pACEBac-ADDomer and epitope-displaying variants were expressed using the MultiBac baculovirus expression system we developed (fig. S1) (23). Briefly, pACEBac-ADDomer and variant plasmids were inserted by Tn7 transposition into an engineered baculoviral genome, EMBacY, in DH10EMBacY cells (27). Transfection, baculovirus amplification, storage, and protein expression in Hi5 insect cell suspension cultures (50 ml) were performed following established protocols (27), resulting in exceptional yields (fig. S1). The EMBacY baculoviral genome encodes a yellow fluorescent protein (YFP) marker. When the YFP signal reached a plateau, cells were harvested by centrifugation, and pellets were flash-frozen in liquid nitrogen and stored at −20°C. Pellets were resuspended in hypotonic lysis buffer [0.33× phosphate-buffered saline (PBS); 1 ml per 2.5 × 107 cells] supplemented by EDTA-free cOmplete protease inhibitor (Roche, Switzerland) and lysed by three cycles of freezing and thawing. Lysate was cleared from debris by centrifugation (6000g for 30 min), and the cleared supernatant was subjected to sucrose gradient (15 to 40%) centrifugation (15). Fractions containing ADDomer (30 to 40% sucrose) were pooled and dialyzed against buffer A [10 mM Hepes (pH 7.4) and 50 mM NaCl]. ADDomer was further purified by ion exchange chromatography using a High Q column (Bio-Rad) using buffer A and applying a linear salt gradient (50 to 500 mM NaCl). Fractions containing highly purified ADDomer (330 to 400 mM NaCl) were pooled and stored at ambient temperature or frozen (−80°C). For animal studies, ADDomer particles were further purified using Detoxi-Gel (Thermo Fisher Scientific) to remove endotoxins, followed by dialysis against PBS.

Following purification, absorbance spectroscopy measurement showed an OD260nm (optical density at 260 nm)/OD280nm ratio below 0.6, evidencing lack of nucleic acid contamination. Before injection into mice, endotoxin levels were quantified using Endosafe PTS (Charles River) evidencing 754 EU (endotoxin units)/mg for ADDomer-tevCHIK and 247 EU/mg for ADDomer, respectively. In our injections, we thus were below 30 EU, the common cutoff value for testing adjuvant properties of any recombinant antigen in a mouse model.

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