used for microencapsulation into LMW chitosan (75–85% deacetylated; Sigma-Aldrich Co., MO, USA).
Chitosan and yeast-derived PCV2-antigen microencapsulation
Preparation of the vaccine formulation included the microencapsulation of the viral antigen to protect it and control its release at the mucosal level. Approximately 30 mg of PCV2 Cap protein was used for each microencapsulation. The amount was calculated by assuming that approximately 10% p/p of the dry weight of the recombinant yeast extract comprised PCV2 VLP (data obtained from a previous report ). The PCV2 VLP antigen was coated with LMW chitosan (Sigma-Aldrich, MO, USA) by ionotropic gelation as previously described , with some modifications. Briefly, 30 ml of LMW chitosan (1% w/v in 2.5% acetic acid) was mixed with 375 mg of dry raw yeast extract (S. cerevisiae/pYES2::opt-cap) with mechanical stirring (1510 rpm). The microencapsulation reaction was initiated by the drop-wise addition of 5 ml of sodium tripolyphosphate (TPP; 3 mg/ml) (Sigma-Aldrich, MO, USA) at a rate of approximately 1 ml/min (with constant stirring). The solution was then stirred for a further 20 min at room temperature.
The resulting microparticle suspension was centrifuged at 3,000 × g for 10 min. The efficiency of the microencapsulation process was 90–95% (estimated by subtracting the total amount of yeast-derived protein remaining in the supernatant from the initial amount of protein added). Protein concentrations were measured using the BCA™ protein assay kit (Pierce, Rockford, IL, USA). The pelleted microparticles were washed twice with Milli-Q water, lyophilized, weighed, and stored. Samples of the lyophilized microparticles were suspended in PBS (pH 7.0; final concentration, 35 mg/ml) and stored at 5°C. These samples were used to test PCV2 antigen delivery in vitro and to induce cellular immune responses in mice following oral administration.
Scanning electron microscopy, size estimation, and measurement of the Zeta potential of chitosan microparticles
Each microparticle formulation was examined under a scanning electron microscope (SEM; Tesla BS 343 operating at 15 KeV; ×3,300 magnification) to examine the morphology and size of the individual microparticles. Briefly, the freeze-dried microparticles were spread onto metallic discs and gold-coated (20 nm thick) using an EMS-550 automated sputter coater.
The Zeta potential of the chitosan microparticles was measured using a Zeta potential analyser (Zeta plus, Brookhaven Instruments Co., NY, USA). All Zeta potential measurements were determined at 25°C in an electric field of 11.00 V/cm. The size and polydispersity index were determined by light scattering using a multi-angle particle sizing option (90PLUS/BI-MAS, Brookhaven Instruments Co.). A stock solution of each chitosan microparticle sample (1.6 mg/ml in ultra-pure water) was used for both Zeta potential and particle size measurements. Ten millilitres of each solution were mixed with 10 ml of bi-filtered KCl (1 mM in ultra-pure water; pH 7).
Immunofluorescence microscopy of chitosan microparticles
For immunofluorescence microscopy, 10 mg of freeze-dried chitosan microparticles were blocked overnight in 200 μl of PBS/5% skim milk at 4°C and then incubated overnight at 4°C with a mouse anti-Cap PCV2-specific monoclonal antibody (isotype IgG2a, Jeno Biotech Inc.) diluted 1:100 in PBS/0.1% Tween-20 (PBST). After washing with PBST, the microparticles were incubated with FITC-conjugated goat anti-mouse IgG (H + L) (Kirkegaard & Perry Laboratories Inc.) for 1 h. After further washing, the microparticles were visualized under a Nikon Eclipse E400 fluorescence microscope interfaced to a PC running capture software (Nis-Element Br, Nikon).
Microparticles loaded with extracts of S. cerevisiae transfected with an empty plasmid (S. cerevisiae/pYES2) were subjected to the same treatment and used as a negative fluorescence control.
