Plasmids, strains, and culture conditions
A modified cry3Aa promoter (P5D), containing a consensus sequence in the -35 region, was used to express and display the target protein on the surface of Bacillus spores during the sporulation phase . The rPA gene was synthesized by a commercial vender (Bioneer, Seoul, Republic of Korea). All DNA manipulations were performed in Escherichia coli JM109 competent cells (Takara bio inc., Tokyo, Japan). The B. subtilis strains DB104  and WB800N  purchased from MoBiTec (Goettingen, Germany) were used as host strains. B. subtilis sporulation was achieved by incubation in Difco sporulation medium (DSM)(Difco, Becton, Dickinson and Company, NJ, USA)(8g nutrient broth, 0.1% KCl, 0.012% MgSO4, and 1% NaOH in 1 L of distilled water, supplemented with 1 mM Ca(NO3)2, 10 μM MnCl2 and 1 μM FeSO4•7H2O) for 24–36 h at 37°C. Ampicillin (100 mg/ml) or chloramphenicol (5 mg/ml) was added to the medium when required.
Construction of PA producing B. subtilis
PA producing B. subtilis was constructed as previously described . Using the plasmid pMar3g as a template, the promoter P5D and the pagA gene were fused and ligated to construct the plasmid pD5D-pagA. The pD5D-pagA plasmid was finally introduced into B. subtilis WB800N, inserting the P5D-pagA expression cassette into the amyE locus.
Spore preparation and verification of PA on the spore surface
The expression of PA and the sporulation of PA-producing B. subtilis were monitored during batch cultivation in DSM at 30°C by measuring the number of spores using a hemocytometer and a microscope. A greater than 70% of sporulation efficiency was observed, spores were purified, as previously described . Briefly, after sufficient sporulation was observed, vegetative cells were lysed and spores were harvested by centrifugation at 10,000 rpm for 10 min, followed by washing twice with phosphate-buffered saline (PBS, pH 7.0). Then, washed spores were treated with lysozyme (50 mg/ml) for 30 min at 37°C to analyze the presence of PA in spore coat protein by a Western blot assay, using a polyclonal anti-PA antibody (Cat.# LS-C19484, WA, US). Also, NaCl (1M) and Triton X-100 (0.1% in NaCl, 1M) were treated on the PA spore and sonicated for decoating purpose. Then, the detached and remained PA was measured by the Western blot assay.
The surface display of PA was also analyzed through flow cytometry. For immunofluorescence staining, the purified spores were washed three times with PBS, resuspended in 1 ml of PBS containing 5% (w/v) skim milk, and incubated for 1 h at 37°C to block nonspecific antibody binding. After being washed with PBS, the spores were incubated for 1 h at 37°C with anti-PA mouse monoclonal antibody (Cat.# MBS190056, MyBioSource.com, CA, US) specifically targeting the PA protein, in PBS supplemented with 0.5% (w/v) skim milk. Then, the spores were washed with PBS and incubated with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (1:200, Sigma-Aldrich, MO, USA) for 1 h at 37°C. The samples were washed three times, resuspended in 1 ml of PBS, and analyzed using a FACSort flow cytometer (Becton Dickinson, CA, USA) and CellQuest ver.1.0 software. In the same way, to amplify the bias effect to the right on the FACS analysis of the PA, PA-displayed spore was adsorbed with concentrated crude PA protein (PA-A spore) and measured by FACS.
Specific-pathogen-free, age-matched male A/J mice (22 ~ 25g in weight; 8 ~ 10 weeks in age) were obtained from Orient Bio (Sungnam, Republic of Korea) and Charles River Laboratories (Hollister, CA, USA), respectively and maintained at a constant temperature (21 ± 2 °C) and a 12–12 h light–dark schedule under specific pathogenic-free conditions in the animal facilities of the Korean Research Institute of Bioscience and Biotechnology (KRIBB; Daejeon, Republic of Korea). All animal experiments were performed blindly in accordance with the Institutional Guidelines for the Care and Use of Laboratory Animals in Research and the approval of the Animal Care and Use Committee of the KRIBB (KRIBB-AEC-13069).
Then, at various time points for each experimental purpose, mice were killed by administration of CO2.
The prepared rPA spores were combined with Cholera toxin B (CTB; Sigma-Aldrich Co., MD, USA), which is a well-known as a mucosal delivery vehicle studied elsewhere and used in this study for the same purpose . The sample size of experimental groups was calculated referring Sample Size Calculator Web application (https://www.surveysystem.com/sscalc.htm).
Groups of six mice were randomly selected to make groups according to weights and immunized either PO, IN, SL or IP with the recombinant PA-displaying spore vaccine. Each group was matched with a corresponding control groups that were treated with either a non-displaying spore vaccine (N spore) or with PA recombinant protein.
Oral doses of 5 × 109 spores/100 μl of PBS were administered via intra-gastric lavage on days 0, 1, 2, 28, 29, 30, and 35, as adapted from the procedure described by Challacombe . Nasal doses of 1 × 109 spores/20 μl of PBS were dropped into the nostrils of mice using a micropipette, at 1-2 second intervals within a 30 second period, on days 0, 14, and 28. For SL doses, mice were lightly anesthetized by injection with Zoletil (100 mg/kg of body weight; Virbac, France) and xylazine hydrochloride (10 mg/kg of body weight; Bayer, Germany) and received a dose of 1 × 109 spores/7 μl of PBS under the tongue on days 0, 14, and 28, as previously described elsewhere . Briefly, forceps were placed under the tongue of the anesthetized mouse, and the vaccine was administered by a micropipette while the mouth was stretched open. After administration, mice were cautiously placed on their backs, to prevent swallowing until awakening from the anesthetic. IP doses of 1 × 109 spores/100 μl of PBS were administered on days 0, 14, and 28. For all administration routes, control groups receiving non-recombinant spores (1 × 109; N spore) and PBS (Naïve) were included.
