WO2014122660A1 - Cd14 inhibitors as an effective treatment for hcv infection - Google Patents

Abstract

This disclosure relates to the prevention and/or treatment of HCV infection and HCV related diseases using CD14 inhibitors. The present disclosure derives from the unexpected finding that blocking CD14 receptor using any type of CD14 inhibitor results in a significant reduction in HCV RNA levels and in almost complete elimination of HCV production.

Description

CD14 INHIBITORS AS AN EFFECTIVE TREATMENT FOR HCV INFECTION

CROSS REFERENCE TO RELATED APPLICATIONS

Benefit is claimed to U.S. Provisional Patent Application No. 61/763,061, filed February 11, 2013, the contents of which are incorporated by reference in their entirety.

FIELD

This disclosure relates to the prevention and/or treatment of HCV infection and HCV related diseases using CD 14 inhibitors. The present disclosure derives from the unexpected finding that blocking CD 14 receptor using any type of CD 14 inhibitor results in a significant reduction in HCV RNA levels and in almost complete elimination of HCV production.

BACKGROUND

Hepatitis C Virus (HCV) is a single- stranded positive RNA virus, which belongs to the family of Flaviviridae, genus Hepacivirus.

The genome of HCV comprises a single positive-stranded RNA that encodes a polyprotein of about 3010 amino acids , flanked at either end by noncoding regions (NCRs). The 5'-NCR and the first part of the polyprotein-encoding region fold into a complex structure of hairpin loops and unpaired regions that can act as an internal ribosome entry site (IRES). The genome-intrinsic IRES results in cap-independent translation of the virus genome; with the initiation of translation directed to the AUG codon at the beginning of the polyprotein, rather than the 5'-terminal AUG.

RNA secondary structures have also been described for the 3 '-untranslated region, and it is thought that these might play a role in the replication of the virus genome, although there is no current direct evidence for this.

The N-terminus of the polyprotein is comprised of four structural proteins (core, El , E2 and p7) and release of these proteins from the polyprotein is dependent upon the signal peptidase associated with the cellular endoplasmic reticulum. Release of the six nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) from the remainder of the polyprotein is mediated by the virus NS2-NS3 and NS3-NS4A proteases.

HCV has extensive genetic heterogeneity reflected by a worldwide distribution of 6 HCV genotypes and more than 70 subtypes. In infected individuals, HCV is presented as a mixture of closely related genomes defined as quasispecies. Globally, at least 170 million people are chronically infected with HCV, with 9 million infected in the United States and Western Europe. Infection with HCV is a major cause of chronic hepatitis and the leading cause of end-stage liver disease including liver cirrhosis and hepatocellular carcinoma. A strong association has also been observed between the development of hepatocellular carcinoma and infection with HCV.

HCV related cirrhosis represents the most common indication for liver transplantation (LT) worldwide. About 40% of liver transplants are performed in patients having HCV and this number is expected to significantly rise during the next decade. Unfortunately, HCV infection invariably recurs after LT, which leads to diminished graft and patient survival (Gane E., Antivir Ther. 2012;17(6 Pt B): 1201-1210).

Treating hepatitis C in this population has a number of major challenges including diminished patient tolerance for side-effects as well as managing the patient's

immunosuppression.

Until May 2011, the standard therapy for chronic HCV infection was the combination of peginterferon-alfa (PEG-IFN) and ribavirin (RBV). However, only approximately 45% of Caucasian patients with HCV genotype 1 infection achieve a sustained virologic response (SVR) to this therapy, and this rate is substantially lower in African Americans and Hispanics, as well as in patients with HIV co-infection. Therefore, there is an effort to develop new therapies against HCV. The main approach is based on the development of direct acting antivirals (DAAs) that target viral proteins directly. These efforts have recently led to the approval of two viral protease inhibitors, boceprevir and telaprevir, for the treatment of genotype 1 chronic HCV infection. However, these protease inhibitors have the significant drawback of rapid emergence of viral resistance; therefore, they must be administrated as a combination therapy with PEG-IFN and RBV. This triple therapy (telaprevir or boceprevir with PEG-IFN/RBV) has increased viral clearance in more patients, but it is still unsuitable for those patients either intolerant of or with contraindications to IFN or RBV. In addition, the triple therapy has limited benefit for patients previously shown to be null responders to PEG- IFN/RBV, and in those with advanced fibrosis.

Thus, there is an urgent need for the development of an IFN-free HCV treatment.

SUMMARY

Disclosed herein is the surprising finding that antagonism or inhibition of CD 14 activity, expression, or availability to HCV can inhibit or even prevent HCV infection in a subject. Accordingly, provided herein are uses of an antagonist or an inhibitor of CD 14 in the manufacture of a medicament for the treatment or prevention of an infection caused by any genotype of hepatitis C virus (HCV) in a subject that is or may become infected by HCV.

It is appreciated that the ability to inhibit HCV infection also provides use of an antagonist or an inhibitor of CD 14 in the manufacture of a medicament for the treatment of an HCV infection, or for the treatment of a disease associated with HCV infection, or for the prevention of related liver diseases in a subject in need thereof, including but not limited to HCV-associated diseases and conditions such as chronic hepatitis C; and including but not limited to HCV-related liver diseases such as liver fibrosis, liver cirrhosis, or hepatocellular carcinoma.

Methods of treating or preventing HCV infection in a subject in need of such treatment or prevention are also provided herein. Such methods include administering to the subject a therapeutically effective amount of a composition comprising a CD 14 inhibitor or antagonist.

Also described herein is a pharmaceutical composition for the prevention or treatment of HCV or a HCV-related liver disease. Such compositions include a CD14 inhibitor or antagonist, for example a small organic molecule which can inhibit CD 14; a peptide which can inhibit CD 14; an anti-CD 14 antibody, its active fragment, or a derivative thereof; an inhibitory CD 14 fragment comprising soluble CD 14 or a fragment thereof; a CD 14 mutant which can inhibit CD 14; an inhibitor of CD 14 expression, such as those described elsewhere herein; or a HCV protein/peptide counterpart directed to bind CD 14.

Additionally provided herein are methods of mapping HCV virus-CD 14 interacting epitopes on the virus for the development of effective vaccines. Such methods include the steps of contacting HCV, the HCV envelope, or a portion thereof with a CD 14 protein, or a fragment thereof; and identifying the virus epitope contacted by the CD 14 protein or a fragment thereof.

The foregoing and other objects, features, and advantages will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows that blocking CD 14 with a CD 14 small molecule antagonist inhibits HCV infection. Fig. 1 A is a chart showing Real-Time RT-PCR analysis of HCV RNA in Huh7.5 cells 24 hours post-infection, in the absence (dil) or presence (Antagonist) of the CD14 small molecule inhibitor IAXO. Figs. IB and 1C show the results of a focus forming assay (FFA) performed 72 hours post-infection in the absence (dil) or presence (Antagonist) of IAXO. Fig. IB is chart illustrating focus forming units (FFU) and Fig. 1C shows immunofluorescent staining of cells infected with HCV in the absence and in the presence of IAXO. Fig. ID and Fig IE are charts showing dose-response inhibition of HCV RNA by CD 14 antagonist molecule IAXO. Fig ID shows RT-PCR measurement of HCV RNA; Fig IE shows cell viability assessment, as measured by crystal violet staining.

Fig. 2 shows that blocking anti-CD 14 antibodies inhibit HCV infection. Figs. 2A and 2C are charts showing the results of RT-PCR to detect viral RNA following HCV infection in the presence or absence of anti-CD 14 antibodies. Fig. 2B is a chart showing %FFU of a HCV infection in the presence and absence of anti-CD 14 antibodies.

Fig. 3 shows the inhibitory effect of CD 14 expression silencing on HCV infection. Fig 3 A is a chart showing RT-PCR analysis of HCV or CD 14 RNA in HCV-infected cells which were transfected with CD 14 siRNA. Fig. 3B is a chart showing RT-PCR analysis of HCV RNA in HCV-infected cells which were transfected with a shRNA-expressing plasmid.

