Immunogenicity assessment
BLOOD, VIRUS, APC
Blood and nasal samples were collected at baseline (day 1, before vaccination), day 15, and day 29 before vaccination. To reduce sample variation, samples from the same participant were run on the same plate in a blinded fashion in all the assays. Serum RSV nAb levels were determined by a qualified RSV A2 microneutralization (MN) assay based on RSV-A2-rLuc reporter virus (RSV F–specific serum IgG and IgA Ab and nasal IgA Ab levels were determined by enzyme-linked immunosorbent assay (ELISA) assay using purified RSV F protein (25 ng per well; SinoBiological, catalog no. 11049-V08B) and twofold serial dilutions in blocking buffer (5% milk/0.5% bovine serum albumin in 1× KPL wash buffer (SeraCare) in duplicate. End-point titer was calculated by 4PL curve fitting using Prism and reported as reciprocal dilution. PIV5-specific IgG and nAb titers were determined by ELISA assay using PIV5 virus-coated plates and by PIV5-rLuc–based MN assay in Vero cells, respectively. The PIV5 IgG end-point titer was calculated by 4PL curve fitting and reported as reciprocal dilution. The PIV5 MN assay was performed similarly to the RSV-rLuc–based MN assay, except in duplicate instead of quadruplicate. Analysis of PIV5 MN was identical to the RSV-rLuc–based MN assay, and the nAb titer was defined as the reciprocal dilution that inhibited at least 50% signal of the virus control.Antigen-specific T cell frequencies were evaluated by intracellular cytokine staining assay using cryopreserved peripheral blood mononuclear cells (PBMCs) isolated from whole blood on day 1 (pre-vaccine dosing), day 15, and day 29. One million cryopreserved PBMCs were thawed in complete 10% fetal bovine serum (FBS) RPMI medium and rested overnight at 37°C before being incubated with an RSV F peptide pool from GenScript at a final concentration of 1 μg/ml in the presence of anti-CD28 ECD (1 μg/ml; Beckman Coulter, clone CD28.2), anti-CD107a fluorescein isothiocyanate (BD Biosciences, clone H4A3), and anti-CD49d (BD Biosciences, clone 9F10). In addition, PBMCs were stimulated with 1 μl of complete medium with 0.5% dimethyl sulfoxide (DMSO; negative control corresponding to the DMSO concentration of the RSV F peptide pool) or 1 μl of phorbol 12-myristate 13-acetate (PMA)/ionomycin [PMA (25 ng/ml) and ionomycin (1 μg/ml)] for negative and positive controls, respectively. After 2-hour incubation at 37°C, brefeldin A (10 μg/ml; BD Biosciences) were added and the cells were incubated for another 4 hours. The cells were washed with phosphate-buffered saline (PBS) and incubated with aqua viability dye (Invitrogen) at room temperature for 15 min. The cells were washed with PBS supplemented with 2% FBS and surface-stained at 4°C for 30 min with anti-CD3 Alexa 700 (BD Biosciences, clone SP34-2), anti-CD4 BV605 (BD Biosciences, clone L200), anti-CD8 BV450 (BD Biosciences, clone RPA-T8), and anti-CD95 phycoerythrin (PE)–Cy5 (BD Biosciences, clone DX2). The cells were washed with PBS with 2% FBS, fixed with Cytofix/Cytoperm (BD Biosciences), permeabilized with 1× Perm/Wash (BD Biosciences), and incubated with anti–IFN-γ PE-Cy7 (BD Biosciences, clone B27), anti–TNF-α allophycocyanin (APC)–Cy7 (BioLegend, clone Mab11), anti–IL-13 PE (Miltenyi Biotec, clone JES10-5A2.2), and anti–MIP-1β APC (eBioscience, clone FL34Z3L) antibodies at 4°C for 30 min. The cells were washed with 1× Perm/Wash and PBS with 2% FBS then resuspended in PBS/2% formaldehyde for acquisition on a BD FACSAria Fusion cell sorter. CD3