Anti-viral agents derived from alpha dystroglycan

Abstract

The present invention relates to anti-viral agents derived from alpha dystroglycan (a-DG), particularly anti-viral agents comprising a polypeptide or fragment of the N-terminal domain of a-DG or a polypeptide or fragment of the 86 domain of a-DG for use in a method of treatment or prophylaxis of an enveloped virus infection in a subject. The enveloped virus may be any enveloped virus with the exception of influenza A virus. Pharmaceutical compositions comprising said anti-viral agents are also considered.

Description

[0001] ANTI-VIRAL AGENTS DERIVED FROM ALPHA DYSTROGLYCAN

[0002] Field of the Invention

[0003] The present invention relates to anti-viral agents derived from alpha dystroglycan (a-DG), particularly antiviral agents comprising a polypeptide or fragment of the N-terminal domain of a-DG or a polypeptide or fragment of the S6 domain of a-DG for use in a method of treatment or prophylaxis of an enveloped virus infection in a subject. The enveloped virus may be any enveloped virus with the exception of influenza A virus. Pharmaceutical compositions comprising said anti-viral agents are also considered.

[0004] Background

[0005] Enveloped viruses include a number of important human and animal pathogenic viruses such as coronavirus, respiratory syncytial virus (RSV), and dengue virus (DENV). Although there are vaccines against some enveloped viruses, for the majority, few therapeutic or prophylactic treatments are available. For example, Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, has limited therapeutic (as opposed to prophylactic) options.

[0006] Dystroglycan (DG) is an adhesion complex expressed in developing and adult tissues, where its main function is to anchor elements of the extracellular matrix (ECM) to the actin cytoskeleton and increase their stability. DG is present as part of the dystrophin glycoprotein complex in a variety of tissues and cell types, including skeletal and cardiac muscle, epithelial cells or neurons and glia in the central and peripheral nervous system (Barresi & Campbell, 2006; Sciandra et al., 2015). The dystroglycan glycoprotein complex is composed of two subunits: the highly glycosylated extracellular a-subunit (a-DG) and the transmembrane p-subunit (p-DG), that interact non-covalently to form a bridge between the extracellular matrix (ECM) and the actin cytoskeleton. a-DG is a receptor for ECM proteins such as laminins, perlecan, neurexins and agrin. It is composed of a N- and a C-terminal globular domain, separated by an elongated, extensively glycosylated mucin-like region (Adams & Brancaccio, 2015). The proprotein convertase furin cleaves the a-DG N-terminal domain, a-DGN (Fig. 5A), during its maturation in the Golgi apparatus (Kanagawa et al., 2004).

[0007] In contrast to the rest of the protein, a-DGN is minimally glycosylated (Brancaccio et al., 1997) and is an autonomous folding unit that constitutes of an N-terminal Ig-like domain connected by a flexible loop to the ribosomal RNA binding protein S6-like domain (Bozic et al., 2004) (Fig.1 A, right). Whilst the a-DGN domain harboured by a-DG has been shown to be important for a-DG decoration with glycans (Okuma et al., 2023), the physiological function of the cleaved a-DGN is unknown, despite its detection in trace amounts in plasma and cerebrospinal fluids (Hesse et al., 201 1 ; Saito et al., 2008; Saito et al., 201 1 ).

[0008] Cao et al. 1998 identified a-DG as a receptor for lymphocytic choriomeningitis virus (LCMV) and Lassa fever virus (LFV). The authors found that native, fully glycosylated a-DG was capable of blocking LCMV and LFV infections, but that this effect was not seen in vesicular stomatitis virus (VSV). Cao et al. 1998 also reported that arenaviruses Oliveros and Mobala were able to bind a-DG protein, but that this effect was not seen with the arenavirus Guanarito. It has more recently been reported that the N-terminal domain of a-DG (referred to herein as a-DGN) exerts antiviral activity against Influenza A virus in mice (de Greet et al., 2019). However, a-DGN itself has not been shown to be a therapeutic option for other types of enveloped viruses.

[0009] Summary of the Invention

[0010] The present inventors have shown that the N-terminal domain of a-DG is a pan-antiviral protein that blocks a wide range of enveloped viruses including SARS-CoV-2 and dengue virus. The inventors have also shown that a smaller fragment of the N-terminal domain of a-DG, the S6-like domain, can block enveloped viruses.

[0011] Thus, a first aspect provides an anti-viral agent comprising an alpha dystroglycan (a-DG) S6-like domain polypeptide or fragment thereof for use in a method of treatment or prophylaxis of an enveloped virus infection in a subject, where the enveloped virus is not influenza A virus (IAV), and where the anti-viral agent does not comprise one or more of an a-DG mucin-like domain, an a-DG Ig-like 2 domain, or an a- DG C-terminal domain.

[0012] The enveloped virus may, for example, be a coronavirus, respiratory syncytial virus (RSV), dengue virus (DENV), vesicular stomatitis virus (VSV), alphavirus (e.g. Semliki Forest virus (SFV) or Chikungunya virus), or tick-borne encephalitis virus (TBEV).

[0013] A second aspect of the invention provides an anti-viral agent comprising an alpha dystroglycan (a-DG) S6-like domain polypeptide or fragment thereof for use in a method of treatment or prophylaxis of an enveloped virus infection in a subject, wherein the enveloped virus is selected from the group consisting of coronavirus, respiratory syncytial virus (RSV), dengue virus (DENV), vesicular stomatitis virus (VSV), alphavirus (e.g. Semliki Forest virus (SFV) or Chikungunya virus), and tick-borne encephalitis virus (TBEV).

[0014] The coronavirus may be severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome-related coronavirus (MERS-CoV), or a common cold-causing coronavirus, e.g., Human coronavirus 229E (HCoV- 229E). The dengue virus may be dengue virus serotype 1 (DENV1 ), dengue virus serotype 2 (DENV2), dengue virus serotype 3 (DENV3), or dengue virus serotype 4 (DENV4).

[0015] The a-DG S6-like domain polypeptide or fragment thereof may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 6 (human S6-I ike domain sequence) or SEQ ID NO: 15 (murine S6-like domain sequence). In some embodiments, the a-DG S6-like domain polypeptide or fragment thereof consists of an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 6 or SEQ ID NO: 15. The anti-viral agent may further comprise an a-DG Ig-like 1 domain polypeptide or fragment thereof. In some embodiments, the a-DG Ig-like 1 domain polypeptide or fragment thereof comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 4 (human Ig-like 1 domain sequence) or SEQ ID NO: 13 (murine Ig-like 1 domain sequence). In some embodiments, the a-DG Ig-like 1 domain polypeptide or fragment thereof consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 4 or SEQ ID NO: 13.

[0016] The anti-viral agent may further comprise a linker sequence positioned between the Ig-like 1 domain polypeptide or fragment thereof and the S6-I ike domain polypeptide or fragment thereof. In some embodiments, the linker sequence comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 5 (human linker sequence) or SEQ ID NO: 14 (murine linker sequence). In some embodiments, the linker sequence consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 5 or SEQ ID NO: 14.

[0017] The anti-viral agent may comprise the a-DG N-terminal domain, a-DGN. An a-DGN polypeptide may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2 (human a- DGN sequence) or SEQ ID NO: 1 1 (murine a-DGN sequence). In some embodiments, the a-DGN polypeptide consists of an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 1 1 .

[0018] The anti-viral agent may comprise a modification in the linker sequence. In some embodiments, the antiviral agent comprises a modification at position corresponding to R168 of the full-length human sequence or R166 of the full-length murine sequence (i.e. numbered by correspondence with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 10 respectively). The linker may have H at that position, i.e. the modification may correspond to R168H or R166H respectively. The modification described herein may stabilise the anti-viral agent.

[0019] The subject to be treated by the anti-viral agent described herein may be human. In some embodiments, the subject is infected with an enveloped virus or is at risk of being infected with an enveloped virus.

[0020] The anti-viral agent may be administered by any suitable route. In some embodiments, the anti-viral agent is administered by inhalation (e.g. intranasally) or subcutaneously.

[0021] A further aspect provides a pharmaceutical composition comprising the anti-viral agent described herein. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier.

[0022] The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided. Summary of the Figures

[0023] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0024] Figure 1. a-DGN and its inhibitory activity on pseudotyped human coronaviruses.

[0025] A) Left: schematic representation of the dystrophin-glycoprotein complex (DGC). This multiprotein complex, of which dystroglycan (DG) is a central element, anchors the extracellular matrix (ECM) to actin and other components of the cytoskeleton. Extracellular a-DG binds to a series of ECM proteins, such as laminin, while transmembrane p-DG binds the actin cytoskeleton via direct interaction with dystrophin. Also depicted are other intracellular molecules associated with the DGC. The N-terminal of a-DG (a- DGN), highlighted in the dashed circle, is liberated in circulation following cleavage by furin. a-Ct: a-DG C-terminal, a-Nt: a-DG N-terminal. Right: cartoon representation of the 3D-structure of a-DGN (PDB: 1 U2C). Indicated are the two domains (N-term Ig-like and C-term S6-like, the latter enclosed into the full circle) and the flexible undefined loop (FUL) connecting them. B) Alignment of the amino acid sequences of ma-DGN (NCBI code: Q62165, top) and ba-DGN (NCBI code: Q141 18, bottom), showing a degree of identity of -93% (-97% homology). The sequences of the Ig-like and S6-like domains are boxed in light grey and dark grey, respectively. Amino acid conservation code: (*) identity, (:) strong similarity, (.) weak similarity. C) SDS-PAGE of the recombinant a-DGN proteins (indicated over each lane) used in this study, as final products of the purification procedure. D) Recombinant a-DGN urea-induced denaturation. Unfolding curves were measured by intrinsic tryptophan fluorescence spectroscopy and fitted to a two- state linear extrapolation model. The mid-point of the transitions (Cm), 2.8M for ma-DGN, 2.7M for ha- DGN and 1 .8M for the shortened version mS6, are indicative of stable polypeptides. E) Dose-response curves for ma-DGN, / ?a-DGN or mS6 pre-treated Caco-2 cells infected with VSV pseudotyped particles expressing the Spike protein from the coronaviruses indicated on top of each panel. Infection was quantified by measuring GFP-positive cells (pseudotyped particles carry a GFP reporter gene) and data are expressed as % infection normalized to an unrelated protein control (means ± SD). IC50 values (from n=3 independent experiments) were calculated using nonlinear regression calculations.

[0026] Figure 2. aDGN blocks SARS-CoV-2 in epithelial cells and human primary gut organoids.

