Ultra 384 luminometer

HIV-1 pseudoviruses were generated, as described previously [86 (link)]. Briefly, 293T cells were cotransfected with plasmids encoding HIV-1 envelope and luciferase-expressing HIV-1 genome using VigoFect reagent (Vigorous Biotech, Beijing, China). The supernatants were replaced with fresh DMEM with 10% FBS at 12 h post-transfection and collected at 48 h post-transfection and stored at -80 °C. The antiviral activities of peptides were determined using U87 CD4⁺ CCR5⁺ cells. Different concentrations of peptide mixed with 100 TCID50 virus and U87 CD4⁺ CCR5⁺ cells (10⁴/well). Changed fresh medium and cultured for additional 48 h at 37 °C, and the luciferase activity was measured by using a luciferase kit (Promega, Madison, WI) and an Ultra 384 luminometer (Tecan).

Avian influenza pseudoviruses expressing H5 HA (HIV (human immunodeficiency virus)/HA (H5)) were produced as described previously [49 (link)]. Briefly, 293T cells were co-transfected with 10 μg plasmid encoding Env-defective, luciferase-expressing HIV-1 (pNL4-3.luc.RE) and 10 μg each of the 6 plasmids, including QH-HA-, XJ-HA-, AH-HA-, HK97-HA-, 1194-HA- and Tailand-HA-pcDNA3.1, respectively, into 100 mm culture dishes using Polyetherimide (PEI). Exogenous bacterial neuraminidase (NA) from Vibrio cholerae (Sigma, St. Louis, MO, USA) was added 24 and 48 h later, and supernatants were harvested 72 h post-transfection for single-cycle infection.
A pseudotyped virus bearing VSV envelope glycoprotein (HIV/VSV-G) was produced in the same way. The pseudovirus titers were quantitated by using Luciferase substrate. For the drug screening process, pseudoviruses were incubated with serially diluted compounds at 37 °C for 15 min. The compound-virus mixture was subsequently added to the cells. 48 h later, cells were lysed by cell lysis buffer (Promega, Madison, WI, USA) and transferred to 96-well luminometer plates. Luciferase substrate (Promega) was added, and relative luciferase activity was determined by Ultra 384 luminometer (GENios Pro, TECAN, Bedford, MA, USA).

To determinate the neutralizing activity of SARS-CoV-2 spike and RBD-specific nanobodies against SARS-CoV-2 infection, a pseudovirus neutralization assay was performed. HEK293T-ACE2 stable cell lines were constructed as previously described [14 (link)]. In brief, HEK293T cells were transfected with a lentivirus vector encoding human ACE2 and puromycin selection marker. Cells were selected with puromycin, and puromycin-resistant clones were expanded and verified by FACS for ACE2 expression. HEK293T-ACE2 cells were plated into 96-well cell-culture plates with 2 × 10⁴ cells/well and cultured overnight at 37 °C with 5 % CO₂. The pseudovirus was preincubated with tenfold serial dilution of Nbs at 37 °C for 1 h before being added to cells. Pseudovirus in culture media without Nbs were used as negative control. Medium were changed the following day after infection. 48 h later, EGFP expression was determined by fluorescent microscopy and flow cytometry to evaluate the neutralization ability. While the cells were lysed using lysis reagent (Promega) after washing with PBS, and relative luciferase activity was measured immediately in the Ultra 384 luminometer (Tecan). The relative luminescence signals (RLU) from the negative control wells were normalized and used to calculate neutralization percentage for each concentration.