Cell and virus
Vero cell (African green monkey kidney cell) was purchased from the American Type Culture Collection (ATCC, USA). Cells were grown in Opti-Eagle`s Minimum Essential Medium (Opti-MEM, Invitrogen) supplemented with 2% heat-inactivated fetal bovine serum (FBS, Invitrogen), incubated at 37℃, and humidified with 5% CO2. The attenuated vaccinia virus strain KVAC103 and the viral vector pVVT1-EGFP-C7L were provided by Korea National Institute of Health (KNIH). This vector contains the vaccine virus C7L gene which encodes interferon antagonist, and this is one of the 26 genes defective in KVAC103 compared to its ancestor strain. This gene is required for enhanced viral reproduction [24].
Construction of anthrax/smallpox dual vaccine candidate vectors
Viral vector constructs were generated using the pVVT1-EGFP-C7L vector [24] as a template (Fig. 1). Human IL-15 gene and human codon-optimized B. anthracis PA and CTA1 genes were synthesized (Bioneer). The synthesized genes were cloned into the vector using SfiI restriction enzyme site. The constructed vectors were mixed with Lipofectamin (Invitrogen) and transfected into KVAC103-infected Vero cells. Single plaques were isolated from the original infected cells and verified using PCR. The primer sequences used for verification were 5’-TTT GAA GCA TTG GAA GCA ACT-3’ and ‘5’-ACG TTG AAA TGT CCC ATC GAG T-3’.
Virus preparation
Viruses were infected to mono-layered Vero cells with 0.01 MOI. The virus-infected cell media were harvested when more than 80% of total cells showed cytopathic effect. From the harvested culture supernatant, viruses were collected by ultra-centrifugation. The pellet was resuspended in 1× PBS, pH 7.0 (Gibco). The concentration of viral particles was determined by the standard plaque assay. The viruses were infected the Vero cells overlaid on 6-well plates for 2 days. The plate were staining with crystal violet and the plaque numbers on each well were counted.
Western blot analysis
Virus-infected Vero cells or their culture supernatants were lysed in 1× RIPA buffer (G-Bioscience) containing 1% PMSF (Theromfisher Scientific) at 4℃. Fifty μg of protein from each cell lysate was resolved on denaturing polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Amersham). Expression levels of PA and CTA1 proteins were detected using mouse monoclonal antibodies against PA and cholera toxin, respectively (Abcam, 1:1000), and horse radish peroxide (HRP)-conjugated secondary antibodies (Abcam, 1:3000).
Mouse immunization and serum collection
Female A/J mice (5-week old) were purchased from SLC, Inc (Japan) and housed in an animal biosafety level 2 (ABL2) facility in KCDC. Mice were immunized with the vaccine candidate virus (5×107 pfu/mouse) with or without the adjuvant-expressing virus (5×107 pfu/mouse) 2-times at 3-week intervals subcutaneously (s.c.) with 8 mice as a group. Mice sera were collected 20 days after final immunization to measure anti-PA IgG and vaccinia virus plaque reduction neutralizing antibody titers. The schematic view of mouse immunization and serum collection is in Fig. 4.
Enzyme-linked immunosorbent assay (ELISA)
The anti-PA IgG titers of mice sera were determined by ELISA as previously described with some modifications [25]. Briefly, 96 well plates were coated with 1μg/ml of recombinant PA (Green Cross, Korea). Mouse sera were diluted from 1:100 to 1:204800 and loaded to each well, incubated for 1 h at 37℃. Horseradish peroxidase-conjugated anti-mouse IgG goat antibody (Invitrogen) and 3,3`,5,5`-tetramethylbenzidine (TMB) substrate were used for detection. The optical density of each well was measured at 450 nm and the half maximal effective concentration (EC50) was calculated by 4-parameter logistic equation regression using SoftMaxPro5.3 (Molecular Device, USA). The data were analyzed and visualized using GraphPad Prism 5.
The IL-15 expression level of KVAC103 derivatives were determined by the IL-15 ELISA kit (Biolegend) according to the manufacturer’s protocol. Vero cells were infected with viruses of 0.01 MOI. Cell lysates were collected 2 days after infection and analyzed.
Plaque reduction neutralization test (PRNT)
Serial two-fold dilutions of heat-inactivated mouse sera were mixed with vaccinia virus Lister strain of approximately 50 plaque forming units (PFU). After 2 h incubation at 37℃, the serum and virus mixtures were inoculated onto monolayered Vero cells. After two days incubation at 37℃ with 5% CO2, cells were fixed and stained using a mixture of crystal violet and formalin for 10 minutes. Stained plates were dried in air at room temperature and the plaque numbers were counted. The neutralizing antibody titer of serum was defined as the reciprocal of dilution fold that reduced plaque number in half (50%) compared to a control (PRNT50).
B. anthracis spore challenge
Immunized mice were challenged with 50-fold of lethal dose 50 (LD50) of B. anthracis Sterne spore by s.c. injections. Survival of the mice was monitored for 14 days as described in Fig.4. Spores were prepared according to a previous study [26]. The LD50 determined by Reed-Muench method [27] in A/J mice model via s.c. route was 1794 spores. Survived animals were euthanized using CO2 gas. Animal study protocols (KCDC-102-16-2A and KCDC-039-17-2A) were approved by the Institutional Animal Care and Use Committee (IACUC) of Korea Centers for Disease Control and Prevention (KCDC). All procedures involved in the housing and care of animal strictly followed guidelines and requirements of the IACUC. The survival graph was generated by GraphPad Prism 5 and log rank test was used to analyze for the curve comparison.