[0027] A) Dose dependent inhibitory activity of ma-DGN (1 pM-10pM) against SARS-CoV-2 infection in HEK293T cells expressing human ACE2 and TMPRSS2. The graph shows the % inhibition of SARS-CoV-2 normalised to the untreated control (means + SD, n=3). B) a-DGN inhibits infection of cells from different SARS-CoV-2 variants, as indicated. VeroE6-TMPRSS2 cells were pre-treated with 10 pM ma-DGN before infection with SARS-CoV-2 Wuhan, Gamma, Delta, Omicron BA.1 or Omicron BA.2 (MOI=0.1 ). Infection was quantified using viral N protein staining (n=3 independent experiments). Bars are means + SEM. p values were determined by one-way ANOVA with multiple comparison w.r.t. control. p***<0.001 , p**<0.01 . C) Left: Inhibitory activity of ma-DGN when pre-incubated with SARS-CoV-2 for 45 min, added at the same time as SARS-CoV-2 or added 2 hours post infection with SARS-CoV-2. After a 16 hours incubation infection was determined by N protein staining. Results are represented as % inhibition normalised to the control (means + SD). Right: ma-DGN inhibits SARS-CoV-2 binding to cells. HeLa ACE-2 cells were preincubated with 10 pM ma-DGN or control before addition of SARS-CoV-2 for 60 min in the cold and fixed. SARS-CoV-2 binding per cell after treatment with ma-DGN (black) or control (grey) was quantified for >100 cells per condition. Significance was determined using a Mann-Whitney test (n=3 independent experiments), p****<0.0001 .

[0028] . D) ma-DGN administered at the same time as SARS-CoV-2 blocks infectivity in gut organoids derived from human primary duodenum tissue. Primary gut organoids were treated with 1 or 10 pM of ma-DGN or vehicle (PBS) control and infected with SARS-CoV-2 at an MOI of 1 for 48h before fixation. The graph shows the percentage reduction of infected (double stranded RNA-positive) organoids upon ma-DGN treatment, as normalised to the vehicle treated control (means + SD, n=4).

[0029] Figure 3. aDGN inhibits SARS-CoV-2 infection in humanised ACE2 mice.

[0030] A) ma-DGN was administered to K18-hACE2 mice at 7.5 pg / mouse or 0.75 pg / mouse at the same time as the lethal dose of the challenging virus (5x104TCID50) via the intranasal (IN) route. Mice were then observed daily for B) survival and C) weight loss. D) Lung viral loads as determined at endpoint. Statistical significance was determined using a Mann Whitney test, p*<0.05. ns: not significant. E) ma- DGN or / ?a-DGN at 7.5 pg / mouse were administered via the IN route 2 hours prior virus challenge and then daily for 3 days. Mice were then observed daily for F) survival and G) weight loss. H) Lung viral loads as determined at endpoint. Statistical significance was determined using a Mann Whitney test, p** <0.01.

[0031] Figure 4. maDGN inhibits enveloped viruses.

[0032] A-F) Inhibitory activity of ma-DGN in infection assays against A) IAV, B) RSV, C) SFV, D) TBEV, E) VSV and F) AdV5 (a non-enveloped virus). Infection was analysed by immunostaining and results are represented as % inhibition normalised to the untreated control. G-J) Inhibitory activity of ma-DGN against the 4 Dengue Virus serotypes G) DENV1 , H) DENV2, I) DENV3 and J) DENV4 in cell assays. DENV RNA was analysed by qRT-PCR. DENV copies were normalised to the internal control protein TBP. Values are means +SD. Statistical significance was determined using an unpaired t-test. ns = not significant, p* < 0.1 , p** <0.01 , p*** <0.001 . K) a-DGN inhibits infection of human primary monocytes by DENV4. Human CD14+ monocytes were pre-treated with ma-DGN for 2h prior to infection with DENV4 at an MOI of 10. Inoculum was washed off after 2h and fresh ma-DGN added. After 48h supernatant was harvested and the viral titer determined by plaque assay. Results are represented as plaque titers (102) of n=6 replicates (means + SD).

[0033] Figure 5. Recombinant a-DGN properties.

[0034] A) Domain structure of recombinant mouse a-DG (ma-DG), from N-terminus (left) to C-terminus (right). SP: signal peptide, Ig-like: immunoglobulin-like domain, S6: ribosomal protein S6-like domain, arrow: furin cleavage site, mucin-like: highly glycosylated central domain of a-DG, C-term: C-terminal portion. The amino acid sequence of the N-terminal domain a-DGN (composed of the Ig-like domain 1 , linker, and S6- like domain) as cloned and expressed in this study, is indicated below the cartoon. The sequence of the Ig-like domain is underlined; the linker sequence is in italics; the sequence of the S6-like domain is shown in bold. Heterologous residues (Gly-Ser) inserted at the N-terminus of recombinant ma-DGN for cloning purposes are circled; the R166H stabilising mutation is in bold italics. B) Electrospray Ionization Liquid Chromatography Mass Spectrometry profile of ma-DG. The recombinant product runs as a single, sharp peak with a MW of 28527Da, matching the MW calculated based on the amino acid sequence (28528Da).

[0035] Detailed Description of the Invention

[0036] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0037] The present inventors have demonstrated that a recombinantly produced N-terminal fragment of a- dystroglycan (a-DG), referred to as a-DGN herein, blocks infection of SARS-CoV-2 in cell culture, human primary gut organoids and lungs of transgenic mice expressing the human receptor angiotensin I- converting enzyme 2 (hACE2). Prophylactic and therapeutic administration of a-DGN reduced SARS- CoV-2 lung titres and protected the mice from clinical respiratory symptoms and death. Recombinant a- DGN was also shown to block, besides different variants of SARS-CoV-2, a wide range of enveloped viruses including the four Dengue virus serotypes, influenza A virus, respiratory syncytia virus, tick-borne encephalitis virus, but not human adenovirus, a non-enveloped virus. Thus, a-DGN has been shown to be a natural candidate therapeutic against enveloped viruses.

[0038] The present inventors have also demonstrated that a smaller fragment of a-DGN, the S6-like domain, is capable of blocking human coronaviruses with a potency comparable to full length a-DGN. Thus, the S6- like domain of a-DG is believed to have the same or substantially similar activity to that shown with a- DGN.

[0039] Dystroglycan is a protein that is encoded by the DAG1 gene in humans. Dystroglycan functions as a transmembrane linkage between the extracellular matrix (ECM) and the cytoskeleton. Dystroglycan is believed to play a role in mediating cell-extracellular matrix communications in a variety of cell types. It is considered to have a role in cell function, including adhesion, integrity of the sarcolemma, neurological development, basement membrane assembly, and epithelial polarisation and morphogenesis.

[0040] Dystroglycan is a complex of two subunits, namely, a-dystroglycan (a-DG) and p-dystroglycan (p-DG). a- DG is extracellular and is highly glycosylated. p-DG is a transmembrane protein which interacts non- covalently with a-DG. a-DG is heavily glycosylated and its glycans are considered to play a role in binding to laminin. a-DG comprises an N-terminal globular domain, a mucin-like domain, and a C-terminal globular domain. The mucin-like domain is the region of a-DG which is highly glycosylated. Typically, the mucin-like domain is highly glycosylated with very specific, long chains of O-mannosyl glycans, collectively called matriglycan. After glycosylation of the mucin-like domain has occurred during processing of a-DG, the N-terminal globular domain is typically cleaved from the remainder of the protein (between the S6-like domain and the mucin-like domain) by furin-like protease.

[0041] The N-terminal domain of a-DG is referred to herein as a-DGN. As shown in Figure 5A, a-DGN comprises (in the N-terminal to C-terminal direction) an immunoglobulin-like domain (“Ig-like 1 ” domain), and ribosomal protein S6-like domain. a-DGN is minimally glycosylated and it is an autonomous folding unit composed of the N-terminal Ig-like domain connected by a flexible loop to the ribosomal RNA binding protein S6-like domain.

[0042] In this specification, the term “alpha-dystroglycan” or “a-DG” refers to the alpha subunit of dystroglycan. The alpha subunit of dystroglycan is composed of an N-terminal globular domain, a mucin-like domain, and a C-terminal globular domain. More specifically, a-DG comprises a signal peptide, an Ig-like 1 domain, an S6-like domain, a mucin-like domain, an Ig-like 2 domain, and a C-terminal domain.

[0043] The “N-terminal domain of a-DG” (also referred to as the “N-terminal globular domain of a-DG”, and “a- DGN”) is the N-terminal fragment of alpha-dystroglycan, composed of an Ig-like 1 domain (e.g., SEQ ID NO: 4 or SEQ ID NO: 13), a flexible loop / linker sequence (e.g., SEQ ID NO: 5 or SEQ ID NO: 14) and an S6-like domain (e.g., SEQ ID NO: 6 or SEQ ID NO: 15). The a-DGN polypeptide sequence may be defined by SEQ ID NO: 2 or SEQ ID NO: 1 1 . The N-terminal domain of a-DG as described herein excludes the alpha dystroglycan signal peptide.

[0044] The “S6-like domain” (also referred to herein as the “S6 domain” of alpha-dystroglycan a-DG, so called because of its similarity to ribosomal protein S6) is located in the N-terminal domain of a-DG (a-DGN). Within the context of the invention, it may therefore be considered as a fragment, portion or sub-domain of either a-DG or a-DGN.

[0045] Table 1. Sequences

[0046] Anti-viral agents

[0047] A first aspect of the invention provides an anti-viral agent comprising an alpha dystroglycan (a-DG) S6- like domain polypeptide or fragment thereof for use in a method of treatment or prophylaxis of an enveloped virus infection in a subject, where the enveloped virus is not influenza A virus (I AV), and where the anti-viral agent does not comprise one or more of an a-DG mucin-like domain, an a-DG Ig-like 2 domain, or an a-DG C-terminal domain.

[0048] Thus, the anti-viral agent according to the first aspect does not encompass full length a-DG. The antiviral agent may comprise any one or any two domains selected from an a-DG mucin-like domain, an a- DG Ig-like 2 domain, and an a-DG C-terminal domain. It does not comprise all three of an a-DG mucinlike domain, an a-DG Ig-like 2 domain, and an a-DG C-terminal domain.

[0049] In some embodiments, at least in its mature form, the anti-viral agent does not comprise an a-DG signal peptide. In some embodiments, the only a-DG sequences present in the anti-viral agent are from a- DGN, i.e. it contains an S6-like domain and optionally an Ig-like 1 domain, but not an a-DG mucin-like domain, an a-DG Ig-like 2 domain, or an a-DG C-terminal domain.

[0050] A mucin-like domain polypeptide may comprise or consist of an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 7 or SEQ ID NO: 16. In some embodiments, a mucin-like domain polypeptide may comprise or consist of an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 7 or SEQ ID NO: 16. An Ig-like 2 domain polypeptide may comprise or consist of an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 8 or SEQ ID NO: 17. In some embodiments, an Ig-like 2 domain polypeptide may comprise or consist of an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 8 or SEQ ID NO: 17.

[0051] A C-terminal domain polypeptide may comprise or consist of an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 9 or SEQ ID NO: 18. In some embodiments, a C-terminal domain polypeptide thereof may comprise or consist of an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 9 or SEQ ID NO: 18.

[0052] A second aspect of the invention provides an anti-viral agent comprising an alpha dystroglycan (a-DG) S6-like domain polypeptide or fragment thereof for use in a method of treatment or prophylaxis of an enveloped virus infection in a subject, where the enveloped virus is selected from the group consisting of: coronavirus, respiratory syncytial virus (RSV), dengue virus (DENV), vesicular stomatitis virus (VSV), Semliki Forest virus (SFV), and tick-borne encephalitis virus (TBEV).

