Investigating the ability of RIG-I agonists to provide protection in mouse and ferret models of respiratory virus infection

Abstract

Respiratory infections caused by influenza A virus (IAV) or respiratory syncytial virus (RSV) lead to substantial morbidity and mortality. Treatment options are limited and there is urgent need for the development of efficient therapeutic and prophylactic treatments. <br /> Pattern recognition receptors (PRRs) such as the cytoplasmic helicase retinoic-acid-inducible gene I (RIG-I) are part of the innate immune system. RIG-I can be activated by recognition of viral nucleic acids, leading to downstream activation of interferon-stimulated genes (ISGs) and restriction of viral replication. We have used synthetic RNA oligonucleotides to stimulate RIG-I to inhibit replication of respiratory viruses using <em>in vitro</em> and <em>in vivo</em> models of infection. <br /> Our <em>in vitro</em> approaches used airway cell lines from humans, mice and ferrets and investigated the effects of RIG-I agonist pre-treatment on subsequent infection with either IAV or RSV. Prophylactic RIG-I agonist treatment induced multiple ISGs and inhibited infection and growth of respiratory viruses in cell lines from each of the different species. <br /> <em>In vivo</em>, we utilised mouse and ferret models to study the antiviral potential of RIG-I agonists against IAV and RSV. In mice, we compared animals which do or do not express a functional Mx1 protein and found that a single prophylactic treatment with RIG-I agonist via the intravenous route resulted in ISG induction in the lungs and this correlated with reduced IAV replication. Of interest, these effects were particularly potent and long-lasting in mice expressing a functional Mx1 confirming an important role of Mx1 for RIG-I agonist-induced protection against IAV. In a mouse model of RSV, we found that a single prophylactic treatment with RIG-I agonist resulted in reduced replication of virus in the lung, as observed using bioluminescence imaging of luciferase-labelled RSV as well as plaque assay for infectious virus. Thus, our studies in mouse models indicate that a single pre-treatment with RIG-I agonists resulted in potent inhibition of two very different respiratory viruses. <br /> In ferrets, after establishing assays to monitor ISG induction in the blood and in airway tissues, we confirmed that a single intravenous injection of RIG-I agonist induced ISG induction in both peripheral blood mononuclear cells (PBMCs) and the lungs. Moreover, a single treatment prior to infection also resulted in reduced replication of both IAV and RSV in ferret lungs, although this treatment had only negligible effects on virus replication in the nasal tissues. A single treatment to animals with an established IAV infection also resulted in reduced virus titres in the lungs, suggesting its potential as a therapeutic antiviral agent. <br /> Myxoma (Mx) proteins are ISGs with potent antiviral effects against IAV. While human and mouse Mx proteins have been studied in detail, ferret Mx proteins have not been characterised. Therefore, we generated different experimental approaches to assess the induction of three endogenous ferret Mx (two splice variants of Mx1 as well as Mx2) in a ferret cell line, as well as <em>in vitro</em> overexpression systems to assess the cellular localisation and antiviral functions of each ferret Mx. Our findings indicate that each ferret Mx localises to the cytoplasm and that particular proteins exhibit antiviral functions against IAV, but not RSV. However, further studies are required to clearly define the antiviral activity of ferret Mx, since our preliminary results indicate that ferret Mx proteins display different antiviral activity following overexpression in human or in ferret cells. <br /> Together, studies described in this thesis demonstrate the potential of RIG-I agonists as antiviral treatments against diverse respiratory viruses both in vitro and in vivo and represent an important step towards the development of novel antiviral treatments in humans.