PCV2 antigen loading and delivery efficiency of the chitosan microparticles
The release of PCV2 VLP from the chitosan microparticles was measured in Tris-HCl (pH 1). After antigen loading, the microparticles were resuspended in Tris-HCl to yield a 1% w/v suspension. Samples (200 μl) were then incubated at 90°C with gentle shaking. After 0, 5, 10, 15 and 30 min, the tubes were centrifuged (10,000 × g for 2 min). Samples of the supernatant (100 μl) were taken and the amount of non-bound PCV2 VLP was determined using the Dot blot method. Briefly, samples were transferred onto a nitrocellulose membrane using a Biodot™ microfiltration apparatus (Bio-Rad, CA, USA). The nitrocellulose membrane was then blocked overnight in 5% skim milk at 4°C and then incubated overnight at 4°C with a mouse anti-Cap PCV2-specific monoclonal antibody (isotype IgG2a, Jeno Biotech Inc.) diluted 1:100 in PBS/0.1% Tween-20 (PBST). After washing with PBST, the membrane was incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (H + L) (1:1,000 dilution; Kirkegaard & Perry Laboratories Inc.) for 1 h. After further washing, the signal was detected using 4-chloro-1-naphthol/H2O2 as directed by the manufacturer (Pierce, Rockford, IL, USA). The concentration of yeast-produced Cap protein was estimated by comparing the signal intensities of the blots with those of known concentrations of a highly purified 6xhis-Cap fusion protein as previously described .
Purification of yeast-derived PCV2 virus-like particles
Clarified yeast extract (500 μl) expressing PCV2 Cap protein was layered onto a discontinuous sucrose gradient (20–50%) and centrifuged at 80,000 × g for 18 h using a Beckman SW-28 rotor. The gradients were fractioned by puncturing the bottom of the centrifuge tube and collecting approximately ten fractions. The fraction densities were determined using a refractometer (32-G110e; Carl Zeiss Jena, Germany). Fractions with a density between 1.2 and 1.27 g/cm3 (three fractions in all) were pooled, and the presence of Cap protein was determined by SDS-PAGE. The VLP preparations were dialyzed against PBS and stored at −20°C until visualization by transmission electron microscopy (TEM).
Purification of the PCV2 6xhis-Cap protein
The complete PCV2 capsid protein gene was subcloned in a pQE80L expression vector (Qiagen, Inc., USA) via the SphI and KpnI restriction sites to generate an in-frame genetic fusion bearing a polyhistidine tag. The bacterial Cap protein was used to produce a purified 6xhis-Cap fusion protein for use as a PCV2 protein standard for blotting as described previously . Briefly, the recombinant E. coli strain, BL21 (Amersham), containing the pqE80L::cap plasmid was grown in Luria Broth medium (10 g l-1 yeast extract, 16 g l-1 tryptone, 5 g l-1 NaCl, 100 μg/ml ampicillin, pH 7.0) and induced for 5 h at 37°C with isopropylthio-b-D-thiogalactoside (IPTG) at a final concentration of 0.1 mM. The cells were pelleted and resuspended in lysis buffer (8 M Urea, 10 mM Tris, 100 mM NaH2PO4, 1% Triton X-100, pH 8.0) and then lysed by sonication on ice for two 60 s cycles using a Branson Digital Sonifier® operated at 10% amplitude. After centrifugation at 10,000 × g for 10 min at 4°C, the supernatant was loaded onto a Ni-NTA affinity column (Ni-NTA Purification System, Invitrogen, CA, USA) according to the manufacturer’s protocol. After washing twice with PBS, the Cap protein was eluted in elution buffer (50 mM Tris-HCl, 10 mM imidazole, pH 8.0) and collected. The collected samples were analysed by SDS-PAGE and Western blotting, as described below. The concentration of Cap protein was determined using a Coomassie (Bradford) Protein Assay Kit (Pierce, Rockford, IL, USA).
Transmission electron microscopy
Yeast-produced VLP preparations (20 μl) were diluted 1/10, adsorbed onto a carbon-coated copper grid, and incubated for 5 min. The grids were then dried using filter paper, negatively stained with 3% phosphotungstic acid (PTA) for 5 min, and viewed using a transmission electron microscope (Zeiss EM 109) operating at 80 kV.
Male C57BL/6 mice (5 weeks old) were obtained from the Faculty of Veterinary Sciences at the University of Chile. The animals were assigned to two experimental groups (n = 3 mice/group) and maintained in a temperature and light-controlled environment with access to food and water ad libitum. One group (group 1) was used to evaluate specific anti-PCV2 cellular responses against PCV2 virions after oral administration of chitosan microparticles loaded with raw extracts of S. cerevisiae expressing the yeast-optimized cap gene (S. cerevisiae/pYES2::opt-cap). The second group comprising untreated (control) mice (group 2) was subjected to the same treatment regimen, but they received PBS alone.
