Intranasal Liposomal Vaccine Formulation

Liposomal vaccines were prepared as per established methods^{11 (link),13 (link)}. Predetermined amounts of J8-palmitic acid, 3D(6-acyl) PHAD (PHAD; Avanti Polar Lipids Inc., USA) and phospholipids—dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol (CHOL) and 1 L-α-phosphatidylglycerol (PG) (Avanti Polar Lipids Inc.) were coated on to round bottom flasks to form a lipid thin film. The thin films were hydrated in 20 mM phosphate-buffered saline (PBS; pH 6) containing trehalose and Diphtheria Toxoid (DT). The resultant liposomal suspension was freeze-dried and vaccines were stored at 4 °C until use. The average particle size (nm) of a liposome was measured using a Nanosizer (Zetasizer Nano Series ZS, Malvern Instruments) as per established method^{11 (link)}. Homogenous size distribution was confirmed by a low polydispersity index (PDI) of 0.08 for J8-Lipo-DT-PHAD with an average size of 1987 nm (SD 104 nm) before freeze-drying and PDI of 0.16 with an average size of 5213 nm (SD 572.2) after reconstitution of freeze-dried liposomes in PBS. For administration, each vaccine vial was resuspended in PBS, and mice were immunized intranasally (i.n.) with a total volume of 20 μl (30 μg/mouse of J8-palmitic acid, 25 μg/mouse of PHAD and 30 μg/mouse of DT—10 μl/nare).