Pseudotype-based virus neutralization assay

VSV pseudotyped with LASV GP, VSV-Lassa-GPc-cFLAG, was used as an additional model for the assessment of neutralization under biosafety level 2 conditions. To generate Lassa-GPc-cFLAG, the sequence for LASV GPC Josiah strain was codon-optimized for human cells and further modified by addition of a 3′ tri-glycine linker (GGGS) and the nucleotide sequence for a FLAG-tag (DYKDDDDK). Lassa-GPc-cFLAG was synthesized and cloned into pcDNA3.1+ (GenScript); accuracy of the construct was confirmed by Sanger sequencing (ACGT). Replication-incompetent pseudotyped VSV particles expressing GFP and luciferase reporters were produced as previously described^73–75.

Sera was gamma-irradiated prior to transfer from biosafety level 4 to biosafety level 2 conditions, complement inactivated at 56 °C for 30 min, and serially diluted (by a factor of 2.5) from 1:10 to 1:2441. Five-fold serial dilutions of human monoclonal antibodies 25.10C, 12.1F, 37.2D, and 8.9F from a starting concentration of 60 µg/mL served as positive controls. A cocktail of all four antibodies, at an initial (total antibody) concentration of 60 µg/mL, was serially diluted in the same way as an additional positive control. Naive guinea pig serum and virus only wells were used as negative controls. Subsequently, 1000 FFU of VSV-Lassa-GPc-cFLAG were added to the diluted sera or antibody and incubated for 1 h at 37 °C and 5% CO₂. Virus-antibody mixtures were added to confluent Vero E6 cells and spin infection was carried out at 4 °C at 1200 × g for 1 h. Infected cells were cultured for 16 h. GFP-positive cells were counted using the ImmunoSpot® 7 Software and CTL Analyzer (CTL Analyzers, LLC). The percent of infected cells was determined relative to uninhibited virus only control wells. Half-maximal inhibition (IC₅₀) values were calculated based on reciprocal sera dilution by GraphPad® Prism 8 using a sigmoidal nonlinear fit model (4PL regression curve). Values above 100% infectivity were converted to 100%.