Production and Purification of SOSIP Trimers

SOSIPv5.2 trimers were produced as previously described [56 (link)]. In brief, 1 L HEK293F cells (Invitrogen, cat no. R79009) were diluted to a cell density of 1 million cells/ml in FreeStyle Medium (Life Technologies) one hour before transfection. Plasmids encoding for SOSIP and furin were co-transfected in a 4:1 ratio using 1 mg/ml Polyethylenimine Hydrochloride (PEImax) (Polysciences Europe GmBH, Eppelheim, Germany) as the transfection agent. After 6 days, the cells were spun down and the supernatant was filtered using 0.22 μm pore size Steritopes (Millipore, Amsterdam, The Netherlands). The supernatant was run overnight at 4°C (0.5–1.0 ml/min) over a PGT145 affinity column, prepared as previously described [9 (link)]. SOSIP trimers were eluted with 3 M MgCl₂ pH 7.2, 20 mM TrisHCl into an equal volume of TN75 buffer (20 mM TrisHCl pH 8.0, 75 mM NaCl). Buffer exchange into TN75 buffer was performed using Vivaspin20 centrifugal filters with 100 kDa MW cut-off (Sartorius, Gӧttingen, Germany).
Viruses were produced by transfection of virus plasmid DNA into HEK293T cells (ATCC, CRL-11268) using lipofectamin2000 (Invitrogen). Supernatant containing virus was harvested 3 days after transfection and used in neutralization assays.