Quil a

All experiments involving mice were conducted in accordance with the guidelines and policies of the Canadian Council on Animal Care and the principles set forth in the Guide for the Care and Use of Laboratory Animals by the Animal Welfare Committee of the University of Montreal (RECH-1748). Housing and husbandry of animals were taken care by the personal of level II facilities at the University of Montreal. Six-week-old CD-1 mice (Charles River Laboratories, Saint-Constant, QC, Canada) were randomly assigned to five groups of 11 or 13 mice for immunization with rSsEno or rDPPIV, respectively, as accepted by the ethical committee. Animals were immunized twice subcutaneously at a 2-week interval with either 50 μg of rSsEno or rDPPIV mixed with one of the following adjuvants: 20 μg of Quil-A® (Brenntag Biosector, Frederikssund, Danemark), 15% of Polygen™ (MVP Laboratories, Omaha, NE, USA), 55% of Stimune® (Thermo Fisher Scientific, Waltham, MA, USA) or 50% of Montanide™ ISA 50 V2 (Seppic, Paris, France), following the manufacturers’ recommendations. A control group received 100 μL of PBS. To follow antibody responses, mice were bled (100 μL) before immunization, and twelve days after the first and the second vaccination doses by the dorsal tail vein. Fifteen days after the second vaccination, animals were intraperitoneally challenged as described below.

The WN-80E protein utilized in these studies was provided by Hawaii Biotech, and has been previously described.^{17 (link)} Briefly, the protein is a carboxy-truncated WNV E-protein which is produced in Drosophila S2 cells, and purified using an antibody affinity-based purification method. Protein was provided in PBS, and stored at −80 °C until use.
SLA, a synthetic lipid adjuvant, is a derivative of GLA and has been previously described.^{49 (link)} QS21 was obtained through in-house purification of Quil A (Brenntag Biosector), or purchased from Desert King (San Diego, CA). QS21 purity was assessed in each lot via a high-performance liquid chromatography-based method established at IDRI and qualified for cGMP release. Liposomes were generally manufactured by microfluidization until a liposome diameter of ~80 nm was achieved.

The immunization regime was based on similar studies performed investigating candidate dengue vaccines in mouse models. Under ketamine hydrochloride (Ceva Animal Health, Glenorie, Australia) and xylazine hydrochloride (Troy Laboratories, Gendenning, Australia) sedation, SV129 mice were immunized with 1 or 0.1 μg tetravalent sE (DENV1: 258848, DENV2: PR159, DENV3: CH53489, DENV4: H241 produced from S2 cells, donated by Prof Mathew Cooper, UQ) formulation by nanopatch or 1–10 μg tetravalent sE by intradermal (ID), subcutaneous (SC), or intra muscular (IM) injection, with or without 3 μg of the saponin adjuvant Quil-A (Brenntag, Essen, Germany). Control groups received PBS delivered via each investigated injection method, while nanopatch control groups received excipients only. Nanopatches containing the dose to be delivered were applied to each ventral ear pinnae using a proprietary applicator at a velocity of 3.1 ms⁻¹ and kept in place for 2 min. Mice received three doses by nanopatch or SC, ID, or IM injection at 28-day intervals, with blood samples collected one day before vaccination and 28 days after final dose.

Seven groups of C57BL/6 mice (n = 5–8) were immunized with 20 μg of AV-1959R (pET11/3Aβ_1-11-MultiTEP) formulated with a selection of adjuvants: Montanide-ISA51 (s.c.; 50/50 ratio; SEPPIC, France), Montanide-ISA720 (s.c.; 50/50 ratio; SEPPIC, France), MPLA-SM (s.c.; 10 μg; Enzo, NY), Alhydrogel^® (s.c.; 70 μg; BRENNTAG, Denmark), Quil-A (s.c.; 20 μg; BRENNTAG, Denmark), Advax™, and Advax^CpG (both i.m.; 1mg; Vaxine Pty Ltd, Adelaide, Australia). All mice were injected four times at biweekly intervals. Sera were collected 14 days after the third immunization and were used to measure anti-Aβ antibody responses. On day 7 after the last injection mice were terminated and cellular immune responses were analyzed in splenocytes.