[0053] The anti-viral agent according to the second aspect may comprise additional a-DG sequences (e.g., one or more domains selected from an Ig-like 1 domain, a mucin-like domain, an Ig-like 2 domain, and / or a C- terminal domain). Thus it may comprise an S6-like domain and an Ig-like 1 domain. It may comprise an S6-like domain, an Ig-like 1 domain, an a-DG mucin-like domain, an a-DG Ig-like 2 domain, and an a-DG C-terminal domain. In some embodiments, at least in its mature form, the anti-viral agent does not comprise an a-DG signal peptide.

[0054] For both aspects of the invention, the a-DG S6-like domain polypeptide or fragment thereof may comprise or consist of an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 6 or SEQ ID NO: 15. In some embodiments, the a-DG S6-I ike domain polypeptide or fragment thereof comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6 or SEQ ID NO: 15. In some embodiments, the S6-like domain polypeptide or fragment thereof comprises or consists of an amino acid sequence of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, or 135 amino acids in length. In some embodiments, the amino acid sequence is no more than 135 amino acids in length. In some embodiments, the a-DG S6-like domain polypeptide is 135 amino acids in length. In some embodiments, the a-DG S6-like domain polypeptide consists of SEQ ID NO: 6.

[0055] In some embodiments, the anti-viral agent further comprises an a-DG Ig-like 1 domain polypeptide or fragment thereof. In some embodiments, the a-DG Ig-like 1 domain polypeptide or fragment thereof comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 4 or SEQ ID NO: 13. In some embodiments, the a-DG Ig-like 1 domain polypeptide or fragment thereof comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4 or SEQ ID NO: 13. In some embodiments, the a-DG Ig-like 1 domain polypeptide or fragment thereof comprises or consists of an amino acid sequence of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, or 111 amino acids in length. In some embodiments, the amino acid sequence is no more than 111 amino acids in length. In some embodiments, the a-DG Ig-like 1 domain polypeptide is 111 amino acids in length. In some embodiments, the a-DG Ig-like 1 domain polypeptide consists of SEQ ID NO: 4. The a-DG Ig-like 1 domain polypeptide or fragment thereof may be linked (directly or indirectly) to the N- terminus of the a-DG S6-like domain polypeptide or fragment thereof. The C-terminus of the a-DG Ig-like 1 domain polypeptide or fragment thereof may be linked (directly or indirectly) to the N-terminus of the a- DG S6-like domain polypeptide or fragment thereof.

[0056] In some embodiments, the anti-viral agent further comprises a linker peptide or sequence. The linker sequence may be a flexible loop connecting the Ig-like domain polypeptide or fragment thereof and the S6-like domain polypeptide or fragment thereof as described herein. The N-terminus of the linker sequence may be linked (directly or indirectly) to the C-terminus of a-DG Ig-like 1 domain polypeptide or fragment thereof and / or the C-terminus of the linker sequence may be linked (directly or indirectly) to the N-terminus of a-DG S6-like domain polypeptide or fragment thereof. Thus, the linker sequence may be positioned between the S6-like domain polypeptide or fragment thereof and the Ig-like 1 domain polypeptide or fragment thereof. In some embodiments, the linker sequence comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 14. In some embodiments, the linker sequence comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 14. In some embodiments, the linker sequence comprises or consists of an amino acid sequence of at least 5, 10, 15, 16, 17, or 18 amino acids in length. In some embodiments, the amino acid sequence is no more than 18 amino acids in length. In some embodiments, the linker sequence is 18 amino acids in length. In some embodiments, the linker sequence consists of SEQ ID NO: 5 or SEQ ID NO: 14. As discussed further below, the linker may comprise a stabilising substitution at the residue corresponding to arginine 6 of SEQ ID NO: 5 or SEQ ID NO: 14 (R168 of full length human a-DG or R166 of full length murine a-DG), which may be a histidine residue. Such a substitution may be designated R6H, R168H or R166H depending on which reference sequence is being used for numbering.

[0057] In some embodiments, the anti-viral agent is a polypeptide or peptide. The peptide or polypeptide may comprise the a-DG S6-like domain polypeptide or fragment thereof or the a-DGN polypeptide or fragment thereof as described herein. The anti-viral agent may comprise or consist of an amino acid sequence at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids in length. The amino acid sequence may be no more than 500 amino acids in length.

[0058] In some embodiments, the anti-viral agent comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 6 or SEQ ID NO: 15. In some embodiments, the anti-viral agent comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6 or SEQ ID NO: 15. In some embodiments, anti-viral agent comprises or consists of an amino acid sequence of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, or 135 amino acids in length. In some embodiments, the amino acid sequence is no more than 135 amino acids in length. In some embodiments, the anti-viral agent is 135 amino acids in length. In some embodiments, the anti-viral agent comprises SEQ ID NO: 6. In some embodiments, the anti-viral agent consists of SEQ ID NO: 6.

[0059] In some embodiments, the anti-viral agent comprises or consists of an a-DGN polypeptide or fragment thereof. The fragment thereof may minimally comprise the S6-like domain polypeptide or a fragment thereof, in particular the S6-like domain polypeptide. In some embodiments, the a-DGN polypeptide or fragment thereof comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 11. In some embodiments, the a-DGN polypeptide or fragment comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 11. In some embodiments, the a-DGN polypeptide or fragment thereof comprises or consists of an amino acid sequence of at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 260, 261 , 262, 263 or 264, amino acids in length, but may be up to 270, 275, 280, 281 , 282, 283, 284, 285, or 286 amino acids in length. In some embodiments, the amino acid sequence is no more than 264, 265 or 270 amino acids in length. In some embodiments, the a-DGN polypeptide is 264 amino acids in length. In some embodiments, the a-DGN polypeptide consists of SEQ ID NO: 2 or SEQ ID NO: 11 . As discussed further below, the a-DGN polypeptide or fragment thereof may comprise a stabilising substitution in the linker region at the residue corresponding to the underlined arginine residue in SEQ ID NO: 2 or SEQ ID NO: 11 above (corresponding to R6 of the linker region, R168 of full length human a-DG, or R166 of full length murine a-DG), which may be a histidine residue.

[0060] It will be understood that “the a-DGN polypeptide or fragment thereof” as used herein refers to the mature form of the protein, without signal peptide, unless the context requires otherwise.

[0061] Percent (%) amino acid sequence identity with respect to a reference sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Various known tools can be used to measure sequence identity, including but not limited to Clustal Omega, Multiple Sequence Alignment (EMBL-EBI). In some embodiments, the % sequence identity is over the length of the polypeptide or the fragment.

[0062] The term “peptide” is used herein to refer to short chains of amino acids consisting of 40 or fewer amino acids linked by peptide bonds. The term “polypeptide” is used herein to refer to large biomolecules, or macromolecules, consisting of one or more long chains of amino acid residues, each being more than 40 amino acids in length. In some embodiments, the peptide or polypeptide is isolated. In some embodiments, the peptide or polypeptide is soluble. The anti-viral agents described herein are capable of inhibiting binding of an enveloped virus to a host cell (e.g., a cell of a subject) and / or infection of the host cell by the virus. In some embodiments, the anti-viral agent are capable of inhibiting binding of an enveloped virus to a receptor site on a host cell.

[0063] In this context, inhibition includes reduction of an enveloped virus binding to a host cell, or infection of a host cell type, to any degree. In other words, binding of an enveloped virus to a host cell may be reduced by around 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% when treated with the anti-viral agent compared to binding of the same virus to an equivalent host cell under otherwise equivalent conditions in the absence of the anti-viral agent.

[0064] Inhibition of enveloped virus binding to a host cell, or inhibition of infection, may be measured in vitro or in vivo by any suitable methodology. In vitro, binding or infection may be assessed by quantifying levels of viral transcript in a cell population (e.g., by using qRT-PCR) by viral plaque assays, or by direct visualisation of bound or infected cells. Visualisation may be performed, for example, by using labelled (such as fluorescently labelled) antibodies directed against viral proteins or other markers of viral infection such as double stranded RNA, or even by using viral particles which are themselves fluorescently labelled. In vivo, viral load of an infected tissue may be determined by any suitable means. The skilled person will be capable of designing a suitable assay depending on the virus in question. Binding to, or infection of, a target cell or tissue type may for example be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% lower in a host cell or tissue treated with the anti-viral agent compared to an untreated equivalent host cell or tissue.

[0065] Modified anti-viral agents:

[0066] The anti-viral agents described herein may further comprise one or more modifications. In some embodiments, the anti-viral agent comprises a stabilising modification.

[0067] The modification may be an amino acid modification. As already discussed above, where the anti-viral agent comprises a linker sequence, e.g. comprising or consisting of SEQ ID NO: 5 or SEQ ID NO: 14, or having homology thereto, the modification may be a modification of the arginine residue at position 6 of SEQ ID NO: 5 or SEQ ID NO: 14, corresponding to R168 of full length human a-DG (SEQ ID NO: 1 ) or R166 of full length murine a-DG (SEQ ID NO: 10). In some embodiments, the residue present at that position is a histidine. Thus the modification may be designated R6H, R168H or R166H depending on which reference sequence is used for numbering.

[0068] Without wishing to be bound by theory it is believed that the modification prevents the action of proteases (such as thrombin) on the anti-viral agent (Bozic et al., 1998). The modification may prevent proteolytic degradation and therefore increase the stability of the anti-viral agent.

[0069] The modification may be the addition of a heterologous moiety. In some embodiments, the anti-viral agent further comprises a heterologous moiety. The heterologous moiety may increase the stability of the agent. The heterologous moiety increases the in vivo half-life of the agent, e.g., the serum half-life of the agent. The heterologous moiety may also be capable of masking the peptide from the host immune system, for example, polyethylene glycol (PEG) or modified PEG.

[0070] The heterologous moiety may be covalently attached to the a-DG S6-like domain polypeptide or fragment thereof or said a-DGN polypeptide or fragment thereof described herein. The heterologous moiety may be non-covalently attached to the a-DG S6-like domain polypeptide or fragment thereof or said a-DGN polypeptide or fragment thereof described herein.

[0071] In some embodiments, the heterologous moiety is a recombinant polypeptide or peptide sequence. The heterologous moiety may be defined as a polypeptide or peptide which is not present in combination (i.e. , not linked to, directly or indirectly) with a-DGN or any of its domains or sequences as described herein in nature. In some embodiments, the heterologous moiety is a non-a-DGN sequence (for example, an a- DG mucin-like domain sequence, an a-DG Ig-like 2 domain sequence, and / or an a-DG C-terminal domain sequence). The heterologous moiety may also be defined as a polypeptide or peptide which is not present in combination (i.e., not linked to, directly or indirectly) with a-DG or any its domains or sequences as described herein in nature. In some embodiments, the peptide heterologous moiety is a non-a-DG peptide sequence.

