Monophosphoryl lipid a

SMNP adjuvant was prepared as described (77 ). Cholesterol (20 mg/ml; Avanti Polar Lipids, catalog no. 700000) and DPPC (1,2-dipalmitoyl-sn-glycero-3-phsophocholine: 20 mg/ml; Avanti Polar Lipids, catalog no. 850355) were dissolved in Milli-Q water containing 20% w/v MEGA-10 (Sigma-Aldrich, D6277) detergent at 60°C. Monophosphoryl lipid A (10 mg/ml; Avanti Polar Lipids, catalog no. 699800P) was dissolved in 20% w/v MEGA-10 at 37°C. Quil-A saponin (InvivoGen; vac-quil) was dissolved in Milli-Q water at a final concentration of 100 mg/ml at 37°C. All components were then mixed at a mass ratio of 10:10:2.5:1 [Quil-A:cholesterol:DPPC:MPLA (Monophosphoryl lipid A)] at 60°C followed by dilution with phosphate-buffered saline (PBS) to a final concentration of cholesterol (1 mg/ml). The solution was allowed to equilibrate over night at room temperature, followed by dialysis against PBS using a 10k molecular weight cutoff (MWCO) membrane. The adjuvant solution was then sterile filtered, concentrated using 50k MWCO centricon spin filters, and further purified by fast protein liquid chromatography using a Sephacryl S-500 HR size exclusion column. Cited concentrations of SMNP refer to the concentration of saponin injected.

Liposomal-monophosphoryl lipid A (LMPLA) were prepared as described.^{65 (link)} Briefly, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, cholesterol, and 1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (Avanti Polar Lipids, Alabaster, AL) were dissolved in freshly distilled chloroform stabilized with 0.75% ethanol at a molar ratio of (9:7.5:1) along with monophosphoryl lipid A (Sigma-Aldrich, St. Louis, MO) for a final concentration of 100 μg/mL. Liposomes were dried under nitrogen stream and placed O/N in a desiccator containing silica-gel desiccant beads (Thermo Fisher Scientific). Liposomes were then resuspended for a final concentration of 10 μg/200 μL of sterile PBS, pH 7.4, containing 20 μg Galα3LN-HSA (3-atom spacer, NGP2334, Dextra Laboratories, Reading, UK) or 20 μg recombinant HSA (Thermo Fisher Scientific), then filtered using a 0.2-μm disc filter (Thermo Fisher Scientific). For immunizations in the absence of LMPLA, a stock solution of 1 mg/mL Galα3LN-HSA in sterile, deionized milli-Q water was prepared and kept in 200-μL aliquots at −20 °C until use.