Er Intrabody-Mediated Inhibition Of Interferon A Secretion By Mouse Macrophages And Dendritic Cells

Interferon alpha (IFN alpha) counteracts viral infections by activating various IFN alpha-stimulated genes (ISGs). These genes encode proteins that block viral transport into the host cell and inhibit viral replication, gene transcription and translation. Due to the existence of 14 different, highly homologous isoforms of mouse IFN alpha, an IFN alpha knockout mouse has not yet been established by genetic knockout strategies. An scFv intrabody for holding back IFN alpha isoforms in the endoplasmic reticulum (ER) and thus counteracting IFN alpha secretion is reported. The intrabody was constructed from the variable domains of the anti-mouse IFN alpha rat monoclonal antibody 4EA1 recognizing the 5 isoforms IFN alpha 1, IFN alpha 2, IFN alpha 4, IFN alpha 5, IFN alpha 6.A soluble form of the intrabody had a K-D of 39 nM to IFN alpha 4. It could be demonstrated that the anti-IFN alpha intrabody inhibits clearly recombinant IFN alpha 4 secretion by HEK293T cells. In addition, the secretion of IFN alpha 4 was effectively inhibited in stably transfected intrabody expressing RAW 264.7 macrophages and dendritic D1 cells. Colocalization of the intrabody with IFN alpha 4 and the ER marker calnexin in HEK293T cells indicated complex formation of intrabody and IFN alpha 4 inside the ER. Intracellular binding of intrabody and antigen was confirmed by co-immunoprecipitation. Complexes of endogenous IFN alpha and intrabody could be visualized in the ER of Poly (I:C) stimulated RAW 264.7 macrophages and D1 dendritic cells. Infection of macrophages and dendritic cells with the vesicular stomatitis virus VSV-AV2 is attenuated by IFN alpha and IFN beta. The intrabody increased virus proliferation in RAW 264.7 macrophages and D1 dendritic cells under IFN beta-neutralizing conditions. To analyze if all IFN alpha isoforms are recognized by the intrabody was not in the focus of this study. Provided that binding of the intrabody to all isoforms was confirmed, the establishment of transgenic intrabody mice would be promising for studying the function of IFN alpha during viral infection and autoimmune diseases.