[0072] In some embodiments, where the heterologous moiety is a polypeptide or peptide, said polypeptide or peptide is a fusion protein with (i.e. part of the same peptide chain as) a peptide or polypeptide comprising said a-DG S6-like domain polypeptide or fragment thereof or said a-DGN polypeptide or fragment thereof as described herein. In other words, a recombinant sequence comprising a heterologous moiety polypeptide or peptide may be fused to a peptide or polypeptide comprising the a- DG S6-like domain polypeptide or fragment thereof or the a-DGN polypeptide or fragment thereof. Nonlimiting example polypeptides include an immunoglobulin Fc region or functional fragment fragment thereof, serum albumin and transferrin. The heterologous moiety may be fused to the N-terminus of the peptide or polypeptide comprising said a-DG S6-like domain polypeptide or fragment thereof or said a- DGN polypeptide or fragment thereof as described herein. Alternatively, the heterologous moiety may be fused to the C-terminus of the peptide or polypeptide comprising said a-DG S6-like domain polypeptide or fragment thereof or said a-DGN polypeptide or fragment thereof as described herein.

[0073] The heterologous moiety may also be a carbohydrate moiety. Non-limiting examples include dextran, glycosylation, polysialylation, hydroxyethylation (HESylation), heparosanylation (HEPylation), and hyaluronic acid polysaccharide (HAylation).

[0074] The heterologous moiety may be a polymer. A non-limiting example includes polyethylene glycol (PEG). The heterologous moiety may be a lipid. Without wishing to be bound by theory, it is believed that a lipid modification improves drug half-life by enabling the binding of albumin that is present in the blood of a subject. Non-limiting examples include laurate, myristate, and palmitate.

[0075] Any heterologous moiety known in the art for use with therapeutic proteins may be used. The heterologous moiety as described herein may be attached directly or indirectly.

[0076] The anti-viral agent described herein is for use in a method of treatment or prophylaxis of an enveloped virus infection in a subject.

[0077] An “enveloped virus” as described herein is a virus comprising an envelope, a lipid bilayer membrane on the outer part of the virus. An enveloped virus may also comprise a capsid, a protein shell encasing the viral genome. The envelope is typically derived from portions of the host cell membranes (phospholipids and proteins) but may also include some (viral) glycostructures such as glycoproteins and / or glycooligopeptides. Viral envelopes are believed to help viruses enter host cells and may also help them to avoid the host immune system. Glycostructures such as glycoproteins and / or glycooligopeptides on the surface of the envelopes are considered to serve to identify and bind to receptor sites on the host's membrane. After identifying and binding to a receptor site, the viral envelope is capable of fusing with the host's membrane, allowing the capsid and viral genome to enter and infect the host.

[0078] The first aspect of the present invention is directed to treating an infection in a subject infected with an enveloped virus. An enveloped virus within the context of the infection may be any virus comprising a lipid bilayer membrane (the “envelope”), with the exception of influenza A virus. The enveloped virus may be an enveloped DNA virus, an enveloped RNA virus, or an enveloped retrovirus. A DNA virus is a virus that has a DNA genome. The DNA may be double stranded (dsDNA) or single stranded (ssDNA). In some embodiments, the enveloped virus is an enveloped double stranded DNA virus. In some embodiments, the enveloped virus is an enveloped single stranded DNA virus. An RNA virus is a virus, other than a retrovirus, that has an RNA genome. The RNA is typically single stranded (ssRNA) but may also be double stranded (dsRNA). In some embodiments, the enveloped virus is an enveloped double stranded RNA virus. In some embodiments, the enveloped virus is an enveloped single stranded RNA virus. A retrovirus is a type of virus that inserts a DNA copy of its RNA genome into the DNA of a host cell that it invades. Briefly, the virus has its own reverse transcriptase making it capable of producing DNA from its RNA genome.

[0079] The enveloped virus infection as described herein does not include an infection with influenza A virus (IAV). Thus, the enveloped viruses described within the context of the invention do not include influenza A virus (IAV), including variants and subtypes. Example subtypes include, but are not limited to, subtype A(H1 N1 ) (also known as A(H1 N1 )pdm09) and A(H3N2) influenza viruses.

[0080] The enveloped viruses may be selected from the group consisting of coronavirus, respiratory syncytial virus (RSV), dengue virus (DENV), vesicular stomatitis virus (VSV), alphavirus (e.g. Semliki Forest virus (SFV) or Chikungunya virus), or tick-borne encephalitis virus (TBEV).

[0081] Other known enveloped viruses that may be treated using the anti-viral agent according to the first aspect of the invention, include but are not limited to, Zika (ZIKV), Ebola (EBOV), Hepatitis B and C (HBV, HCV), Human Immunodeficiency Virus (HIV) and Yellow fever virus (YFV). An infection by any of these enveloped viruses may also be treated by the anti-viral agent according to the first aspect of the invention.

[0082] The second aspect of the invention is directed to treating an infection in a subject infected with an enveloped virus infection in a subject, where the enveloped virus is selected from the group consisting of: coronavirus, respiratory syncytial virus (RSV), dengue virus (DENV), vesicular stomatitis virus (VSV), Semliki Forest virus (SFV), and tick-borne encephalitis virus (TBEV).

[0083] The enveloped virus may be a coronavirus. Coronaviruses are a group of related RNA viruses. They are enveloped viruses with a positive-sense single-stranded RNA genome and a nucleocapsid of helical symmetry. The genome size of coronaviruses typically ranges from approximately 26 to 32 kilobases. The viral envelope is made up of a lipid bilayer comprising membrane protein, envelope proteins and spike proteins. Typically, the spike protein is believed to interact with the host cell.

[0084] The coronavirus may be severe acute respiratory syndrome coronavirus (SARS-CoV). SARS-CoV is known to cause SARS, a respiratory disease associated with symptoms such as fever, muscle pain, lethargy, cough, sore throat. The coronavirus may be severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is believed to be closely related to SARS-CoV. It is known to cause coronavirus disease 2019 (COVID-19). SARS-CoV-2 is the causative agent of the COVID-19 pandemic, for which therapeutic options are limited. Symptoms associated with COVID-19 include fever, cough, headache, fatigue, breathing difficulties, loss of smell, and loss of taste. Also anticipated are known variants SARS-CoV-2, for example the alpha variant, the beta variant, and the delta variant. The coronavirus may be Middle East respiratory syndrome-related coronavirus (MERS-CoV). MERS-CoV is known to cause MERS, a respiratory disease associated with symptoms such as fever, cough, diarrhoea, and shortness of breath. The coronavirus may be a coronavirus associated with mild disease (e.g., “common cold-causing coronavirus”), for example, Human coronavirus OC43 (HCoV-OC43), Human coronavirus HKU1 (HCoV-HKU1 ), Human coronavirus 229E (HCoV-229E), or Human coronavirus NL63 (HCoV-NL63). Accordingly, in some embodiments, the coronavirus is selected from the group consisting of: Severe acute respiratory syndrome coronavirus (SARS-CoV), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome-related coronavirus (MERS-CoV), Human coronavirus OC43 (HCoV-OC43), Human coronavirus HKU1 (HCoV-HKU1 ), Human coronavirus 229E (HCoV-229E) and Human coronavirus NL63 (HCoV-NL63).

[0085] The enveloped virus may be respiratory syncytial virus (RSV). RSV, also known as human respiratory syncytial virus (hRSV) and human orthopneumovirus, is a virus that typically causes infections of the respiratory tract. It is a negative-sense, single-stranded RNA virus. RSV infection can present with a wide range of symptoms from mild upper respiratory tract infections (URTI) to severe and potentially lifethreatening lower respiratory tract infections (LRTI), which typically require hospitalisation and mechanical ventilation. It is known to cause bronchiolitis in infants, common colds in adults and more severe respiratory illnesses in the elderly or immunocompromised. RSV is divided into two antigenic subtypes, A and B. These subtypes are based on the reactivity of proteins present in the lipid envelope (F and G proteins) to monoclonal antibodies. RSV subtype A (RSVA) is considered to be more virulent than RSV subtype B (RSVB). In some embodiments, RSV is RSV subtype A (RSVA). In other embodiments, RSV is RSV subtype B (RSVB).

[0086] The enveloped virus may be dengue virus (DENV). DENV is the cause of dengue fever. It is typically mosquito-borne, and its prevalence has increased dramatically within the last 20 years, now becoming one of the worst mosquito-borne human pathogens in tropical countries. Dengue fever is also known as breakbone fever, vomiting and dandy fever. Dengue haemorrhagic fever and dengue shock syndrome are the severe forms of the disease. Dengue fever is typically associated with symptoms such as severe headache, retro-orbital pain, muscle / joint / bone pain, macular or maculopapular rash, and minor hemorrhagic manifestations, including petechiae, ecchymosis, purpura, epistaxis, bleeding gums, or hematuria. DENV is a single positive-stranded RNA virus. Four serotypes of DENV are known, these are dengue virus serotype 1 (DENV1 ), dengue virus serotype 2 (DENV2), dengue virus serotype 3 (DENV3), or dengue virus serotype 4 (DENV4). In some embodiments, DENV is selected from the group consisting of dengue virus serotype 1 (DENV1 ), dengue virus serotype 2 (DENV2), dengue virus serotype 3 (DENV3), and dengue virus serotype 4 (DENV4).

[0087] The enveloped virus may be yellow fever virus, which is a single stranded RNA virus in the family Flaviviridae, similar to Dengue virus.

[0088] The enveloped virus may be vesicular stomatitis virus (VSV). VSV, also known as Indiana vesiculovirus or Vesicular stomatitis Indiana virus (VSIV) is a virus that leads to a flu-like virus in humans. It is also known to infect insects, cattle, horses, and pigs. In animals, VSV leads to an oral disease which typically appears as mucosal vesicles and ulcers in the mouth, as well as the udder and coronary band. In humans, VSV infection is typically associated with symptoms such as fever, headache, myalgia, weakness and occasionally vesicular lesions of the mouth.

[0089] The enveloped virus may be an alphavirus, such as Semliki Forest virus (SFV). SFV is so called because it was first isolated from mosquitoes in the Semliki Forest in Uganda. SFV is an alphavirus and has a positive-strand RNA with a genome of approximately 13,000 base pairs which encodes nine proteins. The virus is typically spread by mosquitos and can infect humans. Symptoms in humans may include fever, myalgia, and arthralgia. Other alphaviruses include Chikungunya virus, a positive-stranded RNA virus diffused in Africa and Asia, although outbreaks have been reported in Europe and the Americas. The virus is typically spread by mosquitos and can infect humans, with symptoms that include fever, headache, muscle pain, joint swelling and skin rashes, with a risk of death in approximately 1 in 1000 cases.

[0090] The enveloped virus may be tick-borne encephalitis virus (TBEV). TBEV is known to cause tick-borne encephalitis, a disease involving the central nervous system, which typically manifests as meningitis, encephalitis, or meningoencephalitis. TBEV is a positive-strand RNA virus, with a genome approximately 11kb in size. It is known to infect a range of hosts including ruminants, birds, rodents, carnivores, horses, and humans. The disease can also be spread from animals to humans, with ruminants and dogs typically providing the principal source of infection for humans. There are three subtypes of TBEV, which are: Western European subtype (also known as Central European encephalitis virus (CEEV)), Siberian subtype (also known as West Siberian virus), and Far Eastern subtype (also known as Russian Spring Summer encephalitis virus (RSSEV). In some embodiments, TBEV is selected from the group consist of the Western European subtype of TBEV, the Siberian subtype of TBEV, and the Far Eastern subtype of TBEV.