Fig. 4 shows that blocking the TLR4 and MyD88 pathways has no inhibitory effect on

HCV infection. Fig. 4A is a chart showing RT-PCR measurement of HCV RNA expression in infected cells treated with the IAXO CD 14 inhibitor or anti-TLR4 antibodies. Fig. 4B is a series of charts showing the expression of TNFa, TLR4, and CD14 in cells treated with MyD88 inhibitor. Fig. 4C is a chart showing RT-PCR measurement of HCV RNA expression in infected cells treated with MyD88. Fig. 4D is a chart showing the FFU% in the same cells analyzed by RT-PCR in Fig. 4C.

Fig. 5 shows that soluble CD 14 inhibits HCV infection. Fig. 5 A shows Western blots showing expression of soluble CD 14. Fig. 5B is a chart showing RT-PCR measurement of HCV RNA expression in infected cells that are stably expressing CD 14.

BRIEF DESCRIPTION OF THE DESCRIBED SEQUENCES

The nucleic and/or amino acid sequences provided herewith are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the

complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:

SEQ ID NOs: 1 and 2 are forward and reverse PCR primers for detecting HCV infection. SEQ ID NOs: 3 and 4 are forward and reverse PCR primers for detecting expression of GADPH.

SEQ ID NOs: 5 and 6 are forward and reverse PCR primers for detecting expression of CD14.

SEQ ID NO: 7 is a MyD88 homo dimerization inhibitory peptide.

SEQ ID NO: 8 is a control peptide of the MyD88 homo dimerization inhibitory peptide.

SEQ ID NOs: 9 and 10 are forward and reverse PCR primers for cloning CD 14. SEQ ID NO: 11 is the sequence of CD14 cloning vector pcDNA3.1/HisA-CD 14.

DETAILED DESCRIPTION

I. Abbreviations

FFU focus forming units

HCV Hepatitis C Virus

Huh Human hepatoma

PEG-IFN peginterferon-alfa

RBV ribavirin

TLR4 Toll-like Receptor 4 II. Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in Krebs et ah, Lewin 's, Genes XI, published by Jones & Bartlett Learning, 2012; Kendrew et al. (eds.), The

Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995.

Administration: The introduction of a composition into a subject by a chosen route. Administration of an active compound or composition can be by any route known to one of skill in the art. Administration can be local or systemic. Examples of local administration include, but are not limited to, topical administration, subcutaneous administration, intramuscular administration, intrathecal administration, intrapericardial administration, intraocular administration, topical ophthalmic administration, or administration to the nasal mucosa or lungs by inhalational administration. In addition, local administration includes routes of administration typically used for systemic administration, for example by directing intravascular administration to the arterial supply for a particular organ. Thus, in particular embodiments, local administration includes intra- arterial administration and intravenous administration when such administration is targeted to the vasculature supplying a particular organ. Local administration also includes the incorporation of active compounds and agents into implantable devices or constructs, such as vascular stents or other reservoirs, which release the active agents and compounds over extended time intervals for sustained treatment effects.

Systemic administration includes any route of administration designed to distribute an active compound or composition widely throughout the body via the circulatory system. Thus, systemic administration includes, but is not limited to intra- arterial and intravenous administration. Systemic administration also includes, but is not limited to, topical administration, subcutaneous administration, intramuscular administration, rectal, oral administration or administration by inhalation, when such administration is directed at absorption and distribution throughout the body by the circulatory system.

Analog, derivative or mimetic: An analog is a molecule that differs in chemical structure from a parent compound, for example a homolog (differing by an increment in the chemical structure, such as a difference in the length of an alkyl chain), a molecular fragment, a structure that differs by one or more functional groups, a change in ionization. Structural analogs are often found using quantitative structure activity relationships (QSAR), with techniques such as those disclosed in Remington (The Science and Practice of Pharmacology, 19th Edition (1995), chapter 28). A derivative is a biologically active molecule derived from the base structure. A mimetic is a molecule that mimics the activity of another molecule, such as a biologically active molecule. Biologically active molecules can include chemical structures that mimic the biological activities of a compound.

Antagonist: A molecule or compound that tends to nullify the action of another, or in some instances that blocks the ability of a given chemical to bind to its receptor or other interacting molecule, preventing a biological response. Antagonists are not limited to a specific type of compound, and may include in various embodiments peptides, antibodies and fragments thereof, and other organic or inorganic compounds (for example, peptidomimetics and small molecules). The term "inhibitor" is generally synonymous with "antagonist," however for certain processes, it is more common to refer to inhibitors. In a particular example an "anti-sense inhibitor" of CD14 expression will reduce the expression of CD14 on a cell surface, thereby inhibiting HCV infection. In a contrasting example, a CD 14 peptide antagonist can bind to CD 14 and prevent the ability for HCV to infect the cell. Antibody: A protein (or protein complex) that includes one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

The basic immunoglobulin (antibody) structural unit is generally a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one light (about 25 kD) and one heavy chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer, respectively, to these light and heavy chains.

As used herein, the term antibody includes intact immunoglobulins as well as a number of well-characterized fragments produced by digestion with various peptidases, or genetically engineered artificial antibodies. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'₂, a dimer of Fab which itself is a light chain joined to V_H-C_H 1 by a disulfide bond. The F(ab)'₂ may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the F(ab)'₂ dimer into an Fab' monomer. The Fab' monomer is essentially a Fab with part of the hinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press, N.Y., 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, it will be appreciated that Fab' fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies.

Antibodies for use in the methods, compositions, and systems of this disclosure can be monoclonal or polyclonal. Merely by way of example, monoclonal antibodies can be prepared from murine hybridomas according to the classical method of Kohler and Milstein (Nature 256:495-497, 1975) or derivative methods thereof. Detailed procedures for monoclonal antibody production are described in Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988).

The terms bind specifically and specific binding refer to the ability of a specific binding agent (such as, an antibody) to bind to a target molecular species in preference to binding to other molecular species with which the specific binding agent and target molecular species are admixed. A specific binding agent is said specifically to recognize a target molecular species, such as a CD 14 molecule, when it can bind specifically to that target.

A single-chain antibody (scFv) is a genetically engineered molecule containing the V_H and V_L domains of one or more antibody(ies) linked by a suitable polypeptide linker as a genetically fused single chain molecule (see, for example, Bird et al., Science, 242:423-426, 1988; Huston et al, Proc. Natl. Acad. Set, 85:5879-5883, 1988). Diabodies are bivalent, bispecific antibodies in which V_H and V_L domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see, for example, Holliger et al. , Proc. Natl. Acad. Set, 90:6444-6448, 1993; Poljak et al, Structure, 2: 1121-1123, 1994). One or more CDRs may be incorporated into a molecule either covalently or noncovalently to make the resultant molecule an immunoadhesin. An immunoadhesin may incorporate the CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) noncovalently. The CDRs permit the immunoadhesin to specifically bind to a particular antigen of interest. A chimeric antibody is an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies.

An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be different. For instance, a naturally-occurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, while a bispecific or bifunctional antibody has two different binding sites.

A neutralizing antibody or an inhibitory antibody is an antibody that inhibits at least one activity of a target— usually a polypeptide, such as the CD 14 polypeptide— such as by blocking the binding of the polypeptide to a ligand to which it normally binds, or by disrupting or otherwise interfering with a protein-protein interaction of the polypeptide with a second polypeptide, such the interference between CD 14 and an epitope on the HCV envelope. An activating antibody is an antibody that increases an activity of a polypeptide. Antibodies may function as mimics of a target protein activity, or as blockers of the target protein activity, with therapeutic effect derived therein.

Antisense inhibitor: Refers to an oligomeric compound that is at least partially complementary to the region of a target nucleic acid molecule to which it hybridizes, for example a CD14-encoding DNA or RNA sequence. As used herein, an antisense inhibitor (also referred to as an "antisense compound") that is "specific for" a target nucleic acid molecule is one which specifically hybridizes with and modulates expression of the target nucleic acid molecule. As used herein, a "target" nucleic acid is a nucleic acid molecule to which an antisense compound is designed to specifically hybridize and modulation expression. Nonlimiting examples of antisense compounds include primers, probes, antisense oligonucleotides, antisense morpholinos, siRNAs, miRNAs, shRNAs and ribozymes. As such, these compounds can be introduced as single- stranded, double- stranded, circular, branched or hairpin compounds and can contain structural elements such as internal or terminal bulges or loops. Double-stranded antisense compounds can be two strands hybridized to form double- stranded compounds or a single strand with sufficient self- complementarity to allow for hybridization and formation of a fully or partially double- stranded compound.

