The liposomes DOPE and CHEMS were obtained from Sigma-Aldrich Co. Liposome complexes consisting of peptide (50 µg) and CpG-DNA (50 µg) co-encapsulated with DOPE:CHEMS (at a 1:1 ratio) were formulated as reported previously.31 (link) Briefly, DOPE and CHEMS were mixed in 10% ethanol at a molar ratio of 1:1, evaporated with nitrogen gas to produce a solvent-free lipid film, and resuspended in a mixture containing equal volumes of water-soluble MB-ODN 4531(O) (50 µg) and peptide (50 µg), followed by vigorous stirring at room temperature for 30 minutes. After adjusting the pH to 7.0, the peptide and CpG-DNA co-encapsulated with the DOPE:CHEMS [Lipoplex(O)] complex were sonicated lightly for 30 seconds with a sonicator (Sonifier 450; Branson Ultrasonics, Danbury, CT, USA). The complex was filtered with a 0.22 µm filter and freeze-thawed three times with liquid nitrogen.