Detection and activation of HIV broadly neutralizing antibody precursor B cells using anti-idiotypes

Bancroft, Tara; DeBuysscher, Blair L.; Weidle, Connor; Schwartz, Allison; Wall, Abigail; Gray, Matthew D.; Feng, Junli; Steach, Holly R.; Fitzpatrick, Kristin S.; Gewe, Mesfin M.; Skog, Patrick D.; Doyle-Cooper, Colleen; Ota, Takayuki; Strong, Roland K.; Nemazee, David; Pancera, Marie; Stamatatos, Leonidas; McGuire, Andrew T.; Taylor, Justin J.

A c/scFv iglb12 was cloned and expressed using lentiviralbased stable transduction of human embryonic kidney suspension adapted freestyle 293F cells (Thermo Fisher Scientific) as described previously (Bandaranayake et al., 2011) . Briefly, plasmids containing appropriate constructs were incorporated into replication-incompetent lentiviral particles and transduced into human embryonic kidney suspension adapted freestyle 293F cells plated in fresh media at 10 6 /ml in 10 ml and incubated at 37°C with 8% CO 2 and 80% humidity. Cells were supplemented with 25 ml fresh freestyle media 8-16 h after transduction. 3 d following transduction, cells were replated at a cell density of 0.5 × 10 6 /ml in cultures as large as 2 liters. Culture supernatants were harvested by centrifugation at 8,000 rpm to remove cells and clarified by passing through 0.22-μm filter (Thermo Fisher Scientific). Clarified supernatants were supplemented with 150 mM NaCl and concentrated using a 10,000 molecular weight cutoff Amicon Stirred Cell concentrator (EMD Millipore). Recombinant proteins were further purified by size exclusion chromatography using a Superdex 75 and/or Superdex 200 column (GE Healthcare) running in 25 mM piperazine-N,N9-bis(2-ethanesylfonic acid) pH 7.0, 150 mM NaCl, 1 mM EDTA, and 0.02% NaN 3 . Eluted fractions were analyzed using SDS-PAGE to assess purity. Protein concentrations were determined using molar extinction coefficients and absorbance at 280 nm.