Control: A reference standard. A control can be a known value indicative of basal concentration expression of CD14 or a portion of the HCV genome. In particular examples a control sample is taken from a subject that is known not to have a disease or condition such as a HCV related disease or condition. In other examples a control is taken from the subject being diagnosed, but at an earlier time point, either before disease onset or prior to or at an earlier time point in disease treatment.

Efficacy: Refers to the ability of agent to elicit a desired therapeutic effect. Efficacy also refers to the strength or effectiveness of a compound. As used herein, "enhancing efficacy" means to increase the therapeutic action of an agent, such as by inhibiting a HCV infection or preventing the onset of a HCV-related disease or condition.

Effective amount of a compound: A quantity of compound sufficient to achieve a desired effect in a subject being treated. An effective amount of a compound can be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the effective amount of the compound will be dependent on the compound applied, the subject being treated, the severity and type of the affliction, and the manner of administration of the compound.

Morpholino: A morpholino oligo is structurally different from natural nucleic acids, with morpholino rings replacing the ribose or deoxyribose sugar moieties and non-ionic phosphorodiamidate linkages replacing the anionic phosphates of DNA and RNA. Each morpholino ring suitably positions one of the standard bases (A, G, C, T/U), so that a 25-base morpholino oligo strongly and specifically binds to its complementary 25-base target site in a strand of RNA via Watson-Crick pairing. Because the backbone of the morpholino oligo is not recognized by cellular enzymes of signaling proteins, it is stable to nucleases and does not trigger an innate immune response through the toll-like receptors. This avoids loss of oligo, inflammation or interferon induction. Morpholinos can be delivered by a number of techniques, including direct injection to tissues or via infusion pump and intravenous bolus.

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers useful in this disclosure are conventional. Remington 's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the compounds herein disclosed.

In general, the nature of the carrier will depend on the particular mode of

administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Pharmaceutical agent: A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject or a cell.

Preventing or treating a disease: Preventing a disease refers to inhibiting the full development of a disease, for example inhibiting the development of chronic hepatitis in a person who has been infected by HCV. Treatment refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop.

RNA interference (RNA silencing; RNAi): A gene-silencing mechanism whereby specific double- stranded RNA (dsRNA) trigger the degradation of homologous mRNA (also called target RNA). Double-stranded RNA is processed into small interfering RNAs

(siRNA), which serve as a guide for cleavage of the homologous mRNA in the RNA-induced silencing complex (RISC). The remnants of the target RNA may then also act as siRNA; thus resulting in a cascade effect.

Small interfering RNAs: Synthetic or naturally-produced small double stranded RNAs (dsRNAs) that can induce gene-specific inhibition of expression in invertebrate and vertebrate species are provided. These RNAs are suitable for interference or inhibition of expression of a target gene and comprise double stranded RNAs of about 15 to about 40 nucleotides containing a 3' and/or 5' overhang on each strand having a length of 0- to about 5- nucleotides, wherein the sequence of the double stranded RNAs is essentially identical to a portion of a coding region of the target gene for which interference or inhibition of expression is desired. The double stranded RNAs can be formed from complementary ssRNAs or from a single stranded RNA that forms a hairpin or from expression from a DNA vector.

Small molecule inhibitor: A molecule, typically with a molecular weight less than 1000, or in some embodiments, less than about 500 Daltons, wherein the molecule is capable of inhibiting, to some measurable extent, an activity of some target molecule.

Subject: Living multi-cellular organisms, including vertebrate organisms, a category that includes both human and non-human mammals.

Subject susceptible to a disease or condition: A subject capable of, prone to, or predisposed to developing a disease or condition. It is understood that a subject already having or showing symptoms of a disease or condition is considered "susceptible" since they have already developed it. As used herein "subject in need of treatment" is used

synonymously with "subject susceptible to a disease or condition."

Target sequence: A target sequence is a portion of ssDNA, dsDNA, or RNA that, upon hybridization to a therapeutically effective oligonucleotide or oligonucleotide analog (e.g., a morpholino), results in the inhibition of expression of the target. Either an antisense or a sense molecule can be used to target a portion of dsDNA, as both will interfere with the expression of that portion of the dsDNA. The antisense molecule can bind to the plus strand, and the sense molecule can bind to the minus strand. Thus, target sequences can be ssDNA, dsDNA, and RNA.

Therapeutically effective amount: A quantity of compound sufficient to achieve a desired effect in a subject being treated. An effective amount of a compound may be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the effective amount will be dependent on the compound applied, the subject being treated, the severity and type of the affliction, and the manner of administration of the compound. For example, a therapeutically effective amount of an active ingredient can be measured as the concentration (moles per liter or molar-M) of the active ingredient (such as a small molecule, peptide, protein, or antibody) in blood (in vivo) or a buffer (in vitro) that produces an effect. Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes." The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

In case of conflict, the present specification, including explanations of terms, will control. In addition, all the materials, methods, and examples are illustrative and not intended to be limiting.

III. Overview of Several Embodiments

Disclosed herein are compositions and methods for inhibiting HCV infections. The provided compositions and methods were developed in view of the surprising finding that inhibition of CD 14 activity, expression, or other availability to infecting HCV will inhibit and even prevent HCV infection.

A particular embodiment includes use of an antagonist or an inhibitor of CD 14 in the manufacture of a medicament for the treatment of an infection caused by any genotype of hepatitis C virus (HCV) in a subject that is or may become infected by HCV.

It is appreciated that the ability to inhibit HCV infection also provides use of an antagonist or an inhibitor of CD 14 in the manufacture of a medicament for the treatment of an HCV infection, or for the treatment of a disease associated with HCV infection, or for the prevention of related liver diseases in a subject in need thereof, including but not limited to HCV-associated diseases and conditions such as chronic hepatitis C; and including but not limited to HCV-related liver diseases such as liver fibrosis, liver cirrhosis, or hepatocellular carcinoma.

In particular embodiments of the disclosed uses, the antagonist is a small organic molecule which can inhibit CD 14 activity or function, a peptide which can inhibit CD 14 activity or function, an inhibitory CD 14 fragment comprising soluble CD 14 or a fragment thereof, or a CD 14 mutant which can inhibit wild type CD 14 activity or function.

In other particular embodiments of the disclosed uses, the CD 14 inhibitor is an anti- CD 14 antibody, its active fragment, or a derivative thereof; an inhibitor of CD 14 expression; or a HCV protein/peptide/lipoprotein counterpart directed to bind CD 14 or proteins and peptides derived from additional members in CD14-HCV putative complex formation.

In particular embodiments, the inhibitor of CD 14 expression is an anti-CD 14 siRNA, an antisense oligonucleotide, an antisense morpholino, ribozymes, CD 14 competing derivatives, molecules that target the CD 14 promoter, molecules that inhibit CD 14

transcription, or CD14 mimicry substances.

In other embodiments of the described uses, the subject is concurrently undergoing a treatment with interferon-alpha or any other inhibitor of HCV proteases and/or polymerases.

Also described herein are combination compositions for the treatment of HCV infection, or for the treatment of a disease associated with HCV infection, which include a medicament comprising an antagonist of CD14 or an inhibitor of CD14 expression and at least one medicament selected from the group consisting of a medicament comprising interferon-alpha, and an inhibitor of HCV proteases and/or polymerases.

Methods of treating or preventing HCV infection in a subject in need of such treatment or prevention are also provided herein. Such methods include administering to the subject a therapeutically effective amount of a composition comprising a CD 14 inhibitor. The CD 14 inhibitor (which in some examples may be referred to as an antagonist) comprises a small organic molecule which can inhibit CD 14, a peptide which can inhibit CD 14, an anti-CD 14 antibody, its active fragment, or a derivative thereof, an inhibitory CD 14 fragment comprising soluble CD 14 or a fragment thereof, a CD 14 mutant which can inhibit CD 14, an inhibitor of CD 14 expression, or a HCV protein/peptide counterpart which is able to bind CD 14 independently of the whole virion, thereby blocking access to CD 14 by HCV.

