WO2018165720A1 - Agents for treating or preventing viral infections and uses therefor - Google Patents

Abstract

Disclosed are agents and methods for treatment or prevention of Flavivirus infections. In particular, the present invention discloses CD14 antagonist antibodies for use in treating or preventing disease associated with Flavivirus infections, including Dengue Virus infections.

Description

TITLE OF THE INVENTION

"AGENTS FOR TREATING OR PREVENTING VIRAL INFECTIONS AND USES THEREFOR"

FIELD OF THE INVENTION

[0001] This application claims priority to Australian Provisional Application No.

2017900937 entitled "Agents for treating or preventing viral infections and uses therefor" filed 17 March 2017, the contents of which are incorporated herein by reference in their entirety.

[0002] This invention relates generally to agents and methods for treatment or prevention of F/av/v/rus infections. In particular, the present invention relates to CD14 antagonist antibodies for use in treating or preventing disease associated with F/av/v/rus infections, including Dengue virus infections.

BACKGROUND OF THE INVENTION

[0003] Numerous viral diseases are transmitted by insect vectors. For example, many viruses are transmitted by arthropods (predominantly mosquitoes, flies and ticks) and for this reason are commonly referred to as "arboviruses" (arthropod-borne viruses) . Arbovirus infection in mammals can manifest in potentially life-threatening diseases including encephalitis and hemorrhagic fever.

[0004] Members of the Flaviviridae family of arboviruses have been responsible for numerous epidemics in both human and animal populations {see, for example, review by Mlera et a/., 2014. Pathog. Dis. 71(2) : 137-163). For example, Dengue virus (DEN V) is a prevalent mosquito-borne flavivirus causing significant human disease ranging from mild forms of dengue fever to life-threatening severe dengue including dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). It is estimated that 2.5 billion people are at risk of dengue virus infection with 50-100 million cases of dengue fever annually causing approximately 25,000 deaths

(predominantly among children). While dengue fever is an acute, self-limiting illness, DHF/DSS is a plasma leakage syndrome characterized by defects in vascular permeability, marked

thrombocytopenia, hepatomegaly and bleeding diathesis, which can lead to life-threatening shock. Severe dengue also includes patients with hepatitis, neurological disorders, myocarditis or severe bleeding without plasma leakage or shock. If untreated, mortality can be as high as 20%, whereas appropriate case management and intravenous rehydration can reduce mortality to less than 1% (Guzman eta/., 2015. The Lancet 385(9966) : 453-465). Nevertheless, the effects of dengue can be debilitating and over half of disease-affected patients experience persistent symptoms {e.g., arthralgia and fatigue) for up to 2 years after developing the illness (Guzman eta/., 2015 supra). Each of the four dengue virus serotypes I, II, III and IV (also commonly referred to as DENV1, DENV2, DENV3 and DENV4, respectively), can cause the full spectrum of disease. The rapid spread of Dengue virus to most tropical and subtropical countries has led to its classification as an emerging infectious disease and has intensified efforts to prevent infection.

[0005] Japanese encephalitis virus accounts for up to 50,000 cases of encephalitis in humans annually (with case fatality rates of about 25%), and significant epidemics have occurred in India and Nepal in recent years. Yellow fever virus causes a wide spectrum of disease ranging from mild symptoms to kidney/liver failure and hemorrhaging of the gastrointestinal tract. There are an estimated 200,000 cases of yellow fever causing 30,000 deaths worldwide each year and outbreaks are common throughout central Africa and areas of South America.

[0006] West Nile virus (and closely related viruses such as Kunjin virus) are some of the most widely distributed flaviviruses with a geographic range that includes Africa, Australia, Europe, the Middle East, West Asia and the USA. These viruses can infect humans, horses and other mammals as well as birds, causing potentially fatal encephalitis and/or meningitis, and has been responsible for repeated large-scale epidemics worldwide.

[0007] Tick-borne encephalitis virus is transmitted by the bite of infected ticks and can cause severe febrile illness and is frequently associated with encephalitis. Tick-borne encephalitis is the most important tick-borne viral disease of humans in Eurasia with an estimated annual number up to 10,000 cases in Russia and 3,000 cases in Europe.

[0008] Other members of the Flaviviridae family of clinical importance include Murray Valley encephalitis, St. Louis encephalitis, Omsk hemorrhagic fever virus, Bovine viral diarrhea virus, Alfuy virus, Koutango virus, Cacipacore virus, Yaounde virus, Zika virus and Hepatitis C virus.

[0009] The nonstructural glycoprotein NSl is a 352-amino acid polypeptide, which is highly conserved across flaviviruses {see, for example, review by Muller etai, 2013. Antiviral Res. 98(2) : 192-208). NSl exists in multiple oligomeric forms and is found in different cellular locations: a cell membrane-bound form in association with virus-induced intracellular vesicular

compartments, on the cell surface and as a soluble secreted hexameric lipoparticle. Intracellular NSl co-localizes with dsRNA and other components of the viral replication complex and plays an essential cofactor role in replication. Several potential interacting partners for NSl have been identified, particularly for its secreted form, with many being implicated in immune evasion strategies. Both secreted and cell-surface associated forms of NSl are highly immunogenic and the antibodies they elicit have been implicated in the seemingly contradictory roles of protection and pathogenesis in the infected host. NSl also binds to a number of different complement pathway components, leading to complement activation and ensuing systemic generation of anaphylatoxins and membrane attack complex, which contribute to the etiology of vascular leakage (Avitutnan et ai, 2006. J. Infect. Dis. 193 : 1078-1088). Despite its role in complement activation, the main pathway by which vascular leakage is induced by NSl appears to be through pro-inflammatory mediators (Aye etai, 2014. Human Path. 45 : 1221-1233; Chuang etai, 2013. J. Biomed. Sci. 20 : 42). In this regard, it is known that NSl induces an array of cytokines, most pro-inflammatory, as well as chemokines. In dengue patients, levels of TNF-a, IL-Ιβ, IL-6, IL-10, IL-8, IFN-γ and MCP-1 all correlate with disease severity.

[0010] Despite decades of research effort, safe and effective vaccines against most flaviviruses are still not available. Although certain vaccines against Yellow fever virus and

Japanese encephalitis virus exist, these viruses still cause significant disease worldwide.

[0011] In view of the severity of the diseases associated with Flavivirus infection and their pervasiveness in animals and especially man, there is a need for agents and methods for treating a host infected with a flavivirus. SUMMARY OF THE INVENTION

[0012] The present invention arises in part from the discovery that cluster of differentiation 14 (CD14) is a receptor of the soluble form of NS1 (sNSl) (Crooks eta/., 1994. J. Gen. Virol. 75 : 3453-3460; Flammand eta/., 1999. J. Virol. 73 : 6104-6110). CD14 is a glycoprotein that exists both in soluble form (sCD14) and in cell membrane-bound form (mCD14) on the surface of various cells, including immune cells such as macrophages, monocytes, Kupffer cells, neutrophils and B cells, as well as endothelial cells and epithelial cells (Jersmann, HPA, 2005. Immunol Cell Biol. 83 : 462-467). CD14 is also known as a receptor for lipopolysaccharide (LPS) (also known as endotoxin) of Gram-negative bacteria, which receives LPS from LPS binding protein (LBP) in blood to form a complex. LPS also binds to sCD14 and the resulting complex can induce mCD14-independent production of pro-inflammatory mediators, including pro-inflammatory cytokines and chemokines.

[0013] It has also been discovered that blocking the binding of sNSl to mCD14 by a CD14 antagonist antibody prevents NSl-mediated pro-inflammatory cytokine release by immune cells. Since mCD14 is a receptor of sNSl, it is proposed that sNSl will also bind sCD14, which binding could be blocked by a CD14 antagonist antibody, to thereby inhibit sNSl-sCD14-mediated pro-inflammatory cytokine production. Further, it is proposed that endothelial cells, including vascular endothelial cells, will behave in a similar manner, as CD14 antagonist antibody will either bind to mCD14 on these cells or to sCD14, to thereby inhibit or reduce NSl-mediated vascular leakage. As such, CD14 antagonist antibodies are disclosed herein as providing a strategy for therapeutic intervention in Flavivirus disease. These findings have been reduced to practice in methods and compositions for modulating production of pro-inflammatory mediators by immune cells and/or vascular leakage in subjects with Flavivirus infections and for treating or preventing Flavivirus infections or a symptom thereof, as described hereafter.

