Repurposing RIPK3 inhibitor GSK840 as an enhancer of the safety and immunogenicity of lentogenic Newcastle disease virus La Sota used for in ovo and spray vaccination.

Live Newcastle disease (ND) vaccines face major limitations: spray vaccination causes adverse vaccinal reactions in day-old chicks, while in ovo vaccination results in high pathogenicity in embryos. We hypothesized that specific host responses contribute to the pathogenicity of live ND vaccines. In this study, lentogenic NDV La Sota vaccination in ovo significantly reduced the hatchability and post-hatch survival, accompanied by robust upregulation of inflammatory and cell death-related genes, notably the receptor-interacting serine/threonine-protein kinase 2 (RIPK2) in chicken lungs. Given the absence of RIPK3 in chickens and the structural homology of chicken RIPK2 to mammalian RIPK3 (sharing the conserved DFG motif), the RIPK3 inhibitor GSK840 targeting the DFG motif was tested as a potential modulator of La Sota pathogenicity. Co-administration of GSK840 with La Sota in ovo markedly improved the hatchability, post-hatch survival and antibody response. Furthermore, GSK840 pre-treatment at 18 days of embryonation prevented tracheal pathology, particularly the loss of cilium and goblet cells, and boosted antibody responses in day-old chicks receiving spray vaccination. Through biotin-conjugated GSK840 pull-down and mass spectrometry, multiple myosins (∼200 kDa) were identified as its potential targets in chicken trachea, rather than RIPK2. Collectively, our results demonstrate that GSK840 improves the safety and immunogenicity of La Sota in ovo and spray vaccination and that myosins are potential targets of GSK840. These findings present novel insights into the pathogenicity of live ND vaccines and provide important implications for optimization of live ND vaccination.