In particular examples, the methods of treatment or prevention further include sequential or concurrent administration to the subject of any other active ingredient used for treating HCV. In particular examples, such active ingredients include interferon-alpha or any other inhibitor of HCV proteases and/or polymerases.

Also described herein is a pharmaceutical composition for the prevention or treatment of HCV or a HCV-related liver disease. Such compositions include a CD 14 inhibitor or antagonist, for example a small organic molecule which can inhibit CD 14; a peptide which can inhibit CD 14; an anti-CD 14 antibody, its active fragment, or a derivative thereof; an inhibitory CD 14 fragment comprising soluble CD 14 or a fragment thereof; a CD 14 mutant which can inhibit wildtype CD 14 function; an inhibitor of CD 14 expression, such as those described elsewhere herein; or a HCV protein/peptide counterpart directed to bind CD 14 independently of the whole virion, thereby blocking access to CD 14 by HCV.

In particular embodiments, the described pharmaceutical composition can also include any other active ingredient used for treating HCV, such as interferon- alpha or any other inhibitor of HCV proteases and/or polymerases.

Additionally provided herein are methods of mapping HCV virus-CD 14 interacting epitopes on the virus for the development of effective vaccines. Such methods include the steps of contacting HCV, the HCV envelope, or a portion thereof with a CD 14 protein, or a fragment thereof; and identifying the virus epitope contacted by the CD 14 protein or a fragment thereof.

IV. CD14

CD 14 protein was first identified as a differentiation marker on the surface of monocytes and macrophages. It was characterized as a receptor for bacterial endotoxin (LPS) in 1990. CD14 exists in membrane (mCD14) and soluble (sCD14) forms. The mCD14 protein is processed in the endoplasmatic reticulum and expressed on the cell surface via a glycosylphosphatidyl (GPI) anchor. The mCD14 is typically expressed on the surface of monocytes, macrophages and immune cells however, it is also expressed by many non- myeloid cells (Finlay, B.B. & Hancock, R.E. Nat Rev Microbiol 2, 497-504 (2004)), including human hepatocytes which, were reported to express CD14 (hen, Y.C., Wang, S.Y. & King, C.C. J Virol 73, 2650-2657 (1999)).

CD 14 is known to trigger activation of the innate immune response to bacterial components including lipopolysaccharide (LPS) via the Toll-like receptor (TLR) pathway and has an important role in innate immune response facing Gram-negative and Gram- positive sepsis . As such, CD 14 is considered a favorable target for intervention in sepsis and many strategies have been developed for targeting CD 14, including synthetic small molecules, siRNA, antibodies and CD14 mutants.

CD14 together with LPS-binding protein (LBP) plays an essential role in binding of

LPS to the TLR4/MD-2 complex. LBP, which is present in the bloodstream, binds to LPS aggregates and transfers LPS monomers to CD 14. CD 14 associates with TLR4/MD-2 and transfers the LPS monomer to this complex. CD14 exist also in soluble forms (Frey EA, et al.. 1992. J Exp Med 176: 1665-71; Yaegashi Y, et al.. 2005. J Infect Chemother 11: 234-8). sCD14 is found in serum and mediates LPS activation of non-CD 14-bearing cells (Su GL, et al. 1999. J Hepatol 31: 435-42), and it was shown to be also produced by human hepatocytes.

The discovery that CD 14 (both the soluble and membrane forms) is expressed by non- myeloid lineage cells may imply its role in recognition of not only LPS but to other microbial constituents recognized by the pattern receptor CD14 (Su GL, et al. 1999. J Hepatol 31: 435- 42). Indeed, CD 14 have been previously implicated as an attachment factor for Dengue Virus (DENV), a member of the Flaviviridae family in mammalian cells (Chen YC, Wang SY and King CC. 1999. J Virol 73: 2650-7). CD14 was also reported to bind recombinant yeast- derived hepatitis B virus surface antigen (rHBsAg) particles, which contain the S protein only, however, CD 14 did not bind the natural plasma derived HBsAg (pHBsAg) (Vanlandschoot P, et al.. 2002. Journal of General Virology 83: 2279-89.)

CD 14 has been identified as a receptor for LPS. While it is predominantly expressed on monocytes, macrophages, and granulocytes, CD14 was also shown to be expressed in hepatocytes and in human and rat liver (Su GL, et al., 1999. J Hepatol 31: 435-42; Chou MH, et al., 2012. PLoS One 7: e34903). Human hepatocytes were shown to expresses CD14 mRNA by Northern blot analysis and CD 14 protein expression was further confirmed by Western blot analysis and by immunohistochemical staining (Su GL, et al., 1999. J Hepatol 31: 435-42;). Moreover, CD 14 expression was observed on human hepatocytes transplanted into immunodeficient mice (Pan Z, et al., 2000. J Biol Chem 275: 36430-5) as well as in human liver (Cho K, et al., 2004. J Surg Res 122: 36-42). It was also shown that adult hepatocytes are the source of soluble human CD14 in acute phase serum (Cho K, et al., 2004. J Surg Res 122: 36-42; Meuleman P, et al., 2006 Clin Chim Acta 366: 156-62).

As used herein CD 14 may be from human or any non-human mammalian origin.

Accordingly, all discussion of "CD14 antagonists" or "CD14 inhibitors" is generally directed to antagonists and inhibitors of CD 14, and fragments thereof, of human as well as non-human mammalian origin.

V. HCV

The life cycle of HCV is closely tied to lipid metabolism of liver cells, and lipid droplets have emerged as crucial intracellular organelles that support persistent propagation of viral infection. In the blood, viral particles are bound to plasma lipoproteins. These particles named Lipo-Viro-Particles (LVP) appeared as large lipoprotein-like structures enriched in triglycerides containing internal viral capsids and carrying viral envelope proteins at their surface.

Agaugue et al. (PLoS One 2: e330; 2007), have demonstrated that purified LVP from HCV clinical isolate can interfere with the TLR4 pathway. Although the precise mechanism for TLR4 pathway interference has not been identified, it was suggested LVP could interfere directly with the TLR4 pathway via some of its lipid components. HCV infection was shown to directly interfere with TLR4 signaling in hepatocytes, peripheral blood mononuclear cells, Raji cells and dendritic cells (Tamura, R., et al.. Journal of Infectious Diseases 204, 793-801 (2011).

In the course of ongoing studies to elucidate the role of TLR4 signaling pathway in

HCV infection, it was surprisingly found that blocking CD 14, the chaperone of LPS molecules to the TLR4-MD-2 signaling complex, results in a significant reduction in HCV RNA levels and a remarkable inhibition, almost complete elimination of HCV production. Having a role in the early stages of HCV life cycle, targeting CD 14 would have the advantages over those inhibitors that aim to disrupt the viral replication process. It would be unlikely to have cross-resistance with existing drugs and it would be expected to have high activity against currently inhibitor-resistant strains of HCV.

VI. Therapeutic Uses

Disclosed herein is the discovery that inhibition and/or antagonism of CD 14, such as on the surface of hepatic cells, or the inhibition of CD 14 expression, will inhibit or can even prevent HCV infection of cells, and by extension HCV replication. Accordingly, described herein are therapeutic uses for the treatment or prevention of HCV with a composition comprising CD 14 antagonists and inhibitors. Pharmaceutical compositions for such therapeutic uses are also described. Further described are methods of prevention or treatment of HCV infection. It will be appreciated that by inhibiting or even preventing HCV infection in a subject, methods of inhibiting or preventing HCV-related diseases or conditions, which develop as a result of HCV infection, are also described herein, such as liver fibrosis and liver cirrhosis.

Persistent HCV infection is known as a major risk for the development of hepatocellular carcinoma (HCC), the fifth most common cancer responsible for approximately one million deaths each year. It is thought that the malignant transformation of hepatocytes in HCC occurs through a pathway of increased liver cell turnover induced by chronic liver injury and regeneration in a context of inflammation and oxidative DNA damage. Accordingly, inhibiting or preventing HCV infection will also inhibit the development of HCC. Additionally, inhibition of CD 14, which is also an inflammation modulator may can also the inflammation which accompanies HCV infection.

