WO2015162433A1

WO2015162433A1 - Composition

Info

Publication number: WO2015162433A1
Application number: PCT/GB2015/051207
Authority: WO
Inventors: Alexander TARR; Jonathan Ball
Original assignee: The University Of Nottingham
Priority date: 2014-04-23
Filing date: 2015-04-23
Publication date: 2015-10-29
Also published as: GB201407115D0

Abstract

The invention provides a composition comprising a ficolin or a ficolin derived molecule for use in the treatment and/or prevention of infection by an enveloped virus.

Description

COMPOSITION

The present invention relates to compositions for treating or preventing viral infection, and in particular infection by enveloped viruses.

Viruses can be broadly classified as being either 'non-enveloped' or 'enveloped' . Non-enveloped viruses contain a viral genome surrounded by a protein capsid whilst enveloped viruses comprise an additional outer lipid bilayer ('envelope ') encapsulating the capsid. This envelope is acquired as the virus buds from the cell membrane of its host and is derived from the host cell, either from the outer membrane of the host cell or from internal membranes such as the nuclear membrane or endoplasmic reticulum. The envelope comprises components encoded for by the host genome but also by the viral genome, such as viral glycoproteins. Functionally, the viral envelope enables entry of a virus into its host. Viral glycoproteins on the envelope surface recognise and bind to receptor sites on the host's cell membrane. This leads to fusion of the viral envelope with the host' s membrane, release of the viral genome and infection of the host. Enveloped viruses can be further divided into families including Asfaviridae, Hepadnaviridae, Herpesviridae, Poxviridae, Arenaviridae, Arteriviridae, Bunyaviridae, Coronaviridae, Filoviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, Retroviridae, Rhabdoviridae and Togaviridae . Hepatitis C virus (HCV) is a member of the Flaviviridae family. Chronic HCV infection affects approximately 130 million people. Chronic infection is a risk factor for cirrhosis and hepatocellular carcinoma, resulting in the requirement for liver transplantation [ 1 ] . Entry inhibitors are an attractive treatment in the clinical setting of liver transplantation, where they might prevent infection of transplanted tissue. Current therapies target the HCV NS3 protease and the NS5B polymerase [2], but cannot prevent initial infection.

Binding of HCV envelope glycoproteins E l and E2 to cellular CD81 and SR-B 1 is essential for HCV entry, and antibodies that inhibit these interactions neutralize entry (reviewed in [3]). These glycoproteins are targets for therapeutic intervention and host immunity [4,5] . They possess up to 5 and 1 1 N-linked glycosylation sites, respectively [6] . These glycans are structurally heterogeneous [7], and contribute to glycoprotein biosynthesis, modulation of infectivity and evasion of neutralizing antibodies [8,9] . Many glycosylation sites are highly conserved across genetically diverse HCV isolates [ 10] . This makes them attractive targets for anti -viral drug development. The lectins cyanovirin-N [ 1 1], griffithsin [ 12] and mannose binding lectin [ 10] all inhibit entry by binding to these glycans, however clinical use of these agents has been prohibited due to the high concentrations required and/or potential toxicity issues. Ficolins are a family of serum proteins functionally and structurally related to the collectins, sharing quaternary structure with mannose binding lectin (MBL) and complement component C l q They consist of disulfide-linked 35kDa polypeptides organized into trimers. Oligomers of these three polypeptides form functional dodecamers [ 13] . Whilst there are structurally similarities with MBL, the ficolins have markedly different substrate specificities. The ficolins bind glycan-containing pathogen-associated molecular patterns (PAMPs) and activate the complement cascade . The carbohydrate-binding activity is attributed to the C-terminal fibrinogen- like binding domain of the ficolins that has general specificity for N-acetyl groups on the outer walls of microorganisms [ 14] .

In humans, three ficolins have been identified; L-ficolin, M-ficolin, and H-ficolin [ 15] . H-ficolin and L-ficolin are expressed by hepatocytes. L-ficolin has a broad binding specificity for targets including galactose, β-glucan, acetylated compounds, N-acetylglucosamine and N-acetylcysteine. Binding is mediated by four binding sites in the C-terminal fibrinogen-like binding domain, some of which require calcium for interaction [ 16] . L-ficolin associates with MBL-associated serine proteases (MASPs), resulting in complement activation, phagocytosis and clearance of pathogens bearing N-acetylated structures such as N-Acetyl glucosamine (GlcNAc) [ 17, 18], a major component of bacterial cell walls that is also incorporated into virus glycoproteins. This early innate recognition may play a critical step in priming adaptive immune responses to infection [ 19] .

L-ficolin may also be referred to as one of the following: hucolin; P35 ; ficolin-2; ficolin B; ficolin-beta; 37kDa elastin-binding protein; serum lectin P35 ; amd FCNL. M-ficolin is sometimes also referred to as ficolin- 1 ; FCN 1. H-ficolin is sometimes also referred to as ficolin-3 ; FCN3.

