Purification and Formulation of cVLP:IL1β Vaccines

cVLPs and muIL-1β antigens were purified for lipopolysaccharides (LPS) prior to vaccine formulation, as previously described using Triton X-114 [33 (link)]. To formulate the cVLP:IL1β vaccines, the Catcher-cVLP and Tag-IL1β antigens were mixed in a 1:2 molar ratio and incubated O/N at 4 °C. Excess unbound antigen was removed by density gradient ultracentrifugation using an Optiprep™ (Sigma-Aldrich, St Louis, MO, USA) Step gradient (23, 29, and 35%) [31 (link)]. Purified cVLP:IL-1β vaccines were dialysed against sterile PBS, pH 7.4, using a cutoff of 1000 kDa (SpectrumLabs). All vaccines were subjected to a centrifugation stability test (16,000× g for 2 min). An equal number of pre- and post-spin samples were loaded on SDS-PAGE to assess potential precipitation, indicating vaccine aggregation. Antigen concentration on the cVLP and coupling efficiency (calculated as cVLP subunits/coupled cVLP subunits + uncoupled cVLP subunits) were estimated by densitometry from SDS-PAGE gels using ImageQuant TL (Cytiva, Marlborough, MA, USA).