This gene, containing the prefusion SARS-CoV-2 Spike ectodomain, was transiently expressed in HEK293F cells. Cells were first split in 200-ml culture at a density of 0.8 × 106 cells/ml. Fifty micrograms of filtered DNA per 200 ml of culture was mixed with FectoPRO (Polyplus Transfection) in a 1:1 ratio for 10 min at room temperature (RT). The DNAFectoPRO mixture was added to the cells, and the cells were incubated at 125-rpm oscillation, 37°C, 8% CO2, and 70% humidity in a Multitron Pro shaker. After 6 to 7 days, cell suspensions were harvested by centrifugation at 5000g for 15 min. Supernatants were filtered through a 0.22-um Steritop filter and passed through a HisTrap Ni-NTA (nitrilotriacetic acid) column. The protein was eluted with an increasing gradient of imidazole (up to 500 mM). Ni-NTA purification was followed by a Superose 6 10/300 GL size exclusion column in 20 mM phosphate (pH 8.0) and 150 mM NaCl buffer.

The COVID-19 pandemic has spurred interest in potent and thermostable SARS-CoV-2 vaccines. Here, we assess low-dose immunization with lyophilized nanoparticles decorated with recombinant SARS-CoV-2 antigens. The SARS-CoV-2 Spike glycoprotein or its receptor-binding domain (RBD; mouse vaccine dose, 0.1 ug) was displayed on liposomes incorporating a particle-inducing lipid, cobalt porphyrin-phospholipid (dose, 0.4 ug), along with monophosphoryl lipid A (dose, 0.16 ug) and QS-21 (dose, 0.16 ug). Following optimization of lyophilization conditions, Spike or RBD-decorated liposomes were effectively reconstituted and maintained conformational capacity for binding human angiotensin-converting enzyme 2 (hACE2) for at least a week when stored at 60°C in lyophilized but not liquid format. Prime-boost intramuscular vaccination of hACE2-transgenic mice with the reconstituted vaccine formulations induced effective antibody responses that inhibited RBD binding to hACE2 and neutralized pseudotyped and live SARS-CoV-2. Two days following viral challenge, immunized transgenic mice cleared the virus and were fully protected from lethal disease.