Efficacy of Rg1-Oil Adjuvant on Inducing Immune Responses against Bordetella bronchiseptica in Rabbits

Bordetella bronchiseptica (B. bronchiseptica) is an obligately aerobic, oxidase- and catalase-positive, nonfermentative Gram-negative coccobacillus. This study is aimed at examining the immune effects of Rg1, Rg1 plus oil, and other common adjuvants on inactivated B. bronchiseptica vaccine in rabbits. The mechanism underlying the adjuvant effect of Rg1 plus oil on the vaccine was also explored. Rg1 (100 μg) plus oil significantly improved the immune effect of B. bronchiseptica vaccine at both the humoral and cellular levels. Rg1-oil adjuvant increased the levels of IL-2 and IL-4 in rabbits after immunization. Rg1 (100 μg) plus oil also significantly increased TLR2 expression and downregulated NF-κB in splenocytes. Rg1-oil adjuvant may increase the levels of IL-2 and IL-4 via upregulating TLR2, thereby enhancing the immune effect of B. bronchiseptica vaccine. In conclusion, Rg1 plus oil could be used as a potential vaccine adjuvant for rabbit B. bronchiseptica vaccine.


Introduction
Bordetella bronchiseptica (B. bronchiseptica) is an obligately aerobic, oxidase-and catalase-positive, nonfermentative Gram-negative coccobacillus that was first identified in dogs with distemper disease in 1911 [1]. It causes infections in the respiratory tract of rabbits and acts as a precursor for secondary infection with Pasteurella multocida [2]. B. bronchiseptica infection in rabbits occurs all year round, but more commonly in autumn and spring. The nasal secretion from infected animals may contaminate air, food, and water, which facilitates the spread of the disease. B. bronchiseptica infection not only induces pustular pneumonia, bronchitis, and rhinitis in adult rabbits but also leads to acute death in young animals both before and after weaning. The rapid spread and difficulties in complete eradication of this disease often result in serious economic loss [3].
Vaccination is one of the most effective interventions to prevent B. bronchiseptica infection in rabbits. However, the development of adjuvants for B. bronchiseptica vaccine is still challenging. Currently available adjuvants, such as synthetic peptide vaccine and DNA recombinant vaccine, do not meet the demands of vaccines with low immunogenicity. Extensive research on the development of novel adjuvants has been carried out. However, these adjuvants have not been widely used due to poor safety performance. Significant advances have been made to enhance the potency of currently available vaccines by incorporating adjuvants into vaccine formulations. Besides the immune-stimulating effect, adjuvants also reduce the amount of antigens needed for vaccination, thus reducing the cost of production. However, the incorporation of adjuvants into the vaccine should be carefully evaluated to ensure that no adverse side effects occur and the immunogenic effect is stimulated [4]. Ginseng, the root of Panax ginseng C.A. Meyer (Araliaceae), is a herbal tonic used in traditional Chinese medicine for over 2000 years. The adjuvant effects of ginseng saponins (ginsenosides) on immune responses in rats, mice, pigs, and cattle have been widely reported [5].
Rg1 is a ginsenoside that exhibits adjuvant activity in immune responses [6,7]. In this work, we evaluated the immune effects of Rg1 and other common adjuvants on inactivated rabbit B. bronchiseptica vaccine and explored the adjuvant mechanism of Rg1. Our findings provided scientific support for the development of B. bronchiseptica vaccines.

Vaccine Preparation.
Oil-based adjuvants were emulsified for 90 min using an automatic fast-sample grinding instrument (JingXin Technology, Shanghai, China) with an emulsification index of 60 hertz. This procedure was repeated twice. The components of each vaccine are shown in Tables 1  and 2. 2.3. Animals and Study Design. This study was approved by the Ethics Committee of the Zhejiang Academy of Agricultural Sciences (ethics protocol no. 002762) and performed in accordance with the principles and guidelines of the Zhejiang Farm Animal Welfare Council of China. Five-week-old male New Zealand rabbits (weighing 1-1.5 kg) were obtained from the Zhejiang Animal Center and allowed to acclimate for one week before experiments.

Experiment A.
Rabbits were randomly assigned into eight groups (n = 15 per group) and then immunized by subcutaneous injection of 1 mL of B. bronchiseptica (1:5 × 10 10 CFU) alone, B. bronchiseptica (1:5 × 10 10 CFU) dissolved in PBS containing 400 μg Rg1, 400 μg Rg1 plus 750 μL oil, 750 μL oil alone, 50 μg QA, or 200 μg Alum into the neck on Day 1 (Table 1). Blood samples were collected from the ear vein of each animal before and at 5, 10, 15, 21, and 35 days after injection and kept at 4°C. The body weight of each animal was measured before and after immunization on Days 0, 5, 10, 15, and 35. All rabbits were monitored for adverse drug reactions after immunization.