Other known ficolins include Mus musculus ficolin A (NCBI No: 146134471); Xenopus laevis ficolin 1 (NCBI No l48226267 and 148222429 :); Xenopus laevis ficolin 2 (NCBI No: 148230482); Xenopus tropicalis ficolin 3 (NCBI No: 49522423); Rattus norvegicus ficolin B (NCBI No: 16758441); Pig ficolin (NCBI No: 294218); Macaca mulatta ficolin 1 (NCBI No: 297269905); Callithrix jacchus ficolin 3 (NCBI No: 296207192); Pongo abelii ficolin 2 (NCBI No: 297713612); Nomascus leucogenys ficolin 3 (NCBI No: 332245 147); Pan troglodytes ficolin 1 and 2 (NCBI No : 332833290 and 332833287); Enhydris polylepis ficolin (NCBI No: 3841 10807, 3841 10809, 3841 1081 1 , 3841 10813 and 3841 10815); Otolemur garnettii ficolin 1 and 3 (NCBI No: 395844550 and 395854795); Pan paniscus ficolin 1 and 2 (NCBI No: 397492129 and 397492127); Xenopus (Silurana) tropicalis ficolin 3 (NCBI No: 402743787); Saimiri boliviensis boliviensis ficolin 2 and 3 (NCBI No: 403301499 and 403257428); Felis catus ficolin 3 (NCBI No: 410966556); Gorilla gorilla gorilla ficolin 1 , 2 and 3 (NCBI No: 4263635 18, 4263635 14 and 426328544); Nomascus leucogenys ficolin 1 (NCBI No: 441623308); Sus scrofa ficolin 1 (NCBI No : 45 1958120); Orcinus orca ficolin 2 and 3 (NCBI No: 465991534 and 466083768); Tursiops truncatus ficolin 3 (NCBI No: 470655 153); Trichechus manatus latirostris ficolin 1 and 3 (NCBI No: 471414359 and 471379001); Ceratotherium simum simum ficolin 3 (NCBI No: 478503099); Dasypus novemcinctus ficolin 1 , 2 and 3 (NCBI No: 48855 1924, 48855 1920 and 488527941 ); Sorex araneus ficolin 3 (NCBI No: 505780435); Jaculus jaculus ficolin 3 (NCBI No: 507575487); Octodon degus ficolin 3 (NCBI No: 507694803); Mustela putorius furo ficolin 1 (NCBI No: 5 1 1874853); Heterocephalus glaber ficolin 1 and 3 (NCBI No: 5 12857609 and 5 12827572); Gallus gallus ficolin 2 (NCBI No: 5 1321 185 1 ); Cavia porcellus ficolin 1 and 3 (NCBI No: 5 14462306 and 5 1446415 1); Xenopus (Silurana) tropicalis ficolin 1 (NCBI No: 52345777); Mesocricetus auratus ficolin 3 (NCBI No: 5249595 16); Bos taurus ficolin 1 and 3 (NCBI No: 528970649 and 528901220); Spermophilus tridecemlineatus ficolin 1 (NCBI No: 532102789); Chinchilla lanigera ficolin 1 and 3 (NCBI No : 533 191335 and 533 162292); Macaca fascicularis ficolin 1 , 2 and 3 (NCBI No : 544491626, 54449163 1 and 544407920); Equus caballus ficolin 1 and 3 (NCBI No: 545204184 and 545208414); Canis lupus familiaris ficolin 3 (NCBI No: 545492219); Capra hircus ficolin 3 (NCBI No: 548455841); Xenopus tropicalis ficolin B (NCBI No: 5661 1 141); Rattus norvegicus ficolin 1 and 2 (NCBI No: 56789142 and 56971772); Bos taurus ficolin 1 (NCBI No: 58330903); Xenopus (Silurana) tropicalis ficolin 3 (NCBI No: 58332169); and Mus musculus ficolin B (NCBI No: 76827364).

The present invention provides a method of preventing and/or treating infection by one or more enveloped viruses by using a ficolin.

According to a first aspect of the invention, there is provided a composition comprising a ficolin or a ficolin derived molecule for use in the treatment and/or prevention of infection by an enveloped virus.

The use may be therapeutic and/or prophylactic. Preferably the use is prophylactic. The ficolin or ficolin derived molecule may comprise an N-terminal region, a collagen-like domain, and a globular region which is a fibrinogen-like domain. The ficolin may be, or may be derived from, a human ficolin. The ficolin may be L-ficolin, H-ficolin or M-ficolin or a molecule derived therefrom. The ficolin derived molecule preferably comprises at least a fibrinogen-like domain.

The ficolin or ficolin derived molecule is preferably used in an oligomeric form.

In an embodiment the ficolin is L-ficolin or is derived from L-ficolin, preferably the L-ficolin used is in an oligomeric form, preferably in a dodecameric form.

In another embodiment the ficolin may be H-ficolin or may be derived from H-ficolin, preferably the H-ficolin used is in an oligomeric form.

The ficolin may by comprised of one or more monomers each comprising or consisting of the sequence of Seq ID No: 1 or 2 or having at least 50%, 55%, 60%, 65 %, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity with the sequence of Seq ID No: l or 2. Seq ID No: 1 (Figure 1 1) is the protein sequence of a monomer of L- ficolin. The sequence of Seq ID No: 2 (Figure 12) is the protein sequence of the monomer of L-ficolin used in the experiments presented herein. The ficolin may by comprised of one or more monomers each comprising or consisting of the sequence of Seq ID No: 5 or having at least 50%, 55%, 60%, 65%, 70%, 75 %, 80%, 85%, 90%, 95% or more sequence identity with the sequence of Seq ID No: 5 (Figure 15) is the protein sequence of a monomer of H-ficolin.

The ficolin may by comprised of one or more monomers each comprising or consisting of the sequence of Seq ID No: 4 or having at least 50%, 55%, 60%, 65%, 70%, 75 %, 80%, 85%, 90%, 95% or more sequence identity with the sequence of Seq ID No: 4 (Figure 14) is the protein sequence of a monomer of M-ficolin.

The ficolin may by comprised of one or more monomers each comprising or consisting of the sequence of any known ficolin or having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity with any known ficolin. The ficolin derived molecule may comprise or consist of one or more monomers each comprising or consisting of at least the sequence of the fibrinogen-like domain of Seq ID No: 1 , 2, 4 or 5 or having at least 50%, 55 %, 60%, 65%, 70%, 75%, 80%, 85 %, 90%, 95% or more sequence identity with the sequence of the fibrinogen-like domain of Seq ID No: 1 , 2, 4 or 5.

