The viral protein corona directs viral pathogenesis and amyloid aggregation

Artificial nanoparticles accumulate a protein corona layer in biological fluids, which significantly influences their bioactivity. As nanosized obligate intracellular parasites, viruses share many biophysical properties with artificial nanoparticles in extracellular environments and here we show that respiratory syncytial virus (RSV) and herpes simplex virus type 1 (HSV-1) accumulate a rich and distinctive protein corona in different biological fluids. Moreover, we show that corona pre-coating differentially affects viral infectivity and immune cell activation. In addition, we demonstrate that viruses bind amyloidogenic peptides in their corona and catalyze amyloid formation via surface-assisted heterogeneous nucleation. Importantly, we show that HSV-1 catalyzes the aggregation of the amyloid beta-peptide (A beta(42)), a major constituent of amyloid plaques in Alzheimer's disease, in vitro and in animal models. Our results highlight the viral protein corona as an acquired structural layer that is critical for viral-host interactions and illustrate a mechanistic convergence between viral and amyloid pathologies.Peer reviewe

Harnessing the immunoregulatory capacity of single-stranded oligonucleotides to modulate innate immunity

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

Is There a Role for Immunoregulatory and Antiviral Oligonucleotides Acting in the Extracellular Space? A Review and Hypothesis

Here, we link approved and emerging nucleic acid-based therapies with the expanding universe of small non-coding RNAs (sncRNAs) and the innate immune responses that sense oligonucleotides taken up into endosomes. The Toll-like receptors (TLRs) 3, 7, 8, and 9 are located in endosomes and can detect nucleic acids taken up through endocytic routes. These receptors are key triggers in the defense against viruses and/or bacterial infections, yet they also constitute an Achilles heel towards the discrimination between self- and pathogenic nucleic acids. The compartmentalization of nucleic acids and the activity of nucleases are key components in avoiding autoimmune reactions against nucleic acids, but we still lack knowledge on the plethora of nucleic acids that might be released into the extracellular space upon infections, inflammation, and other stress responses involving increased cell death. We review recent findings that a set of single-stranded oligonucleotides (length of 25–40 nucleotides (nt)) can temporarily block ligands destined for endosomes expressing TLRs in human monocyte-derived dendritic cells. We discuss knowledge gaps and highlight the existence of a pool of RNA with an approximate length of 30–40 nt that may still have unappreciated regulatory functions in physiology and in the defense against viruses as gatekeepers of endosomal uptake through certain routes

A Non‐Coding Oligonucleotide Recruits Cutaneous CD11b+ Cells that Inhibit Thelper Responses and Promote Tregs

Abstract Skin‐resident antigen‐presenting cells (APC) play an important role in maintaining peripheral tolerance via immune checkpoint proteins and induction of T regulatory cells (Tregs). However, there is a lack of knowledge on how to expand or recruit immunoregulatory cutaneous cells without causing inflammation. Here, it is shown that administration of a non‐coding single‐stranded oligonucleotide (ssON) leads to CCR2‐dependent accumulation of CD45+CD11b+Ly6C+ cells in the skin that express substantial levels of PD‐L1 and ILT3. Transcriptomic analyses of skin biopsies reveal the upregulation of key immunosuppressive genes after ssON administration. Functionally, the cutaneous CD11b+ cells inhibit Th1/2/9 responses and promote the induction of CD4+FoxP3+ T‐cells. In addition, ssON treatment of imiquimod‐induced inflammation results in significantly reduced Th17 responses. It is also shown that induction of IL‐10 production in the presence of cutaneous CD11b+ cells isolated after ssON administrations is partly PD‐L1 dependent. Altogether, an immunomodulatory ssON is identified that can be used therapeutically to recruit cutaneous CD11b+ cells with the capacity to dampen Th cells

Amelioration of Compound 48/80-Mediated Itch and LL-37-Induced Inflammation by a Single-Stranded Oligonucleotide