In another embodiment, use of CD 14 inhibitors to prevent HCV infection can be used in conjunction with liver transplantation surgery, to reduce the risk of HCV infection in transplant recipients. Administration of CD 14 inhibitor during and after liver transplantation will inhibit HCV reinfection of newly transplanted livers in patients with prior HCV infection.

In still another embodiment, this disclosure encompasses a vaccine composition comprising CD 14 derivatives or fragments thereof for protecting an individual from HCV virus infection. A possible, though non-limiting, mechanism for the action of such vaccines would be by raising antibodies against CD 14 that will bind to native CD 14 or its fragment and so block HCV access to the cell.

CD14 antagonists and inhibitors

Non-limiting examples of CD14 antagonists and inhibitors for use in the described uses, compositions, and methods include: anti-CD 14 antibodies or fragments thereof which are able to bind CD 14; small molecule agents (such as but not limited to IAXO, Vinci Biochem), which interact with CD 14 and interfere with its biological function; CD 14 mimetics; CD14 competing derivatives (peptide and non-peptide based); antisense

oligonucleotides; a nucleic acid which is capable of hybridizing with at least part of a gene encoding CD14, and inhibiting its expression, such as siRNA and miRNA; ribozymes;

molecules that target CD 14 promoter transcription factors; or that bind to the CD 14 promoter, thereby blocking access to such transcription factors; HCV protein/peptide counterpart directed to bind CD 14; and Proteins and peptides derived from additional members in CD 14- HCV putative complex formation (such as LBP and others).

In particular embodiments, the CD 14 antagonist is a small molecule antagonist that interacts with CD 14 and inhibits HCV binding to or entry into hepatocytes. A non-limiting example of a small molecule for use in the current disclosure is the known lipid A antagonist IAXO (methyl 6-deoxy-6-N-dimethyl-N-cyclopentylammonium-2,3-di-0-tetradecyl-a-D- glucopyranoside iodide; available from AdipoGen®).

In other embodiments, the CD 14 antagonist is an anti-CD 14 antibody or fragments thereof which are able to recognize and bind CD 14. Optimally, antibodies raised against CD 14, or a fragment thereof would specifically detect that peptide/protein, and optimally would inhibit the interaction between CD 14 and its HCV-binding eptiope. Antibodies that specifically detect CD 14 would recognize and bind that protein (and peptides derived therefrom) and would not substantially recognize or bind to other proteins or peptides found in a biological sample. The determination that an antibody specifically detects its target protein is made by any one of a number of standard immunoassay methods; for instance, the Western blotting technique (Sambrook et al., In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989). The determination that a CD14-specific antibody inhibits the association between CD 14 and HCV, or the ability of HCV to enter hepatocytes may be made, for example, using any one of the assays described herein, including for instance RT- PCR detection of HCV RNA following viral infection. Monoclonal antibodies that specifically recognizes epitopes of CD 14 can be prepared from murine hybridomas according to the classical method of Kohler and Milstein (Nature 256:495, 1975) or derivative methods thereof. Detailed procedures for monoclonal antibody production are described in Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988). Polyclonal antiserum containing antibodies to heterogeneous epitopes of a single protein can be prepared by immunizing suitable animals with the expressed protein, which can be unmodified or modified to enhance immunogenicity. An effective immunization protocol for rabbits can be found in Vaitukaitis et al. (J. Clin. Endocrinol. Metab. 33:988-991, 1971). Also contemplated are humanized antibodies, for instance humanized equivalents of a murine monoclonal antibodies. A "humanized" immunoglobulin is an immunoglobulin including a human framework region and one or more CDRs from a non-human (for example a mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is termed a "donor," and the human immunoglobulin providing the framework is termed an "acceptor." In one embodiment, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, such as at least about 85- 90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A "humanized antibody" is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. Humanized immunoglobulins can be constructed by means of genetic engineering (see for example, U.S. Patent No. 5,585,089).

In particular examples, monoclonal antibodies produced in hybridomas (e.g. 3C10 and 28C5), which are available from the American Type Culture Collection (ATCC) can be used in the described uses and methods. Such antibodies are characterized as CD14-binding antibodies that recognize the human membrane -bound and soluble isoforms of CD 14. In other examples, the antibody designated as 28C5 can be used is the murine parent of the anti CD 14 monoclonal antibody (IC14), which is a recombinant chimeric (murine/human) monoclonal antibody that has been produced based on that has been already evaluated in phase I clinical trial (Anas, A., van der Poll, T. & d Vos, A.F. Crit Care 14, 209 (2010) and is now under Phase II clinical trials for acute lung injury by Implicit Bioscience Ltd and CMC ICOS.

In further embodiments, the CD 14 antagonist is a CD 14 mimetic or CD 14 competing derivatives (peptide and non-peptide based). Such mimetics or derivatives can be small molecule or peptide based, and will function to block the interaction between native CD 14 and HCV by competing with native CD 14 for binding to its interacting epitope on the surface of the HCV envelope. Other agents that will similarly antagonize native CD 14 are also contemplated, such as soluble CD 14 (sCD14), and sCD14 subtypes, such as Presepsin (sCD14-ST), and mutant CD14 peptides.

Similarly, in other embodiments, the CD 14 antagonist is a HCV protein/peptide counterpart, which binds CD 14 or proteins and peptides derived from additional members in CD14-HCV putative complex formation. As with the CD 14 mimetic or CD 14 competing derivatives, administration of a HCV-derived CD 14 binding agent can blockade the interaction between CD14 and infectious HCV particles.

In still further embodiments, the CD 14 inhibitor is an inhibitor of CD 14 gene expression. It will be appreciated that if the presence of CD 14 on the membrane of hepatocytes is a requirement for HCV infection, then a decreased or elimination of CD 14 expression will inhibit or even prevent HCV infection. CD 14 expression can be inhibited or eliminated at the level of transcription or at the level of translation. In particular

examples,CD14 expression is inhibited by use of antisense oligonucleotides; antisense morpholinos oligonucleotides, or any other nucleic acid which is capable of hybridizing with at least part of a gene encoding CD 14 (or the RNA product thereof), and inhibiting its expression. Such nucleic acids include as siRNA, shRNA, and miRNA. Although the exact mechanism by which antisense RNA molecules interfere with gene expression has not been elucidated, it is believed that antisense RNA molecules bind to the endogenous mRNA molecules and thereby inhibit translation of the endogenous mRNA.

For antisense suppression, a nucleotide sequence from a CD14-encoding sequence, for example all or a portion of the CD 14 cDNA or gene, is arranged in reverse orientation relative to the promoter sequence in the transformation vector. One of ordinary skill in the art will understand how other aspects of the vector may be chosen. The introduced sequence need not be the full length the cDNA or gene, or reverse complement thereof, and need not be exactly homologous to the equivalent sequence found in the cell type to be transformed. Generally, however, where the introduced sequence is of shorter length, a higher degree of homology to the native target sequence will be needed for effective antisense suppression. The introduced antisense sequence in the vector may be at least 20 nucleotides in length, and improved antisense suppression will typically be observed as the length of the antisense sequence increases. The length of the antisense sequence in the vector advantageously may be greater than about 30 nucleotides, or greater than about 100 nucleotides. For suppression of the CD 14 gene itself, transcription of an antisense construct results in the production of RNA molecules that are the reverse complement of mRNA molecules transcribed from the endogenous CD 14 gene in the cell.

Suppression of endogenous CD14 expression can also be achieved using ribozymes. Ribozymes are synthetic molecules that possess highly specific endoribonuclease activity. The production and use of ribozymes are disclosed in U.S. Patent No. 4,987,071 and U.S. Patent No. 5,543,508. The inclusion of ribozyme sequences within antisense RNAs may be used to confer RNA cleaving activity on the antisense RNA, such that endogenous mRNA molecules that bind to the antisense RNA are cleaved, which in turn leads to an enhanced antisense inhibition of endogenous gene expression.

