Neutralization Assay with Pseudotyped VSV

Neutralization assays with replication-incompetent pseudotyped vesicular stomatitis virus (VSV) expressing a luciferase reporter and either WT-EBOV GP (GP_WT) or GP containing the E545D mutation (GP_E545D) were performed as described previously (31 (link)). Briefly, a serial dilution of antibody was incubated with pseudotyped virus in serum-free Eagle’s minimum essential medium (EMEM) for 1 h at room temperature before Vero cells were infected at a multiplicity of infection (MOI) of 0.04. One hour following infection, cells were supplemented with 50% (vol/vol) EMEM containing 2% FBS, 100 IU/ml penicillin, and 100 μg/ml streptomycin. Twenty-four hours later, cells were lysed for 30 min at room temperature with Passive Lysis Buffer (Promega) before the addition of Luciferase Activating Reagent (Promega). Luciferase luminescence was read using a Biotek Plate Reader, and percent neutralization was calculated based on wells containing virus only.