Lipid Nanoparticles, Adjuvants & Vaccine Antigens

Lipid nanoparticle (LNP), alone or encapsulating TLR9-ODN (LNP in TLR9-ODN), were prepared by rapid precipitation process as previously described³³. The lipid components of LNP adjuvants comprised an asymmetric ionizable amino lipid (Lipid D,[47]), distearoylphosphatidylcholine (DSPC), cholesterol, and poly(ethylene glycol)2000-dimyristoylglycerol (PEG2000-DMG). LNP and LNP’s Reduced Cationic Lipid version (RCL), were prepared at varying molar ratios of the constituent lipids. LNP was prepared at a molar ratio of 58:30:10:2 ionizable amino lipid, cholesterol, DSPC and PEG-lipid¹⁶, while RCL was generated at a molar ratio of 33:40:25:2, respectively. For LNP encapsulating TLR9-ODN, the TLR9-ODN was incorporated at a mass ratio as defined by dose for in-vivo experiments. The amino lipid was synthesized at Merck (West Point, PA) according to published methods. DSPC and cholesterol were obtained from Sigma–Aldrich (St. Louis, MO). PEG2000-DMG was manufactured by NOF Corporation (White Plains, NY). Lipid nanoparticle size was determined by dynamic light scattering using a DynaPro particle sizer (Wyatt Technology, SantaBarbara, CA). Amorphous aluminum hydroxylphosphate sulfate based adjuvant termed Merck Aluminum Adjuvant (MAA), was manufactured in-house at Merck & Co., Inc. TLR9 activating agonist, referred to as TLR9-ODN throughout the paper, was originally developed by Idera pharmaceuticals and supplied in bulk under at Merck & Co., Inc⁵⁵. Saponin based ISCOMATRIX^TM adjuvant was manufactured and supplied as sterilized bulk by CSL Limited, Australia under a license agreement. DEN-80E antigen for all four serotypes was expressed using stably transformed Drosophila S2 cells, and purified as previously descried¹³. For mice studies, 1 μg of DEN2-80E antigen was utilized. Tetravalent vaccine formulations of DEN1, DEN2, DEN3, DEN4 serotype specific “80E” env protein (DEN1-4-80E) content were: 3 μg, 3 μg, 3 μg, 6 μg for Guinea pig studies and 10 μg, 10 μg, 10 μg, 20 μg for Rhesus macaques studies.