The ficolin derived molecule may comprise or consist of one or more monomers each comprising or consisting of at least the sequence of the fibrinogen-like domain of any known ficolin or having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity with the sequence of the fibrinogen-like domain of any known ficolin.

The ficolin derived molecule may by a variant of a known ficolin in which one or more residues are added, deleted, inserted or substituted, while having no material effect on the function of the molecule. A residue (or residues) may be added or deleted from either end of the protein, deleted from within the protein, inserted within the protein, or substituted for one or more of the residues within the protein. As would be understood by a person of ordinary skill in art, one or more protein residues may be added, deleted, inserted or substituted while still maintaining the function of the protein. For example, as many as five or more residues may be added to or removed from either end of a protein, or inserted into a protein, and be a ficolin derived molecule within the context of the present invention. In a further example, a conservative substitution of one or more residues within a protein may result in a ficolin derived molecule. As would be well understood to the skilled artisan, a conservative substitution includes a substitution of one amino acid residue with another amino acid residue having one or more similar chemical properties, such as polarity, charge, hydrophobicity, or aromaticity, for example.

A ficolin derived molecule may also comprise only a part of a ficolin molecule, provided that the molecule retains the desired therapeutic and/or prophylactic properties of ficolin.

The ficolin and/or ficolin derived molecule may be provided as a fusion protein.

Percentage sequence identity is defined as the percentage of amino acids in a sequence that are identical with the amino acids in a provided sequence after aligning the sequences and introducing gaps if necessary to achieve the maximum percent sequence identity. Alignment for the purpose of determining percent sequence identity can be achieved in many ways that are well known to the man skilled in the art, and include, for example, using BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) .

Variations in percent identity may be due, for example, to amino acid substitutions, insertions or deletions. Amino acid substitutions may be conservative in nature, in that the substituted amino acid has similar structural and/or chemical properties, for example the substitution of leucine with isoleucine is a conservative substitution.

The enveloped virus may be selected from the group consisting of Asfaviridae, Hepadnaviridae, Herpesviridae, Poxviridae, Arenaviridae, Arteriviridae, Bunyaviridae, Coronaviridae, Filoviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, Retroviridae, Rhabdoviridae and Togaviridae

The enveloped virus may be a Hepatitis C virus; a Vesicular stomatitis virus; an Ebola virus; a Human Immunodeficiency virus; a Machupo virus; or an Influenza virus. The enveloped virus may be a Hepatitis C virus; a Vesicular stomatitis virus; an Ebola virus; a Machupo virus; or an Influenza virus. The enveloped virus may be a Hepatitis C virus; a Vesicular stomatitis virus or an Ebola virus. The enveloped virus may be a Hepatitis C virus.

The enveloped virus may have acetylated glycoproteins.

According to a still further aspect the invention provides a pharmaceutical composition, for use in the treatment and/or prevention of infection of a subj ect by an enveloped virus, comprising a ficolin or a ficolin derived molecule and a pharmaceutically acceptable carrier or excipient.

Suitable acceptable excipients and carriers will be well known to those skilled in the art. These may include solid or liquid carriers. Suitable liquid carriers include water and saline. The proteins of the composition may be formulated into an emulsion or they may be formulated into biodegradable microspheres or liposomes.

The composition may also comprise polymers or other agents to control the consistency of the composition, and/or to control the release of the ficolin or ficolin derived molecule from the composition. The composition may also comprise other agents such as diluents, which may include water, saline, glycerol or other suitable alcohols etc; wetting or emulsifying agents; buffering agents; thickening agents for example cellulose or cellulose derivatives; preservatives; detergents, antimicrobial agents; and the like . Preferably the active ingredients in the composition are greater than 50% pure, usually greater than 80% pure, often greater than 90% pure and more preferably greater than 95 %, 98% or 99% pure . With active ingredients approaching 100% pure, for example about 99.5% pure or about 99.9% pure, being used most often. The pharmaceutical composition may be provided in a liquid form or in a lyophilised form.

Preferably the pharmaceutical composition is capable of preventing infection by an enveloped virus. The phrase "preventing infection" as used herein means that the composition is capable of neutralising the virus and preventing entry of the virus into a host cell and/or is capable of preventing release of virus particles from an infected cell. The extent of protection may result in an about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more reduction in infectivity of the virus. The extent of protection may result in an about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more reduction in viral entry into cells compared to in the absence of the ficolin or a ficolin derived molecule . The extent of protection may result in an about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more reduction in viral release from cells infected with a particular virus compared to in the absence of the ficolin or a ficolin derived molecule.

A composition according to the invention may be for oral, systemic, parenteral, topical, mucosal, intramuscular, intravenous, intraperitoneal, intradermal, subcutaneous, intranasal, intravaginal, intrarectal, transdermal, sublingual, inhalation or aerosol administration.

According to another aspect the invention provides the use of composition comprising a ficolin or a ficolin derived molecule in the preparation of a medicament for the treatment and/or prevention of infection by an enveloped virus. According to a further aspect the invention provides a method of treating and/or preventing a viral infection caused by an enveloped virus in a subject, comprising administering a composition comprising a ficolin or a ficolin derived molecule to a subject in need thereof. If the composition is to be administered for prophylactic purposes preferably it is administered to those at risk of infection prior to exposure to the virus of concern. If the composition is to be administered for therapeutic purposes preferably it is administered after infection by the enveloped virus of concern.

