Expifectamine 293 reagent

HEK-293F cells were grown in suspension and transfected with ExpiFectamine 293 reagent (ThermoFisher, Waltham, MA, USA) according to the manufacturer’s recommendations. Briefly, a total of 75.10⁶ cells were harvested and the pellet was resuspended in 25 mL of Expi293 Expression medium before incubation at 37 °C for 30 min. Twenty-five micrograms of each plasmid were diluted in 1.5 mL of Opti-MEM medium and mixed with ExpiFectamine reagent before incubation at room temperature for 20 min. Finally, the mixed ExpiFectamine/plasmid was added to the cells and further incubated at 37 °C under agitation for 4 days. ExpiFectamine Transfection enhancers were added on day 1 post-transfection. Recombinant proteins were harvested directly from the culture supernatant at day 4 post-transfection. Luciferase activity was quantified onto a Centro XS3 LB 960 luminometer (Berthold Technologies, Thoiry, France) by adding 100 µL of NanoGlo reagent (Promega, Madison, WI, USA) to tenfold dilutions of supernatant.

The N-terminal peptidase domain of human ACE2 (residues 19 to 615, GenBank: BAB40370.1) and the receptor-binding domain (RBD) (residues 319-541) of the SARS-CoV-2 spike (S) protein (GenBank: QHD43416.1) were cloned into phCMV3 vector and fused with C-terminal His-tag. A plasmid encoding stabilized SARS-CoV-2 spike protein S-HexaPro (Hsieh et al., 2020 (link)) was a gift from Jason McLellan (Addgene plasmid #154754;http://addgene.org/154754; RRID: Addgene_154754) and used to express S-HexaPro for the binding assay. The plasmids were transiently transfected into Expi293F cells using ExpiFectamine 293 Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. The supernatant was collected at 7 days post-transfection. The His-tagged ACE2 or S-HexaPro protein were then purified by affinity purification using Ni Sepharose excel resin (Cytiva) followed by size exclusion chromatography.

The N-terminal peptidase domain of human ACE2 (residues 19 to 615, GenBank: BAB40370.1) was cloned into phCMV3 vector and fused with a C-terminal Fc tag. The plasmids were transiently transfected into Expi293F cells using ExpiFectamine™ 293 Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. The supernatant was collected at 7 days post-transfection. Fc-tagged ACE2 protein was then purified with a Protein A column (GE Healthcare) followed by size exclusion chromatography.

The spike glycoprotein was expressed in human embryonic kidney (HEK) cell line 293T and purified from the supernatant of the cells as previously described with certain modifications [12 ]. Briefly, HEK 293T cells were cultivated in 175 cm² cell culture flasks and transfected with the pCAAG-spike vector (kindly provided by Florian Krammer from the Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY), using the ExpiFectamine293 reagent (Thermo Fisher, Waltham, MA).
After 48 hours of transfection, the supernatant was collected, and the recombinant his-tagged proteins were purified through Ni-Sepharose columns (Cytiva, Chicago, IL). The recombinant protein was characterized by western blotting using anti-spike antibody (MyBiosource, San Diego, CA) diluted 1 : 500 (Figure S1). A secondary goat anti-mouse horseradish peroxidase-conjugated antibody was used at 1 : 5000 (Santa Cruz Biotechnology, Dallas, TX). The samples were detected using an Opti-4CN Substrate Kit (Bio-Rad, Hercules, CA). The nucleocapsid protein (N) and RBD fraction from the spike protein were purchased from MyBioSource (San Diego, CA).

VB2129 secretion was assessed by transient transfection of Expi293F suspension cells (Thermo Fisher Scientific, Waltham, MA). Expi293F cells were diluted to 2.8 × 10⁶/mL and incubated overnight in a CO₂ incubator (8% CO₂, 37 °C) with orbital shaking (125 rpm, 19 mm diameter). 2.0 × 10⁶ cells/well) were seeded in deep 96-well plates and transfected with 0.64 µg pDNA complexed with 2 µL ExpiFectamine 293 Reagent (Thermo Fisher) according to the manufacturer’s instructions. Mock transfection with ExpiFectamine 293 Reagent alone served as a control. The cells were incubated for three days in a CO₂ incubator (8% CO₂, 37 °C) with orbital shaking (900 rpm, 3 mm diameter). Supernatants were harvested by centrifugation at 300×g for 5 min and further cleared at 4000×g for 15 min at 4 °C. The supernatants were subjected to sandwich ELISA, Western blotting, and a human (h)CCR5 reporter assay.