Vaccine Adjuvant Evaluation in Mice

C57BL/6, Batf3 KO and Caspase 1 KO mice were purchased from Jackson Laboratories. RIPK3 KO (Ripk3^-/-) (Newton et al., 2004 (link)) and RIPK3 kinase-inactive (Ripk3^K51A/K51A) (Mandal et al., 2014 (link)) were kindly provided by E. Mocarski (Emory University). Tissues from immunized CD169-DTR and LysM-iDTR mice were kindly provided by M. Merad (Icahn School of Medicine at Mount Sinai). ASC KO mice and MLKL KO mice were originally obtained from V. M. Dixit (Genentech) and bred under specific pathogen-free conditions. Mice were immunized subcutaneously at the base of tail with endotoxin-free Ovalbumin (Hyglos) and following adjuvants: MF59 (Novartis), Addavax (Invivogen) and Alum (Alhydrogel; Invivogen). In case of co-administration of cell death inhibitors such as z-VAD-fmk (500 μg/mouse) and Nec-1s (100 μg/mouse) or enzymes including DNaseI (2,000U/mouse), those reagents were mixed with vaccine inoculums right before the subcutaneous injections. In order to deplete LN-resident macrophages, 0.25 mg (50 μl) of Clodrosome (CLL) and Encapsome (control liposome) were subcutaneously injected 5 days prior to each vaccination. Mice were maintained under specific-pathogen-free conditions in the vivarium of Emory Vaccine Center. All animal studies were conducted by following animal protocols reviewed and approved by the Institutional Animal Care and Use Committee of Emory University.