[0091] Medical use

[0092] The first aspect of the invention provides an anti-viral agent comprising an alpha dystroglycan (a-DG) S6- like domain polypeptide or fragment thereof for use in a method of treatment or prophylaxis of an enveloped virus infection in a subject, where the enveloped virus is not influenza A virus (IAV) and where the anti-viral agent does not comprise an a-DG mucin-like domain, an a-DG Ig-like 2 domain, and / or an a- DG C-terminal domain.

[0093] The invention further provides a method of treatment or prophylaxis of an enveloped virus infection in a subject, comprising administering an anti-viral agent comprising an alpha dystroglycan (a-DG) S6-I ike domain polypeptide or fragment to a subject infected with or at risk of being infected with an enveloped virus, where the enveloped virus is not IAV and where the anti-viral agent does not comprise an a-DG mucin-like domain, an a-DG Ig-like 2 domain, and / or an a-DG C-terminal domain. Also provided is the use of an anti-viral agent comprising an alpha dystroglycan (a-DG) S6-I ike domain polypeptide or fragment for the manufacture of a medicament for the treatment or prophylaxis of an enveloped virus infection in a subject, where the enveloped virus is not IAV and where the anti-viral agent does not comprise an a-DG mucin-like domain, an a-DG Ig-like 2 domain, and / or an a-DG C-terminal domain.

[0094] The second aspect of the invention provides an anti-viral agent comprising an alpha dystroglycan (a-DG) S6-like domain polypeptide or fragment thereof for use in a method of treatment or prophylaxis of an enveloped virus infection in a subject, where the enveloped virus is selected from the group consisting of coronavirus, respiratory syncytial virus (RSV), dengue virus (DENV), vesicular stomatitis virus (VSV), alphavirus (e.g. Semliki Forest virus (SFV) or Chikungunya virus), and tick-borne encephalitis virus (TBEV).

[0095] The invention further provides a method of treatment or prophylaxis of an enveloped virus infection in a subject, comprising administering an anti-viral agent comprising an alpha dystroglycan (a-DG) S6-I ike domain polypeptide or fragment to a subject infected with or at risk of being infected with an enveloped virus, where the enveloped virus is selected from the group consisting of coronavirus, respiratory syncytial virus (RSV), dengue virus (DENV), vesicular stomatitis virus (VSV), alphavirus (e.g. Semliki Forest virus (SFV) or Chikungunya virus), and tick-borne encephalitis virus (TBEV). Also provided is the use of an anti-viral agent comprising an alpha dystroglycan (a-DG) S6-I ike domain polypeptide or fragment for the manufacture of a medicament for the treatment or prophylaxis of an enveloped virus infection in a subject, where the enveloped virus is selected from the group consisting of coronavirus, respiratory syncytial virus (RSV), dengue virus (DENV), vesicular stomatitis virus (VSV), Semliki Forest virus (SFV), and tick-borne encephalitis virus (TBEV).

[0096] The anti-viral agent described herein may be used to treat a subject infected with an enveloped virus or at risk of being infected with an enveloped virus. Non-limiting examples include, a subject infected with or at risk of being infected with a coronavirus, a subject infected with or at risk of being infected with respiratory syncytial virus (RSV), a subject infected with or at risk of being infected with dengue virus (DENV), a subject infected with or at risk of being infected with vesicular stomatitis virus (VSV), a subject infected with or at risk of being infected with an alphavirus (e.g. Semliki Forest virus (SFV) or Chikungunya virus), and a subject infected with or at risk of being infected with a tick-borne encephalitis virus (TBEV). This does not include a subject infected with or at risk of being infected with IAV. The subject may have one or more symptoms characteristic of an infection with an enveloped virus.

[0097] As described herein "enveloped viral infection" and "infected with an enveloped virus" as used herein mean that said subject has been exposed to a pathogenic enveloped virus RNA or DNA. The enveloped virus may attach to one or more cells of the host, then penetrate (or may enter) the one or more cells resulting in detrimental effects for at least one cell of the subject.

[0098] The terms “treatment”, “treat”, or “treating” are used herein to refer to the reduction in severity of a disease or condition, the reduction in the duration of a disease; the amelioration or elimination of one or more symptoms associated with a disease or condition, or the provision of beneficial effect to a subject with a disease or condition. The term also encompasses prophylaxis of a disease or condition or its symptoms thereof. “Prophylaxis” is known in the art to mean decreasing or reducing the occurrence or severity of a particular disease outcome, such as delaying progression of a condition in a subject.

[0099] In some embodiments, the agent of the invention is administered to a subject. Depending on the particular agent, they may be administered in any suitable format or by any suitable route, including, but not limited to oral, by inhalation (e.g. intranasally), intravenous, subcutaneous, intradermal, intraperitoneal, intrapleural, intraarticular, intrathecal, locally into the target organ(s) or administered as part of a medical device, e.g., medical or aesthetic implants. Administration by inhalation (e.g. intranasally) or subcutaneously may be particularly preferred, but the most appropriate route may depend on the virus in question.

[0100] Administration is preferably in a “prophylactically effective amount” or a “therapeutically effective amount”, this being sufficient to show benefit to the individual. The actual amount administered, and rate and timecourse of administration, may depend on the individual subject and the nature and severity of their condition.

[0101] As used herein, the term “subject” refers to a human or any non-human animal (e g, mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). In many embodiments, a subject is human. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient.” In some embodiments, the subject is human. In some embodiments, the subject is infected with an enveloped virus or is at risk of being infected with an enveloped virus. The subject as described herein does not include a subject infected with an IAV virus or a subject at risk of being infected with IAV.

[0102] The anti-viral agents described herein can be formulated as a composition or pharmaceutical composition.

[0103] The invention therefore provides a pharmaceutical composition comprising the anti-viral agent according to the first aspect of the invention. The invention also provides a pharmaceutical composition comprising the anti-viral agent according to the second aspect of the invention.

[0104] Pharmaceutical compositions may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other material well known to those skilled in the art. Such substances should be non-toxic and should not interfere with the efficacy of the active ingredient. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0105] Various sterile solutions may be used for administration of the composition, including water, PBS, ethanol, lipids, etc. The concentration of the agent will be sufficient to provide a therapeutic dose, which will depend on the efficiency of transport into the cells.

[0106] Pharmaceutical compositions may be prepared using a pharmaceutically acceptable “carrier” composed of materials that are considered safe and effective. The term “carrier” refers to diluents, binders, lubricants and disintegrants. Those with skill in the art are familiar with such pharmaceutical carriers and methods of compounding pharmaceutical compositions using such carriers. "Pharmaceutically acceptable" refers to molecular entities and compositions that are "generally regarded as safe", e.g., that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset and the like, when administered to a human. In some embodiments, this term refers to molecular entities and compositions approved by a regulatory agency of the US federal or a state government, as the GRAS list under section 204(s) and 409 of the Federal Food, Drug and Cosmetic Act, that is subject to premarket review and approval by the FDA or similar lists, the U.S. Pharmacopeia or another generally recognised pharmacopeia for use in animals, and more particularly in humans.

[0107] The pharmaceutical compositions provided herein may include one or more excipients, e.g., solvents, solubility enhancers, suspending agents, buffering agents, isotonicity agents, antioxidants or antimicrobial preservatives. When used, the excipients of the compositions will not adversely affect the stability, bioavailability, safety, and / or efficacy of the active ingredients. Thus, the skilled person will appreciate that compositions are provided wherein there is no incompatibility between any of the components of the dosage form. Excipients may be selected from the group consisting of buffering agents, solubilizing agents, tonicity agents, chelating agents, antioxidants, antimicrobial agents, and preservatives.

[0108] The composition described herein may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

[0109] ***

[0110] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

[0111] While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

[0112] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

[0113] Any section headings used herein are for organisational purposes only and are not to be construed as limiting the subject matter described.

[0114] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0115] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example + / - 10%. Examples

[0116] Materials and Methods a-DGN production

[0117] The murine and human proteins (variants R166H and R168H) were produced in E.coli BL21 CodonPlus(DE3) RIL (Agilent Technologies) carrying the plasmid pHisTrx-ma-DGN and pHisTrxha-DGN, respectively, as described in Bozzi et al., 2015. The original pHisTrx plasmid was modified to replace the thrombin cleavage site downstream of the thioredoxin fusion protein with a TEV proteolytic site, to yield the pHisTrx(TEV) plasmid. A multi-sites mutagenesis protocol adapted from the Quickchange single-step mutagenesis protocol was employed successfully to modify the original plasmid (Liu and Naismith, 2008). Hereinafter, the mouse and human a-DGN proteins will be referred to as ma-DGN and / ?a-DGN, respectively. The gene of the mouse S6-like domain (for the amino acid sequence see Fig. 5A), as synthetically produced by GeneArt, was cloned into the pHis-Trx(TEV) plasmid for protein production in E.coli downstream a His-tagged thioredoxin fusion partner. The mS6 protein was expressed and purified following the same protocol as the full-length protein.

[0118] Cell lines and antibodies

[0119] African Green Monkey kidney epithelial cell line Vero E6 expressing TMPRSS2 (VeroE6-TMPRSS2), human embryonic epithelial cells 293T expressing ACE2 and TMPRSS2 (HEK-Ace2-TMPRSS2)

[0015] , human cervical epithelial cell line HeLa and HeLa cells expressing ACE2, human lung epithelial cell line A549 and A549 cells expressing ACE2 and TMPRSS2 (AAT) were cultured in Dulbecco’s Modified Eagle’s Medium with 4500 mg / L glucose (Sigma-Aldrich, FIN) supplemented with 10 % fetal bovine serum (FBS; Gibco, FIN, UK), 2 mM L-glutamine (Sigma-Aldrich, FIN, UK), 100 units of penicillin and 0.1 mg / ml streptomycin (PenStrep; Sigma-Aldrich, Fin, UK), and non-essential amino acids (NEAA; Sigma- Aldrich, FIN, UK). Human neuroblastoma cell line SK-N-SH (ATCC HTB-11) was cultured in Dulbecco’s Modified Eagle’s Medium with 1g / L glucose (Sigma-Aldrich, FIN) supplied with 10 % FBS, L-glutamine and PenStrep. The colorectal adenocarcinoma Caco-2 cell line and Caco-2 expressing ACE2 were maintained in DMEM+GlutaMAX containing 1 mM Sodium Pyruvate (Gibco™, Thermo Fisher, UK), 10% FBS (Gibco™, Thermo Fisher, UK), PenStrep, and 0.1 mM NEAA (Sigma Aldrich, UK). Huh7 (Duke Cell Repository) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% FBS. All cell lines were cultured in a 37°C incubator with 5 % CO2 and passaged 1 :8 (VeroE6-TMPRSS2, HeLa, HeLa-ACE2, A549, AAT), 1 :5 (SK-N-SH) or 1 :4 (Caco-2, Caco-2-ACE2) every three to four days.