Group 1 received four 200 μl doses of a solution containing 35 mg of chitosan microparticles dissolved in 1 ml of PBS (approximately 7 mg of microparticles per mouse) via oral gavage, with a 14 day interval between doses. The concentration of PCV2 Cap protein in each dose (approximately 300 μg) was determined by densitometric analysis of Dot blots generated using standard dilutions of known concentrations of a bacterially produced Cap-6 × his fusion protein. Group 2 received four 200 μl doses of PBS.
These immunization experiments were repeated twice under the same conditions; thus the total number of animals analysed was 12.
An additional control group (n = 3) was used to examine the immune response elicited by a commercially available PCV2 vaccine (Ingelvac® CircoFLEX™, Boehringer Ingelheim Vetmedica GmbH), which was administered subcutaneously. Briefly, 0.1 ml of formulated vaccine, containing approximately 100 μg of PCV2 antigen, was injected subcutaneously, followed by a booster immunization (with the same dose) 2 weeks later. A third and final immunization was performed 2 weeks after boosting .
Animals were sacrificed by an overdose of a mixture of isoflurane/O2. The experimental protocol was approved by the institutional animal bioethics committee as stipulated in the guide to the care and use of experimental animals of the Canadian Council on Animal Care.
Analysis of T-cell proliferation
Animals were euthanized on Day 42 of the experiment as described above, and the spleens were aseptically removed and ground through a sterile cuprous mesh into PBS. A suspension of individual cells was then obtained by repeated passage through a 21G syringe. The splenocytes were then centrifuged and resuspended in erythrocyte lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 1.3 mM EDTA). After washing with PBS, the cells were stained with CFSE (CellTrace™; CFSE Cell Proliferation Kit, Molecular Probes) as previously described , with some modifications. Briefly, cells (5 × 107) were incubated with 1 ml of PBS containing 10 μM CFSE at 37°C for 10 min. The cells were then washed twice with PBS/5% FBS (Foetal Bovine Serum) resuspended (at 2 × 106 cells/ml) in RPMI medium (Thermo Scientific™, MA, USA) supplemented with 10% FBS and then seeded into 96-well plates at a density of 4 × 105 cells/well. The stained splenocytes were then re-stimulated with PCV2 virions (10 TCID50 in 50 μl of MEM-α) obtained from PCV2-positive PK-15 cells (ATCC CCL-33) . Non-stimulated splenocytes were used as a negative control (vehicle). As a positive control for non-specific lymphocytic proliferation, splenocytes were incubated in 96-well plates coated with anti-CD3 antibodies (polyclonal stimulators). The cultures were incubated for 96 h and lymphocyte proliferation was examined by acquiring 100,000 events in a FACSCalibur® flow cytometer (Becton Dickinson Immunocytometry Systems, CA, USA). Data were analysed using Flowing Software, version 2.5.
Initially, the population of interest was defined by gating on SSC (cellular complexity) and FSC (cell size). The population of interest was further defined as viable mature and immature lymphocytes, as previously described . This population contained the highest percentage of CD3+ cells. Further population analysis was performed by gating on FSC and the CD4 or CD8 lymphocyte markers.
Measurement of IFN-γ secretion
A mouse IFN-γ enzyme-linked immunosorbent assay (ELISA) Kit (Thermo Fisher Scientific Inc, MA, USA) was used to measure the concentration of IFN-γ in T-cell culture supernatants according to the manufacturer’s instructions. Briefly, culture supernatants from splenocytes derived from immunized and controls mice were diluted 1:50 in PBS, and 100 μL of the resulting solution was added to triplicate wells of the ELISA plate. The absorbance was measured at 550 nm and at 450 nm in a Microplate Reader (Bio-Rad Instruments, CA, USA). The former value was then subtracted from the latter. A standard curve was constructed using a set of standards provided by the manufacturer and the experimental values were read off this curve.
The IFN-γ ELISA assay results were expressed as the mean ± standard deviation. Differences between groups were analysed by ANOVA with Tukey’s post-test. A p-value < 0.01 was considered significant. Analyses were performed using GraphPad Prism software.