Chemical adjuvant treatment

For chemical adjuvant treatment, hair-removed lateral dorsal skin of mice was intradermally injected with 20 μL AddaVax (1:1 vol/vol mixed with phosphate buffered saline (PBS)), Alum (Alhydrogel, 160 μg aluminum hydroxide), MPL (25 μg), or MPL (25 μg)/Alum adjuvant (Alhydrogel, 100 μg aluminum hydroxide) (n = 3). AddaVax was prepared by 1:1 (vol/vol) mixing with PBS. Alum adjuvant was prepared by 4:1 (vol/vol) mixing with PBS. MPL (1 mg) was dissolved in 100 μL DMSO and then mixed with 300 μL endo-free H₂O. Before injection, MPL was mixed with PBS at 1:1 (vol/vol) ratio. MPL/Alum adjuvant was prepared by mixing MPL (2.5 mg/mL) with Alum at 1:1 (vol/vol) ratio to simulate the combinatorial AS04 adjuvant.⁴⁷