293f cells

One mg of plasmid DNA per 1 liter of cells was diluted in DMEM and mixed with PEI. PEI:DNA mixtures were added to 293F cells (ThermoFisher, catalog #R79007) for 4 h. 293F cells were subsequently washed and diluted to 1.25 million cells per mL in Freestyle293 media (ThermoFisher). The cells were cultured for 5 days, and on the fifth day, the cell culture media was cleared of cells by centrifugation and filtration with a 0.8 μm cellulose membrane (Nalgene). The cell culture media was concentrated with a vivaflow 50 with a 10 kD molecular weight cutoff. The concentrated cell culture supernatant was rotated with lectin beads (Vistar Labs) in MES pH 7.0 buffer overnight at 4 °C. The beads were pelleted by centrifugation the next day and resuspended in MES pH 7.0 wash buffer. The lectin beads were washed twice and the glycosylated HIV-1 gp120 was eluted with 0.5 M methyl-α-pyranoside. The protein was buffer-exchanged into phosphate-buffered saline and stored at –80 °C. Monomeric recombinant gp120 was purified by size exclusion chromatography (SEC) in phosphate-buffered saline using a Superdex200 10/300 gel filtration column (GE Healthcare) and stored at –80 °C. The glycans on recombinant gp120 Envs have been shown to be more complex residues compared to glycans on virus associated gp160 Envs^{47 (link)}.

The Bma-LAD-2-Renilla reniformis luciferase (Ruc) construct was inserted into a pREN2 vector by GenScript. The predicted signal sequence was removed prior to gene synthesis. The vector was cloned into TOP10 cells (Thermo Fisher Scientific) and amplified on agarose plates with kanamycin at 50 μg/mL. Plasmid DNA was isolated and purified using a Miniprep kit (Qiagen) per the manufacturer’s guidelines. 293F cells (Thermo Fisher Scientific) were transfected with 30 μg of Bma-LAD-2 plasmid at a concentration of 1 × 10⁶ cells per mL. The 293F cells were collected 72 h later and homogenized. The lysate was stored at −80°C for later use.

Peripheral blood mononuclear cell (PBMC) isolation was performed via density gradient centrifugation over Ficoll-Paque, then IgG⁺ memory B cells were isolated from a cryopreserved COVID-19 patient’s PBMC by using CD22 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and immortalized with Epstein–Barr virus (EBV) as previously described (16 (link)). Culture supernatants were tested for their ability to bind SARS-CoV-2 proteins using enzyme-linked immunosorbent assay (ELISA). Positive cultures were collected and expanded. The VH and VL sequences from positive cultures were retrieved by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into human IgG1 and Ig kappa or Ig lambda expression vectors as previously described (17 (link)). Monoclonal antibodies were produced by transient transfection of 293F cells (Invitrogen-Life technologies, Grand Island, USA). Supernatants from transfected cells were collected after 4 days, and IgG was affinity purified by protein A chromatography (GE Healthcare, Chicago, USA) and desalted against PBS.

The receptor knock-out Huh-7 cell lines were a kind gift from Professor Yoshiharu Matsuura (Osaka University). Huh-7 cells were acquired from Apath LLC and Caco-2 from ATCC. All cells were grown at 37 °C in DMEM (Gibco) supplemented with 10 % FCS, 1 % non-essential amino acids and 1 % penicillin/streptomycin (P/S) (DMEM/FCS). For soluble E2 production, 293T (ATCC) and 293T 293T^CD81KO cells were initially maintained in DMEM/FCS. For large-scale purifications, these cells were adapted to suspension culture by growing the cells in a mix of FreeStyle 293 Expression medium (Gibco) and DMEM/FCS and decreasing the concentration of DMEM/FCS at each passage. Finally, both cell lines were grown in pure FreeStyle medium. 293-F cells (Invitrogen) were maintained in FreeStyle 293 Expression. All suspension cell lines were maintained in a shaking incubator (125 r.p.m.) at 37 °C and 8 % CO₂.