Trimers were transiently expressed in HEK293 F or ExpiCHO cells (Thermo Fisher Scientific) except for crystallographic analysis in which HEK293 F cells were treated with kifunensine. The protocol used for trimer production in HEK293 F cells has been described previously (36, 56). For cleaved HR1-redesigned trimers, the furin plasmid was added during transfection. The protocol for trimer and nanoparticle production in ExpiCHO cells is as follows. Briefly, ExpiCHO cells were thawed and incubated with ExpiCHO Expression Medium (Thermo Fisher Scientific) in a shaker incubator at 37°C, with 135 rpm and 8% CO2. When the cells reached a density of 10 × 106 ml−1, ExpiCHO Expression Medium was added to reduce cell density to 6 × 106 ml−1 for transfection. The ExpiFectamine CHO/plasmid DNA complexes were prepared for 100-ml transfection in ExpiCHO cells following the manufacturer’s instructions. For SOSIP and HR1-redesigned trimers as well as the I3-01 nanoparticles presenting an HR1-redesigned trimer of the BG505 strain, 80 μg of antigen plasmid, 30 μg of furin plasmid, and 320 μl of ExpiFectamine CHO reagent were mixed in 7.7 ml of cold OptiPRO medium (Thermo Fisher Scientific), whereas for UFO trimers (including UFO-BG and UFO-C) as well as UFO-BG-FR nanoparticles, 100 μg of antigen plasmid was used without furin. After the first feed on day 1, ExpiCHO cells were cultured in a shaker incubator at 32°C, with 120 rpm and 8% CO2 following the Max Titer protocol with an additional feed on day 5 (Thermo Fisher Scientific). Culture supernatants were harvested 13 to 14 days after transfection, clarified by centrifugation at 4000 rpm for 20 min, and filtered using a 0.45-μm filter (Thermo Fisher Scientific). For trimers, Env protein was extracted from the culture supernatants using a GNL column (Vector Labs), whereas for nanoparticles, Env fusion protein was purified using a 2G12 affinity column. Some trimers were further purified by SEC on a Superdex 200 Increase 10/300 GL column or a HiLoad 16/600 Superdex 200 PG column (GE Healthcare). The purity of I3-01 nanoparticles was characterized by SEC on a Superose 6 10/300 GL column. For both trimers and nanoparticles, protein concentration was determined using UV280 absorbance with theoretical extinction coefficients.

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