2.6. Adjuvant Preparation and Formulation Characterization

Aluminum hydroxide gel (Alhydrogel 2%) was purchased from Croda. The SWE adjuvant (squalene-in-water emulsion) was co-developed by the Vaccine Formulation Institute (Plan-les-Ouates, Switzerland) and Seppic (La Garenne Colombes, France) and is available at GMP grade (Sepivac SWE™) under an open-access model. The SQ, SMQ, LQ, and LMQ adjuvant formulations were manufactured at the Vaccine Formulation Institute (VFI) [16].

The SQ adjuvant was prepared by combining a solution of QS21 saponin (Desert King International, San Diego, CA, USA) with a squalene-in-water emulsion containing cholesterol. The SMQ adjuvant was prepared similarly, but with the incorporation of a synthetic TLR4 agonist, 3D(6acyl)-PHAD (3D6AP) (Avanti Polar Lipids, Alabaster, AL, USA). The LQ adjuvant was prepared by adding a QS21 solution to neutral liposomes composed of dioleoyl phosphatidylcholine (DOPC) and cholesterol. The LMQ adjuvant is based on LQ with the inclusion of 3D6AP. Injectable formulations were prepared with LQ, SQ, LMQ, and SMQ at 2 μg of the TLR4 agonist 3D6AP and/or 5 μg of QS21 saponin per injected dose. The SWE, SQ, and SMQ emulsion adjuvants contained 1 mg of squalene per injected dose. The compatibility of R_M and R_T antigens with the panel of adjuvants was assessed through a comprehensive physicochemical characterization of each adjuvant in formulations prepared at a 1:1 volume ratio and stored for 24 h at 5 °C. Integrity of the R_M and R_T antigens in adjuvanted formulations was monitored using a sandwich ELISA that was essentially as previously described [17]. Briefly, R_M and R_T adjuvanted formulations were added to 96-well plates coated with human mAb CR3022 anti-SARS-CoV-2 RBD (Abcam, Cambridge, UK) and bound antigen was detected with a biotinylated human ACE2-Fc fusion protein (Institute of Protein Design, Seattle, WA, USA).