Swine testis (ST) cells were obtained from the Institute of Veterinary Immunology & Engineering, Jiangsu Academy of Agricultural Sciences (Nanjing, China), and maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with heat-inactivated 10% fetal bovine serum (FBS, HyClone, Thermo Scientific, USA), penicillin (100 U/mL) and streptomycin (100 µg/mL), at 37°C in a humidified atmosphere containing 5% CO₂. The PRV Bartha-K61 strain was also provided by the Institute of Veterinary Immunology & Engineering and was amplified in ST cells. PRV Bartha-K61 titers were determined to be 10^8.0 tissue culture infectious dose 50% (TCID₅₀)/mL using the Reed-Muench assay. PRV virus was inactivated by adding methyl aldehyde (1:2000 v/v) (13, 14).

C-di-GMP (MedChemExpress, China) immunopotentiator was prepared as a water-in-oil emulsion formulation. Briefly, c-di-GMP (100 mg/mL)was dissolved in sterile water as the aqueous phase, mixed with inactivated PRV, and then mixed thoroughly with mineral adjuvant ISA 206 (Seppic, France).

PRV live virus (hereafter referred to as LV) was prepared by diluting live virus with DMEM to 1×10^6.0 TCID₅₀/mL, then emulsifying with an equal volume of ISA 206. PRV inactivated virus vaccine (KV) was prepared by diluting inactivated virus with DMEM and emulsified as above. To prepare PRV inactivated virus + c-di-GMP vaccine (KV-c-di-GMP), inactivated virus was diluted with DMEM as above, mixed with 100 µg c-di-GMP (100 µg/1mL), then emulsified with an equal volume of ISA 206. A blank control vaccine was prepared by emulsifying phosphate-buffered saline (PBS) with an equal ISA 206.

Five-week-old BALB/c female mice were purchased from Yang Zhou University (Yangzhou, Jiangsu, China). The study and protocol were approved by the Jiangsu Academy of Agricultural Sciences Experimental Animal Ethics Committee of the Science and Technology Agency of Jiangsu Province (approval ID NKYVET 2015-0066). All animal studies were performed according to the guidelines of Jiangsu Province Animal Regulations (Government Decree No. 45). Eighty mice were randomly divided into four groups of 20 mice each. Each group was subcutaneously vaccinated in the neck with one of the vaccines mentioned above (100 µL per mouse).

Serum samples from individual mice were collected at 7, 14, 28, 56, 90, and 120 days post-immunization (dpi) and evaluated for gB antibodies using an Aujeszky gB Antibody Test Kit (BioChek, Holland) following the manufacturer’s instructions. Briefly, 100 µL of 1:50 dilutions of serum samples were added to the appropriate wells and incubated at room temperature for 30 min. After four washes, plates were incubated with 100 µL of 1:5000 dilutions of sheep anti-mouse IgG/horseradish peroxidase for 30 min at room temperature. Plates were washed four times, 100 µL prepared substrate reagent was added to each well, then incubated for 15 min at room temperature in the dark. Finally, 50 µL stop solution was added to each well, and absorbance at 405 nm was measured within 30 min.

To evaluate the frequencies of the CD3⁺CD4⁺ and CD3⁺CD8⁺ subsets of T cells induced by vaccination, samples of approximately 1×10⁶ cells/well were prepared from pools of superficial cervical lymph nodes (LNs) excised from three mice/group. Cells were incubated with Fc blocking reagent (Miltenyi Biotec, USA) in PBS plus 1% FBS (HyClone, Thermo Scientific, USA) for 10 min at 4°C, then stained with the following monoclonal antibodies (mAbs): anti-CD3 FITC, anti-CD4 PerCP and anti-CD8 PE (BD Biosciences, USA). Fluorescence-activated cell sorting (FACS) controls (1×10⁶ cells) included unstained cells or only stained with anti-CD3 FITC, anti-CD4 PerCP, or anti-CD8 PE, and appropriate isotype controls.

To characterize Tfh cell responses, superficial cervical LN single-cell suspensions were prepared by homogenization and then incubated with Fc blocking reagent in PBS plus 1% FBS for 10 min at 4°C. Approximately 1×10⁶ LN cells were stained for 30 min at 4°C with the following mAbs: anti-CD4 PerCP, anti-CXCR5 PE, and anti-ICOS Alexa648 (BD Biosciences).