According to a yet further aspect the invention provides a kit for use in treating and/or preventing a viral infection caused by an enveloped virus, wherein the kit comprises a ficolin or a ficolin derived molecule and instructions relating to administration.

According to a further aspect the invention provides a protein, or a composition comprising a protein, having the sequence of Seq ID No:2, the protein may be provided in monomeric or oligomeric form. The skilled man will appreciate that any of the preferable features discussed above can be applied to any of the aspects of the invention.

Preferred embodiments of the present invention will now be described, merely by way of example, with reference to the following figures and examples.

Figure 1 shows recombinant, oligomeric human L-ficolin. Figure 1 A illustrates schematically the structure of L-ficolin. Each polypeptide monomer possesses a cysteine-rich N-terminal domain, a collagen-like domain, a neck region, and a C-terminal fibrinogen-like domain. Monomers oligomerize into dodecamers formed of trimeric subunits. Figure IB shows L-ficolin was purified from human serum using GlcNAc-Sepharose . The flow-through (F), mannose wash (M), and L-ficolin eluate fractions (lanes 1 -7) were detected by western blot with mAb GN5 following non-reducing (NR) or reducing (R) SDS-PAGE. Serum L-ficolin oligomers appeared as a 35kDa monomer, 70kD dimers and ~250kD oligomers, as well as higher-order multimers. Figures 1 C & ID show expression of recombinant L-ficolin. In vitro expressed L-ficolin was purified using an anti-FLAG M2 affinity resin. L-ficolin samples were resolved using non-reducing PAGE and western blotting with anti L-ficolin antibody, GN5 (Figure 1 C) or anti-FLAG mAb M2 (Figure ID). Each blot presents reactivity with start material (S), flow through (F) and eluted L- ficolin (E). Monomeric (35kDa), dimeric (~70kDa), and multimeric forms (>250kD) of recombinant L-ficolin were observed. Figure IE shows eluted material was analyzed under non-reducing conditions (NR) or reduced in the presence of dithiothreitol (R), demonstrating the reduction of oligomers (o) to monomers (m).

Figure 2 shows that recombinant L-ficolin binds to HCV glycoproteins El and E2 in an acetyl-specific manner. Figure 2A shows L-ficolin binding to recombinant HCV glycoproteins as evaluated by ELISA. E2 was captured using mAb GN5 -immobilised L-ficolin and detected by anti-E2 antibody. Equivalent, dose-dependent interaction was observed between the soluble ectodomain of HCV E2 (sE2₆₆ i) and both recombinant L-ficolin (■) or purified serum L-ficolin (O). The highest concentration of sE2₆₆ i was incubated with mAb GN5 in the absence of L-ficolin as a control (♦). Figure 2B shows the dose-dependent binding of L-ficolin to 293T cell lysates from cells transfected with E 1/E2 (■), or mock-transfected cells (A). Figure 2C shows immobilized L-ficolin pre-incubated with the indicated concentrations of L-ficolin ligands GlcNAc ( T ),CysNAc (A), or D-Mannose (■), prior to binding sE2. Binding is presented as a proportion of binding in the absence of inhibitor. IC₅₀ values were 260mM (GlcNAc), and 1.8mM (CysNAc). No inhibition by D-Mannose was observed.

Figure 3 shows that L-ficolin mediates neutralisation of HCV and VSV entry. In Figure 3A retroviral pseudoparticles possessing envelope glycoproteins E 1/E2 from strains of HCV representing genotypes la (H77c), 3a (UKN3A 13.6), or 4a (UKN4. 1 1. 1) or the VSV G protein were treated for one hour at room temperature with purified recombinant L-ficolin (grey bars) or a control fraction from purification containing no L-ficolin (black bars), before infecting Huh7 cells. All of the HCVpp and the VSV G pseudoparticles were neutralized by L-ficolin. Data is presented as the proportion of infectivity in the absence of inhibitor. In figure 3B FLAG-tagged L-ficolin expressed into the supernatants of HEK293T cells was fractionated by affinity purification on FLAG resin and pooled into three independent fractions (fractions F l , F2 and F3), as well as a negative fraction containing no L-ficolin (N) These fractions contained 157 μgmL^"1, 46 μgmL^"1, 10 μgmL^"1 and 0 μgmL^"1 total protein, respectively. Figure 3C shows that when analyzed by staining an SDS-PAGE gel, protein was only detected in sample F l . Figure 3D shows that fractions diluted to 1 μgmL^"1 total protein and assessed for neutralization of entry of HCVpp bearing JFHl E 1/E2 glycoproteins. Data is presented as a proportion of an uninhibited control. Significant neutralization was only exhibited for the fraction containing detectable oligomeric L-ficolin (* * p<0.01). E) The F l fraction was found to neutralize entry of the JFHl strain of cell-cultured HCV in a dose dependent manner (IC₅₀= l ^gmL^"1), while no inhibition was observed with the negative control sample.

Figure 4 shows that neutralisation of HCV entry by L-ficolin requires interaction with virus particles. Protein fractions containing oligomeric L- ficolin, or a control with no detectable L-ficolin diluted equivalently, were incubated with either HCVpp (strain H77), or target Huh7 cells. Following washing, cells were infected with HCVpp. >50% neutralization was only observed when L-ficolin was pre-incubated with virus particles. A significant difference was observed between virus and cells treated with L-ficolin, compared to cells treated alone ( 1 -way ANOVA; * p<0.05). No significant differences in entry were observed when cells were treated with L-ficolin or a negative control.

Figure 5 shows that inhibition of HCV entry is calcium-dependent.

