IFN-β-inducible activity of γ-PGA was further investigated in mice to ascertain the prophylactic antiviral efficacy of orally delivered γ-PGA. Initial experiments revealed production of both IFN-α and IFN-β at levels below the detection limits of ELISA at 2, 4, 8, 12, and 24 h post-administration of γ-PGA (50 mg/kg body weight per day) to BALB/c mice (Supplementary Fig. 4a,b). These results suggest weak IFN-inducing activity or inefficient delivery of the 2,000-kDa γ-PGA in an intact conformation to the ileum (Fig. 3b,c). When γ-PGA (50 mg/kg body weight) was administered to mice, once a day for five days, serum IFN-β level increased starting from day 3 (Fig. 4a). Induction of IFN-α was negligible (Fig. 4b and Supplementary Fig. 4b). Production of IFN-β was induced by 2,000-kDa γ-PGA but not by 500-kDa γ-PGA (Supplementary Fig. 4c,d), suggesting the importance of its structural entity in activating TLR4 signaling. Furthermore, IFN-β levels (mean values) neither decreased nor increased from day 3 to day 5, indicating that multiple administrations of γ-PGA do not induce TLR4 tolerance to blunt the responsiveness to repetitive challenges with γ-PGA.Figure 4Analysis of cytokines induced by the oral administration of γ-PGA. BALB/c mice (6 weeks old, n = 10 per group) were given 2,000-kDa γ-PGA (50 mg/kg of body weight) in 100 µl saline once daily for five days. Blood samples were collected before (day 0) or 4 h after administering γ-PGA on day 1, 3, or 5 to measure titers of IFN-β (a), IFN-α (b), and other cytokines (c) by ELISA. In all panels, data are mean ± s.d. **P < 0.01; ***P < 0.001; n.s., not significant; by two-tailed unpaired Student’s t-test. LOD, limit of detection.