Construction of plasmids
The sequences of BoHV-1 gB, gC, and gD genes were obtained from GenBank (Accession No. NC_001847). The genes flanked with restriction sites were synthesized by TSINGKE Biological Technology Company (Beijing, China) and then cloned into eukaryotic expression vector pcDNA-Myc-hisB, resulting in recombinant plasmids pcDNA-gB-hisB, pcDNA-gC-hisB, and pcDNA-gD-hisB. The recombinant plasmids were validated by sequencing.
Preparation of the DNA vaccine
The eukaryotic expression plasmids pcDNA-gB-hisB, pcDNA-gC-hisB, and pcDNA-gD-hisB were mixed at a mass ratio of 1:1:1. Subsequently, the plasmids were mixed with PEI magnetic beads (Enriching Biotechnology, Shanghai, China) of 50 nm in a maximal adsorption manner. The procedures for identifying the maximal adsorption of PEI magnetic beads and plasmids were: (1) PEI magnetic beads (50 μL) were mixed with 5, 10, 15, 20, 25, and 30 μg plasmid mixture, respectively. The mixture of PEI and plasmids were incubated at room temperature (RT) for 30 min to ensure complete adsorption. (2) The PEI magnetic beads were centrifuged at 13,000 × g for 30 min. The OD260 values of DNA in the supernatants were measured. (3) Identification of the maximal DNA adsorption by curve plotting, where the beginning of curve reaching parallel was taken as the maximal adsorption level. (4) The maximal DNA adsorption of magnetic beads was calculated by removing DNA in the supernatant from the total DNA.
The PEI magnetic beads were mixed with 60% of maximal adsorption plasmids to prepare the DNA vaccine. Then, the PEG600 was added into the mixture at a ratio of 1:100 (V/V) to neutralize the positive charge on the surface of PEI and became a protective layer to prevent DNA degradation induced by respiratory mucosa.
The 8-week-old healthy female Kunming mice were randomly assigned into five groups. The mice were classified into PBS, PEI, DNA, PEI + DNA, and PEI + DNA + PEG groups. In the DNA-containing groups, the mice were vaccinated with 5 μg twice at an interval of 1 month via the intranasal route. In the DNA-free groups, the dose of the drop was kept consistent with DNA vaccine components.
The lung and spleen were stored in 30% sucrose for 24 h after fixation with 4% PFA. The specimens were sectioned in a cryostat (Microm, Heidelberg, Germany) at 4 μm. IHC was used to detect the expression of viral proteins in the tissues from immunized mice. Briefly, the tissue sections were fixed with absolute ethanol for 30 min. Then, the sections were treated with 1% Triton X-100 for 1 h and followed by treatment of 1% sodium borohydride solution for 30 min at RT. Subsequently, the sections were inoculated with rabbit anti-BoHV-1 polyclonal antibody (prepared in our lab) for 1 h at RT. The sections were rinsed 4 times with PBS-Tween 20 (PBST) and then detected with FITC-conjugated goat anti-rabbit secondary antibody. The expression of viral proteins in each tissue was captured by confocal microscopy (Leica Stellaris 5, US).
Safety evaluation of DNA vaccine in mice
The prepared DNA vaccine was used to vaccinate mice via the intranasal route. In each group, 8-week-old female Kunming mice were included for anal temperature examination each day after vaccination. Three mice in each group were sacrificed to examine the tissue inflammation, and the sera were collected for detection of phosphorus (PHOS), creatine kinase (CK), aspartate aminotransferase (AST), UREA, and alkaline phosphatase (ALKP) levels from 1 to 5 weeks after vaccination.
Hematoxylin–eosin (HE) staining
The tissues obtained from the liver, spleen, lung, and kidney were fixed with 4% paraformaldehyde (PFA). The 6 µm thick paraffin tissue sections were prepared for HE staining.
To perform HE staining, the paraffin was removed from tissue sections using dimethyl benzene and then orderly immersed into 100%, 90%, 80%, 70%, and 60% ethanol for 1 min, respectively. Next, the sections were rinsed with pure water and followed by hematoxylin and eosin staining for 3 and 2 min, respectively. Subsequently, the sections were immersed into a 1% hydrochloric acid alcohol solution for differentiation processing. Finally, the stained sections were orderly immersed into 60%, 70%, 80%, 90%, 100% ethanol, and dimethyl benzene for dehydration.
The blood samples (approximately 300 μL) were collected from mouse eyeball using a heparin sodium anticoagulant tube. The triple volume of red blood cell lysis buffer (Solarbio, Beijing, China) was added into the blood for 10 min and then centrifuged at 400 × g for 15 min. The supernatant was discarded, and the sedimentary leukomonocytes were resuspended with 300 μL PBS twice. Subsequently, 1 μL FITC-conjugated anti-CD4 antibody (BD, US) and 2 μL PE-conjugated anti-CD8 antibody (BD, US) were incubated with leukomonocytes for 30 min in a dark box. Then, leukomonocytes were rinsed with PBS 3 times. A total of 10,000 cells were counted for calculating the percentages of CD4 and CD8 positive cells using flow cytometry (CytoFLEX S, Beckman Coulter, US).
Evaluation of humoral immune responses
The enzyme-linked immunosorbent assay (ELISA) was employed to identify anti-BoHV-1 antibody levels. BoHV-1 Cooper strain was purified by ultracentrifugation and then inactivated by ultrasonication (28 kHz, 600 W) for 30 min . The inactivated viruses were used as coating antigens. The ELISA was established in our laboratory. Briefly, 100 ng of antigens were coated in each well of a 96-well plate at 37 °C for 1 h and followed by blocking with 1% BSA at 37 °C for 2 h. The mouse sera were inoculated at 37 °C for 1 h and followed by three washes with PBST. The HRP-conjugated goat anti-mouse IgG secondary antibody (Abcam, UK) was used to detect the anti-BoHV-1 antibody level. The serum antibody level was monitored per week after the second vaccination.
Evaluation of cellular immune responses
The sera collected from vaccinated mice were used for cytokine detection. According to the manufacturer's instructions, the cytokines IFNγ, IL2, IL4, and IL10 were detected using commercial kits (R&D system, USA).
Total IgA detection
The supernatant (~ 100 μL) of crushed lung liquid was used for ELISA as described above. The total IgA level was detected with HRP-conjugated goat anti-mouse IgA alpha chain antibodies (Abcam, UK). The difference in IgA level was analyzed among each group.
The statistical data were expressed as mean ± SEM. Statistical analyses were performed using GraphPad Prism 6.01 software (GraphPad Software, Inc.). The difference in biochemical indicators, cytokines, total antibody, total IgA, and CD4/CD8 between DNA vaccine vaccinated and control groups were analyzed by two-way ANOVA post-Sidak's multiple comparison test. The difference in the neutralizing antibody level between DNA and DNA-vaccinated mice was analyzed by unpaired Student's t-test. Throughout the manuscript, P < 0.05 was taken as the criteria for statistical significance (*: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001, ns: no statistical significance).