As the first line of defense, the innate immune system is exceptionally efficacious against invading pathogens but must be tightly regulated to avoid immune-related pathogenesis. Accordingly, the innate immune system recognizes highly conserved components associated with infections and damaged cells using a limited number of pattern-recognition receptors (PRRs), which are differentially expressed across cell types. Importantly, tissue damage and viral infections can induce the release and production of nucleic acids, which can be sensed by nucleic-acid sensing Toll-like-receptors (TLRs), a subset of PRRs located in cellular endosomes that encompass the receptors TLR3,7,8 or 9. It is imperative that these receptors can differentiate “self” nucleic acids from “non-self” in order to avoid autoimmune reactions. However, increasing data has shown that over-active signaling from these receptors can contribute to TLR-mediated inflammatory and auto-immune consequences. 

Previously, a 35 bases long single-stranded oligonucleotide (ssON) was revealed to inhibit TLR3 activation in dendritic cells (DCs). Hence, the research presented in this thesis aims to harness the capacity of certain immunosuppressive oligonucleotides to modulate the innate immune response to inflammation and viral infection. In Paper I we demonstrated that ssON inhibits certain receptor-mediated endocytosis, thereby preventing activation from TLR3/4/7 signaling endosomes Additionally, ssON modulated TLR3-mediated activation in vivo in the skin. Therefore, in Paper II, we further assessed ssON’s effect on prominent symptoms of inflammatory skin disorders. We demonstrated that ssON inhibits non-IgE-mediated degranulation of mast cells induced by certain ligands of the pseudo-allergic receptor MRGPRX2, thereby alleviating the respective downstream itch and inflammation. 

There is currently an urgent need to develop new broad-spectrum antivirals against acute respiratory tract infections, which at present remain the deadliest communicable diseases. Since ssON inhibits clathrin-mediated and caveolin-dependent endocytosis, which are cellular entry pathways utilized by many viruses, we investigated ssON’s potential as an anti-viral agent against two major respiratory viruses. In Paper III we aimed to assess the effect of ssON on influenza A virus (IAV) infection. We demonstrated that ssON inhibits the infection of IAV, and more specifically the pandemic H1N1, in vitro in DCs and reduces viral load and disease in murine models of IAV infection. In Paper IV we aimed to assess the anti-viral capacity of ssON on respiratory syncytial virus (RSV) infection. We discovered that ssON acts as an efficient antiviral agent against RSV in vitro, seemingly by competing with the virus for binding to the entry receptor nucleolin. Additionally, ssON inhibits viral infection in vivo in a murine model of RSV infection, and the combination of ssON treatment and RSV infection leads to an upregulation of immune-related genes in the lungs, which most likely aid in viral clearance.

In summary, the research presented in this thesis uncovers novel immunomodulatory functions of synthetic, noncoding ssONs to modulate the innate immune responses in the context pseudo- allergic itch and inflammation as well as in IAV and RSV infection.  Furthermore, these studies unveil prospective therapeutic possibilities for ssON as an antiviral agent or as a treatment for certain inflammatory dermatoses.

Respiratory syncytial virus (RSV) is an enveloped RNA virus of the Pneumoviridae family. RSV is reported to infect host cells by receptor-mediated endocytosis, also called clathrin-dependent endocytosis. Many reports indicate that the virus utilizes the host receptor nucleolin for entry. RSV is one of the leading causes of acute lower respiratory tract infection in children, the elderly and immunocompromised individuals. It is therefore important to find viable treatments against RSV infections. To this date, the development of an RSV vaccine has unfortunately been unsuccessful. Therefore, a lot of research is instead focusing on developing entry or replication inhibitors against RSV.

We have previously discovered that a 35 bases long single-stranded oligonucleotide (ssON) can inhibit certain endocytic pathways, such as clathrin- and caveolin-dependent endocytosis. Based on this, the overall aim of the projects in this thesis is to investigate if ssON can work as an entry inhibitor against RSV and to study the overall effect that ssON has on RSV infection in vivo in a murine RSV challenge model. Furthermore, we aim to study the effect that proteins present in the surrounding extracellular environment of the virus have on the viral infectivity and pathogenesis.

In Paper I we show that single-stranded oligonucleotides can inhibit RSV infection in vitro and in vivo. We demonstrate that ssON shields nucleolin in vitro. Nucleolin is a receptor involved in RSV entry and by shielding nucleolin present in the cell membrane, ssON prevents RSV from binding to the cells. Furthermore, we show that ssON inhibits RSV infection in vivo. ssON treatment of RSV infection was associated with enhanced expression of RSV-induced Interferon-stimulated genes (ISGs), suggesting that interferon responses likely contributed to reduced RSV infection. As a continuation to Paper I, in Paper II we investigate if small non-coding RNAs (sncRNAs) exhibit the same antiviral capacity as ssON. We demonstrate that sncRNAs, of similar size as ssON, can inhibit RSV infection in vitro and similarly to ssON, they bind to the entry co-receptor nucleolin. In Paper III we study the pathogenesis of RSV. We show that proteins from the extracellular environment of the virus can bind to the virus and affect the viral infectivity as well as the interaction of the virus with host cells. We determine that RSV accumulates a rich and distinctive protein corona in different biological fluids including human plasma (HP), human bronchoalveolar lavage fluid (BALF), non-human primate plasma (MP) and fetal bovine serum (FBS). We show that RSV incubated with BALF has an increased viral infectivity in epithelial HEp-2 and monocyte-derived dendritic cells (moDCs). Moreover, RSV surrounded by a BALF-derived corona induce the activation of moDCs as assessed by increased expression of co-stimulatory molecules.

In summary, the projects in this thesis aim to assess the hypothesis that single-stranded oligonucleotides of DNA or RNA origin can be used to inhibit RSV infection. Furthermore, we aim to determine the effects, in terms of viral infectivity and pathogenesis, of the differential protein compositions present in biological fluids surrounding viruses.