| dc.description.abstract | The innate immune response is the first line of defense against viral infections. Upon infection, viruses are sensed by cells which triggers a signaling cascade that leads to the production of interferons (IFNs). IFNs induce the expression of IFN-stimulated genes, thereby causing cells to enter an antiviral state. Recently, auto-antibodies (auto-Abs) neutralizing the antiviral activity of IFNs have been detected in patients suffering from severe viral diseases, and these anti-IFN auto-Abs have been associated with disease severity caused by several viruses. To better understand the molecular mechanisms of anti-IFN auto-Abs and their relationship to virus replication, an initial in vitro model system to study the effect of inhibition of the IFN system on influenza A virus (IAV) replication was developed, involving A549 cells and the avian IAV A/mallard/New York/6750/1978 (A/mallard/NY/78). In addition, tools and assays were set-up with the aim of studying the effect of anti-IFN auto-Abs on virus replication in vitro. Replication of A/mallard/NY/78 was enhanced upon treatment of A549 cells with ruxolitinib (a JAK inhibitor). In contrast, no consistent enhancement of A/mallard/NY/78-replication was observed in A549 cells treated with anti-IFNAR and/or anti-IFNLR Abs. Next, an anti-IFNα auto-Ab was cloned and produced, and commercial neutralizing Abs against IFNα, IFNß, and IFNω were characterized. Additionally, CRISPR/Cas9-mediated knockout of IFNAR1, IFNLR1, and type I IFNs was performed. The genomic, functional and western blot data of selected IFNAR1 and IFNLR1 single cell clones showed inconsistencies, and further validation of individual single cell knockout clones is therefore required. Initial virus replication kinetics experiments with A/mallard/NY/78 and GFP-expressing viruses in IFNAR1 and IFNLR1 single cell knockout clones did not show enhanced virus replication compared to wild-type A549 2D8 cells. Overall, this report described efforts to set-up assays and a model system to elucidate the molecular characteristics of anti-IFN auto-Abs and their relationship to virus replication. | |