Figure 5A shows the results of infection assays prepared with cell-culture grown HCV. Neutralization was performed with approximately 50% neutralizing concentrations of oligomeric L-ficolin in the presence of 2mM CaCl₂ (black bars) or 7mM CaCl₂ (grey bars). Neutralization was compared to a positive uninhibited control, and a sample treated with an equivalent L- ficolin-negative fraction from purification. Greater neutralization of HCVcc was observed with L-ficolin fractions in the presence of 7mM CaCl₂. (One-way

ANOVA; * * p<0.01 , * * * p<0.001). In figure 5B L-ficolin was incubated in PBS +/-7mM CaCl₂ before analysis by SDS-PAGE/western blotting with mAb GN5. No difference in oligomerization was observed in the presence of CaCl₂.

Figure 6 shows the effect of CysNAc on neutralisation of HSV pseudoparticles. In figure 6A neutralization assays using HCVpp representing strain H77 were performed with L-ficolin in the presence or absence of l OmM CysNAc, previously demonstrated to inhibit binding of L-ficolin to HCV E2. While neutralization was observed in the preparation containing L-ficolin, CysNAc unexpectedly did not inhibit neutralization, but augmented it. A similar effect of the presence of CysNAc was observed when incubating with a negative control preparation that had little effect on entry, demonstrating that the inhibition of entry by CysNAc was independent of the action of L-ficolin. Significant neutralization was observed with L-ficolin, as determined by Oneway ANOVA (* p<0.05). Figure 6B illustrates that incubation of CysNAc with pseudoparticles possessing either HCV or VSV glycoproteins inhibited entry. Figure 6C shows Huh7 cells incubated with increasing concentrations of CysNAc and then stained with trypan blue to assess toxicity of this compound on the cells. No appreciable cell death occurred as a result of increased CysNAc concentration, but changes in cell morphology and size were observed with increasing CysNAc concentration.

Figure 7 illustrates the concentration of circulating serum L-ficolin. The concentration of L-ficolin was assayed in 40 HCV-infected individuals and 30

HCV RNA/antibody-negative healthy donors. Serum L-ficolin concentrations ranged between l - 12^g/mL. The median value for healthy controls was 4^g mL- 1 (S .D. ± 1.5) and 4.2μ& mL- 1 (S .D. ± 1.9) in chronic HCV infections. No significant difference between the groups was evident.

Figure 8 shows neutralisation of HCV entry by genetic variants of L- ficolin. Single amino acid substitutions were introduced into the recombinant L-ficolin protein, and the activity of these proteins was assessed in a retroviral pseudovirus assay using the glycoproteins of strain 1A20.8. Inhibition was compared to an uninhibited control. The mutation T236M had little effect on the neutralizing potency of the protein.

Figure 9 shows the S1/S4 binding site highlighted on the crystal structure of L-ficolin fibrinogen-like domain (PDB ID 2J1G). The polymorphic amino acid at site 258 is highlighted in red. Amino acids known to make up the S4 binding site are highlighted in white, and S I binding site in orange on the primary amino acid backbone of the L-ficolin fibrinogen-like domain. The calcium ion required for binding is represented as a green ball. The side chains of the two binding sites are highlighted to illustrate the proximity of amino acid 258 with these two sites.

Figure 10A shows the inhibition of Ebola Virus and Machupo Virus entry by L-ficolin. Preparations of L-ficolin expressed in human embryonic kidney cells (HEK293T) were purified and incubated with Ebola pseudoviruses or Machupo Virus for 1 hour before infecting target human hepatoma (HuH7.5) cells with the mixture. Infectivity was determined by assaying delivery of a luciferase reporter gene to infected cells. Infectivity is expressed as a proportion of an uninhibited control in each case . Three Ebola virus strains were tested: Sudan, Zaire and Reston. Machupo Virus was included as a member of the the Arenavirus family. Figure 10B shows inhibition of Ebola Virus entry by H-ficolin. Preparations of H-ficolin expressed in human embryonic kidney cells (HEK293T) were purified and incubated with Ebola pseudoviruses for 1 hour before infecting target human hepatoma (HuH7.5) cells. Infectivity was determined by assaying delivery of a luciferase reporter gene to infected cells. Infectivity is expressed as a proportion of an uninhibited control in each case. Three Ebola virus strains were tested: Sudan, Zaire and Reston. Figure IOC shows the inhibition of Hepatitis C Virus entry by H-ficolin.

Preparations of H-ficolin expressed in human embryonic kidney cells (HEK293T) were purified and incubated with HCV pseudoviruses for 1 hour before infecting target human hepatoma (HuH7.5) cells. Infectivity was determined by assaying delivery of a luciferase reporter gene to infected cells. Infectivity is expressed as a proportion of an uninhibited control in each case.

The genotype la HCV strain H77 was used in this assay.

Figure 11 shows the sequence of an L-ficolin monomer. This sequence is also referred to herein as Seq ID No: 1. The sequence in bold italics is the signal sequence. The sequence in italics in normal font is the cys rich region. The sequence underlined is the collagen-like domain. The sequence in plain font is the neck region. The sequence in bold is the fibrinogen-like domain.

Figure 12 shows the sequence of the L-ficolin monomer used in the experiments. This sequence is also referred to herein as Seq ID No: 2. The sequence includes the L-ficolin sequence of Figure 1 1 followed by a synthetic spacer and a linker (containing a cloning site), followed by two tags - one epitope tag and one poly-histidine. The sequence of Figure 1 1 is shown in plain text, the synthetic spacer is in italics, the linker is in bold, the epitope tag is underlined and the poly-histidine tag is in italics and bold.

Figure 13 shows the nucleotide sequence which encodes the L-ficolin monomer of Figure 12. This sequence is also referred to herein as Seq ID No: 3. Figure 14 shows the sequence of an M-ficolin monomer. This sequence is also referred to herein as Seq ID No: 4.