[0014] Accordingly, in one aspect, the present invention provides methods for modulating production of a pro-inflammatory mediator {e.g., a cytokine such as IL-6, TNF-a, IL-Ιβ and IFN-β) in a subject with a Flavivirus infection. These methods generally comprise, consist or consist essentially of contacting CD14 {e.g., mCD14 and/or sCD14) in the subject with a proinflammatory mediator-modulating amount of a CD14 antagonist antibody. In specific

embodiments, a cell that comprises mCD14 is contacted with the CD14 antagonist antibody, representative examples of which include immune cells {e.g., a macrophage or monocyte) or endothelial cells {e.g., a vascular endothelial cell).

[0015] Another aspect of the present invention provides methods for modulating vascular leakage in a subject with a Flavivirus infection. These methods generally comprise, consist or consist essentially of contacting CD14 {e.g., mCD14 and/or sCD14) in the subject with a vascular leakage-modulating amount of a CD14 antagonist antibody. In specific embodiments, an endothelial cell {e.g., a vascular endothelial cell) that comprises mCD14 is contacted with the CD14 antagonist antibody.

[0016] In a related aspect, the present invention provides methods for inhibiting sNSl- mediated disease symptoms {e.g., production of a pro-inflammatory mediator, vascular leakage, etc.) associated with a Flavivirus infection. These methods generally comprise, consist or consist essentially of contacting CD14 {e.g., mCD14 and/or sCD14) with a CD14 antagonist antibody suitably in an amount sufficient to inhibit binding of sNSl to CD14.

[0017] Yet another aspect of the present invention provides methods for treating or preventing a Flavivirus infection or a symptom thereof in a subject. These methods generally comprise, consist or consist essentially of administering an effective amount of a CD14 antagonist antibody to the subject.

[0018] In related aspects, the present invention provides the use of a CD14 antagonist antibody for inhibiting the binding of sNSl to CD14 {e.g., mCD14 and/or sCD14), or for modulating production of a pro-inflammatory mediator {e.g., a cytokine such as IL-6, TNF-a, IL-Ιβ and IFN-β) that is associated with a Flavivirus infection, or for modulating vascular leakage that is associated with a Flavivirus infection, or for inhibiting the binding of sNSl to a cell that comprises CD14 and that mediates disease symptoms associated with a Flavivirus infection, or for treating or preventing a Flaviviridae virus infection. In some embodiments, the CD14 antagonist antibody is manufactured as a medicament for any one or more of those applications.

[0019] Suitably, the Flavivirus is a virus selected from the group consisting of Dengue virus (DENV), Japanese encephalitis virus (JEV), Yellow fever virus (YFV), Murray Valley encephalitis virus (MVEV) , West Nile virus (WNV) , Tick-borne encephalitis virus (TBEV), St Louis encephalitis virus (SLEV), Alfuy virus (AV), Koutango virus (KV), Cacipacore virus (CV), Yaounde virus (YV) and Zika virus (ZV). In specific embodiments, the Flavivirus s selected from Dengue virus serotype I, II, III, or IV. In some embodiments, the methods further comprise identifying that the subject has or is at risk of developing a Flavivirus infection, suitably prior to administration of the CD14 antagonist. In illustrative examples of this type, the methods comprise determining the presence of NS1 {e.g., soluble or non-soluble forms of NS1) in the subject {e.g., in a biological sample of the subject, illustrative examples of which include blood, serum, plasma, saliva, cerebrospinal fluid, urine, skin or other tissues, or fractions thereof), suitably prior to

administration of the CD14 antagonist antibody. The presence of NS1 is suitably determined by detecting an expression product of an NS1 gene {e.g., NS1 mRNA or NS1 polypeptide) in the biological sample.

[0020] In preferred embodiments, the CD14 antagonist antibody is IC14, or an antigen- binding fragment thereof.

[0021] The CD14 antagonist may be administered alone or in combination with one or more ancillary agents that treat or ameliorate the symptoms of a Flavivirus infection. Accordingly, in still another aspect, the present invention provides pharmaceutical compositions, suitably for treating a Flavivirus infection or symptom thereof. These compositions comprise, consist or consist essentially of a CD14 antagonist antibody and an ancillary anti- Flaviviridae virus agent, optionally together with a pharmaceutically acceptable carrier or diluent.

[0022] In a related aspect, the present invention provides methods for treating or preventing a Flavivirus infection or symptom thereof in a subject. These methods generally comprise, consist or consist essentially of administering concurrently to the subject an effective amount of a CD14 antagonist antibody and an effective amount of an ancillary anti- Flavivirus agent. Suitably, the CD14 antagonist antibody and the ancillary anW- Flavivirus agent are administered in synergistically effective amounts. [0023] In specific embodiments, the ancillary ax\W- Flavivirus agent is selected from interferons, illustrative examples of which include interferon alpha {e.g., interferon alpha 2a and interferon alpha 2b) and interferon beta {e.g., interferon beta la and interferon beta lb), as well as ax\W- Flavivirus antibodies and small molecules, or nucleic acid constructs from which an ancillary ax\W- Flavivirus agent is expressible.

[0024] In another related aspect, the present invention provides the use of a CD14 antagonist antibody and an ancillary ax\W- Flavivirus agent for treating or preventing a Flavivirus infection or symptom thereof. In some embodiments, the CD14 antagonist antibody and the ancillary ax\W- Flavivirus virus agent are manufactured as a medicament for this application.

Suitably, the CD14 antagonist antibody and the ancillary ax\M- Flavivirus agent are formulated for concurrent administration.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a graphical representation showing the effect of IC14 (Axtelle eta/., 2001. J Endotoxin Res 1 ': 310-314) on NS1 and LPS induction of IL-6 in human cells. Human PBMCs (peripheral blood mononuclear cells) were incubated with IC14 or isotype control antibody (a human IgG4 control monoclonal antibody) at concentrations indicated in serum free conditions (A) or in the presence of serum (B). Data were analyzed using a four parameter dose response model and IC50 values along with 95% confidence intervals are summarized below each graph.

[0026] Figure 2 is a graphical representation showing the dose response of human PBMCs to varying concentrations of LPS and NS1, and that a CD14-antagonist antibody blocks IL-6 production over a range of ligand concentrations. PBMCs from two donors were treated with a control human IgG4 antibody (Ab) or IC14 for lhr. Cells were then treated with the indicated concentrations of LPS (top panel) or NS1 (bottom panel) overnight and IL-6 secretion quantified by ELISA. Data show the mean and range for duplicate cell treatments.

[0027] Figure 3 is a graphical representation showing that CD14 is required for NS1- and low dose LPS-mediated induction of cytokine mRNA expression in murine bone marrow-derived macrophages (BMMs). BMMs were isolated from WT and CD14-deficient mice and were untreated or treated with LPS (at 10 ng/mL or 1 pg/mL) or NS1 (10 pg/mL) for 3 hrs. Cytokine mRNA was quantified by qRT-PCR and is expressed relative to hypoxanthine phosphoribosyltransferase {HPRT) mRNA levels.

[0028] Figure 4 is a graphical representation showing that CD14 is required for NS1 to induce colony-stimulating factor 1 receptor (CSF-1R) down-regulation in murine BMMs. CSF-1- starved WT and CD14-deficient BMMs were treated with varying concentrations of NS1 (top panel) or LPS (bottom panel) for lhr and stained with a PE-conjugated anti-CSF-lR antibody and analyzed by flow cytometry.

