RSV Neutralization Assay Protocol

Neutralization assays were performed as previously described [37 (link)]. Briefly, HEp-2 cells were seeded at a density of 2.4 × 10⁴ cells/well in 384-well black optical bottom plates from ThermoFisher (Cat#142761). Serial 2-fold dilutions were performed on samples, either NHP sera or monoclonal antibodies, in a final volume of 40 µL. The starting dilution was 1:10 for sera and 1:10, 1:200, or 1:500 for the monoclonal antibodies. An equal volume of recombinant mKate-RSV subtype A (strain A2) was added to the diluted samples and incubated at 37 °C for 1 h [38 (link)]. After incubation, 50 µL of each diluted sample-virus mixture was added to the HEp-2 cells and incubated for 37 °C for 24 h. Fluorescence endpoints were recorded at 24 h using excitation at 588 nm and emission at 635 nm (SpectraMax M2e, Molecular Devices, San Jose, CA, USA). The IC₅₀ titer for each sample was determined using a four-parameter non-linear regression curve fit with GraphPad Prism version 7 (GraphPad Software Inc., San Diego, CA, USA), which was then used to calculate the IC₅₀ concentration of each monoclonal antibody or NHP sera.