Figure 15 shows the sequence of an H-ficolin monomer. This sequence is also referred to herein as Seq ID No: 5.

Figure 16 illustrates schematically oligomeric human H-ficolin.

INTRODUCTION

L-ficolin is a liver-expressed soluble pattern recognition molecule that contributes to innate immune defense against microorganisms. It is well described that binding of L- ficolin to specific pathogen-associated molecular patterns activates the lectin complement pathway resulting in opsonization and lysis of pathogens. It has been demonstrated that in addition to this indirect effect, L-ficolin has a direct neutralizing effect against enveloped virus entry as illustrated here with reference to HCV, VSV and Ebola. Specific, dose-dependent binding of recombinant L-ficolin to HCV glycoproteins E l and E2 has been observed. This interaction is inhibited by soluble L- ficolin ligands. Interaction of L-ficolin with E l and E2 potently inhibits entry of retroviral pseudoparticles bearing these glycoproteins. L-ficolin also inhibits entry of cell-cultured HCV in a calcium-dependent manner. Neutralizing concentrations of L- ficolin have been found to be circulating in the serum of HCV-infected individuals. This is the first description of direct neutralization of HCV entry by a ficolin and highlights a novel role for L-ficolin as a virus entry inhibitor.

RESULTS

In vitro expressed recombinant L-ficolin forms oligomers similar to serum- purified L-ficolin. Natural L-ficolin exists as oligomers of a 35kD subunit, up to and including a dodecamer form (Figure 1A). These oligomers were observed for fractions of L-ficolin purified from human serum after passage through a GlcNAc-Sepharose matrix and elution with soluble GlcNAc (Figure IB, left panel). Proteins observed at 35 kDa, 70 kDa, and above 250 kDa represented monomers, dimers, and higher order oligomers, respectively. Consistent with previous reports [3 1], proteins were also observed at molecular masses greater than the expected dodecamer, suggesting that L- ficolin is able to form covalently-linked higher molecular weight oligomers. Under reducing conditions the oligomers of serum L-ficolin were reduced to a molecular mass of 35 kDa (Figure IB, right panel), with traces of dimer or trimer. Recombinant FLAG-tagged, affinity purified L-ficolin was observed as a similar mixture of monomers and oligomers, both when probed with anti-L-ficolin mAb (Figure 1 C), and with anti-FLAG mAb (Figure ID), indicating that the recombinant protein possesses similar structure to the in vivo form. Under these non-reducing conditions, higher oligomers including a possible dodecamer complex were observed. Treatment with dithiothreitol resulted in only the monomeric recombinant protein resolved by western blot (Figure IE).

Recombinant L-ficolin interacts with HCV glycoproteins. Two recombinant glycoprotein constructs derived from the HCV infectious clone H77c [32] were used to model the interaction between L-ficolin and HCV virions. Binding of both recombinant L-ficolin and L-ficolin purified from serum to the E2 glycoprotein ectodomain (aa363-661) was comparable (Figure 2A). This was confirmed by dose- dependent binding of recombinant L-ficolin to E 1/E2 heterodimers (aal 70-746) (Figure 2B).

Pre-incubating L-ficolin with ligands resulted in competition for E2 binding (Figure 2C). Both N-acetyl-cysteine (CysNAc) and GlcNAc inhibited the binding interaction between recombinant L-ficolin and the E2 ectodomain in a dose-dependent manner. Fifty per cent inhibition was achieved with 1.8 mM CysNAc, and 260 mM GlcNAc. At the greatest concentration tested ( 1 M), the MBL ligand D-Mannose had no effect on binding.

L-ficolin neutralizes genetically diverse HCV strains. Having demonstrated an interaction between recombinant L-ficolin and the HCV glycoproteins, the effect of L- ficolin on entry of HCVpp possessing the HCV glycoproteins isolated from patient viruses was investigated. Before performing these experiments the expression of L- ficolin by target Huh7 cells was assessed. No cellular expression of L-ficolin in the target cells was observed, either by immunofluorescence or by quantitative RT-PCR of L-ficolin mRNA (data not shown). Glycoproteins derived from genetically diverse HCV viruses were tested in a pseudoparticle entry assay (Figure 3A). Active L-ficolin in the preparation was quantified using a functional assay binding to acetylated-BSA. At a concentration of ^gmL^"1 active protein almost complete inhibition of entry of pseudoparticles reconstituted with strains H77 (genotype la), UKN3A 13.6 (genotype 3a) and UKN4.1 1. 1 (genotype 4) was observed (Figure 3 A). A control fraction from the purification procedure containing no L-ficolin (as determined by western blot and BCA assay) had no inhibitory effect when diluted equivalently. Entry of pseudoparticles bearing VSV G was also neutralized by L-ficolin. Recombinant L-ficolin was further separated into three fractions containing a mixture of oligomers/monomers, or only monomers, as analyzed by western blot (Figure 3B) and stained SDS-PAGE (Figure 3C). Consistent with the greater sensitivity of western blotting, protein was only observed in sample F l in the stained gel. No contaminating protein was observed. Total protein was quantified in these samples by BCA assay. Samples F l , F2 and F3 possessed 157μgmL^~1, 46μgmL^"1, and 10μgmL^"1 protein, respectively. Each sample was diluted to ^gmL^"1 total protein and assessed for neutralizing potency against HCVpp possessing the glycoproteins from strain JFH 1 (Figure 3D). Fraction 1 neutralized HCVpp entry by >60%. Fractions 2 and 3 demonstrated no significant inhibition. A negative control fraction possessed no neutralizing activity. To exclude the possibility that the small neutralizing effect of fractions 2 and 3 might be due to the presence of residual FLAG peptide in the eluted material, neutralization experiments with this peptide demonstrated no effect on the entry of HCV pseudoparticles (not shown) . Given that the negative fraction was treated identically and had no effect on entry it is most likely that the neutralizing effect was due to small quantities of oligomeric L-ficolin in these fractions. The sample possessing detectable oligomeric L-ficolin consistently had significantly greater neutralizing effect than any other sample tested. This sample was further found to inhibit the entry of cell-cultured HCV (HCVcc) of strain JFH- 1. Sample F l , at a stock of 157μgmL^"1 was serially diluted in parallel with the negative fraction before incubation with HCVcc. Neutralization by F l was dose-dependent, with an EC₅₀ of l ^gmL^"1 (Figure 3F), while no inhibition was observed for the negative fraction.

