A Solution with Ginseng Saponins and Selenium as Vaccine Diluent to Increase Th1/Th2 Immune Responses in Mice

Abstract

Pseudorabies is an important infectious disease of swine, and immunization using attenuated pseudorabies virus (aPrV) vaccine is a routine practice to control this disease in swine herds. This study was to evaluate a saline solution containing ginseng stem-leaf saponins (GSLS) and sodium selenite (Se) as a vaccine adjuvant for its enhancement of immune response to aPrV vaccine. The results showed that aPrV vaccine diluted with saline containing GSLS-Se (aP-GSe) induced significantly higher immune responses than that of the vaccine diluted with saline alone (aP-S). The aP-GSe promoted higher production of gB-specific IgG, IgG1, and IgG2a, neutralizing antibody titers, secretion of Th1-type (IFN-γ, IL-2, IL-12), and Th2-type (IL-4, IL-6, IL-10) cytokines, and upregulated the T-bet/GATA-3 mRNA expression when compared to aP-S. In addition, cytolytic activity of NK cells, lymphocyte proliferation, and CD4⁺/CD8⁺ ratio was also significantly increased by aP-GSe. More importantly, aP-GSe conferred a much higher resistance of mice to a field virulent pseudorabies virus (fPrV) challenge. As the present study was conducted in mice, further study is required to evaluate the aP-GSe to improve the vaccination against PrV in swine.

Figure 1

Effects of GSLS and/or Se on PrV gB-specific IgG response. Mice (n = 6/group) received twice i.m. injections of an aPrV vaccine (1000 TCID₅₀) in saline or saline with Se (2 μg), GSLS (6 μg), or GSLS (6 μg) mixed with Se (2 μg) at two weeks apart. Sera were collected 1 week after the booster for analysis of PrV gB-specific IgG by a blocking ELISA. Data are expressed as the mean ± SE. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, among the groups as indicated by one-way ANOVA with LSD test.

Figure 2

Effect of aP-GSe on antibody responses and survival of mice challenged with fPrV. (1) Mice (n = 6/group) received twice i.m. injection of aP-GSe or aP-S at two weeks apart. Mice injected with saline served as control. Sera were collected 1, 2, 4, 6, 8, and 10 weeks after the boost injection for analysis of PrV gB-specific IgG, IgG1, and IgG2a by ELISA (a–c). Serum samples collected 2 weeks after the booster immunization were analyzed for PrV-specific neutralizing antibody titers (d). Data are expressed as the mean ± SE. (2) Mice (n = 10/group) received twice i.m. injections of saline or aP-S, GSLS-Se, or aP-GSe at two weeks apart and challenged 2 weeks after boost immunization by intraperitoneal injection of fPrV at a lethal dose of 5 × 10⁵ TCID₅₀. The animals were monitored during 240 h and data are expressed as percent survival (e). ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗∗p < 0.0001 vs. aP-S group as indicated by two-way ANOVA with Tukey's multiple comparisons test (a–c), Student's t-test (d) or log-rank test (e).

Figure 3

Lymphocyte proliferative response to Con A (a), LPS (b), and PrV antigen (c). Mice (n = 6/group) received twice i.m. injections of aP-GSe or aP-S at two weeks apart. Mice injected with saline were served as a control. Splenocytes were prepared 10 weeks after boost immunization and cultured with Con A, LPS, or PrV antigen for 48 h for analysis of lymphocyte proliferation using an MTT method and a stimulation index (SI) was calculated. Data are expressed as the mean ± SE. ^∗∗∗∗p < 0.0001 vs. aP-S group as indicated by one-way ANOVA with the LSD test.

Figure 4

Quantification of splenic CD3⁺CD4⁺ and CD3⁺CD8⁺ T cell subpopulations and the CD3⁺CD4⁺/CD3⁺CD8⁺ratio. Mice (n = 6/group) received twice i.m. injection of aP-GSe or aP-S at two weeks apart and animals injected with saline served as a control. Splenocytes were prepared 2 weeks after boost immunization for analysis of CD3⁺CD4⁺ and CD3⁺CD8⁺ T cell subpopulations by flow cytometry. Data are expressed as the mean ± SE. ^∗∗p < 0.01 vs. aP-S group as indicated by one-way ANOVA with the LSD test.

Figure 5

Production of IFN-γ, IL-4, IL-2, IL-6, IL-12, and IL-10 by splenocytes. Mice (n = 6/group) received twice i.m. injection of aP-GSe or aP-S at two weeks apart. Mice injected with saline served as control. Splenocytes were prepared 2 weeks after boost immunization and cultured with PrV antigen for 48 h. The supernatants were harvested for analysis of (a) Th1-type cytokines (IFN-γ, IL-2, IL-12) and (b) Th2-type cytokines (IL-4, IL-6, IL-10) by ELISA. Data are expressed as the mean ± SE. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001 vs. aP-S group as indicated by one-way ANOVA with the LSD test.

Figure 6

T-bet (a) and GATA-3 (b) mRNA expressed by splenocytes. Mice (n = 6/group) received twice i.m. injection of aP-GSe or aP-S at two weeks apart and mice injected with saline served as a control. Splenocytes were prepared 2 weeks after boost immunization and cultured with PrV antigen for 15 h. Fold change of relative mRNA expression of T-bet and GATA-3 was analyzed by RT-qPCR. Data are expressed as the mean ± SE. ^∗∗p < 0.01, ^∗∗∗∗p < 0.0001 vs. aP-S group as indicated by one-way ANOVA with the LSD test.

Figure 7

Effect of administration routes of GSLS-Se on IgG response. (a) Mice (n = 6/group) were i.m. injected with saline with or without GSLS-Se on their right hind limb (R) before immunization on the left hind limb (L). (b) Mice (n = 6/group) were i.m. injected with saline with or without GSLS-Se for 3 days before immunization. Immunization was i.m. administered with aP-GSe or aP-S at two weeks apart. Control animals were not immunized but injected with saline. Sera were collected 2 weeks after the booster for analysis of PrV gB-specific IgG. Data are expressed as the mean ± SE. ^∗p < 0.05, among the groups as indicated by one-way ANOVA with LSD test.

Figure 8

Cytotoxicity of NK cells (a) and IFN-γ response (b). Mice (n = 6/group) received i.m. injection of aP-GSe or aP-S, or just injection of saline with or without GSLS-Se. In twenty-four-hour postimmunization, splenocytes were isolated for analysis of cytolytic activity to YAC-1 cells by an MTT method, and blood samples were collected for analysis of IFN-γ by an ELISA. Data are expressed as the min to max (a) or mean ± SE (b). ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗∗p < 0.0001 vs. aP-S group or control as indicated by two-way ANOVA with Tukey's multiple comparisons test (a) or one-way ANOVA with LSD test (b).