Shark squalene

The nanoemulsions were produced as previously described^{23 (link)} and were measured for their physiochemical properties. A mixture of 5% triterpene oil (v/v), 0.5% Tween-80 (w/v), and 0.5% Span-85 (w/v) suspended in a 20 mM citrate buffer (pH 6.0) was passed through an Avestin EmulsiFlex-C5 high pressure homogenizer at 12,000 PSI at least eight times. The nanoemulsion was then passed through a 0.2 µm filter and the particle size was measured using a Malvern Nano Zetasizer Dynamic Light Scattering (DLS) spectrophotometer. Shark squalene was obtained from Sigma-Aldrich and Addavax (a squalene nanoemulsion commercially available adjuvant) from InvivoGen. The adjuvants were combined 1:1 with the quadrivalent influenza antigen that had been diluted with 2 × PBS to create the adjuvanted vaccine. The influenza antigen used was the 2018–2019 Afluria vaccine (Seqirus), a non-adjuvanted quadrivalent influenza vaccine (H1N1A, H3N2, B/Maryland/15/2016, and B/Phuket/3073/2013-BVR-1B). The antigen-only control was formulated by mixing 20 mM citrate buffer (pH 6.0) with an equal amount of Afluria antigen. The final concentration of total antigens in each vaccine was 0.36 µg (0.09 µg per individual antigen). The Pierce Chromogenic Endotoxin Quant kit (Thermo Fisher) was used for endotoxin detection.

Methyl methacrylate (MMA, 99%), ethyl methacrylate (EMA, 99%), butyl methacrylate (BMA, 99%), lauryl methacrylate (LMA, 96%), stearyl methacrylate (SMA, 89.5%), methyl acrylate (MA, 99%), iron(II) bromide (FeBr₂, 98%), 2,2-azobis(isobutyronitrile) (AIBN, 98%), glyoxal solution (40 wt% in H₂O), 2,6-diisopropylaniline (97%), 2,4,6-trimethylaniline (98%), and shark squalene were purchased from Sigma-Aldrich (Gillingham, UK). Cobalt(II) bromide (CoBr₂, 98%) and dimethyl glyoxime (DMG, 99%) were purchased from Acros Organics (Verona, Italy). Diphenyl glyoxime (DPG, 99%) was purchased from Alfa Aesar (Heysham, UK). Bis[(difluoroboryl) diphenylglyoximato]cobalt(II) (PhCoBF) was supplied by DuPont (Wilmington, DE, USA). Technical-grade toluene, propan-2-ol, and diethyl ether were obtained from campus departmental stores. Amaranth squalene was purchased from Wilshire Technologies (Princeton, NJ, USA). Napa Valley Naturals (Stonewall Kitchen, York, ME, USA) grapeseed oil was purchased from a local grocery store. 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was obtained from Lipoid (Ludwigshafen, Germany). Poloxamer 188 and glycerol were obtained from Spectrum Chemical (New Brunswick, NJ, USA). Buffer components were obtained from J.T.Baker (Phillipsburg, NJ, USA) or Fluka (Charlotte, NC, USA). All materials were used as supplied without further purification.