Abstract: Objective To study the effects of MF59 (an oil-in-water emulsion composed of small droplets of squalene surrounded by a monolayer of nonionic detergents (Span85 and Tween 80)) as adjuvant on immune responses to the heat-killed Bacillus Calmette-Guérin (hBCG) to prove MF59 enhancing the cellular immune responses to hBCG. Methods BALB/c mice were divided into seven groups, which were immunized with MF59 (group A), low doses of hBCG (0.025 mg/ml） (group B), middle doses of hBCG (0.25 mg/ml) (group C), high doses of hBCG (2.5 mg/ml) (group D), MF59+ low doses of hBCG (group E), MF59+ middle doses of hBCG (group F) and MF59+ high doses of hBCG (group G) for three times at intervals of two weeks, respectively. The mice were sacrificed two weeks after the last immunization. The splenic lymphocytes and peritoneal macrophages were isolated and cultured with BCG-PPD. Sandwich ELISA and ELISPOT assay were used to detect BCG-PPD-specific cytokines in culture supernatants and the number of IFN-γ, IL-2, IL-4 producing spot forming cells (SFCs), respectively. One-way analysis of variance with least-significant-difference (LSD) comparisons was used to test differences among the levels of cytokines and the number of SFCs. P values of <0.05 were considered statistically significant. Results The number of IFN-γ, IL-2, IL-4 producing SFCs in group G (151.3±66.6, 247.8±58.0, and 65.8±24.6) was higher than that in other groups (the biggest means of IFN-γ, IL-2, IL-4 producing SFCs were (30.4±13.0), (37.1±10.8), and (16.4±9.7), respectively) (t=3.2, P=0.007 for IFN-γ producing SFCs；t=3.6, P=0.003 for IL-2 producing SFCs；t=3.0, P=0.01 for IL-4 producing SFCs). The levels of IL-1β in the supernatants from BCG-PPD-stimulated peritoneal macrophages were higher in groups E (663.3±177.5 pg/ml), F (813.3±193.4 pg/ml), and G (742.3±316.1 pg/ml) than that in groups A (104.7±65.8 pg/ml), B (82.9±54.8 pg/ml) and C (66.5±53.5 pg/ml) (group E vs A, t=2.9, P=0.01; group F vs A, t=3.5, P=0.004; group G vs A, t=2.5, P=0.031; group E vs B, t=3.1, P=0.007; group F vs B, t=3.6, P=0.003; group G vs B, t=2.6, P=0.024; group E vs C, t=3.0, P=0.01；group F vs C, t=3.5, P=0.004；group G vs C, t=2.5, P=0.031). The levels of IL-12 were lower in groups A (36.3±20.5 pg/ml), C (94.5±20.6 pg/ml) and G (128.2±54.6 pg/ml) than that in groups E (545.7±97.2 pg/ml) and F (665.5±295.4 pg/ml) (group A vs E, t=－5.1, P=0.000; group C vs E, t=－4.5, P=0.000; group G vs E, t=－3.7, P=0.002; group A vs F, t=－4.4, P=0.001; group C vs F, t=－3.7, P=0.002; group G vs F, t=－2.9, P=0.012). Conclusion MF59 combined with high dose of hBCG adjuvant can induce BCG-PPD specific IFN-γ, IL-2, and IL-4 secretion from splenic lymphocytes, and MF59 combined with low or middle doses of hBCG adjuvant can increase the levels of IL-1β and IL-12 secreted from the macrophage.

Key words: Polysorbates, Squalene, BCG vaccine, Adjuvants, immunologic, Cytokines