Cells and virus
Swine testis (ST) cells were maintained in Dulbecco's modified eagle medium (DMEM, Gibco) with 10% fetal bovine serum (FBS, Gibco) at 37°C in a CO2 incubator and used to serially passage and propagate TGEV. The monolayers of ST cells were maintained in DMEM with 0% FBS and TGEV were propagated by inoculating at a multiplicity of infection (MOI) of 0.1. The virulent TGEV used in our research was the passage 15 (P15) of TGEV HN-2012 strain that isolated and identified by our laboratory. The propagated TGEV culture was harvested when the cytopathic effect (CPE) was >80%, and the virus titer was determined to be 10 -8.0 /0.1 mL. The cell debris were removed by 8 000 rpm centrifugation for 30 min at 4°C and then filtered by 0.2µL pore size filter.
Inactivation protocols of TGEV
Three inactivating agents were selected (FA, BPL and BEI) in our study. For FA inactivation agent, 40% FA (Sinopharm Chemical Reagent Co, Ltd) was used at a final concentration of 0.1%, 0.2%, 0.3% (v/v) respectively. Different concentrations of FA were incubated with TGEV at 37°C and collected after 6, 12, 18, 24, 30 and 36h, respectively. The reactions were stopped by adding sterilized 1M sodium thiosulfate (Sinopharm Chemical Reagent Co, Ltd) at the concentration 10 times of the FA final concentration. For BPL (Acros Organics, Geel, Belgium), three concentrations of 0.01%, 0.02%, 0.03% (v/v) were used as the final concentrations respectively. The three concentrations of BPL were incubated with TGEV at 4°C and collected after 6, 12, 18, 24, 30 and 36h, respectively. The reactions were stopped in water bath at 37°C for 2 h. BEI was prepared as described below. Briefly, 2-bromo-ethylamine HBr (BEA) (Sigma-Aldrich, USA) was dissolved in 0.2 mol /L NaOH (Sinopharm Chemical Reagent Co, Ltd) to get the final concentration of 0.1 M of BEA. The solution was then incubated at 37°C for 1 h and BEI was formed. The three concentrations of 0.03%, 0.04%, 0.05% (v/v) of BEI were incubated with TGEV at 37°C and were collected after 6, 12, 18, 24, 30 and 36h, respectively. The reactions were stopped by adding sterilized 1M sodium thiosulfate at the concentration of 10 times of the BEI final concentration. The experiments were performed 3 times.
Tests of infectivity and sterility of TGEV
In order to determine the infectivity of TGEV after three inactivation reagents treatment, inactivated TGEV was collected and cultured in ST cells for three passages. The CPE was observed and TCID50 titers were calculated. Untreated TGEV was used as the positive control, and DMEM was used as the negative control. Sterility tests were conducted in common nutrient agar and ordinary broth. Bacterial growth was observed on the culture medium after 30 h at 37°C.
Preparation of inactivated TGEV vaccine
To produce the inactivated TGEV vaccine, the inactivated viral antigens were emulsified with Freund's complete adjuvant (Sigma-Aldrich, USA) at a ratio of 1:1 (v/v), and this vaccine was used for the first immunization of mice. For the second and the third immunization, the inactivated TGEV antigen was emulsified with Freund's incomplete adjuvant (Sigma-Aldrich, USA) at a ratio of 1:1 (v/v).
Vaccines immunization of mice
To detect the immunization effects of vaccines, sixty-eight female healthy 6-8-week-old BALB/c mice were purchased from the Henan Province Laboratory Animal Management Committee in China. Before injection, all mice were detected by the enzyme-linked immunosorbent assay (ELISA) kit (Wuhankeqian Animal Biological Products Co., Ltd.) to make sure the TGEV antibody was negative. The mice were then randomly divided into 4 groups (n = 17/group), and housed separately. Mice were subcutaneously injected into the backs with 200μL of TGEV vaccine inactivated with FA (group 1), 200 μL of TGEV vaccine inactivated with BPL (group 2), 200 μL of TGEV vaccine inactivated with BEI (group 3), and 200 μL PBS (group 4) as the negative control. In this research, vaccine injections were performed three times at two-week intervals. Animal experiments in this study were carried out in accordance with the Health guide for the care and use of Laboratory animals of Henan Agricultural University.