Experiment B.
Rabbits were randomly divided into six groups (n = 8 per group) and then subcutaneously injected with 1 mL of B. bronchiseptica (1:5 × 10 10 CFU) alone, B. bronchiseptica (1:5 × 10 10 CFU) dissolved in PBS containing 100, 200, or 400 μg Rg1 plus 750 μL oil, or 750 μL oil alone into the neck on Day 1 ( Table 2). Blood samples were     (Table 3) were designed according to rabbit genome sequences shown in GenBank. ACTB was used as an internal control. The 2 -ΔΔCT method was used for relative quantification. Each sample was analyzed at least twice.
2.9. Western Blot. Frozen spleen tissue samples were homogenized using RIPA buffer and then centrifuged at 12000 rpm for 10 min at 4°C to obtain supernatants. Protein concentration was determined by the BCA method.
Samples were added with 5x SDS loading buffer, boiled at 100°C for 5 min, separated on SDS-PAGE, and finally transfected to the PVDF membrane. After blocked with 5% milk at room temperature for 1 h, the membrane was incubated with anti-TLR2 antibody  Journal of Immunology Research and then dropped on the sealing film. The results were analyzed using a gel imaging system.

Immunofluorescence Analysis of NF-κB.
HeLa cells were cultured in 10% serum medium in a 37°C/CO 2 incubator until 70% confluence. Then, cells were digested by trypsin, collected, and evenly plated into a 24-well plate covered with aseptic climbing tablets. Rg1 (1 mg) was dissolved in 1 mL PBS, which was then diluted in gradient with culture medium. Cells were incubated with medium containing with or without Rg1 at 10, 20, 40, 100, 200, and 400 μg/mL for 2 h. After treatment, cells were fixed with 4% paraformaldehyde for 20 min, followed by 15 min incubation with 0.5% Triton X-100 (prepared by 1x PBS) at room temperature. After washing, 50 μL of diluted ProteinFind® anti-NF-κB p65 mouse monoclonal antibody (1 : 100, Cat No. HA106) was added to each cell climbing tablet and incubated at 4°C overnight. Subsequently, climbing tablets were gently washed with 1x PBST for 5 min and incubated with a fluorescent Pro-teinFind® secondary anti-rabbit IgG (H+L) antibody conjugated to AF488 (1 : 100, Cat No. HS131) at 37°C for 1 h. After three washes with 1x PBST, samples were incubated with 40 μL of Hoechst 33342 for 15 min to stain the nuclei. Then, the cell climbing sheet was sealed with sealing liquid and cells were photographed under a fluorescence microscope.

Statistical
Analysis. Data were shown as the mean ± SD. The differences among groups were compared using Tukey's HSD test and one-way ANOVA. A P value of <0.05 was considered statistically significant.

Measurements of the B. bronchiseptica-Specific Serum IgG
Level and Antibody Agglutination. In Experiment A, the serum levels of B. bronchiseptica-specific IgG in rabbits after immunization were monitored over time (Figure 1) (S-OD450nm). The IgG concentration started to increase at 5 days postinjection in all groups. From Days 10-21, the serum IgG level in rabbits immunized with B. bronchiseptica dissolved in Rg1-oil adjuvant was significantly increased com-pared to that in other adjuvant groups (P < 0:05). On Days 10 and 15, the serum IgG concentration in the oil group was significantly higher than that in other adjuvant groups (P < 0:05), except the Rg1-oil group. Compared to other adjuvant groups, the antibody agglutination in animals immunized with B. bronchiseptica was significantly enhanced by Rg1-oil adjuvant and oil alone from Days 10 to 35 (P < 0:05). No significant difference was observed between the Rg1-oil and oil groups (Figure 1) (S-Bb antibody agglutination).
In Experiment B, the serum levels of B. bronchisepticaspecific IgG in rabbits after immunization were monitored over time (Figure 2) (S-IgG). Compared to other adjuvant groups, the serum IgG level in rabbits immunized with B. bronchiseptica was effectively increased by Rg1 (100, 200, and 400 μg) plus oil and oil alone from Days 15 to 30 (P < 0:05).  3.5. RT-PCR and Western Blot. Total RNA and protein were extracted from spleen tissue samples from rabbits in Experiment B at 30 days postimmunization. The mRNA expression of NF-κB2 in the oil group was significantly higher compared to that in other groups (P < 0:05) on Day 30 after immunization ( Figure 5) (S-NF-κB RT-PCR). The protein levels of NF-κB2 in the Rg1 (100 μg) plus oil and antigen groups were significantly higher compared to those in others on Day 30  Figure 5(a)). The Rg1-oil (100 μg) group showed significantly increased TLR2 expression on Day 30 compared with other groups (P < 0:05, Figure 5(b)) (S-TLR2 RT-PCR). The protein level of TLR2 in the Rg1-oil (100, 200, and 400 μg) group was also higher compared to that in other groups on Day 30 ( Figure 5(c)).