[0029] Figure 5 is a graphical representation showing the dose response of human PBMCs to varying concentrations of a CD14-antagonist antibody in the presence of NS1 from DENV1, DENV2 and DENV3. Human PBMCs were incubated with the IC14 antibody or control IgG4 antibody at concentrations ranging from 0.001 to 10 pg/mL for 1 h. CHO cell medium containing NS1 was added to the cells at 1 in 5 dilution, and incubated overnight. Secreted IL-6 was quantified by ELISA. All data is with the same donor and error bars represent the range for duplicate treatment wells. [0030] Figure 6 is a graphical representation showing the effect of a CD14-antagonist antibody on NS1 and LPS-induced gene expression. Human PBMCs were incubated with the IC14 antibody or control human IgG4 antibody at 3 pg/mL for 1 h. Either DENV2 NSl-containing CHO cell medium giving a final concentration of 1.8 pg/mL NS1 or LPS at 10 ng/mL was added to the cells and incubated for a further 2 h. Levels of expression of the indicated genes was determined by real time PCR, and is shown relative to the housekeeping gene HPRT for each sample. Shown are the mean and range of data from two donors for NS1 treatments, and the result of a single donor for LPS treatments.

[0031] Figure 7 is a graphical representation showing the effect of IC14 on viral infection and replication. Human monocyte-derived macrophages were pretreated with either IC14 antibody or control human IgG4 for one hour. Cells were then infected with DENV2 virus at a multiplicity of infection (MOI) of 10; this was either virus alone ("DENV"), or virus pre-incubated with antibody to generate immune complex capable of mediating antibody-dependent

enhancement ("DENV-ADE"). Culture supernatant was assessed for viral titer immediately after infection on day 0, and on days 1-2. Results are from a single sample for each cell treatment.

DETAILED DESCRIPTION OF THE INVENTION

1. Definitions

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

[0033] The articles "a" and "an" are used herein to refer to one or to more than one {i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0034] As used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0035] The terms "administration concurrently" or "administering concurrently" or "co- administering" and the like refer to the administration of a single composition containing two or more agents, or the administration of each agent as separate compositions and/or delivered by separate routes either contemporaneously or simultaneously or sequentially within a short enough period of time that the effective result is equivalent to that obtained when all such agents are administered as a single composition. By "simultaneously" is meant that the agents are administered at substantially the same time, and desirably together in the same formulation. By "contemporaneously" it is meant that the agents are administered closely in time, e.g., one agent is administered within from about one minute to within about one day before or after another. Any contemporaneous time is useful . However, it will often be the case that when not administered simultaneously, the agents will be administered within about one minute to within about eight hours and suitably within less than about one to about four hours. When administered

contemporaneously, the agents are suitably administered at the same site on the subject. The term "same site" includes the exact location, but can be within about 0.5 to about 15 centimeters, preferably from within about 0.5 to about 5 centimeters. The term "separately" as used herein means that the agents are administered at an interval, for example at an interval of about a day to several weeks or months. The agents may be administered in either order. The term "sequentially" as used herein means that the agents are administered in sequence, for example at an interval or intervals of minutes, hours, days or weeks. If appropriate the agents may be administered in a regular repeating cycle.

[0036] The term "agonist" refers to a ligand that stimulates a receptor to which it binds. An agonist, by classical definition, whether an orthosteric, allosteric, inverse or a co-agonist has a property to bind to a receptor, alter its receptor state and result in a biological action, including activation, whether directly or indirectly, of a chemical or physical signaling cascade, which results in a definable change in the behavior, physical or biological state of a cell . Consequently, agonism is defined as a property of an agonist to produce a biological action. In the context of the present invention, a CD14 agonist includes, but is not limited to, a flavivirus NS1 protein and LPS.

[0037] The term "antagonist" is used in its broadest sense, and includes a ligand of a receptor, which does not provoke a biological response itself upon binding to the receptor, but blocks or dampens a response mediated by an agonist of the receptor, or prevents or reduces agonist binding and, thereby, agonist-mediated responses. Accordingly, a CD14 antagonist refers to an agent that inhibits or abrogates binding of a CD14 agonist such as sNSl to CD14, and/or that binds to CD14 and inhibits a CD14 agonist-mediated response. For example, a CD14 antagonist may compete with an agonist {e.g., sNSl) for binding to CD14, thereby inhibiting an action of the agonist on CD14, which normally accompanies the binding of the agonist to CD14. Inhibition of CD14 activity by an antagonist suitably reduces or inhibits production of pro-inflammatory mediators including pro-inflammatory cytokines by the cell or the modulation of other cellular elements that are associated with Flavivirus disease symptoms. An antagonist of the present invention is suitably a direct antagonist of CD14.

[0038] The term "direct antagonist" of CD14 refers to an antagonist that binds or otherwise interacts with CD14 or specifically inhibits expression of a nucleic acid encoding CD14. In specific embodiments, the direct antagonist does not detectably bind or displays negligible binding to a binding partner of CD14, such as a CD14 binding partner that is located on the cell membrane, representative examples of which include MD2 or TLR4. Such antagonists are referred to herein as "CD14 specific antagonists".

[0039] The term "antagonist antibody" is used in the broadest sense, and includes an antibody that inhibits or decreases the biological activity of an antigen to which the antibody binds {e.g., CD14). For example, an antagonist antibody may partially or completely block interaction between a receptor {e.g., CD14) and a ligand {e.g., sNSl), or may practically decrease the interaction due to tertiary structure change or down regulation of the receptor. Thus, a CD14 antagonist antibody encompasses antibodies that bind to CD14 and that block, nullify, antagonize, suppress, decrease or reduce (including significantly), in any meaningful degree, a CD14 agonist activity, including activation of downstream pathways such as Toll-like receptor (TLR) signaling pathways {e.g., TLR4 signaling pathway) and the TIR-domain-containing adapter-inducing IFN-β (TRIF) pathway, or elicitation of a cellular response {e.g., production of pro-inflammatory mediators including pro-inflammatory cytokines) to CD14 binding by a CD14 ligand {e.g., sNSl). [0040] The term "antibody" herein is used in the broadest sense and specifically covers naturally occurring antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies {e.g., bispecific antibodies), antibody fragments, or any other antigen-binding molecule so long as they exhibit the desired immuno-interactivity. A naturally occurring "antibody" includes within its scope an immunoglobulin comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised specific CH domains {e.g., CHI, CH2 and CH3). Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementary determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions of the antibodies may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system {e.g., effector cells) and the first component (Clq) of the classical complement system. The antibodies can be of any class (isotype) {e.g., IgG, IgE, IgM, IgD, IgA and IgY), subclass {e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), or modified version thereof {e.g., IgGl isotype, which carries L234A and L235A double mutations (igGl-LALA). The antibodies can be of any species, chimeric, humanized or human. In other embodiments, the antibody is a homomeric heavy chain antibody {e.g., camelid antibodies) which lacks the first constant region domain (CHI) but retains an otherwise intact heavy chain and is able to bind antigens through an antigen-binding domain. The variable regions of the heavy and light chains in the antibody-modular recognition domain (MRD) fusions will contain a functional binding domain that interacts with an antigen of interest.

[0041] The "variable domain" (variable domain of a light chain (VL), variable domain of a heavy chain (VH)) as used herein denotes each of the pair of light and heavy chain domains which are involved directly in binding the antibody to the antigen. The variable light and heavy chain domains have the same general structure and each domain comprises four FRs whose sequences are widely conserved, connected by three CDRs or "hypervariable regions". The FRs adopt a β-sheet conformation and the CDRs may form loops connecting the β-sheet structure. The CDRs in each chain are held in their three-dimensional structure by the FRs and form together with the CDRs from the other chain the antigen binding site.

[0042] The term "antigen-binding portion" when used herein refer to the amino acid residues of an antibody which are responsible for antigen-binding generally, which generally comprise amino acid residues from the CDRs. Thus, "CDR" or "complementarity determining region" (also referred to as "hypervariable region") are used interchangeably herein to refer to the amino acid sequences of the light and heavy chains of an antibody which form the three- dimensional loop structure that contributes to the formation of an antigen binding site. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated "CDR1", "CDR2", and "CDR3", for each of the variable regions. The term "CDR set" as used herein refers to a group of three CDRs that occur in a single variable region that binds the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat eta/., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as "Kabat CDRs". Chothia and coworkers (Chothia and Lesk, 1987. J. Mo/. Biol. 196: 901-917; Chothia eta/., 1989. Nature 342 : 877-883) found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as "LI", "L2", and "L3", or "HI", "H2", and "H3", where the "L" and the "H" designate the light chain and the heavy chain regions, respectively. These regions may be referred to as "Chothia CDRs", which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan (1995. FASEB J. 9 : 133-139) and MacCallum (1996. J. Mo/. Biol. 262(5) : 732- 745). Still other CDR boundary definitions may not strictly follow one of these systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding.

