Recombinant RSV variant engineering, microneutralization, and serum neutralization susceptibility assays
infection
HEAT, SECONDARY, INFECTION
Recombinant RSV variants were engineered using reverse genetics to contain individual and co-occurring (for polymorphisms within the binding sites) amino acid substitutions that were observed in the Phase 2b and MELODY trials in the full-length RSV F protein. Briefly, amino acid substitutions were introduced into the complementary DNA (cDNA) of the F gene of full-length recombinant RSV A (A2-000; containing the E66 residue) or recombinant RSV B (B9320-000; containing the N197 residue) using molecular cloning techniques. Recombinant RSV variants were rescued by co-transfecting full-length antigenomic cDNA encoding the polymorphic change(s) in the F gene along with supporting plasmids encoding the ribonucleocapsid genes N, P, L, and M2-1, and plaque-purified as previously described in refs. Microneutralization assays were conducted by Viroclinics Biosciences BV (Rotterdam, Netherlands) in technical triplicates by serially diluting nirsevimab or palivizumab in a 96-well plate and incubating with recombinant RSV reference strain or test variant at an input of 50–1700 tissue culture infectious dose 50 per well for 1 h at 37 °C prior to the addition of Hep-2 cells (Hep-2 CCL-23, ATCC, Gaithersburg, MD, USA). Following incubation for 5 days at 37 °C, infected cells were fixed in 80% acetone and stained with a mouse anti-RSV F primary (clone 133-1H; Millipore, Cat. No. MAB858-1) and peroxidase-conjugated goat anti-mouse secondary antibody (Life Technologies, Cat. No. A16072). Tetramethylbenzidine substrate was added to the wells and RSV F expression was determined by measuring absorbance at 450 nm using a Biotek Synergy H1M plate reader. The assay was validated through the characterization of intermediate precision and repeatability. The ICSerum neutralization assays were performed in technical duplicates using the microneutralization protocol described above with minimal modification. Briefly, serum from 17 donors were heat inactivated at 56 °C for 30 min prior to serial dilution and incubation with recombinant RSV test variants. Following infection on Hep-2 cells and RSV F staining, the half-maximal neutralization titer (NTAs the Hep-2 cell line is commonly misidentified, cells were authenticated using PCR to identify the presence of the following markers: amelogenin, CSF1PO, D13S317, D16S539, D5S818, TH01, TPOX, and vWA.