[0120] Immunostaining was done using polyclonal rabbit anti-SARS-CoV Nucleocapsid (N) protein antibody (Rockland, UK), polyclonal rabbit anti-SARS-CoV-2 (2019-nCoV) Spike antibody (SinoBiological, UK), in house prepared monoclonal mouse hybridoma anti-NP (HB-65) antibody, rabbit polyclonal antibody commercially raised against TBEV M, goat anti-rabbit Alexa Fluor-488 (Thermo Fisher, UK), goat antimouse Alexa Fluor-488 (Thermo Fisher, UK), goat anti-rabbit Alexa Fluor-594 (Thermo Fisher, UK), Wheat Germ Agglutinin (WGA) Alexa Fluor-647 (Thermo Fisher, UK), phalloidin Alexa Fluor-594 (Thermo Fisher, UK), Hoechst 3342 (Thermo Fisher, UK). Viruses

[0121] The following SARS-CoV-2 ancestral early passage virus isolates and variants of concern (VOCs) were used in this study: ancestral SARS-CoV-2 isolates Finland / 1 / 2020 (GenBank ID: MT020781 .2), SARS- CoV-2 / hCoV-19 / Singapore / 2 / 2020 (SG12) (GISAID ID: EPI_ISL_407987) (Gan et al., 2021 ) , SARS-CoV- 2 / human / GBR / liverpool_strain / 2020 (Genbank accession number MW041 156.1 , (Daly et al., 2020) and SARS-CoV-2 VOCs, Gamma (hCoV-19 / England / 520336_B1_P0 / 2021 , GISAID ID: EPI_ISL_2080492); Delta (hCoV-19 / England / BRS-UoB-233 / 2021 , GISAID ID: EPI_ISL_15250227 (Erdmann et al., 2022) Omicron BA.1 (hCoV-19 / England / BRS-UoB-942 / 2022, GISAID ID: EPI_ISL_18150332) and Omicron BA.4 (hCoV-19 / England / BRS-UoB-942 / 2022, GISAID ID: EPI_ISL_18151340). The Delta, Omicron BA.1 and BA.4 VOCs were isolated from clinical research samples collected through the Bristol Biobank (UK NHS Research Ethics Committee Ref 20 / WA / 0273). The Gamma VOC was kindly provided by Professor Wendy Barclay, Imperial College, London and Professor Maria Zambon, UK Health Security Agency). Viral stocks were produced by infecting VeroE6-TMPRSS2 cells at a MOI of 0.01 in infection medium (Minimal Essential Medium (MEM) supplemented with 2 % FBS, L-glutamine, PenStrep and NEAAs.

[0122] Virus containing supernatant was collected at 24-72 h post infection and clarified either by centrifugation for 10 min at 300 x g or filtration through a 0.2 pM membrane, stored at -80°C and used for infections.

[0123] Tick-borne encephalitis virus (TBEV) strain Kuutsalo-14_lxodes_ricinus_Finland-2017 (GenBank MG589938.1 ) was produced by infecting SK-N-SH cells at MOI (multiplicity of infection) 0.001 in infection medium (DMEM, 2 % FBS, L-glutamine, PenStrep, 0.35 pM rapamycin (SelleckChem, S1039)). Viruscontaining supernatant was collected at 72 hours post infection (h.p.i), pre-cleared and stored as described above. Virus titres were determined using plaque assays or 50% tissue culture infectious dose (TCID50) titrations in VeroE6-TMPRSS (CoV2) or SK-N-SH (TBEV) cells. SFV-ZsG, RSV-GFP, and VSV- GFP were produced in BHK-21 , Hela Hep2, and Vero-E6 cells respectively. Cells were infected at an MOI of 0.1 for 22 h (SFV-ZsG and VSV-GFP) or 48h (RSV-GFP), in MEM supplemented with 0.2% BSA, 2 mM L-glutamine, penstrep and NEAA. The collected media were centrifuged twice at 300xg for 10 min, at 4C and the supernatants aliquoted and stored at -80C.

[0124] DENV2 (ST) was a clinical isolate obtained from Singapore General Hospital while DENV1 -2402, DENV3-863, and DENV4-2270 were clinical isolates obtained from the Early Dengue Infection and Outcome study (Low et al. 2006)

[0125] Infection inhibition assays

[0126] Pre-incubation of ma-DGN with virus

[0127] For infections with SARS-CoV-2 Finland / 1 / 2020, RSV, SFV, TBEV, VSV and Ad5, cells were seeded in a ViewPlate-96 (Perkin Elmer, FIN) at the concentration of 104cells per well one day prior to the assay. The virus was mixed with 10 pM ma-DGN or buffer control, incubated for 5 minutes at room temperature and used to infect the cells. For each virus, the viral dose was calibrated to obtain 20-30% infected cells in buffer control treated cells. Following a 16-hour (TBEV, RSV-GFP, hAdV5) or 6-hour (SFV-ZsG, VSV- GFP) infection, the cells were fixed with 4 % formaldehyde, washed with PBS and infected cells were identified by immunostaining using antibodies raised against dsRNA (SCICONS) for TBEV, or by direct detection of fluorescence produced by reporter fluorescent proteins expressed by recombinant viruses (SFV-ZsG, VSV-GFP, RSV-GFP, hAdV5-RFP), and quantified using the open source Cell Profiler-4 software. The inhibitory effect of a-DGN was determined by comparing the percentage of infected cells in the presence of the protein or buffer control.

[0128] Pre-incubation of ma-DGN with cells

[0129] For infections with SARS-CoV-2 Wuhan, Gamma, VOC Delta Clin94, Omicron BA.1 and Omicron BA.4, and influenza A virus (X31 ) cells were seeded in clear 96-well Microplates (Greiner Bio-one, UK) at the concentration of 104cells per well one day prior to the assay, Cells were pre-treated with 1 OpM ma-DGN for 30 minutes at 37°C prior to the addition of virus in infection media (MEM, 2% FBS, 0.1 % NEAA) at a MOI of 0.1 . Following a 16-hour incubation at 37°C and 5 % CO2, cells were fixed with 4% formaldehyde. The fixed cells were blocked in PBS, 1% BSA for 30 min followed by permeabilization for 3 min (PBS, 1% BSA, 0.1% Triton-X 100). Thereafter the cells were stained with anti-N (1 :2,000) (SARS-CoV-2) or with anti-NP (1 :15) (IAV) for 60 min at room temperature. The cells were washed and stained with goat antirabbit Alexa Fluor-488 (1 :2,500, SARS-CoV-2) or goat anti-mouse Alexa Fluor-488 (1 :2,500, IAV) for 30 min at room temperature. Hoechst was used to stain the nuclei. Cells were imaged using an automated spinning-disc microscope CQ1 (Confocal Quantitative Image 130 Cytometer, Yokogawa, Japan). Images were analysed using cell Path Finder (Yokogawa, Japan) software and graphed in Prism 9.4 (GraphPad). The inhibitory effect of a-DGN was determined by comparing the percentage of infected cells in the presence or absence of the protein.

[0130] Generation and viral infection of organoids

[0131] Human jejunal samples were obtained from patients undergoing Roux-en-Y gastric bypass surgery. The study regarding relevant samples and associated ethical regulations were approved by Helsinki University Hospital review board. Written and informed consent was obtained before enrolment. Crypts from human jejunal biopsies were isolated by vigorous shaking after 1 -h incubation in ice cold PBS with 10 mM EDTA. To enrich crypts, tissue suspension was filtered through 70-pm nylon mesh. Enriched crypts were washed once with cold PBS and plated into 60% Matrigel (BD Biosciences) overlaid with epidermal growth factor / Noggin / R-spondin1 (hENR) medium; (Advanced DMEM / F12 (Gibco), 1 x Glutamax (Gibco), 10 mM Hepes (AdDF++), 1 x B-27 (Gibco), 1 x N-2 (Gibco), 50 ng / ml of mouse EGF (RnD), 100 ng / ml noggin (Peprotech), 500 ng / ml of R-spondin-1 (RnD), 10 nM gastrin (Sigma-Aldrich), 100 ng / ml Wnt3A (RnD), 10 mM nicotinamide (Sigma-Aldrich), 500 nM A-83-01 (Sigma-Aldrich) and 10 pM SB202190 (Sigma-Aldrich). Organoids were collected into cold adDF++ medium and washed once to remove excess Matrigel. Organoids were then sheared using a P1000 pipet tip and trituration and onto multi-well tissue culture plates at approximately 500,000 cells per well. SARS-CoV-2 was mixed with 1 or 10 pM ma-DGN or buffer, incubated for 5 min at room temperature and used to infect the broken organoids at MOI=1 for 2 h at 37°C and 5 % CO2, after which the unbound virus was removed by washing with AdDF++ and centrifugation for 5 min at 300g. Organoids were embedded in Matrigel incubated for 20 min at 37°C and 5 % CO2 to allow matrigel solidification and then overlayed with hENR. Organoids were fixed with 4 % formaldehyde processed for immunofluorescence using an antibody against dsRNA. Pseudovirus particles generation and infection

[0132] Pseudovirus particles expressing the surface spike protein of interest (SARS-S, SARS-2-S, MERS-S, HCoV-229E-S) were generated using the VSVAG system as previously described (Hoffmann et al., 2020; Berger Rentsch & Zimmer, 2011 ). ). In Brief, HEK293T cells were transfected with pCG1_SARS-S, pCG1_SARS-2-S, pCAGGS_MERS-S or pCAGGS_HCoV-229E-S using Polyethylenimin (PEI) Max (MW = 40,000KDa, Polysciences, DE) as transfection reagent at a ratio of 4:1 (PEI:DNA) in serum free DMEM for 4 hours at 37°C. Subsequently the cells were washed with PBS and cultured in DMEM supplemented with 5% FBS at 37°C overnight. The cells were then subjected to the replication deficient VSV*AG-fLuc vector, whose glycoprotein (G) gene is substituted by an expression cassette for enhanced green fluorescent protein (eGFP) and firefly luciferase for 2 hours at 37°C. The cells were washed once with PBS and cultured in medium supplemented with anti-VSV-G 11 antibody (Clone 8G5F11 , Absolute Antibody, UK) for a further 24 hours at 37°C. The supernatant was harvested and concentrated by ultracentrifugation through a sucrose cushion (10%-30%) at 100,000 x g for 2 hours. The pellet was resuspended in PBS supplemented with 10%FBS, aliquoted and stored at -80°C until use.