[0043] As used herein, the term "framework region" or "FR" refers to the remaining sequences of a variable region minus the CDRs. Therefore, the light and heavy chain variable domains of an antibody comprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDRs and FRs are typically determined according to the standard definition of Kabat, E. A., eta/., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991) and/or those residues from a

"hypervariable loop".

[0044] As used herein, the terms "light chain variable region" ("VL") and "heavy chain variable region" (VH) refer to the regions or domains at the N-terminal portion of the light and heavy chains respectively which have a varied primary amino acid sequence for each antibody. The variable region of an antibody typically consists of the amino terminal domain of the light and heavy chains as they fold together to form a three-dimensional binding site for an antigen. Several subtypes of VH and VL, based on structural similarities, have been defined, for example as set forth in the Kabat database.

[0045] The term "chimeric antibody" refers to antibodies that comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies having murine heavy and light chain variable regions linked to human constant regions.

[0046] "Humanized" forms of non-human {e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Thus, the FRs and CDRs of a humanized antibody need not correspond precisely to the parental {i.e., donor) sequences, e.g., a donor antibody CDR or the consensus framework may be mutagenized by substitution, insertion, and/or deletion of at least one amino acid residue so that the CDR or FR at that site does not correspond to either the donor antibody or the consensus framework. Typically, such mutations, however, will not be extensive and will generally avoid "key residues" involved in binding to an antigen. Usually, at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the humanized antibody residues will correspond to those of the parental FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework region in the consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences {see, for example, Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, 1987)). A "consensus immunoglobulin sequence" may thus comprise a "consensus framework region(s)" and/or a "consensus CDR(s)". In a family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones eta/. (1986. Nature 321 : 522-525), Riechmann eta/. (1988. Nature 332 : 323- 329) and Presta (1992. Curr. Op. Struct. Biol. 2 : 593-596). A humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any subclass, including without limitation IgGl, IgG2, IgG3, and IgG4. A humanized antibody may comprise sequences from more than one class or isotype, and particular constant domains may be selected to optimize desired effector functions using techniques well known in the art. As used herein, the term "key residue" refers to certain residues within the variable region that have more impact on the binding specificity and/or affinity of an antibody, in particular a humanized antibody. A key residue includes, but is not limited to, one or more of the following : a residue that is adjacent to a CDR, a potential glycosylation site (can be either N- or O-glycosylation site), a rare residue, a residue capable of interacting with the antigen, a residue capable of interacting with a CDR, a canonical residue, a contact residue between heavy chain variable region and light chain variable region, a residue within the Vernier zone, and a residue in the region that overlaps between the Chothia definition of a variable heavy chain CDR1 and the Kabat definition of the first heavy chain framework.

[0047] As used herein, "Vernier" zone refers to a subset of framework residues that may adjust CDR structure and fine-tune the fit to antigen as described by Foote and Winter (1992. J. Mo/. Biol. 224: 487-499). Vernier zone residues form a layer underlying the CDRs and may impact on the structure of CDRs and the affinity of the antibody.

[0048] As used herein, the term "canonical" residue refers to a residue in a CDR or framework that defines a particular canonical CDR structure as defined by Chothia etai. (1987. J. Mol. Biol. 196: 901-917; 1992. J. Mol. Biol. 227: 799-817), both are incorporated herein by reference). According to Chothia etai, critical portions of the CDRs of many antibodies have nearly identical peptide backbone confirmations despite great diversity at the level of amino acid sequence. Each canonical structure specifies primarily a set of peptide backbone torsion angles for a contiguous segment of amino acid residues forming a loop.

[0049] As used herein, the terms "donor" and "donor antibody" refer to an antibody providing one or more CDRs to an "acceptor antibody". In some embodiments, the donor antibody is an antibody from a species different from the antibody from which the FRs are obtained or derived. In the context of a humanized antibody, the term "donor antibody" refers to a non-human antibody providing one or more CDRs.

[0050] As used herein, the terms "acceptor" and "acceptor antibody" refer to an antibody providing at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% of the amino acid sequences of one or more of the FRs. In some embodiments, the term "acceptor" refers to the antibody amino acid sequence providing the constant region(s). In other

embodiments, the term "acceptor" refers to the antibody amino acid sequence providing one or more of the FRs and the constant region(s). In specific embodiments, the term "acceptor" refers to a human antibody amino acid sequence that provides at least 80%, preferably, at least 85%, at least 90%, at least 95%, at least 98%, or 100% of the amino acid sequences of one or more of the FRs. In accordance with this embodiment, an acceptor may contain at least 1, at least 2, at least 3, least 4, at least 5, or at least 10 amino acid residues that does (do) not occur at one or more specific positions of a human antibody. An acceptor framework region and/or acceptor constant region(s) may be, for example, derived or obtained from a germline antibody gene, a mature antibody gene, a functional antibody {e.g., antibodies well-known in the art, antibodies in development, or antibodies commercially available).

[0051] The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences {e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0052] The terms "heavy chain variable region CDR1" and "H-CDR1" are used interchangeably, as are the terms "heavy chain variable region CDR2" and "H-CDR2", the terms "heavy chain variable region CDR3" and "H-CDR3", the terms "light chain variable region CDR1" and "L-CDR1"; the terms "light chain variable region CDR2" and "L-CDR2" and the terms "light chain variable region CDR3" and "L-CDR3" antibody fragment. Throughout the specification, complementarity determining regions ("CDR") are defined according to the Kabat definition unless specified otherwise. The Kabat definition is a standard for numbering the residues in an antibody and it is typically used to identify CDR regions (Kabat eta/., (1991), 5th edition, NIH publication No. 91-3242) .

[0053] Antigen binding can be performed by "fragments" or "antigen-binding fragments" of an intact antibody. Herein, both terms are used interchangeably. Examples of binding fragments encompassed within the term "antibody fragment" of an antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; an Fd fragment consisting of the VH and CHI domains; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a single domain antibody (dAb) fragment (Ward eta/., 1989. Nature 341 : 544-546), which consists of a VH domain; and an isolated complementary determining region (CDR).

[0054] A "single chain variable Fragment (scFv)" is a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird eta/., 1988. Science 242 :423-426; and Huston eta/., 1988. Proc. Natl. Acad. Sci. 85 : 5879- 5883). Although the two domains VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by an artificial peptide linker that enables them to be made as a single protein chain. Such single chain antibodies include one or more antigen binding moieties. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0055] The term "monoclonal antibody" and abbreviations "MAb" and "mAb", as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each mAb is directed against a single determinant on the antigen. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies may be produced, for example, by a single clone of antibody- producing cells, including hybridomas. The term "hybridoma" generally refers to the product of a cell-fusion between a cultured neoplastic lymphocyte and a primed B- or T-lymphocyte which expresses the specific immune potential of the parent cell.

[0056] An antibody "that binds" an antigen of interest {e.g., CD14) is one that binds the antigen with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting a cell or tissue expressing the antigen, and does not significantly cross-react with other proteins. In such embodiments, the extent of binding of the antibody to a "non-target" protein will be less than about 10% of the binding of the antibody, oligopeptide or other organic molecule to its particular target protein as determined, for example, by fluorescence activated cell sorting (FACS) analysis, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation or radioimmunoprecipitation (RIA). Thus, an antibody that antagonizes CD14 to which it binds suitably inhibits production of pro-inflammatory mediators, including pro-inflammatory cytokines/chemokines and vascular leakage. With regard to the binding of an antibody to a target molecule, the term "specific binding" or "specifically binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide target means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target. The specific region of the antigen to which the antibody binds is typically referred to as an "epitope". The term "epitope" broadly includes the site on an antigen which is specifically recognized by an antibody or T-cell receptor or otherwise interacts with a molecule. Generally epitopes are of active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and generally may have specific three-dimensional structural characteristics, as well as specific charge characteristics. As will be appreciated by one of skill in the art, practically anything to which an antibody can specifically bind could be an epitope.