Serum and saliva samples were obtained at 0, 2, 4, and 6 weeks post vaccination (wpv). Blood was collected via the retro-orbital plexus after isoflurane induced anesthetization. The isolated serum samples were stored at -20 °C until analysis. Saliva was collected as previously described . Briefly, salivation was stimulated through the IP injection of 1 μg/g mouse pilocarpine hydrochloride (Sigma, MO, USA) in 100 μl of PBS. After 2 to 5 min, a micropipette was placed under the tongue to collect the salivary flow. The collected saliva was centrifuged at 10,000 ×g to remove any debris and stored at -70 °C until analysis.
Pretreatment to detect IgM and IgA
To improve the sensitivity and specificity of an indirect IgM and IgA ELISA, IgG was removed using recombinant protein G, according to the method described by elsewhere . Briefly, 25 μl of serum was mixed with 100 μl of 50% Protein G coupled beads (50% [w/v] in PBS; Roche, Basel, Switzerland) and incubated for 1 h at 37 °C on a Nutator Mixer (BD Clay Adams, GA, USA). Then, the sample was centrifuged for 1 min at 1,000 ×g and the 75 μl of supernatant, representing an approximately 1/3 dilution of the initial serum sample, was used for IgM and IgA analysis.
Determination of anti-PA-specific antibody endpoint titration by the indirect ELISA
ELISA methods were used to measure antibody concentrations. First, 96-well microplates were coated with 50 μl of recombinant purified PA antigen (4 μg/ml in carbonate-bicarbonate buffer) per well and incubated at 4 °C overnight. After blocking with 2% BSA at room temperature for 1h, serum samples were applied as a 2-fold dilution series, starting with a 1/40 dilution in assay dilution buffer (10 mM PBS [pH 7.4], 1% [w/v] BSA, 0.05% Tween 20). Every plate included replicate wells of a negative control (a 1/40 diluted preimmune serum).
The plate was incubated for 2 hours at 37°C, followed by washing with a washing buffer (10 mM PBS [pH 7.4], 0.05% Tween 20) for three times. Then, an anti-mouse IgG conjugated with FITC as a secondary antibody was diluted to 1:50 and distributed according to the manufacturer’s instruction. The plate was incubated for 1 hour at 37°C, followed by washing with a washing buffer (10 mM PBS [pH 7.4], 0.05% Tween 20) for three times. Absorbances were read on a spectrophotometry at 450 nm.
Dilution curves were extracted for each sample, and the endpoint titer for each sample was determined as the reciprocal of the dilution resulting in an optical density that was 0.1 U greater than that of the background value, as established by a 1/40 dilution of a pooled preimmune serum.
Toxin neutralization assay (TNA)
The TNA was performed as described elsewhere with slight modifications . Briefly, the same sera used in the antibody ELISA titration were measured using a RAW 264.7 cell line (ATCC® TIB-71™) instead of J774A.1. The RAW264.7 cell line was proved to be sensitive to the anthrax lethal toxin by others . The anthrax lethal toxin (LeTx) consisted of 0.1 µg of PA and 0.08 µg of LF per ml in cell culture medium. The cell viability was measured using the MTT assay (Vybrant® MTT Cell Proliferation Assay Kit, ThermoFisher Scientific, MA, US) according to the manufacturer’s instruction. TNA titers were expressed as the reciprocal of the highest serum dilution which absorbance higher than the median absorbance of control wells (medium + LeTx)/2. Preliminarily, A 4-parameter sigmoid regression curve (Sigma Plot) was used to determine the dilution of the antisera that resulted in 50% neutralization (ED50) of anthrax LeTx. The neutralization ratio was determined by dividing the test sample ED50 by the reference sample ED50. A 4-parameter sigmoid regression curve was used to determine the dilution of anthrax LeTx that resulted in 50% cytotoxicity in the absence of serum from the toxin titration curve.
Subcutaneous challenge with B. anthracis spores
Groups of ten male A/J mice (8~12 weeks old) were challenged subcutaneously with approximately 6×107 CFU (Tox+ Cap−) spores of the B. anthracis Sterne strain (equivalent to 100 50% median lethal doses (MLD50s) per animal . The animals were observed everyday for 14 days to determine their protected status. The challenge experiment was performed in a blinded fashion, and humane endpoints were strictly observed, such that any animal that displayed a collection of clinical signs that indicating a lethal infection was culled, and death was recorded. Individuals showing no symptoms after 14 days were considered immune.
Statistical analysis was completed using IBM SPSS Statistics 24. Summary statistics were performed for all groups to assess the overall quality of the data, including normality. Obtained data were evaluated by a repeated measurement analysis of variance (ANOVA). If the test indicated significance, a one-way ANOVA with pair-wise testing using Tukey’s adjustment was performed for each time point. A value of P < 0.05 was considered significant.