Virus, cells, plasmid, and animal
JEV (strain P3) was donated by Professor Min Cui from Huazhong Agricultural University and stored at -80 °C. It was used as the coating antigen and stimulation for in vitro experiments and used for challenge experiments. Vero cells are used for plasmid transfection and plaque assay to detect virus titers, plaque reduction neutralization test (PRNT) are used to detect nAb titers. C6/36 cells are used for virus proliferation. The pV-JP3ME plasmid was constructed by introducing the BamHI enzyme digestion site, Kozak sequence and signal sequence upstream of the prM/E sequence of P3 strain, and introducing XhoI digestion site downstream into the eukaryotic expression vector pV. Specific pathogen-free 6-8-week-old female BALB/c mice were purchased from Beijing Vital River Experimental Animal Technology Co., Ltd. and used for immunity, sera and splenocytes collection and challenge tests.
Reagents and instruments
Restriction enzymes BamHI and XhoI, eukaryotic expression vector pV, nuclear staining agent 4',6-diamidino-2-phenylindole (DAPI), and transfection reagent Lipofectamine 3000 were purchased from Thermo Scientific, USA. Minimal essential medium (MEM) and RPMI-1640 medium were purchased from Gibco, USA. Methylcellulose was purchased from Sigma, USA. Goat anti-mouse fluorescein isothiocyanate (FITC)-IgG antibody was purchased from Beijing TransGen Biotech, China. Goat anti-mouse horseradish peroxidase (HRP)-IgG antibody was purchased from Abcam, USA. Tetramethylbenzidine (TMB) substrate color solution was purchased from MabTech company, USA. Enzyme-Linked ImmunoSpot (ELISPOT) kit, streptavidin and AEC color development kit were purchased from BD company, USA. The gene introduction instrument was purchased from Shanghai Teresa Corporation, China. The enzyme-linked immune-sorbent assay (ELISA) plate reader and cell culture incubator were purchased from Thermo, USA. The ELISPOT plate reader was purchased from CTL, USA.
Transfection and immunofluorescence experiments
pV-JP3ME or pV were transfected into Vero, respectively. After 5 h, the transfection plasmid/reagent mixture was discarded and replaced with a complete culture medium. After 40 h, the medium was discarded, and the two groups of cells were simultaneously fixed. Then, cells were incubated with JEV antiserum (1:1,000) as primary antibody and goat anti-mouse FITC-IgG as a secondary antibody. Observation of specific green fluorescence under a fluorescence microscope indicates that the plasmid was successfully transfected and expressed in mammalian cells in vitro.
Animal experiments
The mice were randomly divided into two groups. The vaccine group was vaccinated with the DNA vaccine pV-JP3ME, and the control group was vaccinated with the empty vector plasmid pV. Immunization was performed three times, each immunization dose was 50 μg, intramuscular injection with electroporation was used. 1 and 3 weeks after the last immunization, the splenocytes and sera of the two groups of mice were taken, respectively. The next day, mice in the vaccine and the control group were challenged with JEV. The body weight changes and survival rate were measured daily after the challenge, and the observation was for 12 consecutive days. The animal experiment schedule was showed in Fig. 1.
Plaque assay
Vero cells were cultured into 24-well plates, and the cell density must be more than 95% before performing the virus infection. The serially diluted virus was serially 10-fold-diluted from the original solution (1:1), for a total of seven dilutions, namely 1:1 to 1:106. 200 μL of the virus dilution was added to each well and incubated at 37 °C for 1 h. Gently shake the plate once every 15 min. After discarding the dilution, add 5 mL of MEM medium containing 1.2% methylcellulose to each well. After 4 d of culture, to discard the medium and add 1 mL of crystal violet staining solution to each well and stain for 30 min at room temperature. The number of plaques per well was counted and the virus titer was calculated. The titer of the virus solution needs to be repeatedly measured three times and an average value is taken, which is expressed as a plaque-forming unit (PFU)/mL.
ELISA
Heat-inactivated JEV was coated into a 96-well plate at 105 PFU per well at 4 °C overnight. The coating antigen was discarded and blocked with 1% bovine serum albumin (BSA) at 37 °C for 2 h. The blocking solution was discarded. The sera of each group of mice were started from 1:100, and was serially diluted at a two-fold ratio, for a total of 12 dilutions, namely from 1:100 to 1:204,800, and added to the wells in turn as a primary antibody, 4 °C overnight. The next day, the primary antibody was discarded. After washing the plate five times, HRP-labeled goat anti-mouse IgG antibody (1:4,000) was added as a secondary antibody. After incubation at 37 °C for 1 h, the plate was discarded. The substrate solution developed color for 20 min, and the reaction was stopped with H2SO4. To take 1/2 of the A450 nm value at the 1:100 dilution of the control group as the cut-off value, and the maximum dilution greater than this cut-off value is the serum IgG antibody titer.
PRNT
Vero cells were cultured into 24-well plates as described previously [11, 12]. The serum is diluted from 1:10, and is serially diluted at a 2-fold ratio. There are seven consecutive dilutions, that is, 1:10 to 1:640. Each dilution of serum is mixed with an equal volume of virus solution (containing 100 PFU). To incubate at 37 °C for 1 h, and set serum-free virus samples at 4 °C and 37 °C, respectively, for excluding temperature factor and referencing positive virus count. Then, the serum/virus mixture was added to the wells in order and incubated at 37 ° C for 1 h. During the period, the plate was shaken gently every 15 min. The subsequent step is the same as described in 2.5. The serum dilution corresponding to a 50% reduction in the number of plaques in the positive wells incubated at 37 °C was recorded as the PRNT50 value, which is the nAb titer.
ELISPOT assay
IL-2 and IFN-γ capture antibody diluted 1:200 were coated in a 96-well plate at 4 °C overnight. To discard the coating solution and block with RPMI-1640 medium containing 10% FBS at room temperature for 2 h. Then, splenocytes of two groups were added to each well, 2 × 105 per well, and 105 PFU heat-inactivated JEV was added as a stimulus, and cultured at 37 °C for 72 h. After discarding the cultured splenocytes, IL-2 and IFN-γ detection antibodies were added, respectively, and then spot forming unit (SFU) was counted by adding streptavidin and AEC chromogenic solution.
Statistical analysis
All experimental data were recorded using Excel 2016 software, statistical analysis was performed using SPSS 17.0 software (USA), body weight change was analyzed by repeated analysis of multivariate analysis of variance, survival rates were compared using Log-rank test, and the differences between the other groups were compared using one-way ANOVA analysis, quantitative data are expressed as mean ± standard deviation. P < 0.05 was considered statistically significant.