Suppression can also be achieved using RNA interference, using known and previously disclosed methods. Several models have been put forward to explain RNAi, in particular the mechanisms by which the cleavage derived small dsRNAs or siRNAs interact with the target mRNA and thus facilitate its degradation (Hamilton et al, Science 286, 950, 1999; Zamore et al, Cell 101, 25, 2000; Hammond et al, Nature 404, 293, 2000; Yang et al, Curr. Biol 10, 1191, 2000; Elbashir et al, Genes Dev. 15, 188, 2001; Bass Cell 101, 235, 2000). dsRNAs can be formed from RNA oligomers produced synthetically (for technical details see material from the companies Xeragon and Dharmacon, both available on the internet). Small dsRNAs and siRNAs can also be manufactured using standard methods of in vitro RNA production. In addition, the Silencer™ siRNA Construction kit (and components thereof) available from Ambion (Catalog # 1620; Austin, TX), which employs a T7 promoter and other well known genetic engineering techniques to produce dsRNAs. Double stranded RNA triggers could also be expressed from DNA based vector systems.

Inhibition of gene expression also can be accomplished using morpholino

oligonucleotides, as described herein, and which are employed by similar methods as standard antisense oligonucleotides, and which comprise the same or similar nucleotide sequence as anti-CD 14 antisense oligonucleotide.

In another embodiment, the CD 14 inhibitor includes an oxidized phospholipid such as l-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (OxPAPC) that has been shown to bind and block the interaction of LPS with CD14 (Bochkov VN, et al., Nature.

2002;419(6902):77-81).

It will be appreciated that when administered to a subject, the CD 14 inhibitory and antagonist compounds described herein are formulated in standard formulations known to the art for pharmaceutical compositions. Such compositions can further include any standard pharmaceutically acceptable salts, carriers, and excipients know in the art which are appropriate for any particular mode of administration.

VII. Combination Therapies

The therapeutic uses disclosed herein further encompass combination therapies for the treatment or inhibition of HCV infection, and inhibition of the development of HCV-related diseases and conditions. Such combinations include one or more of the CD 14 inhibitor or antagonist agents to be used in conjunction with a known anti-HCV treatment. In particular examples, the known anti-HCV treatment can include, but is not limited to a type I interferon (e.g., IFN-a or IFN-β), ribavirin, or a combination thereof. In other examples, the

combination treatments embraces treatments or compositions that include at least (1) a CD14 antagonist, (2) a type I interferon or ribavirin, (3) an additional agent that inhibits hepatic virus infection such as but not limited to the newly available protease inhibitors the such as oral protease (PI), boceprevir (BOC) or telaprevir (TVR), along with pegylated IFN (PeglFN) and ribavirin (RBV) (if not used as component #2). In further embodiments the combinations can include (4) an additional agent that inhibits hepatic virus infection such as HCV polymerase inhibitors.

In particular embodiments, the combinations described herein are administered simultaneously, in separate or combined formulations, and in multiple possible combinations. In other embodiments, the described combinations are administered as part of a treatment regimen, wherein first one anti-HCV therapeutic agent (or subcombination of the above agents) is administered, followed by administration of another.

In still other embodiments, the combination therapies contemplated herein are administered concurrently until a defined treatment goal is achieved (e.g. a particular viral titer or measurement of viral RNA), followed by the extension of anti-HCV therapy with a single particular agent (such as a CD 14 inhibitor or antagonist) for a defined length of time to further reduce viral titer or eliminate virus from a subject entirely.

VII. Vaccine Development

The ability to inhibit HCV infection by blockading the interaction between HCV and CD 14 indicates that HCV envelope epitopes or HCV- specific interacting protein/lipoprotein may be good targets for vaccine development. In this respect, the CD 14 inhibitory compounds described herein may be useful tools in elucidating mechanistic information about the biological pathways involved in viral diseases, which can lead to the development of new combinations or single agents for treating, preventing, or reducing a viral disease. For example, the inhibitors described herein will be useful for identification of the interaction between HCV and CD 14, and for mapping virus specific epitopes for the development of effective vaccines and to provide a decoy receptor for viral neutralization.

The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described.

EXAMPLES

Example 1: A CD14 antagonist small molecule blocks HCV infection

This example shows that pre-incubation of hepatocyte cell line with a CD- 14 small molecule antagonist can block HCV infection.

The small molecule CD 14 inhibitor, IAXO (methyl 6-deoxy-6-N-dimethyl-N- cyclopentylammonium-2,3-di-0-tetradecyl-a-D-glucopyranoside iodide; available from AdipoGen®) was used to test the effects of CD 14 on HCV infection. IAXO acts as a Lipid A antagonist and inhibits CD 14 and Toll-like Receptor 4 (TLR4) receptor in vitro and in vivo. It is a drug candidate with therapeutic activity in rodent models of inflammatory diseases, neuropathic pain and LPS (endotoxin) induced septic shock.

Huh7.5 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin (Biological Industries, Israel) in a humidified 5% C02 incubator at 37°C, as specified, cultured cells were treated with IAXO for three hours and then infected with HCV virus (HCV HJ3-5) at a multiplicity of infection (moi) of 0.1-0.01. The effects of IAXO on HCV infection was determined 24 hours post-infection by detecting HCV RNA by Real -Time RT-PCR, according to standard protocols.

Briefly, real-time PCR was carried out with PerfeCTa® SYBR® Green FastMix

(Quanta Biosciences, USA) in a 10 μΐ reaction volume prepared in a 96-well format, using 1 μΐ of template cDNA, 5μ1 of 2* SYBR® Green FastMix , 10 pmol of forward primer HCV-F (nts 82-103) 5'-GCCATGGCGTTAGTATGAGTGT-3' (SEQ ID NO: 1) and reverse primer HCV-R (nts 281-301)- 5'-CCCTATCAGGCAGTACCACAA-3' (SEQ ID NO: 2) (HCV sequences will amplify the HCV nontranslated region; nucleotide numbering is from the HCV genomic sequence on file at GenBank accession number: AF139594). GAPDH was used as the internal control gene (primers: GAPDH-F (nts 176-194) 5'-

G A AGGTG A AGGTC GG AGTC- 3 ' (SEQ ID NO: 3) and GAPDH-R (nts 492-511) 5'-

GAAGATGGTGATGGGATTTC-3' (SEQ ID NO: 2). Primer sequences are derived as indicated from the GADPH gene sequence on file as GenBank Accession No.

NM_001289745). PCR thermal cycling was carried out using ABI 7000 (Applied

Biosystems) detection system

PCR results are shown in Fig 1 A. The effects of IAXO on HCV production was determined 72 hours post-infection using the FFU assay essentially as previously described (Shapira A, et al. (2012); PLoS ONE 7(2): e32320) (Fig. IB). Immunofluorescent staining of cells infected with HCV in the absence and in the presence of IAXO was also assayed using human anti-HCV polyclonal antibodies, followed by staining with 1: 100 diluted Cy3- conjugated goat anti-human IgG

(Jackson ImmunoResearch Laboratories, USA), essentially as previously described (Shapira A, et al. (2012); PLoS ONE 7(2): e32320) (Fig. 1C).

Dose-response inhibition of HCV RNA by CD 14 antagonist molecule IAXO was assayed by incubation of Huh7.5 cells with 0-0.5 μΜ IAXO prior to HCV infection. HCV RNA was assayed by RT-PCR as above. Control (non-treated) cells were assigned a value of 1. Results are normalized to untreated infected cells (Fig. ID). The effect of IAXO on cell viability was also assayed. Huh7.5 cells treated with 0-0.5 μΜ of IAXO was assessed for cell viability by staining with crystal violet, essentially as described previously (Gal-Tanamy M et al., 2011; Hepatology ;54(5): 1570-9. Percent of viable cells was determined compared to non-treated cells set as 100%. Fig. IE shows that cells remained viable even at the highest concentration IAXO tested.

This results shown in Fig. 1 show that IAXO treatment prior to infection resulted in remarkable inhibition of HCV as determined by monitoring HCV RNA (Fig. 1 A) and by FFU assays (Fig. 1B-1C). The results also show that IAXO inhibited HCV in a dose dependent manner (Fig. ID) and this inhibitory effect did not result from a cytotoxic effect as determined by cell viability (Fig IE).

