Application of red clover isoflavone extract as an adjuvant in mice

Abstract

In the present study, the safety of red clover isoflavone extract (RCIE) and its potential adjuvant effects on the cellular and humoral immune responses to ovalbumin (OVA) were evaluated using an ICR mouse model. On day 1, the mice were first subcutaneously immunized with 100 µg OVA, 100 µg OVA + 200 µg aluminum hydroxide gel (alum) or OVA + 50, 100 or 200 µg RCIE (RCIE + OVA), following which booster immunization was performed on day 15. After 2 weeks, the stimulation of splenocyte proliferation and levels of serum antibodies were measured. No notable stress responses were observed after the initial and booster immunization. Splenocyte proliferation was significantly increased in mice immunized with OVA + 100 µg RCIE (P<0.01). The levels of IgG, IgG1 and IgG2a antibodies in serum were also significantly increased in OVA + RCIE groups compared with the OVA control group (P<0.05). In the OVA + RCIE groups, serum levels of interleukin (IL)-2, interferon-γ (IFN-γ) and IL-10 were increased, and the mRNA expression levels of IL-2, IFN-γ, IL-4, IL-10, T-bet and GATA-3 were also significantly increased compared with the OVA control group (P<0.05) in splenocytes. In addition, as an adjuvant, RCIE significantly increased the survival rates of mice inoculated with an E. coli vaccine and enhanced the early immune protection against pathogenic E. coli. In conclusion, these findings suggest that RCIE can be used as a safe vaccine adjuvant and supports its use in clinical applications.

Keywords: adjuvants; antibody; ovalbumin; red clover isoflavone extract.

Figure 1.

Effect of OVA, LPS or ConA stimulation on the cell viability of splenocytes isolated from mice immunized with OVA only, OVA + RCIE or OVA + alum. Values are presented as mean ± SEM (n=10). *P<0.05 and **P<0.01 vs. OVA. OVA, ovalbumin; LPS, lipopolysaccharide; ConA, concanavalin A; alum, aluminum hydroxide gel; RCIE, red clover isoflavone extract.

Figure 2.

Effect of RCIE or alum adjuvant on the serum levels of OVA-specific IgG, IgG1 and IgG2a antibodies in OVA-immunized mice. Values are presented as mean ± SEM (n=10). *P<0.05 and **P<0.01 vs. OVA. OVA, ovalbumin; alum, aluminum hydroxide gel; RCIE, red clover isoflavone extract; IgG, immunoglobulin G.

Figure 3.

Effect of RCIE or alum adjuvant on the serum levels of Th1 or Th2 cytokines in OVA-immunized mice. Values are presented as mean ± SEM (n=10). *P<0.05 vs. OVA. IL, interleukin; IFN-γ, interferon-γ; TNF-α; tumor necrosis factor-α; OVA, ovalbumin; alum, aluminum hydroxide gel; RCIE, red clover isoflavone extract; Th, T helper.

Figure 4.

Effect of RCIE or alum adjuvant on CD4⁺ and CD8⁺ T cell populations in the peripheral blood of OVA-immunized mice. Representative flow cytometry dot plots showing the population of CD4⁺ and CD8⁺ T cells in the peripheral blood from mice in the (A) Saline, (B) OVA, (C) OVA + alum, (D) OVA + 50 µg RCIE, (E) OVA + 100 µg RCIE and (F) OVA + 200 µg RCIE groups. (G) Quantified flow cytometry data. Values are presented as mean ± SEM (n=10). *P<0.05 vs. OVA. OVA, ovalbumin; alum, aluminum hydroxide gel; RCIE, red clover isoflavone extract; PE, phycoerythrin.

Figure 5.

Effect of RCIE or alum adjuvant during immunization with inactivated E. Coli on the survival in mice challenged with pathogenic E. coli. Mice were challenged with pathogenic porcine E. coli 3 days following inoculation with the E. coli vaccine alone or in combination with alum or RCIE, following which survival was monitored for 48 h. Alum, aluminum hydroxide gel; RCIE, red clover isoflavone extract.