Numerous inflammatory skin disorders display a high prevalence of itch. The Mas-related G protein coupled receptor X2 (MRGPRX2) has been shown to modulate itch by inducing non-IgE-mediated mast cell degranulation and the release of endogenous inducers of pruritus. Various substances collectively known as basic secretagogues, which include inflammatory peptides and certain drugs, can trigger MRGPRX2 and thereby induce pseudo-allergic reactions characterized by histamine and protease release as well as inflammation. Here, we investigated the capacity of an immunomodulatory single-stranded oligonucleotide (ssON) to modulate IgE-independent mast cell degranulation and, more specifically, its ability to inhibit the basic secretagogues compound 48/80 (C48/80)-and LL-37in vitroandin vivo. We examined the effect of ssON on MRGPRX2 activationin vitroby measuring degranulation in a human mast cell line (LAD2) and calcium influx in MRGPRX2-transfected HEK293 cells. To determine the effect of ssON on itch, we performed behavioral studies in established mouse models and collected skin biopsies for histological analysis. Additionally, with the use of a rosacea mouse model and RT-qPCR, we investigated the effect on ssON on LL-37-induced inflammation. We reveal that both mast cell degranulation and calcium influx in MRGPRX2 transfected HEK293 cells, induced by the antimicrobial peptide LL-37 and the basic secretagogue C48/80, are effectively inhibited by ssON in a dose-dependent manner. Further, ssON demonstrates a capability to inhibit LL-37 and C48/80 activationin vivoin two mouse models. We show that intradermal injection of ssON in mice is able to block itch induced via C48/80 in a dose-dependent manner. Histological staining revealed that ssON inhibits acute mast cell degranulation in murine skin treated with C48/80. Lastly, we show that ssON treatment ameliorates LL-37-induced inflammation in a rosacea mouse model. Since there is a need for new therapeutics targeting non-IgE-mediated activation of mast cells, ssON could be used as a prospective drug candidate to resolve itch and inflammation in certain dermatoses

Single-Stranded Oligonucleotide-Mediated Inhibition of Respiratory Syncytial Virus Infection

International audienceRespiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in young children. Currently, there is no RSV vaccine or universally accessible antiviral treatment available. Addressing the urgent need for new antiviral agents, we have investigated the capacity of a non-coding single-stranded oligonucleotide (ssON) to inhibit RSV infection. By utilizing a GFP-expressing RSV, we demonstrate that the ssON significantly reduced the proportion of RSV infected A549 cells (lung epithelial cells). Furthermore, we show that ssON’s antiviral activity was length dependent and that both RNA and DNA of this class of oligonucleotides have antiviral activity. We reveal that ssON inhibited RSV infection by competing with the virus for binding to the cellular receptor nucleolin in vitro. Additionally, using a recombinant RSV that expresses luciferase we show that ssON effectively blocked RSV infection in mice. Treatment with ssON in vivo resulted in the upregulation of RSV-induced interferon stimulated genes (ISGs) such as Stat1, Stat2, Cxcl10, and Ccl2. This study highlights the possibility of using oligonucleotides as therapeutic agents against RSV infection. We demonstrate that the mechanism of action of ssON is the inhibition of viral entry in vitro, likely through the binding of the receptor, nucleolin and that ssON treatment against RSV infection in vivo additionally results in the upregulation of ISGs

Single-Stranded Nucleic Acids Regulate TLR3/4/7 Activation through Interference with Clathrin-Mediated Endocytosis

Abstract Recognition of nucleic acids by endosomal Toll-like receptors (TLR) is essential to combat pathogens, but requires strict control to limit inflammatory responses. The mechanisms governing this tight regulation are unclear. We found that single-stranded oligonucleotides (ssON) inhibit endocytic pathways used by cargo destined for TLR3/4/7 signaling endosomes. Both ssDNA and ssRNA conferred the endocytic inhibition, it was concentration dependent, and required a certain ssON length. The ssON-mediated inhibition modulated signaling downstream of TLRs that localized within the affected endosomal pathway. We further show that injection of ssON dampens dsRNA-mediated inflammatory responses in the skin of non-human primates. These studies reveal a regulatory role for extracellular ssON in the endocytic uptake of TLR ligands and provide a mechanistic explanation of their immunomodulation. The identified ssON-mediated interference of endocytosis (SOMIE) is a regulatory process that temporarily dampens TLR3/4/7 signaling, thereby averting excessive immune responses