Cells and viruses
Chick embryo fibroblast cells (CEF) were obtained from Charles River (Charles River Laboratories International, Inc., Wilmington, MA) and maintained in Dulbecco’s modified Eagle medium (DMEM; Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 2.5 µg/ml amphotericin B, and incubated at 37 °C in 5% CO2. MVA virus used in these studies was obtained through BEI Resources (NIAID, NIH, ref # NR-727). A recombinant MVA virus expressing GFP (MVA-GFP) was based on the wild-type MVA, and its construction has been previously described 20,23.
Generation of recombinant viruses
Five recombinant MVA viruses were constructed. Four of them were bearing the ASFV p30 gene being expressed under the control of different promoters such as: 1) poxvirus PrMVA-13.5L (MVA-PrMVA-13.5L-p30), 2) pHyb (MVA-pHyb-p30), 3) S E/L (MVA-S E/L-p30), and 4) PrS5E (MVA-PrS5E-p30). A fifth MVA recombinant contained the ASFV p30 gene under the control of the S E/L promoter and in-frame with the vaccinia secretory signal C13L (MVA- S E/L-C13L-p30) (Fig. 1). DNA cassettes containing these sequences, the mCherry gene under the control of a late p11 promoter, and flanking sequences from the MVA hemagglutinin gene (HA) were synthesized 24. Expression of the mCherry protein allows for visual-based selection and permits an easy distinction between recombinant (red) and wild-type (green) viruses. We used IBS 1.0.3 software (Illustrator for Biological Sequences) to illustrate all expression cassettes (Fig. 1) 25.
All cassettes were PCR amplified using Phusion High-Fidelity DNA polymerase (New England Biolabs, # M0530S, Ipswich, MA), and then used for homologous recombination. Recombinant MVA viruses were generated as described previously 26,27. Briefly, CEF cells were seeded into six-well plates the day before transfection and then infected at a multiplicity of infection (MOI) of 0.05 PFU/cell for 1.5 hours (hr) with wild-type MVA-GFP virus. Following the manufacturer's protocol, cells were then transfected with appropriate PCR fragments using FuGENE HD (Roche Diagnostics, # E2311, Indianapolis, IN). At 48–72 hr post-transfection, monolayers were harvested, centrifuged at 500xg for 5 minutes (min) at 4°C, and cells were disrupted by freeze-thaw (3 times) followed by sonication (2 times for 15 s using a cup sonicator). The disrupted cell extracts were plated again onto fresh CEF cells and overlaid with 1% agarose. After 48–72 hr, virus-generated plaques expressing mCherry were picked into 500 mL media with a sterile filter pipette tip, then subjected to freeze-thaw, sonication (as described above), and plated to continue passaging. Plaques were passaged until no wild-type (GFP expressing) virus was observed, at which point PCR was performed to confirm the presence of only the recombinant virus 28. Viral DNA was extracted using the quick-DNA Viral kit from Zymo Research (Zymo Research, # D3015, Irvine, CA), and PCRs were then performed using Phusion High-Fidelity DNA polymerase with specific primers that bind to the flank sequences (HA gene).
High titer virus stocks were produced and further characterized by PCR and DNA sequencing to ensure genetic homogeneity and stability.
In vitro characterization of recombinant MVA viruses
p30 and mCherry mRNA expression analysis in DF-1 cells using quantitative reverse transcription PCR (RT-qPCR).
Briefly, DF-1 cells were infected with an MOI of 5 PFU/cell for 24 hr at 37°C and 5% CO2. Infected cells were collected, and RNA was extracted using Direct-zol RNA MicroPrep (Zymo Research, #R2062). One nanogram of RNA was used as the template for the reverse transcription into complementary DNA (cDNA). cDNA was produced using Verso cDNA Synthesis Kit (Thermo Fisher Scientific, #AB1453A, Waltham, MA), and anchored oligo dT was used for priming. iTaqTM Universal SYBRR Green Supermix (BioRad, #1725120) and primers ASFV-p30_F1 (5’-ctgctgagcaccgtgaagta-3’), ASFV-p30_R2 (5’-gtctcctcctcgaacagcac-3’), mCherry-F1 (5’-agggcgaggaggataacatg-3’), and mCherry-R1 (5’-cttggtcaccttcagcttgg-3’) were used for the amplification of the cDNA template. To ensure the correctness of the comparison, the ACTB (β-actin gene) was used as normalizing control and primers designed previously 29. The following amplification conditions were used: 95°C for 30 sec, 40 cycles of 95°C for 15 sec, 60°C for 1 min. Sample reactions, including negative controls (MVA-GFP), were performed in triplicate and two independent repetitions. Fold change data was obtained using the delta-delta CT method as previously described 17.
