Neutralization of ebolavirus GP-pseudotyped HIV

To detect neutralizing activity in human blood, sera of donor vaccines were diluted in DMEM at a starting ratio of 1:5, followed by 3-fold serial dilutions. Next, 50 μL of diluted sera were incubated with an equal volume of HIV-EBOV GP-Luc (luciferase units were adjusted to 20,000 ~ 100,000 in the absence of mAbs) for 1 h at 37°C. Then, 2 × 10⁴ 293 T cells in 100 μL DMEM were added to the virus-antibody mixtures. After infection at 37°C, 5% CO₂ for 36 h, medium was removed, and cells were incubated with 50 μL lysis buffer (E1531; Promega) for 10 min at room temperature. A 20-μL volume of cell lysate was added to 96-well white assay plates (3599; Costar, Corning) and light intensity was read immediately on a GloMax 96 Microplates Luminometer (Promega) after addition of 50 μL luciferase assay reagent to each well. The neutralizing ability of mAbs was calculated by comparing the light intensity of wells in the presence of mAbs to that of wells containing virus only, and the IC₅₀ was calculated by fitting to a four-parameter curve using GraphPad Prism 7 software.

To examine neutralizing activity of purified mAbs or Fabs, 50 μL antibodies or Fabs in serial dilutions starting at 100 μg/mL were incubated with 50 μL diluted HIV pseudotype ebolavirus in 96-well plates at 37°C for 1 h, followed by the same procedures described above.