To characterize GC B cell responses, superficial cervical LN single-cell suspensions were prepared by homogenization and then incubated with Fc blocking reagent in PBS plus 1% FBS for 10 min at 4°C. Approximately 1×10⁶ LN cells were stained for 30 min at 4°C with the following mAbs: anti-B220 APC (Miltenyi Biotec), and anti-GL-7 PE and anti-CD38 PE-Cyanine7 (eBioscience, USA).

For the characterization of bone marrow (BM) plasma cells (PCs), BM single-cell suspensions were generated. Cell suspensions were incubated with Fc blocking reagent in PBS plus 1% FBS for 10 min at 4°C. Approximately 1×10⁶ cells were stained for 30 min at 4°C with the following mAbs: anti-B220 APC and anti-CD138 PE (BD Biosciences).

Cells were analyzed with a BD Accuri C6 (BD Biosciences). Data analyses were performed using FlowJo version 7.6.1 software.

The expression levels of eight genes (Bcl-6, Il-21, BCMA, Irf4, Bach2, Pax5, Bcl-2, Mcl-1) were measured by real-time RT-PCR following vaccination of the mice with LV, KV, KV-C-di-GMP and PBS (three mice/group). TRIzol-extracted RNA from lymphocytes (1×10⁶) from superficial cervical LNs was used for reverse transcriptase (RT)-PCR with a Prime ScriptTM II Strand cDNA Synthesis kit (Takara, Dalian, China) to the manufacturer’s protocol. Real-time RT-PCR was performed for each cDNA sample in duplicate using Bright Green 2× qPCR Master Mix in a Roche Light Cycler^® 480 (Roche, Basel, Switzerland) with the following thermal cycling program: an initial 10 min at 95°C, followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. The expression level of β-actin was used as the internal control, and relative gene expression was measured. Primer sequences are shown in Table 1.

To quantify relative levels of cytokines induced by vaccination, samples of approximately 1×10⁶ cells/well were prepared from pools of spleen tissue (28 dpi) excised from three mice/groups. Cells were stimulated with PRV (5×10⁵ TCID₅₀/mL), or medium, overnight at 37°C, in the presence of 5% CO₂. Supernatant samples were collected and evaluated for expression of four cytokines (interferon [IFN]-γ, interleukin [IL]-2, IL-4, IL-6) using commercially available ELISA kits (Angle Gene Technologies, Nanjing, China).

Statistical analysis was performed using GraphPad Prism version 5 (GraphPad Software, San Diego, CA, USA). Statistical analyses were performed by one-way analysis of variance followed by Tukey’s t-test and Student’s t-test. Results are expressed as the mean ± standard deviation. A P-value of < 0.05 was considered to be statistical significance; P-values of < 0.01 and < 0.001 indicated greater degrees of significance.

To evaluate the efficacy of c-di-GMP to serve as an immunopotentiator to PRV inactivated vaccine, BALB/c mice were immunized with KV vaccine with or without c-di-GMP and compared with mice immunized with LV or a PBS control. All mice tolerated the vaccine formulations with no adverse effects. To first assess the ability of c-di-GMP to induce T cell responses, subset of lymphocytes from LNs collected from all groups at 7, and 14 dpi were analyzed using FACS. At both time points, the percentages of CD3⁺CD4⁺ lymphocytes in the KV-c-di-GMP vaccine group were significantly higher than those receiving the LV, KV, or control vaccines (Figures 1A, C). By contrast, there were no significant differences between the vaccine groups in the percentages of CD3⁺CD8⁺ lymphocytes at either 7 or 14 dpi (Figures 1B, D). These results indicated that CD4+ and CD8+ T cell responses elicited by a PRV inactivated vaccine with c-di-GMP were higher and comparable, respectively, to responses elicited by a live and inactivated virus vaccination.

We next examined the capacity of KV with c-di-GMP to augment humoral responses. PRV-specific humoral responses were examined over time in sera from mice inoculated with the LV, KV, and KV-c-di-GMP vaccines or PBS control (Figure 2). As expected, LV-immunized mice possessed substantially higher antibody responses than mice receiving KV at all time points examined. The inclusion of c-di-GMP resulted in substantial augmentation of these responses. There were significant differences in the PRV-specific gB antibody response between the KV-c-di-GMP and KV groups at each sampling point and between the KV-c-di-GMP and LV groups at 28, 56, 90, and 120 dpi. Lower gB antibody responses were detected in the PBS control group at each sampling point. Collectively, we found that c-di-GMP was sufficient to raise antibody responses of a KV vaccine to levels equal to or exceeding those detected for LV-immunized mice.