Example 2: Blocking anti-CD14 antibodies inhibit HCV infection

This example shows that, similar to the small molecule inhibitor IAXO, anti-CD 14 antibodies, can inhibit HCV infection.

Huh7.5 cells were treated with an anti CD14 antibody (Santa Cruz or R&D) or supernatant of hybridoma 3C10 (ATCC® TIB228™) for 3 hours and then infected with HCV as described above and in Shapira A, et al. (2012); PLoS ONE 7(2): e32320 . HCV RNA was collected and analyzed by Real -Time RT-PCR after 24 hours as described in Example 1. The control non-treated cells were assigned a value of 1 and results are normalized to untreated infected cells.

The finding that the CD 14 small molecule antagonist IAXO strongly inhibits HCV infection indicated a general ability to inhibit HCV by blocking the activity of CD 14. This hypothesis was tested using anti-CD 14 polyclonal antibodies (Santa Cruz Biotechnology, Inc. USA), raised against amino acids 17-321 of human CD14. Treating cells with anti-CD14 antibodies resulted with more than 90% inhibition in HCV RNA expression level as determined by Real -time RT-PCR (Fig. 2A), and resulted in a complete elimination of viral production as determined by FFU assay (Fig. 2B). Anti CD81 antibody was used as a positive control since it is known to inhibit HCV and anti-rabbit IgG antibodies were used as negative control. Similar results were obtained using a different commercial antibody (R&D systems) raised against amino acids 20-352 of humanCD14, and with supernatant collected from 3C10 (ATCC® TIB228™) hybridoma producing monoclonal antibody that bind CD14 (Fig. 2C). Example 3: CD14 silencing limits of HCV infection

This example shows the effect of siRNA against CD 14 using transiently transfected siRNA or shRNA stably expressed in Huh 7.5 cells. Huh7.5 cells were transfected with a 50nM pool of siRNAs, ON-TARGETplus

Human CD14 siRNA (Catalog No.# L011121-02-0005; Dharmacon, USA) or ON- TARGETplus Non-targeting Control siRNAs (catalogue no# D-001810-01-05; Dharmacon, USA) . SiRna transfection was performed in a 96 well plate, 7500 cells/well. 1 micro-liter of INTERFERin (Polyplus-transfection SA, France) was added to 50nM siRNA diluted in Opti- MEM® (Gibco, USA) , mixed and incubated for 15 minutes at room temperature. Cells were then added to the INTERFERin®/siRNA complexes in a final volume of 175 μΐ and incubated incubate at 37°C for 72 hours. At 72 hours post-transfection, cells were infected with HCV and after 24 hours CD 14 and HCV mRNA expression levels were analyzed by Real Time PCR as described above. For CD 14 mRNA expression CD 14 specific primers were used: CD14-F (nts 359-381) -5'- GGTTCGGAAGACTTATCGACCAT-3' (SEQ ID

NO: 5) and CD14-R (nts 445-465) -5'-TCGTCCAGCTCACAAGGTTCT-3' (SEQ ID NO: 6) (CD 14 amplification primers were derived as indicated from the CD 14 sequence deposited at GenBank as Accession No. NM_000591). The results are shown in Fig 3A.

To test the effects of a stably expressed shRNA on CD 14 expression and HCV infection, the CD 14 gene was targeted with lug of a pool of CD 14 shRNA plasmid (Catalog No.# SC-29248-SH) or Control shRNA Plasmid-A (catalogue no# SC- 108060) consisting of 4 target- specific different shRNAs (Santa Cruz Biotechnology, Inc. USA). Huh7.5 cells were seeded in a six well tissue culture plate, in antibiotic-free normal growth medium

supplemented with FBS. For each transfection, we used 6 μΐ of FuGENE® 6 Transfection Reagent (Roche Applied Science, Germany) mixed with 1 μg of shRNA Plasmid DNA added to Opti-MEM® transfection medium (Gibco, USA) and incubated for 15 minutes at room temperature. The transfection/ shRNA complexes were added to Huh7.5 cells. For selection of stably transfected cells 48 hours post-transfection the medium was replaced with ^g/ml puromycin. Upon selection the stable transfected cells were infected with HCV and after 24 hours CD 14 and HCV mRNA expression levels were analyzed by Real Time PCR as described in the previous examples, and the results shown in Fig. 3B. As shown in the figures, expression of inhibitory RNA molecules inhibited HCV infection, with stably expressed shRNA having a greater effect. Example 4: Blocking TLR4 and MyD88 pathway has no inhibitory effect on HCV infection

Blocking CD 14 by a lipid- A antagonist (IAXO), by specific anti-CD 14 antibodies or by SiRNA and shRNA targeting CD 14 resulted in inhibition of HCV infection. This example explores the possibility that the observed HCV inhibition may result from blocking a required CD 14 downstream signaling molecule, such as TLR4 or the MyD88 dependent pathway.

Methods

Huh7.5 cells were treated with CD14 antagonist (IAXO) or TLR4 specific monoclonal antibody and then infected with HCV virus. 24 hours post infection HCV RNA was analyzed by Real-Time RT-PCR.

Huh7.5 cells were treated with MyD88 inhibitor, control peptide or TLR4 specific monoclonal antibody and then infected with HCV. After 72 hours, HCV RNA was analyzed by Real-Time RT-PCR as described above. The control non-treated cells (HCV) were assigned a value of 1 and results are normalized to untreated infected cells.

Results

Anti-TLR4 specific antibodies showed that blocking TLR4 with anti-TLR4 antibodies (Catalog no# IMG-417EIMGENEX, USA) does not inhibit HCV, and that HCV RNA levels were even higher in the presence of TLR4 antibodies (Fig. 4A).

It was also determined whether the HCV inhibitory effect of blocking CD 14 is directed through the MyD88 dependent pathway (It is noteworthy, that in Huh7.5 cells, the only highly permissive cell line for HCV production, neither TLR3 nor RIG-I pathways are functional). MyD88 is an essential adaptor protein with function in the activation and regulation of the immune system. A specific MyD88 homo dimerization inhibitory peptide that inhibits MyD88 dependent TLR signaling activity thus functions as a decoy by binding to the MyD88 TIR domain (peptide: DRQIKIWFQNRRMKWKKRDVLPGT (SEQ ID NO: 7); Catalog No.# IMG-2005-1; IMGENEX, USA). Huh7.5 cells were treated with MyD88 inhibitor peptide (ΙΟΟμΜ), control peptide (peptide: DRQIKIWFQNRRMKWKK (SEQ ID NO: 8); IMGENEX, USA) for 24 hours and then infected with HCV. After 72 hours, HCV RNA was analyzed by Real-Time RT-PCR as described above. The MyD88 inhibiting peptide that inhibits MyD88 pathway (as can be seen by its inhibitory effect on LPS activation (Fig. 4B) had no inhibitory effect on HCV infection, as analyzed by HCV RNA (Fig. 4C) and FFU (Fig. 4D) as described in the previous examples. These results indicate that the inhibition of HCV is not due to a direct inhibition of TLR4 receptor or TLR4 pathway.

Example 5: Soluble CD14 inhibits HCV infection

This example shows inhibition of HCV infection in cells overexpressing soluble

CD14.

Methods

A plasmid overexpressing CD 14 was constructed by standard methods. Briefly, The human CD 14 coding sequence was amplified by PCR from a cDNA template derived from Huh7.5 mRNA using the following primers: CD14-F EcoRI : 5'-

CCAGAATTCTTATGGAGCGCGCGTCCTGCTTGT-3' (SEQ ID NO: 9), and CD14-R Notl : 5'-GGTGCGGCCGCTTAGGCAAAGCCCCGGGC -3' (SEQ ID NO: 10). The PCR product was digested with EcoRI and Notl (restriction sites are in bold in the primer sequences) and was cloned between the corresponding sites in mammalian expression plasmid pcDNA3.1/HisA (Invitrogen, USA). The sequence of the resulting plasmid (CD14-His) was determined and is set forth herein as SEQ NO: 11. In SEQ ID NO: 11, the hexahistidine tag is encoded by nucleotides 13-30; the cloned CD 14 coding sequence is encoded by nucleotides 125-1251.