Differential Blood Cell Count. In Experiment
3.6. Immunofluorescence Analysis of NF-κB. The expression of NF-κB in HeLa cells was dose-dependently increased in the presence of Rg1 (10-200 μg), while decreased when exposed to Rg1 at 400 μg ( Figure 6). The expression of Hoechst 33342 showed the same change when compared with the immunofluorescence staining of NF-κB ( Figure 6).  Journal of Immunology Research

Discussion
The rapid spread of B. bronchiseptica infection often leads to huge economic loss [8]. The application of attenuated B. bronchiseptica vaccines is limited due to safety issues during storage and transportation. Inactivated vaccines are safe, but the immunogenicity of B. bronchiseptica antigens is low [8]. Therefore, it is urgent to find an effective vaccine adjuvant for B. bronchiseptica vaccine. Since the original report by Espinet et al. [9] showing that saponins can be used as a vaccine adjuvant, considerable efforts have been expended to evaluate the adjuvant activity of saponins. On the one hand, saponin-based adjuvant stimulates cell-mediated immune responses and enhances antibody production at low doses. On the other hand, undesirable side effects caused by some saponins limited their application as vaccine adjuvants [10,11]. Panax ginseng is a tonic medicine commonly used in Asian countries. Rg1 extracted from ginseng shows low hemolytic activity [12] and high immunomodulating activity [6,13] and therefore could be used as an adjuvant for veterinary vaccines. The syringe extrusion method has a good background for immunization [14]. The water-in-oil emulsion is prepared using syringe extrusion, vortex, or high-speed  7 Journal of Immunology Research homogenization for laboratory and clinical peptide-based vaccination. In this study, we showed a synergistic effect between Rg1 and oil, consistent with previous studies showing that a mixture of saponins and oil improved the immune effect of vaccines [15,16]. We also evaluated the effects of other adjuvants on inactivated B. bronchiseptica vaccine. The results indicated that ELISA was more sensitive than the agglutination test. The serum antibody level began to increase in all groups of rabbits since Day 5 postimmunization. The Rg1 (400 μg) plus oil group showed a significantly increased antibody level and antibody titer compared to other groups, indicating the synergistic effect of Rg1-oil adjuvant on boosting humoral responses in rabbits. Moreover, the cellular immune test showed that Rg1, Rg1-oil, oil, and QA groups had a higher number of WBC compared to the aluminum glue and control groups since Day 10 postimmunization, but no significant difference was observed among Rg1, Rg1-oil, oil, and QA groups. At 15 and 35 days postimmunization, the number of WBC in the Rg1-oil group was significantly higher than that in other groups, suggesting that Rg1-oil adjuvant significantly improved cellular immune responses in rabbits. At 21 days postimmunization, the Rg1-oil group showed a significantly higher number of SCC compared to other groups. On Day 35 after immunization, the Rg1-oil group showed significantly more SCC than other groups (except the Rg1 group). Previous evidence demonstrated that Rg1 increased the proportion of T helper cells among all T cells and promoted the expression of IL-4 and IL-2 in murine splenocytes [17]. In Experiment B, the levels of IL-4 and IL-2 in the Rg1 (100 μg) plus oil group were significantly higher than those in other groups, while no significant difference was observed between the oil group and other groups, indicating that Rg1 in Rg1-oil adjuvant promoted the production of IL-4 and IL-2 in immunized rabbits.
Rg1 usually exerts an inhibitory effect on NF-κB in inflammation. For example, Rg1 extracted from white ginseng plays an anti-inflammatory role by reducing the expression of NF-κB [18]. It was also found that Rg1 effectively suppressed allergic airway inflammation of asthma partly through the TNF-α/NF-κB pathway [19]. Our results showed that the expression of NF-κB in the Rg1 (100-400 μg) plus oil group was significantly lower than that in the oil group, implying that Rg1-oil adjuvant downregulated NF-κB in noninflammatory condition. The in vitro study demonstrated that Rg1 promoted the expression of NF-κB in HeLa cells at doses between 10 μg and 200 μg but inhibited NF-κB expression at 400 μg, suggesting that Rg1 had an inhibitory effect on HeLa cells. These findings were also consistent with the RT-PCR results, which showed the splenic expression of NF-κB in the Rg1 (100-400 μg) plus oil group. TLRs are key transmembrane pattern recognition receptors that play an important role in initiating immune responses against invading microbial pathogens. Our data showed that Rg1 (100 μg) plus oil significantly increased the expression of TLR2 in splenocytes, which was consistent with a previous report [20]. It has also been shown that active components of Panax ginseng possess a putative TLR ligand in different cell models [21,22].

Conclusion
Rg1 (100 μg) plus oil significantly improved the immune effect of rabbit B. bronchiseptica vaccine at both the humoral and cellular levels. Rg1-oil adjuvant increased the production of IL-2 and IL-4 in rabbits after immunization. Rg1 (100 μg) plus oil significantly upregulated TLR2 expression but decreased the level of NF-κB in splenocytes. Rg1-oil adjuvant may increase the levels of IL-2 and IL-4 by upregulating TLR2, thus enhancing the immune effect of B. bronchiseptica vaccine. Thus, Rg1-oil adjuvant could be used as a potential vaccine adjuvant for rabbit B. bronchiseptica vaccine.

Data Availability
Data could be found in supplemental files.

Conflicts of Interest
The authors declare no conflict of interest.