Tfh cells play an important role in GC response and formation of PCs from the BM (16). To assess the capacity of c-di-GMP to modulate these cells, percentages of CXCR5⁺ICOS⁺CD4⁺ Tfh cells in LNs at 28 dpi were analyzed by FACS in each group (Figure 3A). Compared with the KV and LV vaccines, KV-c-di-GMP induced production of a significantly higher percentage of Tfh cells in mice (Figure 3B), supporting the immunopotentiator activity of c-di-GMP.

The transcriptional repressor B-cell lymphoma (BCL)-6 protein is known to instruct naive CD4⁺ T cells to commit to a Tfh fate. Tfh cells then signal B cells that recognize cognate antigens through the surface and secreted molecules, including CD40L, inducible costimulator (ICOS), and IL-21 (17, 18). To further examine the Tfh response induced by the KV-c-di-GMP vaccine, we quantified the expression levels of Bcl-6 and Il-21 in the lymphocytes of LNs using real-time RT-PCR. As shown in Figure 3C, Bcl-6 expression was significantly higher in the group of mice vaccinated with KV-c-di-GMP than in the group receiving KV or LV. The expression levels of Il-21 were significantly higher in the KV-c-di-GMP group compared with those in the KV group (P < 0.001), but there was no significant difference between the KV-c-di-GMP and LV groups (Figure 3D).

Because GCs are the sites responsible for the activation and maturation of B cells, we also analyzed the B cell response in terms of the percentage of B220⁺GL-7⁺CD38^- cells in the LNs at 28 dpi (Figure 4A). There was a significantly higher B cell response in the KV-c-di-GMP group than in the KV and LV groups (P < 0.001, P < 0.01; Figure 4B), further supporting c-di-GMP immunopotentiator capacity when adjuvanted to the KV vaccine.

Long-lived humoral immunity is manifested by the ability of BM PCs to survive for extended periods. FACS analysis of the differentiation of BM PCs revealed a significantly higher percentage of B220⁻CD138⁺ BM PCs in the KV-c-di-GMP group compared with the percentages in the KV and LV groups (P < 0.001, P < 0.001; Figure 5).

Transcription factors including BCMA, Pax5, Bach2, IRF4, Bcl-2, and Mcl-1 have recently been shown to play integral roles in developing mature B cells and the differentiation or survival of activated B cells (19, 20). Using real-time RT-PCR, we assessed the effects of the vaccines on relative mRNA expression levels of these six transcription factors in lymphocytes from LNs at 28 dpi (Figure 6). Compared with those in the PBS control group, the expression of BCMA and Irf4 were upregulated, while Pax5 and Bach2 were downregulated, in all three vaccine groups, with significant differences between the KV-c-di-GMP group and the KV and LV groups. Anti-apoptotic genes Bcl-2 and Mcl-1 were also significantly upregulated in the KV-c-di-GMP group compared with those in the KV and LV groups, indicating that inclusion of c-di-GMP to a KV vaccine was capable of supporting the production of transcription factors at or higher than those observed with an LV vaccination approach.

Enhancement of the cellular immune response can improve the ability of animals to ward off viral infection. We used ELISA to examine the vaccine-mediated induction of cytokines in spleen cells isolated at 28 dpi and re-stimulated with PRV in vitro (Figure 7). The highest levels of all four cytokines were observed in the KV-c-di-GMP group. Specifically, the levels of IFN-γ in the KV-c-di-GMP, KV, and LV groups were 326 ± 10.98 pg/mL, 201 ± 8.56 pg/mL, and 297 ± 10.62 pg/mL, respectively. The levels of IL-2 in the KV-c-di-GMP, KV, and LV groups were 63 ± 6.2 pg/mL, 24.6 ± 4.8 pg/mL, and 56.2 ± 5.7 pg/mL, respectively. The levels of IL-4 in the KV-c-di-GMP group (61.8 ± 5.97 pg/mL) were significantly higher than those in the KV group (31.6 ± 4.83 pg/mL), but not the LV group (55.3 ± 5.87 pg/mL). The levels of IL-6 in the KV-c-di-GMP, KV, and LV groups were 51.6 ± 4.87 pg/mL, 26.8 ± 4.32 pg/mL, and 39.0 ± 4.98 pg/mL, respectively. Collectively, these data support the efficacy of c-di-GMP to serve as an immunopotentiator to the KV vaccine to elicit Th1 and Th2 immune responses comparable to or exceeding those of an LV vaccine.