Application of red clover isoflavone extract as an adjuvant in mice

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.