Plasmids encoding the extracellular domain of HA (Influenza A/New Calendonia/20/1999) C-terminally fused to the trimeric Foldon of T4 fibritin and a hexahistidine affinity tag proteins were transfected into the human embryonic kidney cell line 293F and isolated from expression supernatants 96 h post-transfection. HA trimers, either wildtype (WT) or a mutant HA that masks the RBS by glycosylation at residue 190 (dRBS HA), were purified as previously described. The trimeric assembly of these viral proteins was confirmed by size exclusion FPLC on a prepacked Superdex 200 10/300 column (Life Technologies). Virosome construction was as described. Briefly, 1 g 1,2-dioleoyl-sn-glycero-3-phosphocholine: 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl) iminodiacetic acid)] succinyl [DOPC: DGS-NTA(Ni)] (Avanti Polar Lipids Inc.) in a 1:1 molar ratio was evaporated under a stream of nitrogen for 1 h. The lipid film was then rehydrated in 1 mL liposome buffer [50 mM HEPES, 150 mM NaCl, pH 7.25 (HBS)] and shaken for 40 min, above the Tm of the lipid mixture. The suspension was then subjected to 10 freeze-thaw cycles and then extruded 21 times through a 100 nm polycarbonate membrane using a mini-extruder (Avanti Polar Lipids Inc.). HA virosomes/proteoliposomes were produced by incubating the resultant liposomes with either His-tagged HA or His-tagged DRBS HA (HA trimer-lipid molar ratio of 1/900). To isolate HA-virosomes, the sample was adjusted to 15% iodixanol (in 1.25 mL HBS) and overlaid with 1.75, 0.5, and 0.5 mL of 10%, 2.5%, and 0% iodixanol in HBS, respectively. Samples were then centrifuged at 200,000 g in a TH660 rotor (Sorvall) for 2 h.