To eliminate the possibility that the observed neutralizing effect was a result of L- ficolin interacting with the target cells, pre-incubation experiments were performed with a preparation of L-ficolin. Neutralization was only observed when pseudoparticles were pre-incubated with the L-ficolin-containing sample (Figure 4). L-ficolin neutralizes cell cultured HCV in a calcium-dependent manner. L-ficolin binding has been described to be partially calcium-dependent [30] . To determine if this is the case for recognition of HCV, neutralization experiments were performed using the HCVcc in the presence of different concentrations of CaCl₂. HCVcc particles were prepared in media containing a basal level of 2mM CaCl₂, or media supplemented to a final concentration of 7mM CaCl_2. Using l ^gmL^"1 L-ficolin in this assay, enhanced neutralization was observed with increasing calcium chloride concentration, indicating that this activity is, at least in part, calcium-dependent. This is likely to be caused by interactions between the S I binding site and the GlcNAc present on the surface of HCV particles. When L-ficolin treated with 7mM CaCl₂ was analyzed by western blot, no difference in patterns of oligomerization was observed (Figure 5B). Consistent with previous reports [3 1], the action of calcium is likely to be on binding activity, rather than disulphide-mediated oligomerization. Inibition of HCV or VSV entry by L-ficolin ligands. To further assess the binding specificity to the observed neutralization, inhibition experiments with GlcNAc and CysNAc were performed. At the concentrations demonstrated to inhibit interaction of E2 with L-ficolin, both GlcNAc (data not shown) and CysNAc (Figure 6) were also found to inhibit entry in the absence of L-ficolin. As such, any blocking effect of L- ficolin neutralization was not resolvable. This unexpected result could be evidence that cellular receptors involved in HCV entry recognize similar acetyl-containing molecular entities as the ficolin, so that acetyl-containing inhibitors (GlcNAc and CysNAc) also competitively inhibit these interactions. To investigate this further, CysNAc was incubated with either HCV or VSV pseudoviruses, or target cells, prior to infection (Figure 6B). Treatment of both HCVpp and VSVpp with CysNAc resulted in reduction in infectivity. The lack of inhibition of pseudovirus entry with CysNAc pre-treated cells suggests that this blocking effect is mediated on the virus, rather than the cells, and is not attributed to cell cytotoxicity. Visual inspection of Huh7 cells treated with CysNAc (Figure 6C) confirmed that no cell death occurred in treated cells, but revealed a visible change in cell morphology and size, which may be linked to altered receptor expression and resistance to HCV entry.

L-ficolin neutralizes HCV entry at physiologically relevant levels. As many proteins can inhibit viruses at high concentrations, experiments were undertaken to determine if the concentration of L-ficolin that resulted in in vitro neutralization was physiologically relevant in both healthy donors and a HCV-infected cohort of patients. Serum L-ficolin was quantified by ELISA in the sera taken from healthy donors and patients with chronic HCV infection (Figure 7) . There was no significant difference between the median L-ficolin concentrations in healthy donors (4^g mL- 1 S .D . ± 1.5) or chronic HCV infections (4.2μg mL- 1 S .D. ± 1.9) (Mann-Whitney U test, p=0.29). This confirmed that the concentration of L-ficolin that neutralizes HCV entry in vitro is biologically relevant, and that individuals with chronic HCV infection do not have impaired capacity to produce L-ficolin. Differential neutralization of HCV by mutant forms of L-ficolin. The L-ficolin gene FCN2 is known to possess multiple alleles. Two non-synonymous substitutions occur as well-represented polymorphisms in the exon 8 region in healthy populations. These result in the amino acid changes T236M and A258S. It was postulated that these substitutions could alter interactions with ligands such as hepatitis C virus glycoproteins. To investigate the importance of amino acid mutations in the binding domain of L-ficolin on the recognition of HCV, Both the wild type variant (T236/A258) and the M236/A258 variant neutralized HCV entry effectively.

L-ficolin neutralizes Ebola and Machupo virus entry. Figure 10A illustrates that in addition to HCV and VSV, L-ficolin can also neutralise Ebola and Machupo virus entry.

H-ficolin neutralizes Ebola virus and HCV entry. Experiments analogous to those performed with L-ficolin were undertaken with H-ficolin expressed and isolated from a human cell line . The results shown in Figure 10B and I OC demonstrate that H- ficolin, like L-ficolin, also neutralises viral entry into cells.

DISCUSSION The data presented here demonstrates that a recombinant, oligomeric L-ficolin or H- ficolin, mediates direct neutralization of enveloped virus entry, as exemplified with HCV, VSV and Ebola.

A key advance was the expression of correctly-folded oligomeric recombinant L- ficolin in human cells, in contrast to previous studies using bacterially-expressed protein that yielded only monomers [22] . The monomeric form of L-ficolin was described to activate the complement cascade and facilitate complement-mediated lysis of HCV infected cells, but not to inhibit HCV entry. The N-terminal FLAG-tag used for purification had no significant effect on the oligomerization of the recombinant L-ficolin polypeptides, and this oligomer possessed binding equivalent to serum-purified protein. This construct provides a useful tool for further investigations of the direct anti-viral properties of L-ficolin.