[0133] For inhibition studies using VSV pseudotyped particles (VSVpp) expressing different spike proteins (VSVpp-SARS-S, VSVpp-SARS-2-S, VSVpp-MERS-S and VSVpp-HCoV-229E-S), Caco-2 cells were grown in clear 96-well Microplates (Greiner Bio-one, UK). Cells were pre-treated with the respective compounds (ma-DGN, fta-DGN and mS6) for 30 minutes at 37°C prior to the addition of VSVpp in infection media (DMEM, 2% FBS). Following a 16-hour incubation at 37°C, cells were fixed with 4% formaldehyde, washed and Hoechst was used to stain for the nuclei. Infection was quantified by measuring GFP-positive cells using an automated spinning-disc microscope CQ1 (Confocal Quantitative Image 130 Cytometer, Yokogawa, Japan). Images were analysed using cell Path Finder (Yokogawa, Japan), graphed in Prism 9.4 (GraphPad), and IC50 values were calculated by nonlinear regression analysis using the dose-response (variable slope) equation.

[0134] Plaque assays

[0135] Cells were grown in a 6-well plate and infected with 200 ul of 10-fold serial dilutions of virus in infection medium. Following 1 hour incubation at 37°C and 5 % CO2 with rocking, cell monolayers were covered with 3 ml of overlay medium (MEM, 2 % FBS, L-glutamine, penstrep, 1 .2 % Avicel). Cells were fixed with 10 % formaldehyde at 60 (CoV2) or 72 (TBEV) h.p.i. and plaques were visualized by staining with a crystal violet solution (0.2 % crystal violet, 1 % methanol, 20 % ethanol, 3.6 % formaldehyde). Viral titers were determined as plaque-forming units per ml of stock. ma-DGN binding to cells

[0136] For these experiments ma-DGN was conjugated with Alexa Fluor-647 succinimidyl (NHS) esther (ThermoFisher Scientific) following an established protocol (Hayashi-Takanaka et al., 2014). A549- ACE2-TMPRSS2 or Caco-2-ACE2 cells were seeded into clear 96-well Microplates (Greiner Bio-one, UK) at a concentration of 6,000 or 10,000 cells per well respectively the day before the binding assay. The cells were washed once with media and then incubated with 4 pM of Alexa Fluor-647 labelled ma-DGN for 45 min on a chilled metal plate on ice, after which cells were immediately fixed with 4 % formaldehyde.. The nucleus was stained with Hoechst and phalloidin Alexa Fluor-594 (1 :2,000) was used to stain for actin filaments. The cells were imaged using an automated spinning-disc microscope CQ1 (Confocal Quantitative Image 130 Cytometer, Yokogawa, Japan) with a 40x / 0.95na objective. Images were generated using ImageJ.

[0137] SARS-CoV-2 binding inhibition assay

[0138] HeLa cells stably expressing ACE2 were seeded into clear 96-well Microplates (Greiner Bio-one, UK) at the concentration of 6x103cells per well the day before the assay. For the assay the cells were washed once with media and then pre-treated with 10 pM ma-DGN for 30 minutes at 37°C. Following the incubation, SARS-CoV-2 was added at an MOI=50 and allowed to bind on chilled metal plates for 45 minutes, after which cells were immediately fixed with 4% formaldehyde. Fixed cells were then blocked with PBS, 1%BSA for 30 minutes at room temperature. Cells were stained with a rabbit anti-S (1 :200) antibody for 60 minutes at room temperature, washed and stained with a goat anti-rabbit Alexa Fluor-594 (1 :2,500) secondary antibody for 30 minutes at room temperature. The nucleus was stained with Hoechst and Wheat Germ Agglutinin (WGA) Alexa Fluor-647 (1 :200) was used to stain the cell membrane. The cells were imaged using an automated spinning-disc microscope CQ1 (Confocal Quantitative Image 130 Cytometer, Yokogawa, Japan) with a 40x / 0.95na objective. The images were analysed by a pipeline created in Cell Path Finder (Yokogawa, Japan). Briefly, SARS-CoV-2-S-AF594 labelled particles within the boundary of a segmented cell were counted to quantify the number of bound viral particles per cell. More than 700 cells (100-250 cells per well) were analysed for each condition.

[0139] Infection inhibition assays using DENV

[0140] Infection inhibition in Huh7 cells

[0141] Huh7 cells were pre-treated with ma-DGN or an unrelated protein control for 2 h before each of the four DENVs were inoculated at MOI 1 and incubated for 2 hours at 37°C. The virus inoculum was then removed and the cells were maintained in DMEM containing 9% FBS for a further 5 days. Cells and supernatants were collected at the specified time point and stored at -80°C before DENV infection levels were measured by real time-PCR. RNA extraction of cell lysates and supernatant were done using the RNeasy Mini Kit and QIAamp Viral RNA Mini Kit, respectively (Qiagen) according to the manufacturer’s instructions. Next, cDNA synthesis was performed using the qScript cDNA Synthesis Kit (Quantas Biosciences). Viral replication was measured by RT-qPCR using the SYBR Green Supermix Kit (Roche) with primers for pan-serotype DENV and for the TATA box binding protein as a reference (Sun et al., 2017). All reactions were run on a Roche LightCycler 480, and data analysis was performed with LightCycler 480 software.

[0142] Infection inhibition in human primary CD14+ monocytes

[0143] Human CD14+ monocytes were harvested by negative selection from peripheral blood mononuclear cells (PBMCs) of healthy donors according to a protocol approved by the National University of Singapore’s Institutional Review Board (reference no. B-15-227) as described previously (Chan et al., 201 1 ). Monocytes were pre-treated with ma-DGN or the protein control for 2 h. Following this incubation monocytes were infected with DENV4 at an MOI of 10 and incubated for 2 h at 37°C. The virus was then washed off and fresh ma-DGN or unrelated protein control was added. 48 h later the supernatant was harvested for the determination of DENV titres by plaque assay, as previously described (Chan et al., 2011 ).

[0144] Animal experiments

[0145] K18-hACE2 C57 / BL / 6 mice were obtained from InVivos, Singapore. All animal procedures were performed under approved protocols by the Institutional Animal Care and Use Committee at Singapore Health Services (protocol no: 2020 / SHS / 1554) and were in accordance with guidelines provided by the National Advisory Committee for Laboratory Animal Research (NACLAR) in Singapore.

[0146] Treatment with ma-DGN, / ?a-DGN and buffer control, as well as the SARS-CoV-2 challenges were done via the intranasal (IN) route. For the IN administrations mice were slightly anaesthetised with Isoflurane. In the first instance maDGN was administered at 7.5 pg / mouse or 0.75 pg / mouse via the IN route, immediately followed by IN administration of a lethal (5x10A4 TCID50 in 25 pl PBS) SARS-CoV-2 (brain derived SG12-B; (hCoV-19 / Singapore / 2 / 2020)) challenge dose. In the second experiment animals received 7.5 pg maDGN or baDGN / mouse 2 h prior to the SARS-CoV-2 (lung derived SG12-L; (hCoV- 19 / Singapore / 2 / 2020)) IN challenge (2x10A4 PFU in 25 pl PBS)

[0019] and then 7.5 pg maDGN or AraDGN / mouse once a day for the 3 consecutive days after the challenge. Mice were observed daily for weight loss, clinical signs of disease and death. Mice were sacrificed when they reached 20% loss in bodyweight or a clinical score of 10. At the endpoint lung and brain tissues were collected and analysed for viral loads by plaque assay or qRT-PCR that targeted the ORFI ab gene of SARS-CoV-2 , as previously described (Gan et al., 2021 ; Zhu et al., 2020).

[0147] EXAMPLE 1: a-DGN and its S6-like domain block pseudotyped human coronaviruses infection in Caco-2 cells a-DGN lacking the signal peptide from M. musculus (residues 50-313) and H. sapiens (residues 52-315) as cloned into a modified version of the pHis-Trx expression vector (Sciandra et al., 2001 ) whereby the original thrombin cleavage site was replaced by a TEV protease one (see Materials and Methods), was recombinantly expressed in E.coli The proteins were stabilised by the modification R166H (Bozic et al., 2004) and R168H, respectively (Fig. 5A), located in the loop region connecting the Ig- and S6-like domains (Fig.1 A, right). Hereinafter, the mouse and human a-DGN proteins will be referred to as maDGN and ha-DGN, respectively. Both a-DGN proteins, which share highly homologous sequences (identity: 92.7%, strong similarity: 97.5%, Fig.lB), ran as homogeneously pure electrophoretic bands at an apparent molecular weight of 30 kDa (Fig. 1C). The homogeneity of the protein preps was further confirmed by Electrospray Ionization Liquid Chromatography Mass Spectrometry (Fig. 5B), and the proteins are stable as measured by intrinsic tryptophan fluorescence upon urea-mediated denaturation (Fig. 1D).

[0148] The antiviral activity of ma-DGN and / ?a-DGN was tested against infection of intestinal cancer Caco-2 cells with vesicular stomatitis virus (VSV) pseudotyped with the surface Spike proteins of human coronaviruses (CoVs) SARS-CoV-2, SARS-CoV, middle east respiratory syndrome coronavirus (MERS- CoV) and the seasonal cold strain human coronavirus 229E (HCoV-229E). Both ma-DGN and / ?a-DGN provided potent antiviral activity against all pseudoviruses tested, with IC50 values for SARS-CoV-2-S, SARS-CoV-S, MERS-CoV-S and HCoV-229E-S of 1 .99, 4.58, 2.74, and 1 .85 pM for maDGN, and 2.18, 3.86, 3.69, and 2.76 pM for Ara-DGN, respectively (Fig. 1E). The ECM protein agrin, recombinantly expressed from E.coli, was used as a non-specific protein control. These results established that a-DGN has broad antiviral activity against VSV pseudotyped with different coronavirus spikes. ma-DGN was dissected into its two main domains, the Ig-like and the S6-like domains, to analyse their possible inhibitory potency. They were cloned separately into the pHisTrx(TEV) vector and produced each as described for the full-length protein. The Ig-like domain could not be purified efficiently due its low stability. However, the purified S6-like domain (hereafter mS6) displayed a good degree of stability in solution (Fig. 1D) as a ~15KDa protein, and was further tested for its antiviral activity as described above. mS6 blocks VSV pseudotyped with CoV-S proteins from SARS-CoV-2-S, SARS-CoV-S, MERS-CoV-S and HCoV-229E-S with IC50 values of 1 .64, 2.54, 1 .15 and 1 .49 pM respectively. (Fig. 1E). This shows that mS6 blocks them with a potency comparable to full-length ma-DGN.

[0149] EXAMPLE 2: a-DGN blocks different SARS-CoV-2 variants in susceptible cell lines

[0150] In the first instance ma-DGN was tested against the clinical SARS-CoV-2 isolate Finland / 1 / 2020 in HEK 293T cells stably expressing human angiotensin l-converting enzyme 2 (hACE2) and human transmembrane serine protease 2 (TMPRSS2) (Cantuti-Castelvetri et al., 2020; Daly et al., 2020). Here ma-DGN blocked SARS-CoV-2 infection in a dose-dependent manner, with a reduction in infection of SARS-CoV-2 by 87.2% compared to the vehicle control at the highest concentration tested (10 pM) (Fig. 2A). The inhibitory activity of ma-DGN was further tested against infection of VeroE6-TMPRSS2 cells with different SARS-CoV-2 variants. The tested variants included the original Wuhan strain, and four variants of concern (VOCs) including Gamma, Delta, Omicron BA.1 and Omicron BA.4 that appeared during the SARS-CoV-2 pandemic. Pre-treatment of cells with 10pM ma-DGN blocked infection of VeroE6- TMPRSS2 cells with SARS-CoV-2 Wuhan, Gamma, Delta, Omicron BA.1 and Omicron BA.4 by 80.4, 61 .7, 86.4, 84.3 and 82.7% respectively (Fig. 2B). These data further support the broad-band antiviral activity of a-DGN against authentic SARS-CoV-2 VOCs circulating in humans.

