0.45 μm filter flasks

293T cells were plated in 175-mm flasks and transfected with 80 μg SHIV-AD8-EO by calcium phosphate technique. At 12 h post-transfection, medium was replaced with fresh DMEM (10% FBS). Medium was harvested at 48 h post-transfection, and debris was cleared by centrifugation for 10 min at 1,500 × g and filtered using 0.45-μm filter flasks (Millipore Sigma, Billerica, MA). Virus stocks were aliquoted and frozen at −80°C. Virus titers were determined by an SIV p27 ELISA kit (ABL, Rockville, MD).

Production of recombinant ITS01 was performed as previously described (12 (link)). Briefly, HEK293T cells in 175 mm plates were transfected with 60 μg total DNA/plate at 80% confluency with PEIpro transfection reagent (Polyplus). Cells were co-transfected with the AAV ITS01 transfer plasmid and a plasmid encoding furin at a 4:1 ratio. At 16 hrs post-transfection, 10% FBS-DMEM media was replaced with serum-free 293 Freestyle media (Invitrogen). Media was collected after 48 hrs, debris was cleared by centrifugation for 10 min at 1,500 g and filtered using 0.45 μm filter flasks (Millipore Sigma). Proteins were isolated with HiTrap columns (Cytiva) and eluted with IgG Elution Buffer (Thermo Scientific) into 1M Tris-HCl Buffer, pH 9.0 (G Biosciences, St. Louis, MO). Buffer was exchanged with PBS and protein concentrated to 1-2 mg/mL with Amicon Ultra Centrifugation Filters (Millipore Sigma). Heavy and light chains of the antibodies were assessed by Coomassie-stained SDS-PAGE. Antibodies were stored at 4°C before use and frozen at -80C for long term storage.

Production of antibodies was performed as previously described.⁵² (link) Briefly, HEK293T cells in 175-mm plates were transfected with 80 μg total DNA/plate at 50% confluency with a calcium phosphate transfection kit (Takara, Mountain View, CA). For human antibody production, cells were co-transfected with both heavy-chain vector and light-chain vector at a 1:1 ratio. For rhesus antibody production, cells were co-transfected with AAV transfer plasmid and a plasmid encoding furin at a 4:1 ratio. At 12–16 hr post-transfection, 10% fetal bovine serum (FBS)-DMEM was replaced with serum-free 293 Freestyle media (Invitrogen, Carlsbad, CA). Media were collected after 48 h, and debris was cleared by centrifugation for 10 min at 1,500 × g and filtered using 0.45-μm filter flasks (Millipore Sigma, Billerica, MA). Proteins were isolated with HiTrap columns (GE Healthcare, Pittsburgh, PA) and eluted with IgG Elution Buffer (Thermo Scientific, Waltham, MA) into 1 M Tris-HCl Buffer (pH 9.0) (G Biosciences, St. Louis, MO). Buffer was exchanged with PBS and protein concentrated to 1 mg/mL with Amicon Ultra Centrifugation Filters (Millipore Sigma, Billerica, MA). Antibodies were stored at 4°C.