[0057] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term "comprising" and the like indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By "consisting of" is meant including, and limited to, whatever follows the phrase

"consisting of". Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0058] By "effective amount", in the context of treating or preventing a condition is meant the administration of an amount of an agent or composition to an individual in need of such treatment or prophylaxis, either in a single dose or as part of a series, that is effective for the prevention of incurring a symptom, holding in check such symptoms, and/or treating existing symptoms, of that condition. The effective amount will vary depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. Non-limiting symptoms of Flavivirus infections include acute febrile illness, malaise, headache, flushing, diarrhoea, nausea, vomiting, abdominal pain, myalgias and, in severe disease, production of pro-inflammatory mediators, including pro-inflammatory cytokines and vascular leakage.

[0059] The term "endothelial cell" as used herein refers to cells that line the inside surfaces of body cavities, blood vessels, and lymph vessels and making up the endothelium.

Endothelial cells are typically but not necessarily thin, flattened cells.

[0060] "Hybridization" is used herein to denote the pairing of complementary nucleotide sequences to produce a DNA-DNA hybrid or a DNA-RNA hybrid. Complementary base sequences are those sequences that are related by the base-pairing rules. In DNA, A pairs with T and C pairs with G. In RNA U pairs with A and C pairs with G. In this regard, the terms "match" and

"mismatch" as used herein refer to the hybridization potential of paired nucleotides in

complementary nucleic acid strands. Matched nucleotides hybridize efficiently, such as the classical

A-T and G-C base pair mentioned above. Mismatches are other combinations of nucleotides that do not hybridize efficiently. In the present invention, the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. Hybridization can occur under varying

circumstances as known to those of skill in the art.

[0061] As used herein, the term "immune cell" refers to a cell belonging to the immune system. Immune cells include cells of hematopoietic origin such as but not limited to T lymphocytes (T cells), B lymphocytes (B cells), natural killer (NK) cells, granulocytes, neutrophils, macrophages, monocytes, dendritic cells, and specialized forms of any of the foregoing, e.g., plasmacytoid dendritic cells, Langerhans cells, plasma cells, natural killer T (NKT) cells, T helper cells, and cytotoxic T lymphocytes (CTL).

[0062] Reference herein to "immuno-interactive" and its grammatical equivalents, includes reference to any interaction, reaction, or other form of association between molecules and in particular where one of the molecules is, or mimics, a component of the immune system.

[0063] By "isolated" is meant material that is substantially or essentially free from components that normally accompany it in its native state.

[0064] The term "ligand", as used herein, refers to any molecule which is capable of binding a receptor.

[0065] As used herein, the terms "modulating", "regulating" and their grammatical equivalents refer to an effect of altering a biological activity or effect {e.g., cytokine production, vascular leakage, binding of an agonist to CD14, etc.). For example, an agonist or antagonist of a particular receptor modulates the activity of that receptor by either increasing / stimulating {e.g., agonist, activator), or decreasing / inhibiting {e.g., antagonist, inhibitor) the activity or effect {e.g., cytokine production, vascular leakage, binding of an agonist to CD14, etc.) of the receptor.

[0066] The terms "patient", "subject", "host" or "individual" used interchangeably herein, refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, for whom therapy or prophylaxis is desired. Suitable vertebrate animals that fall within the scope of the invention include, but are not restricted to, any member of the subphylum Chordata including primates {e.g., humans, monkeys and apes, and includes species of monkeys such as from the genus Macaca {e.g., cynomolgus monkeys such as Macaca fascicularis, and/or rhesus monkeys {Macaca mulatta)) and baboon {Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (species from the genus Saguinus), as well as species of apes such as chimpanzees {Pan troglodytes), rodents {e.g., mice rats, guinea pigs), lagomorphs {e.g., rabbits, hares), bovines {e.g., cattle), ovines {e.g., sheep), caprines {e.g., goats), porcines {e.g., pigs), equines {e.g., horses), canines {e.g., dogs), felines {e.g., cats), avians {e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc.), marine mammals {e.g., dolphins, whales), reptiles {e.g., snakes, frogs, lizards etc.), and fish. In specific embodiments, the subject is a primate such as a human. However, it will be understood that the terms "patient", "subject", "host" or "individual" do not imply that symptoms are present.

[0067] By "pharmaceutically acceptable carrier" is meant a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject along with the selected active agent without causing any or a substantial adverse reaction. Carriers may include excipients and other additives such as diluents, detergents, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, transfection agents and the like.

[0068] Similarly, a "pharmacologically acceptable" salt, ester, amide, prodrug or derivative of a compound as provided herein is a salt, ester, amide, prodrug or derivative that this not biologically or otherwise undesirable.

[0069] The terms "polynucleotide", "genetic material", "genetic forms", "nucleic acids" and "nucleotide sequence" include RNA, cDNA, genomic DNA, synthetic forms and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art.

[0070] As used herein, the terms "prevent", "prevented", or "preventing", refer to a prophylactic treatment which increases the resistance of a subject to developing the disease or condition or, in other words, decreases the likelihood that the subject will develop the disease or condition as well as a treatment after the disease or condition has begun in order to reduce or eliminate it altogether or prevent it from becoming worse. These terms also include within their scope preventing the disease or condition from occurring in a subject which may be predisposed to the disease or condition but has not yet been diagnosed as having it.

[0071] The term "pro-inflammatory mediator" means an immunoregulatory agent that favors inflammation. Such agents include, cytokines such as chemokines, interleukins (IL), lymphokines, and tumor necrosis factor (TNF) as well as growth factors. In specific embodiments, the pro-inflammatory mediator is a "pro-inflammatory cytokine". Typically, pro-inflammatory cytokines include IL-la, IL-Ιβ, IL-6, and TNF-a, which are largely responsible for early responses. Other pro-inflammatory mediators include LIF, IFN-γ, IFN-β, I FN -a, OSM, CNTF, TGF-β, GM-CSF, TWEAK, IL-11, IL-12, IL-15, IL-17, IL-18, IL-19, IL-20, IL-8, IL-16, IL-22, IL-23, IL-31 and IL-32 (Tato etal., 2008. Cell 132 : 900; Ce// 132 : 500, C*?// 132 : 324). Pro-inflammatory mediators may act as endogenous pyrogens (IL-1, IL-6, TNF-a), up-regulate the synthesis of secondary mediators and pro-inflammatory cytokines by both macrophages and mesenchymal cells (including fibroblasts, epithelial and endothelial cells), stimulate the production of acute phase proteins, or attract inflammatory cells. In specific embodiments, the term "pro-inflammatory cytokine" relates to any one or more of TNF-a, IL-6, IFN-β, IL-Ιβ and IL-8. In other specific embodiments, the term "proinflammatory cytokine" relates to any one or more of IL-Ιβ, IL-6, TNF-a, IFN-γ and IFN-β.

[0072] The term "receptor" denotes a cell-associated protein that binds to a bioactive molecule termed a "ligand". This interaction mediates the effect of the ligand on the cell. In general, the binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the receptor and other molecule(s) on the surface of the cell or in the interior of the cell, which in turn leads to an alteration in the metabolism of the cell. Metabolic events that are often linked to receptor-ligand interactions include gene transcription,

phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids, hydrolysis of phospholipids and modulation of a cellular pathway {e.g., stimulation or inhibition of production of one or more pro-inflammatory mediators). [0073] The terms "salts", "derivatives" and "prodrugs" includes any pharmaceutically acceptable salt, ester, hydrate, or any other compound which, upon administration to the recipient, is capable of providing (directly or indirectly) a compound of the invention, or an active metabolite or residue thereof. Suitable pharmaceutically acceptable salts include salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulfuric, phosphoric, nitric, carbonic, boric, sulfamic and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulfonic, toluenesulfonic, benzenesulfonic, salicylic, sulfanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids. Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium. Also, basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides;

dialkyl sulfates like dimethyl and diethyl sulfate; and others. However, it will be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the invention since these may be useful in the preparation of pharmaceutically acceptable salts. The preparation of salts and prodrugs and derivatives can be carried out by methods known in the art. For example, metal salts can be prepared by reaction of a compound of the invention with a metal hydroxide. An acid salt can be prepared by reacting an appropriate acid with a compound of the invention.