Acetylated sugars are defined ligands for L-ficolin [ 14] . It is likely that the high mannose oligosaccharides present on the surface of E 1/E2 possessing a GlcNAc₂ stem are binding targets for L-ficolin [34,35] . There is evidence that two of the N-linked glycosylation sites might possess complex glycans containing terminal GlcNAc residues at residues 423 and 430 [7] . These asparagines have been implicated in the entry of HCVpp [8], and HCVcc [9], respectively. The neutralization of entry of HCVcc and HCV pseudoparticles representing HCV genotypes 1 , 2, 3 and 4 is consistent with a role for conserved E 1/E2 glycans in HCV entry. The genotype 3a clone used in this study has previously been described to be resistant to neutralization by broadly-neutralizing antibodies [27,36] . L-ficolin effectively neutralized this isolate, indicating possible therapeutic application for inhibiting entry of antibody neutralization-resistant HCV strains. Consistent with previous reports demonstrating interaction of L-ficolin with GlcNAc [ 17] and CysNAc [30], both ligands inhibited interaction of recombinant L-ficolin with glycoprotein E2, suggesting interaction of the fibrinogen-like domain and N-linked glycans. Four discrete binding sites in the L- ficolin fibrinogen-like domain have been identified (S I -4), which possess different binding specificities [37] . This may account for the difference observed in competition assays with the ligands GlcNAc and CysNAc; although they both bind in the S2 site, GlcNAc also binds around the S I site, while CysNAc is able to bind to site S3, and at high concentrations, to other sites with little structural interaction The calcium-dependent nature of the neutralizing activity suggested that the accessibility of the binding site on L-ficolin is modulated by the presence of a Ca²⁺ ion as previously described [ 16] .

The data presented demonstrates that recombinant oligomeric L-ficolin and H-ficolin inhibit entry of HCV, VSV and Ebola pseudoviruses, illustrating that- ficolinneutralization has potential for application as an entry inhibitor for other enveloped viruses, particularly those with acetylated glycoproteins.

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Claims

1. A composition comprising a ficolin or a ficolin derived molecule for use in the treatment and/or prevention of infection by an enveloped virus.

2. A pharmaceutical composition, for use in the treatment and/or prevention of infection of a subj ect by an enveloped virus, comprising a ficolin or a ficolin derived molecule and a pharmaceutically acceptable carrier or excipient.

3. Use of composition comprising a ficolin or a ficolin derived molecule in the preparation of a medicament for the treatment and/or prevention of infection by an enveloped virus.

4. A method of treating and/or preventing a viral infection caused by an enveloped virus in a subject, comprising administering a composition comprising a ficolin or a ficolin derived molecule to a subject in need thereof.

5. A kit for use in treating and/or preventing a viral infection caused by an enveloped virus, wherein the kit comprises a ficolin or a ficolin derived molecule and instructions relating to administration.

6. The composition, use, method or kit of any preceding claim for use as a therapeutic and/or prophylactic agent.

7. The composition, use, method or kit of any preceding claim wherein the ficolin or ficolin derived molecule comprises at least a fibrinogen-like domain.

8. The composition, use, method or kit of any preceding claim wherein the ficolin is L-ficolin, H-ficolin, M-ficolin or a molecule derived therefrom.

9. The composition, use, method or kit of any preceding claim wherein the ficolin or ficolin derived molecule is in an oligomeric form.

10. The composition, use, method or kit of any preceding claim wherein the ficolin is L-ficolin or is derived from L-ficolin and is in an oligomeric form, preferably in a dodecameric form.

1 1. The composition, use, method or kit of any of claims 1 to 9 wherein the ficolin is H-ficolin or is derived from H-ficolin and is in an oligomeric form.

12. The composition, use, method or kit of any preceding claim wherein the ficolin or ficolin derived molecules comprises one or more monomers each comprising or consisting of the sequence of Seq ID No: 1 , 2, 4 or 5 or that of any known ficolin; or having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity with the sequence of Seq ID No: l , 2, 4 or 5 or that of any known ficolin.

13. The composition, use, method or kit of any preceding claim wherein the ficolin or ficolin derived molecules comprises one or more monomers each comprising or consisting of at least the sequence of the fibrinogen-like domain of Seq ID No: 1 , 2, 4 or that of any known ficolin; or having at least 50%, 55%, 60%, 65%, 70%, 75 %, 80%, 85%, 90%, 95% or more sequence identity with the sequence of the fibrinogen- like domain of Seq ID No: 1 , 2, 4 or that of any known ficolin.

14. The composition, use, method or kit of any preceding claim wherein the enveloped virus is selected from the group consisting of Asfaviridae, Hepadnaviridae, Herpesviridae, Poxviridae, Arenaviridae, Arteriviridae, Bunyaviridae, Coronaviridae, Filoviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, Retroviridae, Rhabdoviridae and Togaviridae

15. The composition, use, method or kit of any preceding claim wherein the enveloped virus is a Hepatitis C virus; a Vesicular stomatitis virus; an Ebola virus; a Human Immunodeficiency virus; a Machupo virus; or an Influenza virus.

16. The composition, use, method or kit of any preceding claim wherein virus is a Hepatitis C virus; a Vesicular stomatitis virus; an Ebola virus or a Machupo virus..

17. A protein, or a composition comprising a protein, having the sequence of Seq ID No:2.

18. The protein or composition of claim 17 may be provided in monomeric or oligomeric form.