Huh7.5 cells were stably transfected with CD14-His using FuGENE® 6 Transfection Reagent (Roche Applied Science, Germany) or with control empty vector (pcDNA3.1/HisA ; Invitrogen, USA) as described above. Cells were grown in culture medium containing 40μg/ml G418 for selection for stably-transfected cells. Cells containing stably expressing CD 14 or empty plasmids were grown to confluence in 6 well plates for 24 hours, after which medium was changed to serum free for an additional 24 hours. CD14 expression was assayed (analysis was done on two clones (CD 14 c-2 and CD 14 c-4)) by Western blot analysis of all cell lysates and supernatant collected after 24 hours, essentially as described previously (Gal- Tanamy M et al., 2011; Hepatology ;54(5): 1570-9). Briefly, for Cdl4 cell expression, cells were washed with PBS, scraped and lysed in SDS sample buffer. Soluble CD14 secreted from cells grown in serum free medium for 24 hours was collected. For immunoblotting, protein samples (cell lysate and secreted CD14) were separated on 12% SDS/polyacrylamide gel, transferred to nitrocellulose and detected using rabbit polyclonal anti-CD 14 antibody (catalog no# sc-9150; Santa Cruz Biotechnology, Inc. USA), and mouse monoclonal anti-β actin (Abeam, UK) for loading control, followed by goat anti-rabbit and goat anti-mouse antibodies respectively, labeled with Infrared fluorophore (LI-COR Biosciences, Lincoln, NE, USA). Western blots were analyzed with the Odyssey infrared imaging system (LI-COR

Biosciences). The images were scanned on the Odyssey system and signal intensities were quantified.

The effects of soluble CD 14 expression on HCV infection was tested as in the above examples. The CD14 transfected cells were infected with HCV virus. 24 hours post infection, HCV RNA was analyzed by Real-Time RT-PCR. The control cells (transfected with empty plasmid) were assigned a value of 1 and results were normalized to control cells.

Results and Discussion

Although, predominantly expressed on monocytes, macrophages and granulocytes, CD 14 was also shown to be expressed in hepatocytes and in human and rat liver (Su GL, et al., 1999. J Hepatol 31: 435-42; Chou MH, et al., 2012. PLoS One 7: e34903). It was also shown that adult hepatocytes are the source of soluble human CD 14 in acute phase serum (Cho K, et al., 2004. J Surg Res 122: 36-42; Meuleman P, et al., 2006 Clin Chim Acta 366: 156-62). In attempt to further elucidate the role of CD14 in HCV life cycle we established Huh7.5 cells over expressing the CD14 gene. For that end the authors cloned the cDNA coding sequence of CD14 in a mammalian expression plasmid and established Huh7.5 cells stably expressing the CD 14. Western blot analysis performed on all cell lysates (Fig. 5A, lanes 1-3) and supernatant (lanes 4-5), and revealed a higher expression of CD14 protein then in the empty vector transfected cells. Analysis of HCV infection in these cells showed that HCV infection was inhibited in both clones as analyzed by HCV RNA (Fig. 5B). This inhibitory effect could be explained by the extensive expression of the recombinant sCD14 in those cells (Fig. 5B).

Soluble receptors usually retain their ligand binding capacity and may compete with the membrane-bound forms for free ligand. In regard to CD 14 it was shown that soluble CD 14 induces inhibition of antigen-mediated peripheral blood mononuclear cells (PBMC) proliferation and has the capacity to negatively regulate T lymphocyte activation and function by interacting directly with activated T cells (Nores JE, et al. 1999, Eur. J. Immunol. 29: 265- 276). It was also shown that at high concentrations, recombinant sCD14 can neutralize LPS, possibly by competing with mCD14 (Frey EA, et., al., 1992, J Exp Med 176: 1665-71).

These results show that soluble receptor CD 14 at high concentration can inhibit HCV infection. In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. Use of an antagonist or an inhibitor of CD 14 in the manufacture of a medicament for the treatment or prevention of an infection caused by any genotype of hepatitis C virus (HCV) in a subject in need thereof.

2. Use of an antagonist or an inhibitor of CD 14 in the manufacture of a medicament for the treatment of an HCV infection, or for the treatment of a disease associated with HCV infection, or for the prevention of related liver diseases in a subject in need thereof, wherein the disease associated with HCV comprises chronic hepatitis C, and wherein the related liver diseases comprise liver fibrosis, liver cirrhosis, or hepatocellular carcinoma.

3. The use according to claim 1 or 2, wherein the antagonist is a small organic molecule which can inhibit CD 14.

4. The use according to claim 1 or 2, wherein the antagonist is a peptide which can inhibit CD 14.

5. The use according to claim 1 or 2, wherein the inhibitor is an anti-CD14 antibody, its active fragment, or a derivative thereof.

6. The use according to claim 1 or 2, wherein the antagonist comprises an inhibitory CD 14 fragment comprising soluble CD 14 or a fragment thereof.

7. The use according to claim 1 or 2, wherein the antagonist is a CD 14 mutant which can inhibit CD 14.

8. The use according to claim 1 or 2, wherein the CD 14 inhibitor is an inhibitor of CD 14 expression.

9. The use according to claim 8, wherein the inhibitor of CD 14 expression comprises anti-CD 14 siRNA, an antisense oligonucleotide, an antisense morpholino, ribozymes, CD 14 competing derivatives, molecules that target the CD 14 promoter, molecules that inhibit CD14 transcription, or CD14 mimicry substances.

10. The use according to claims 1 or 2, wherein the inhibitor of CD 14 is an HCV protein/peptide/lipoprotein counterpart directed to bind CD 14 or proteins and peptides derived from additional members in CD14-HCV putative complex formation.

11. The use according to any one of claims 1 to 5, wherein the subject is concurrently undergoing a treatment with interferon- alpha or any other inhibitor of HCV proteases and/or polymerases.

12. A combination composition for the treatment of HCV infection or for the treatment of a disease associated with HCV infection, comprising a medicament comprising an antagonist of CD 14 or an inhibitor of CD 14 expression and at least one medicament selected from the group consisting of a medicament comprising interferon- alpha, and an inhibitor of HCV proteases and/or polymerases.

13. A method of treating or preventing HCV infection in a subject in need of such treatment or prevention, comprising administering to the subject a therapeutically effective amount of a composition comprising a CD 14 inhibitor.

14. The method of claim 13, wherein the CD 14 inhibitor comprises a small organic molecule which can inhibit CD 14, a peptide which can inhibit CD 14,

an anti-CD 14 antibody, its active fragment, or a derivative thereof, an inhibitory CD 14 fragment comprising soluble CD 14 or a fragment thereof, a CD 14 mutant which can inhibit CD 14, an inhibitor of CD 14 expression, or a HCV protein/peptide counterpart directed to bind CD14.

15. The method of claim 13, further comprising sequential or concurrent administration to the subject any other active ingredient used for treating HCV.

16. The method of claim 15, wherein the other active ingredient comprises interferon-alpha or any other inhibitor of HCV proteases and/or polymerases.

17. A pharmaceutical composition for the prevention or treatment of HCV or a HCV-related liver disease comprising a composition comprising a CD 14 inhibitor.

18. The pharmaceutical composition of claim 17, wherein the composition comprises a small organic molecule which can inhibit CD 14, a peptide which can inhibit

CD 14, an anti-CD 14 antibody, its active fragment, or a derivative thereof, an inhibitory CD 14 fragment comprising soluble CD 14 or a fragment thereof, a CD 14 mutant which can inhibit CD 14, an inhibitor of CD 14 expression, or a HCV protein/peptide counterpart directed to bind CD14.

19. The pharmaceutical composition of claim 18, further comprising any other active ingredient used for treating HCV.

20. The pharmaceutical composition of claim 19, wherein the other active ingredient comprises interferon- alpha or any other inhibitor of HCV proteases and/or polymerases.

21. A method of mapping HCV virus-CD14 interacting epitopes on the virus for the development of effective vaccines, the method comprising:

contacting HCV, the HCV envelope, or a portion thereof with a CD 14 protein, or a fragment thereof; and

identifying the virus epitope contacted by the CD 14 protein or a fragment thereof.