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Respiratory syncytial virus (RSV) is a commonly occurring pathogen that can cause severe disease in children, the elderly, and immunocompromised individuals with a large, unmet clinical need. We developed a high-throughput, primary cell-based antiviral RSV assay to enable identification of small molecules using cytopathic effect (CPE) as a phenotypic end point. To provide increased biological relevance, we developed our assay with primary human small airway epithelial cells (SAECs), which originate from known sites of RSV infection and replication instead of a more traditional immortalized cell line. Using purchased low-passage cells, cost-effective large-scale culture methods were developed to provide assay-ready frozen SAECs. A high-throughput screening campaign using the GSK Screening Collection was performed. The screen was executed in 384-well plates over a 12-week period with an average Z' of 0.5. The screen yielded 17 post-entry hits with activity in the primary cells, which were not active in immortalized cells. Potencies for this class of compounds were equal between the primary and immortalize cell lines. For entry inhibitors, the number was much lower, with increased potency observed in immortalized cells. This is the first known use of frozen primary human cells for an RSV high-throughput screening phenotypic campaign.

A Phenotypic High-Throughput Screen with RSV-Infected Primary Human Small Airway Epithelial Cells (SAECs)