[0074] The term "sequence identity" as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base {e.g., A, T, C, G, I) or the identical amino acid residue {e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison {i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, "sequence identity" will be understood to mean the "match percentage" calculated by an appropriate method. For example, sequence identity analysis may be carried out using the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software.

[0075] "Similarity" refers to the percentage number of amino acids that are identical or constitute conservative substitutions as defined in Table 1.

TABLE 1

ORIGINAL RESIDUE EXEMPLARY SUBSTITUTIONS

Ala Ser

Arg Lys

Asn Gin, His

Asp Glu

Cys Ser

Gin Asn

Glu Asp Gly Pro

His Asn, Gin

He Leu, Val

Leu He, Val

Lys Arg, Gin, Glu

Met Leu, He,

Phe Met, Leu, Tyr

Ser Thr

Thr Ser

Trp Tyr

Tyr Trp, Phe

Val He, Leu

[0076] Similarity may be determined using sequence comparison programs such as GAP (Deveraux eta/., 1984. Nucleic Acids Res. 12, 387-395). In this way, sequences of a similar or substantially different length to those cited herein might be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.

[0077] As used herein a "small molecule" refers to a molecule or compound that has a molecular weight of less than 3 kilodaltons (kDa), and typically less than 1.5 kDa, and more preferably less than about 1 kDa. Small molecules may be nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic (carbon-containing) or inorganic molecules. As those skilled in the art will appreciate, based on the present description, extensive libraries of chemical and/or biological mixtures, often fungal, bacterial, or algal extracts, may be screened with any of the assays of the invention to identify compounds that modulate a bioactivity. A "small organic molecule" is an organic compound (or organic compound complexed with an inorganic compound {e.g., metal)) that has a molecular weight of less than 3 kDa, less than 1.5 kDa, or even less than about 1 kDa.

[0078] Terms used to describe sequence relationships between two or more

polynucleotides or polypeptides include "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity" and "substantial identity". A "reference sequence" is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence {i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may comprise additions or deletions {i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package

Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment {i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul eta/., 1997. Nucleic Acids Res. 25 : 3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel eta/., "Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998, Chapter 15.

[0079] "Stringency" as used herein refers to the temperature and ionic strength conditions, and presence or absence of certain organic solvents, during hybridization. The higher the stringency, the higher will be the observed degree of complementarity between sequences. "Stringent conditions" as used herein refers to temperature and ionic conditions under which only polynucleotides having a high proportion of complementary bases, preferably having exact complementarity, will hybridize. The stringency required is nucleotide sequence dependent and depends upon the various components present during hybridization, and is greatly changed when nucleotide analogues are used. Generally, stringent conditions are selected to be about 10° C to 20° C less than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a target sequence hybridizes to a complementary probe. It will be understood that a polynucleotide will hybridize to a target sequence under at least low stringency conditions, preferably under at least medium stringency conditions and more preferably under high stringency conditions. Reference herein to low stringency conditions include and encompass from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization at 42° C, and at least about 1 M to at least about 2 M salt for washing at 42° C. Low stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHP0₄ (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 2xSSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHP0₄ (pH 7.2), 5% SDS for washing at room temperature. Medium stringency conditions include and encompass from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization at 42° C, and at least about 0.5 M to at least about 0.9 M salt for washing at 42° C. Medium stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHP0₄ (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHP0₄ (pH 7.2), 5% SDS for washing at 42° C. High stringency conditions include and encompass from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization at 42° C, and at least about 0.01 M to at least about 0.15 M salt for washing at 42° C. High stringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 M NaHP0₄ (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 0.2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, ImM EDTA, 40 mM NaHP0₄ (pH 7.2), 1% SDS for washing at a temperature in excess of 65° C. One embodiment of high stringency conditions includes hybridizing in 6 x SSC at about 45° C, followed by one or more washes in 0.2 x SSC, 0.1% SDS at 65° C. One embodiment of very high stringency conditions includes hybridizing 0.5 M sodium phosphate, 7% SDS at 65° C, followed by one or more washes at 0.2 x SSC, 1% SDS at 65° C. Other stringent conditions are well known in the art. A skilled addressee will recognize that various factors can be manipulated to optimize the specificity of the hybridization. Optimization of the stringency of the final washes can serve to ensure a high degree of hybridization. For detailed examples, see CURRENT PROTOCOLS IN MOLECULAR BIOLOGY {supra) at pages 2.10.1 to 2.10.16 and MOLECULAR CLONING. A

LABORATORY MANUAL (Sambrook eta/., eds.) (Cold Spring Harbor Press 1989) at sections 1.101 to 1.104. [0080] As used herein, the terms "treatment", "treating", and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be therapeutic in terms of a partial or complete cure for a disease or condition {e.g., a hematologic malignancy) and/or adverse effect attributable to the disease or condition . These terms also cover any treatment of a condition or disease in a mammal, particularly in a human, and include: (a) inhibiting the disease or condition, i. e. , arresting its development; or (b) relieving the disease or condition, i. e. , causing regression of the disease or condition.

[0081] As used herein, underscoring or italicizing the name of a gene shall indicate the gene, in contrast to its protein product, which is indicated by the name of the gene in the absence of any underscoring or italicizing. For example, "CD14" shall mean the CD J 4 gene, whereas "CD14" shall indicate the protein product or products generated from transcription and translation and/or alternative splicing of the "CDJ4' gene.

[0082] Each embodiment described herein is to be applied mutatis mutandis - to each and every embodiment unless specifically stated otherwise.

2. Compositions and methods for modulating cytokine production and vascular leakage in Flavivirus infections

[0083] The present invention is based in part on the determination that ( 1) Flavivirus sNSl binds to CD14, (2) Flavivirus sNSl is an agonist of CD14-mediated production of proinflammatory mediators, (3) blockage of sNSl agonist interaction with mCD14 by a CD14 antagonist antibody inhibits NSl-mediated production by i mmune cells of pro-inflammatory cytokines such as IL-6, TN F-a, IL-Ιβ and IFN-β, (4) sNSl will bind to sCD14 and blockage of this interaction by a CD14 antagonist antibody will inhibit sNSl-sCD14-mediated pro-inflammatory cytokine production, and (5) blockage of sNSl agonist interaction with mCD14 or sCD14 by a CD14 antagonist antibody will inhibit NSl-mediated pro-inflammatory cytokine production by endothelial cells, including vascular endothelial cells . Accordingly, the present invention provides methods and compositions that include a CD14 antagonist antibody for modulating the production of proinflammatory mediators and/or vascular leakage in Flavivirus infections, and for treating or preventing any of symptoms associated with such infections, such as acute febrile illness, malaise, headache, flushing, diarrhea, nausea, vomiting, abdominal pain, myalgias and, in severe disease, production of pro-inflammatory mediators, including pro-inflammatory cytokines and vascular leakage.