Recombinant protein expression patterns of MVA constructs using immunoblotting.
We seeded CEF cells at 5 x 105 cells/well into six-well plates and 24 hr later infected at an MOI of 5 PFU/cell in OptiMEM (Thermo Fisher Scientific, # 22600134, Waltham, MA) in the absence of FBS. At 24 hr after infection, cells were harvested and lysed using RIPA lysis buffer (Thermo Fisher Scientific, # 89900, Waltham, MA) at 4°C for 30 min under gentle agitation. Lysates were then centrifuged for 10 min at 13,000 rpm, diluted into 4x Laemmli buffer (Bio-Rad, Richmond, #1610747 CA), heated at 95 °C for 5 min, and 30 μl were loaded into a Bio-Rad 4-20% precast gel. Gels were transferred to a nitrocellulose membrane using Trans-Blot Turbo Blotting System following the manufacturer’s instructions. Swine serum from a convalescent animal inoculated with ASFV and anti-b-actin antibody (Sigma, # A5441, St Louis, MO) were used to probe blots at a dilution of 1:5000. Goat anti-porcine IgG-HRP-conjugated (Southern biotech, # 6050-05, Birmingham, AL) and goat anti-mouse IgG-HRP-conjugated (Thermo Fisher Scientific, # 31430, Waltham, MA) were used to detect p30 and b-actin, respectively. Membranes were developed using a 1-Step Ultra TMB-Blotting Solution (Thermo Fisher Scientific, #37574, Waltham, MA).
Detection of specific anti-p30 antibodies.
ELISA assay was performed as described by Brewoo et al., 2010 23. Briefly, 96-well ELISA plates were coated with 4ng of p30 protein (baculovirus-expressed) diluted in 100 μl carbonate-bicarbonate buffer, pH 9.6 per well, at 4°C overnight. Coated plates were washed three times with PBS-T (0.05% Tween-20) and incubated with blocking buffer (5% BSA in PBS) at room temperature (RT) for 1 hr. Serum samples were serially diluted in triplicate from 1:50 – 1:1,600 in blocking buffer, added to the ELISA plates, and incubated at RT for 2 hr. Negative serum samples collected at day 0 (pre-prime) were used as the negative control. After washing, 100 μl of a 1:5,000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-porcine IgG (Southern biotech, #6050-05, Birmingham, AL) was added to each well and incubated at RT for 1 hr. Plates were washed six times, and 100 μl of tetra-methyl-benzidine (TMB) chromogen (Sigma, #37574, St Louis, MO) was added to each well and incubated in the dark for 5 min. The reaction was then stopped by adding 100 μl per well of 2mM H2S04. Absorbance was measured using an ELx800 microplate reader (BioTek, Winooski, VT) at test wavelength of 450 nm and a reference wavelength of 630 nm. The highest dilution that was positive (exceeded the mean of known negative serum samples plus three standard deviations) was considered the endpoint, and its reciprocal value was transformed in geometric mean titers (GMT-log10) to calculate of the antibody titers of each group.
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The IACUC protocol (Protocol #V005220) was approved by the Institutional Animal Care and Use Committee of the University of Wisconsin. Additionally, we confirm that this study is reported in accordance with ARRIVE guidelines.
Groups of five- to six-week-old female BALB/c mice (N=4) (Harlan Sprague Dawley, Indianapolis, IN) received primary and two booster immunizations (21 and 35 days apart) with each MVA construct via intradermal injection into the hind, left footpad. A dose of 1 x 107 PFU in 50ml was used for all MVA-p30 constructs. Negative control groups were immunized with MVA-GFP at the same dose. Mice were bled via the maxillary vein at days 0, 21, 28, and 42 post-vaccination (p.v.) to monitor levels of anti-p30 antibodies.
Statistical Analysis Statistical
Statistical analyses were performed using GraphPad Prism 9 software (La Jolla, CA, USA). The significance of the difference in the p30 and mCherry mRNA expression and the anti-p30 antibody titers among the groups was determined by one-way ANOVA, followed by Tukey’s multiple-comparison test and a P-value of P<0.05 was considered significant.