Detection of TGEV-specific IgG by indirect ELISA
Blood samples were randomly collected from 5 mice in each group at 0, 1, 2, 3, 4, 5, 6, 7 and 8 week after the initial immunization, and placed at 37°C for 1 h. After centrifugation at 3500 rpm for 10 min, the serum from each mouse was collected. IgG was detected by TGEV antibody-IgG ELISA kit (Wuhankeqian Animal Biological Products Co., Ltd.). All the steps in the kits were followed, and the optical density (OD) 450 was measured. When OD450 values of the experimental groups were greater than or equal to 2.1 times of the values of the control group, the samples considered to be positive. Values ˂ 0.05 were excluded.
Detection of CD4+, CD8+ T lymphocytes
300 μL of blood samples were randomly collected from eyeballs of 3 mice in each group at 21 day post-inoculation (dpi) and 35 dpi of the first immunization, respectively. The positive rates of CD4+, CD8+ T lymphocyte subsets were analyzed by flow cytometry. Briefly, 3 mL volume of red blood cell lysis buffer (Solarbio) was added to each sample to crack red blood cells totally. Then the samples were washed and re-suspended with DMEM to 1×106 cells/mL. Cells were transferred to 48-well plates with 100μL volume of each well, 2μL of cell activation cocktail (Bio legend) and 1μL of BrefeldinA (Bio legend) were added to each sample and incubated at 37°C for 6h. After washed and re-suspended with PBS, samples were incubated with specific fluorescent antibodies (Bio legend) of Brilliant Violet 510 (BV510) conjugated anti-mouse CD3 antibody (0.4 μg/sample), PerCP/Cyanine 5.5 conjugated anti-mouse CD4 antibody (0.2 μg/sample), and fluorescein isothiocyanate (FITC) conjugated anti-mouse CD8a antibody (1 μg/sample) for 30 min at room temperature in the dark according to the manufacturer’s guidelines. All samples were stained in triplicate. The samples were analyzed by flow cytometry with a BD FACS Canto plus (BD Biosciences, US). Data were analyzed using Canto diva software (BD Biosciences, US).
Detection of CD4+IFN-γ+, CD4+IL-4+ T lymphocytes
To further determine the levels of CD4+IFN-γ+, CD4+IL-4+ T lymphocyte subsets, cells treated in 2.7 were fixed with 500 μL of 4% FA for 20 min in the dark, ruptured with 1 mL of Permeabilization Wash Buffer (Bio legend). Then the cells were stained with allophycocyanin (APC) conjugated anti-mouse IFN-γ antibody (0.8 μg/sample), phycoerythrin (PE) conjugated anti-mouse IL-4 antibody (0.2 μg/sample) (Biolegend) for 30 min. All samples were stained in triplicate. These samples were analyzed by flow cytometry, and data were analyzed.
Lymphocyte proliferation assay
At 14, 21 and 35 dpi, lymphocytes were isolated randomly from the spleens of three mice in each group. The protocol of lymphocyte isolation from spleen was modified from the previous study [13]. And the lymphocytes were re-suspended in DMEM and adjusted to 1.0×106 /mL. Cells were cultured in 96-well flat-bottom plates for 100μL per well, and stimulated with concanavalin A (ConA, Sigma) with the final concentration of 50 μg/mL and 20μL of inactivated TGEV antigen (1×105 TCID50/mL) in each well, respectively. DMEM was used as a negative control. All treatments were in triplicate. The plates were incubated at 37°C for 20 h, and then added methylthiazoltetrazolium (MTT) (5 mg/mL) with 10 μL/well, and incubated the cells further at 37°C for 4 h. 100 μL of 10% dimethyl sulfoxide (DMSO, Solarbio) was added to stop the reaction. The OD492 value of the solution was determined. The stimulation index (SI) was calculated with the following formula: SI = (OD sample well − OD blank well)/(OD negative well − OD blank well).
Gross pathology and histopathology
At 35 dpi, 3 mice in each group were randomly selected and killed. The major organs which including heart, liver, spleen, lung, kidney, small intestines (jejunum and ileum) and muscles of back that injected with vaccine were examined grossly, and then fixed by 10% formalin for 48 h. To evaluate whether the vaccine had adverse reactions to mice, these fixed tissues were stained with Mayer’s H.E for histopathological examination.
Statistical analysis
Data of the three experimental groups and control group were evaluated by SPSS 17.0 software, and error bars represented standard deviations. The results were unpaired two-way analysis of variance (ANOVA). The results were expressed as mean ± standard deviation (SD), with P-values<0.05, P-values<0.01 and P-values<0.001 considered to be statistically high, significantly high and extremely high, respectively.