[0151] EXAMPLE 3: a-DGN acts at the early stapes of viral infection

[0152] To test what stage of viral infection is hindered by ma-DGN, its inhibitory activity was analysed as a function of time of administration. Pre-incubation of SARS-CoV-2 with ma-DGN before addition to the cells resulted in 50% inhibition, while adding ma-DGN and SARS-CoV-2 at the same time to the cells reduced infection by 76.4%. In contrast addition of ma-DGN 2 h after SARS-CoV-2 inoculation resulted in little or no reduction of infection (Figure 2C, left). When HeLa cells expressing hACE2 were preincubated with ma-DGN before virus addition, SARS-CoV-2 binding was blocked by 50% compared to the vehicle control (Figure 2C, right, providing further evidence that a-DGN interferes with the binding of viruses to cells. To test whether this interference could depend on the steric hindrance caused by ma- DGN binding to the cells, protein-cell binding assays were performed. Incubation of AAT cells with 4 pM Alexa Fluor-648 labelled ma-DGN in the cold for 45 minutes resulted in a fluorescent signal diffused at the cell periphery, indicating that a-DGN binds to the cell surface. Similar experiments on different cell lines (Caco-2-ACE2) had the same outcome, suggesting that the interaction is not cell-type specific and a-DGN binds to yet unidentified host cell-surface components. (Data not shown.)

[0153] EXAMPLE 4: a-DGN blocks SARS-CoV-2 in human primary put orpanoids

[0154] Organoids are miniaturized three-dimensional organs assembled in vitro with stem cells or adult cells from specific tissue types. In COVID-19 patients, SARS-CoV-2 infects the digestive tracts and has been detected in the small intestine, in the colon and in the ileum (Larners et al., 2020). Therefore, human primary gut organoids are employed as a model system in SARS-CoV-2 studies for their superior capacity to mimic the complexity of human tissue and organs in physiological as well as pathological conditions, as compared to 2D cell culture models. To analyse the antiviral activity of ma-DGN in these organoids, duodenum tissue explants of human donors were first amplified in vitro as a three- dimensional multi-crestal organoids. After mechanical disruption to expose the cellular apical surface, the gut organoids were used for inhibition studies. Pre-treatment of SARS-CoV-2 Finland / 1 / 2020 isolate with 10pM ma-DGN decreased the number of infected organoids by 74.9% (from 20.7% in the vehicle control to 5.2% in ma-DGN-treated organoids) (Figure 2D). This shows that ma-DGN efficiently inhibits SARS- CoV-2 infection in in an organoid model.

[0155] EXAMPLE 5: a-DGN inhibits SARS-CoV-2 in humanised ACE2 mice

[0156] The therapeutic and prophylactic potential of aDGN was examined in vivo using the SARS-CoV-2 infection model in K18-hACE2 transgenic mice expressing angiotensin l-converting enzyme 2 (hACE2) (Gan et al., 2021 ). First, ma-DGN was administered at 10pM and 1 pM (25 pi I per dose, i.e.7.5 and 0.75pg / animal, respectively) at the same time as a lethal dose (5x10A4 TCID50) of the challenging SARS- CoV-2 (SG12 strain) (Gan, et al., 2021 ) virus through the intranasal route. All animals were monitored daily for weight loss and clinical signs of disease (Fig. 3A). After one intranasal administration of a-DGN, mice were protected from a lethal SARS-CoV-2 infection, with a survival rate increase of 20% and delayed weight loss (Fig. 3B and 3C). Moreover, treated mice had significantly reduced viral loads in their lungs compared to animals receiving only a vehicle control (Fig. 3D). The prophylactic potential was determined by administering a-DGN (25pl of 10pM, i.e.7.5pg / dose) two hours before virus challenge, followed by one daily administration of the same dose for the next three consecutive days (Fig. 3E). Animals treated with a-DGN showed increased time of survival despite no evidence of sensible weight loss delay (Fig. 3F and 3G) as well as significantly reduced viral loads (Fig. 3H). Taken together, these studies demonstrate that aDGN increased survival and reduced respiratory infection in humanised ACE2 mice upon challenge with a lethal dose of SARS-CoV-2.

[0157] EXAMPLE 6: a-DGN blocks a broad ranpe of enveloped viruses

[0158] The inhibitory effect of a-DGN on the infectivity of pseudotyped CoVs and authentic SARS-CoV-2 prompted the investigation of its broad antiviral potential against other human viruses that cause disease. Specifically, ma-DGN was further tested for its antiviral activity against other enveloped viruses including influenza A virus (IAV), respiratory syncytia virus (RSV), semliki forest virus (SFV), tick-borne encephalitis virus (TBEV) and vesicular stomatitis virus (VSV). ma-DGN significantly reduced infection by all viruses tested, specifically IAV by 80%, RSV by 91 %, SFV by 57.3 %, TBEV by 90 %, VSV by 99 % (Fig. 4A-E). Strikingly, ma-DGN blocked Dengue virus serotypes 1 , 2, 3 and 4 in Huh-7 cells, reducing viral replication by 57, 58, 50 and 42.5%, respectively, as detected by qRT-PCR (Fig. 4G-J-I). ma-DGN further blocked infection of DENV4 in human primary monocytes by 91 .3% (Fig. 4K)

[0159] Interestingly it did not inhibit infection of the human Adenovirus serotype 5 (hAd5), a non-enveloped virus (Fig. 4F), suggesting that ma-DGN is a broad-range inhibitor against enveloped viruses.

[0160] Taken together, these data provide strong evidence that the natural compound a-DGN has broadspectrum antiviral activity against enveloped viruses. In particular we showed that ma-DGN potently inhibits SARS-CoV-2 and other coronaviruses infection in vitro. Strikingly, it is also able to delay mortality and significantly reduce SARS-CoV-2 viral loads in humanised ACE2 mice after challenge with a lethal virus dose. Moreover, we could pinpoint the inhibitory activity of ma-DGN to its S6-like domain, that alone displays a strong antiviral capacity against all the coronaviruses tested. Therefore, a-DGN (and its shortened version) shows potential for further development as a broad-spectrum antiviral against enveloped viruses and to complement vaccine strategies in current and future pandemics.

[0161] References

[0162] A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein.

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Claims

Claims:1 . An anti-viral agent comprising an alpha dystroglycan (a-DG) S6-I ike domain polypeptide or fragment thereof for use in a method of treatment or prophylaxis of an enveloped virus infection in a subject, wherein the enveloped virus is not influenza A virus (IAV) , and wherein the anti-viral agent does not comprise one or more of n a-DG mucin-like domain, an a-DG Ig-like 2 domain, or an a-DG C-terminal domain.

2. The anti-viral agent for use according to claim 1 , wherein the enveloped virus is selected from the group consisting of coronavirus, respiratory syncytial virus (RSV), dengue virus (DENV), vesicular stomatitis virus (VSV), alphavirus (e.g. Semliki Forest virus (SFV) or Chikungunya virus), and tick-borne encephalitis virus (TBEV).

3. An anti-viral agent comprising an alpha dystroglycan (a-DG) S6-I ike domain polypeptide or fragment thereof for use in a method of treatment or prophylaxis of an enveloped virus infection in a subject, wherein the enveloped virus is selected from the group consisting of coronavirus, respiratory syncytial virus (RSV), dengue virus (DENV), vesicular stomatitis virus (VSV), alphavirus (e.g. Semliki Forest virus (SFV) or Chikungunya virus), and tick-borne encephalitis virus (TBEV).

4. The anti-viral agent for use according to claim 2 or claim 3, wherein the coronavirus is selected from the group consisting of Severe acute respiratory syndrome coronavirus (SARS-CoV), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome-related coronavirus (MERS-CoV), and Human coronavirus 229E (HCoV-229E).

5. The anti-viral agent for use according to claim 2 or claim 3, wherein DENV is selected from the group consisting of dengue virus serotype 1 (DENV1 ), dengue virus serotype 2 (DENV2), dengue virus serotype 3 (DENV3), and dengue virus serotype 4 (DENV4).

6. The anti-viral agent for use according to any one of claims 1 to 5, wherein the a-DG S6-like domain polypeptide or fragment thereof comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 6 or SEQ ID NO: 15.

7. The anti-viral agent for use according to claim 6, wherein the a-DG S6-I ike domain polypeptide or fragment thereof consists of an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 6 or SEQ ID NO: 15.

8. The anti-viral agent for use according to any one of claims 1 to 7, wherein the anti-viral agent further comprises an a-DG Ig-like 1 domain polypeptide or fragment thereof.

9. The anti-viral agent for use according to claim 8, wherein the a-DG Ig-like 1 domain polypeptide or fragment thereof comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 4 or SEQ ID NO: 13.

10. The anti-viral agent for use according to claim 9, wherein the a-DG Ig-like 1 domain polypeptide or fragment thereof consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 4 or SEQ ID NO: 13.11 . The anti-viral agent for use according any one of claims 7 to 10, wherein the anti-viral agent further comprises a linker sequence positioned between the Ig-like 1 domain polypeptide or fragment thereof and the S6-like domain polypeptide or fragment thereof.

12. The antiviral agent for use according to claim 11 , wherein the linker sequence comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 5 or SEQ ID NO: 14.

13. The anti-viral agent for use according to claim 12, wherein the linker sequence consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 5 or SEQ ID NO: 14.

14. The anti-viral agent for use according to any one of claims 1 to 13, wherein the anti-viral agent comprises an a-DGN polypeptide.

15. The anti-viral agent for use according to claim 14, wherein the a-DGN polypeptide comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 11 .

16. The anti-viral agent for use according to claim 14, wherein the a-DGN polypeptide consists of an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 11 .

17. The anti-viral agent for use according to any one of claims 12 to 16, wherein the anti-viral agent comprises a modification at position R168 numbered according to SEQ ID NO: 1 .

18. The anti-viral agent for use according to claim 17, wherein the modification is R168H.

19. The anti-viral agent for use according to claim 17 or claim 18, wherein the modification stabilises the anti-viral agent.

20. The anti-viral agent for use according to any one of claims 1 to 19, wherein the subject is human.21 . The anti-viral agent for use according to any one of claims 1 to 20, wherein the subject is infected with an enveloped virus or is at risk of being infected with an enveloped virus.

22. The anti-viral agent for use according to any one of claims 1 to 21 , wherein the anti-viral agent is administered by inhalation or subcutaneously.

23. A pharmaceutical composition comprising the anti-viral agent for use according to any one of claims 1 to 19.

24. The pharmaceutical composition according to claim 23, wherein the composition comprises a pharmaceutically acceptable carrier.

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