2.1 Conservation of NS1 across Flaviviruses and use of CD14 antagonist therapy

[0084] The Flavivirus NS1 glycoprotein comprises about 350 amino acids and has a molecular weight of 48-55 kDa, depending on its glycosylation status. It exists in multiple oligomeric forms and is found in different cellular locations : a cell membrane-bound form in association with virus-induced intracellular vesicular compartments, on the cell surface and as a soluble secreted hexameric lipoparticle {i. e. , sNSl) . Because NS1 is structurally conserved across all members of the genus, it is proposed that Flavivirus NS1 glycoproteins are generally recognized as PAMPs by CD14 and that CD14 antagonists, especially CD14 antagonist antibodies, and are useful therefore for treating or preventing disease associated with any Flavivirus species including but not li mited to : Aroa virus, Bussuquara virus, Iguape virus, Naranjal virus, Dengue virus group, Dengue virus, Dengue virus 1, Dengue virus 2, Dengue virus 3, Dengue virus 4, Japanese encephalitis virus group, Japanese encephalitis virus, Japanese encephalitis virus strain

JAOARS982, Japanese encephalitis virus strain Nakayama, Japanese encephalitis virus strain SA(V), Japanese encephalitis virus strain SA-14, Koutango virus, Murray Valley encephalitis virus, Alfuy virus, Murray valley encephalitis virus (strain MVE-1-51), St. Louis encephalitis virus, St. Louis encephalitis virus (strain MS 1-7), Usutu virus, West Nile virus, Kunjin virus, West Nile virus crow/New York/3356/2000, West Nile virus H442, West Nile virus SA38 l/OO, West Nile virus SA 93/01, West Nile virus SPU116/89, West Nile virus strain 385-99, West Nile virus strain PT5.2, West Nile virus strain PT6.16, West Nile virus strain PT6.39, West Nile virus strain PT6.5, West Nile virus strain PTRoxo, Kokobera virus group, Kokobera virus, New Mapoon virus, Stratford virus, unclassified Kokobera virus group, CY1014 virus, Modoc virus group, Cowbone Ridge virus, Jutiapa virus, Modoc virus, Sal Vieja virus, San Perlita virus, mosquito-borne viruses, Ilheus virus, Rocio virus, Sepik virus, Ntaya virus group, Bagaza virus, Israel turkey meningoencephalomyelitis virus, Ntaya virus, Tembusu virus, Sitiawan virus, Yokose virus, Rio Bravo virus group, Apoi virus, Bukalasa bat virus, Carey Island virus, Dakar bat virus, Entebbe bat virus, Rio Bravo virus, Saboya virus, Potiskum virus, Seaborne tick- borne virus group, Mcaban virus, Saumarez Reef virus,

Tyuleniy virus, Spondweni virus group, Zika virus, Spondweni virus, tick- borne encephalitis virus group, Kyasanur forest disease virus, Alkhurma hemorrhagic fever virus, Langat virus, Langat virus (strain TP21), Langat virus (strain Yelantsev), Louping ill virus, Louping ill virus (strain 31), Louping ill virus (strain K), Louping ill virus (strain Negishi 3248/49/P10), Louping ill virus (strain Norway), Louping ill virus (strain SB 526), Omsk hemorrhagic fever virus, Phnom Penh bat virus, Powassan virus, Deer tick virus, Tick-borne powassan virus (strain lb), Royal Farm virus, Karshi virus, Tick-borne encephalitis virus, Kumlinge virus, Negishi virus, Tick-borne encephalitis virus (strain HYPR), Tick-borne encephalitis virus (STRAIN SOFJIN), Tick-borne encephalitis virus (WESTERN SUBTYPE), Turkish sheep encephalitis virus, Yaounde virus, Yellow fever virus group, Banzi virus, Bouboui virus, Edge Hill virus, Uganda S virus, Wesselsbron virus, Yellow fever virus, Yellow fever virus 17D, Yellow fever virus 1899/81, Yellow fever virus isolate Angola/14FA/1971 , Yellow fever virus isolate Ethiopia/Couma/1961 , Yellow fever virus isolate Ivory Coast/1999, Yellow fever virus isolate Ivory Coast/85-82H/1982, Yellow fever virus isolate Uganda/A7094A4/1948, Yellow fever virus strain French neurotropic vaccine, Yellow fever virus strain Ghana/Asibi/1927, Yellow fever virus Trinidad/79A/1979, unclassified Flavivirus, Aedes flavivirus, Batu Cave virus, Cacipacore virus, Cell fusing agent virus, Chaoyang virus, Chimeric Tick-borne encephalitis virus/Dengue virus 4, Culex flavivirus, Flavivirus CbaAr4001, Flavivirus FSME, Flavivirus SST-2008, Gadgets Gully virus, Greek goat encephalitis virus, Jugra virus, Kadam virus, Kamiti River virus, Kedougou virus, Montana myotis leukoencephalitis virus, Ngoye virus, Nounane virus, Quang Binh virus, Russian Spring-Summer encephalitis virus, Sokoluk virus, Spanish sheep encephalitis virus, T'Ho virus, Tai forest virus B31 , Tamana bat virus, Tick-borne flavivirus, Wang Thong virus, and Flavivirus sp..

2.2 CD14 antagonist antibodies

[0085] The present invention contemplates any CD14 antagonist antibody that binds to CD14 and blocks the binding of sNSl to CD14 and/or that binds to CD14 and inhibits or abrogates a CD14 agonist-mediated response such as the production of pro-inflammatory mediators, including the production of pro-inflammatory cytokines and/or chemokines. In some embodiments, a CD14 antagonist antibody of the present invention inhibits binding of a CD14 agonist, suitably sNSl, to CD14. In illustrative examples of this type, the CD14 antagonist antibody is selected from the 3C10 antibody that binds an epitope comprised in at least a portion of the region from amino acid 7 to amino acid 14 of human CD14 (van Voohris et al., 1983. J. Exp. Med. 158: 126-145; Juan eta/., 1995. J. Biol. Chem. 270(29) : 17237-17242), the MEM-18 antibody that binds an epitope comprised in at least a portion of the region from amino acid 57 to amino acid 64 of CD14 (Bazil et al, 1986. Eur. J. Immunol. 16(12) : 1583-1589; Juan etai, 1995. J. Biol. Chem. 270(10) : 5219- 5224), the 4C1 antibody (Adachi etai, 1999. J. Endotoxin Res. 5 : 139-146; Tasaka etai, 2003. Am. J. Respir. Cell. Mol. Biol; 2003. 29(2) : 252-258), as well as the 28C5 and 23G4 antibodies that inhibit binding of LPS and suppress release of pro-inflammatory cytokines, and the 18E12 antibody that partly inhibits binding of LPS and suppresses release of pro-inflammatory cytokines (U.S. Patent Nos. 5,820,858, 6,444,206 and 7,326,569 to Leturcq etai). In some embodiments, a CD14 antagonist antibody of the present invention inhibits binding of CD14 to a TLR such as TLR4, thereby blocking CD14-agonist mediated response, illustrative examples of which include the F1024 antibody disclosed in International Publication WO2002/42333. Each of the above references relating to CD14 antagonist antibodies is incorporated herein by reference in its entirety. The CD14 antagonist antibody may be a full-length immunoglobulin antibody or an antigen-binding fragment of an intact antibody, representative examples of which include a Fab fragment, a F(ab')2 fragment, an Fd fragment consisting of the VH and CHI domains, an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, a single domain antibody (dAb) fragment (Ward etai, 1989. Nature 341 : 544-546), which consists of a VH domain; and an isolated CDR. Suitably, CD14 antagonist antibody is a chimeric, humanized or human antibody.

[0086] In some embodiments, the CD14 antagonist antibody is selected from the antibodies disclosed in U.S. Pat. No. 5,820,858 :

[0087] (1) an antibody comprising :

a VL domain comprising, consisting or consisting essentially of the sequence:

QS PAS LAVS LG Q RATIS C RASESVDSFGNSFMH WYQQKAGQPPKSSIY RAANLES

GIPARFSGSGSRTDFTLTINPVEADDVATYFC QQSYEDPWT FGGGTKLGNQ [SEQ ID NO: 1] (3C10 VL); and

a VH domain comprising, consisting or consisting essentially of the sequence:

LVKPGGSLKLSCVASGFTFS SYAMS WVRQTPEKRLEWVA SISSGGTTYYPDNVKG RFTISRDNARNILYLQMSSLRSEDTAMYYCAR GYYDYHY WGQGTTLTVSS [SEQ ID NO: 2] (3C10 VH);

[0088] (2) an antibody comprising :

a VL domain comprising, consisting or consisting essentially of the sequence:

QS PAS LAVS LG Q RATIS C RASESVDSYVNSFLH WYQQKPGQPPKLLIY RASNLQS

GIPARFSGSGSRTDFTLTINPVEADDVATYCC QQSNEDPTT FGGGTKLEIK [SEQ ID NO: 3] (28C5 VL); and

a VH domain comprising, consisting or consisting essentially of the sequence:

LQQSG PG LVKPSQSLS LTCTVTGYSIT SDSAWN WIRQFPGNRLEWMG YISYSGSTSYNPSLKS

RISITRDTSKNQFFLQLNSVTTEDTATYYCVR GLRFAY WG QGTLVTVS A [SEQ ID NO: 4] (28C5 VH); and

[0089] (3) an antibody comprising :

a VL domain comprising, consisting or consisting essentially of the sequence:

QTPSSLSASLGDRVTISC RASQDIKNYLN WYQQPGGTVKVLIY YTSRLHS

GVPSRFSGSGSGTDYSLTISNLEQEDFATYFC QRGDTLPWT FGGGTKLEIK [SEQ ID NO: 5] (18E12 VL); and a VH domain comprising, consisting or consisting essentially of the sequence:

LESG PG LVAPSQS LSITCTVSG FS LT NYDIS WIRQPPGKGLEWLG VIWTSGGTN YN SAFM S RLSITKDNSESQVFLKMNGLQTDDTGIYYCVR GDGN FYLYNFDY WGQGTTLTVSS [SEQ ID NO: 6] (18E12 VH);

[0090] Also contemplated are antibodies that comprise the VL and VH CDR sequences of the above antibodies, representative embodiments of which include:

[0091] (1) an antibody that comprises: a) an antibody VL domain, or antigen binding fragment thereof, comprising L-CDR1, L-CDR2 and L-CDR3, wherein : L-CDR1 comprises the sequence RASESVDSFGNSFMH [SEQ ID NO: 7] (3C10 L-CDR1); L-CDR2 comprises the sequence RAANLES [SEQ ID NO: 8] (3C10 L-CDR2); and L-CDR3 comprises the sequence QQSYEDPWT [SEQ ID NO: 9] (3C10 L-CDR3); and b) an antibody VH domain, or antigen binding fragment thereof, comprising H-CDR1, H-CDR2 and H-CDR3, wherein : H-CDR1 comprises the sequence SYAMS [SEQ ID NO: 10] (3C10 H-CDR1); H-CDR2 comprises the sequence SISSGGTTYYPDNVKG [SEQ ID NO: 11] (3C10 H-CDR2); and H-CDR3 comprises the sequence GYYDYHY [SEQ ID NO: 12] (3C10 H- CDR3);

[0092] (2) an antibody that comprises: a) an antibody VL domain, or antigen binding fragment thereof, comprising L-CDR1, L-CDR2 and L-CDR3, wherein : L-CDR1 comprises the sequence RASESVDSYVNSFLH [SEQ ID NO: 13] (28C5 L-CDR1); L-CDR2 comprises the sequence RASNLQS [SEQ ID NO: 14] (28C5 L-CDR2); and L-CDR3 comprises the sequence QQSNEDPTT [SEQ ID NO: 15] (28C5 L-CDR3); and b) an antibody VH domain, or antigen binding fragment thereof, comprising H-CDR1, H-CDR2 and H-CDR3, wherein : H-CDR1 comprises the sequence SDSAWN [SEQ ID NO: 16] (28C5 H-CDR1); H-CDR2 comprises the sequence YISYSGSTSYNPSLKS [SEQ ID NO: 17] (28C5 H-CDR2); and H-CDR3 comprises the sequence GLRFAY [SEQ ID NO: 18] (28C5 H-CDR3); and

[0093] (3) an antibody that comprises: a) an antibody VL domain, or antigen binding fragment thereof, comprising L-CDR1, L-CDR2 and L-CDR3, wherein : L-CDR1 comprises the sequence RASQDIKNYLN [SEQ ID NO: 19] (18E12 L-CDR1); L-CDR2 comprises the sequence YTSRLHS [SEQ ID NO: 20] (18E12 L-CDR2); and L-CDR3 comprises the sequence QRGDTLPWT [SEQ ID NO: 21] (18E12 L-CDR3); and b) an antibody VH domain, or antigen binding fragment thereof, comprising H-CDR1, H-CDR2 and H-CDR3, wherein : H-CDR1 comprises the sequence

NYDIS [SEQ ID NO: 22] (18E12 H-CDR1); H-CDR2 comprises the sequence VIWTSGGTNYNSAFMS [SEQ ID NO: 23] (18E12 H-CDR2); and H-CDR3 comprises the sequence GDGNFYLYNFDY [SEQ ID NO: 24] (18E12 H-CDR3).

[0094] In some embodiments, the CD14 antagonist antibody is humanized. In illustrative examples of this type, the humanized CD14 antagonist antibodies suitably comprise a donor CDR set corresponding to a CD14 antagonist antibody {e.g., one of the CD14 antagonist antibodies described above), and a human acceptor framework. The human acceptor framework may comprise at least one amino acid substitution relative to a human germline acceptor framework at a key residue selected from the group consisting of: a residue adjacent to a CDR; a glycosylation site residue; a rare residue; a canonical residue; a contact residue between heavy chain variable region and light chain variable region; a residue within a Vernier zone; and a residue in a region that overlaps between a Chothia-defined VH CDR1 and a Kabat-defined first heavy chain framework. Techniques for producing humanized mAbs are well known in the art (see, for example, Jones eta/., 1986. Nature 321 : 522-525; Riechmann eta/. 1988. Nature 332 : 323-329; Verhoeyen eta/., 1988. Science 239 : 1534-1536; Carter eta/., 1992. Proc. Natl. Acad. Sci. USA 89 : 4285-4289; Sandhu, JS., 1992. Crit. Rev. Biotech. 12 : 437-462, and Singer eta/., 1993. J. Immunol. 150 : 2844-2857). A chimeric or murine monoclonal antibody may be humanized by transferring the mouse CDRs from the heavy and light variable chains of the mouse

immunoglobulin into the corresponding variable domains of a human antibody. The mouse framework regions (FR) in the chimeric monoclonal antibody are also replaced with human FR sequences. As simply transferring mouse CDRs into human FRs often results in a reduction or even loss of antibody affinity, additional modification might be required in order to restore the original affinity of the murine antibody. This can be accomplished by the replacement of one or more human residues in the FR regions with their murine counterparts to obtain an antibody that possesses good binding affinity to its epitope. See, for example, Tempest etai. (1991.

Biotechnology 9 : 266-271) and Verhoeyen etai (1988 supra). Generally, those human FR amino acid residues that differ from their murine counterparts and are located close to or touching one or more CDR amino acid residues would be candidates for substitution.

[0095] In a preferred embodiment, the CD14 antagonist antibody is the IC14 antibody (Axtelle etai, 2001. J. Endotoxin Res. 7: 310-314; and U.S. Pat. Appl. No. 2006/0121574, which are incorporated herein by reference in their entirety) or an antigen-binding fragment thereof. The IC14 antibody is a chimeric (murine/human) monoclonal antibody that specifically binds to human CD14. The murine parent of this antibody is 28C5 noted above {see, Patent Nos. 5,820,858, 6,444,206 and 7,326,569 to Leturcq etai, and Leturcq etai, 1996. J. Clin. Invest. 98: 1533- 1538). The IC14 antibody comprises a VL domain and a VH domain, wherein:

[0096] the VL domain comprises the amino acid sequence:

METDTILLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASESVDSYVNSFLHWYQQKPGQPPKLLIYRA SNLQSGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKS GTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC [SEQ ID NO: 25]; and

[0097] the VH domain comprises the amino acid sequence:

MKVLSLLYLLTAIPGILSDVQLQQSGPGLVKPSQSLSLTCTVTGYSITSDSAWNWIRQFPGNRLEWMGYISYSGS TSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCVRGLRFAYWGQGTLVTVSSASTKGPSVFPLAPCSRST SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTK VDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLSLGK [SEQ ID NO: 26] .

3. Other direct antagonists of CD14

[0098] As disclosed herein, antagonist antibodies that bind to CD14 directly, inhibit NSl-mediated production of pro-inflammatory cytokines, and as such, it is proposed that any direct antagonist of CD14 function will modulate the production of pro-inflammatory mediators and/or vascular leakage in Flavivirus infections including in methods of treating or preventing Flavivirus infections. Accordingly, the present invention further contemplates the use of a direct CD14 antagonist, including non-antibody direct antagonists of CD14, in methods and compositions for inhibiting the binding of sNSl to CD14 {e.g., mCD14 or sCD14), or for modulating production of a pro-inflammatory mediator {e.g., a cytokine such as IL-6, TNF-a, IL-Ιβ and IFN-β) that is associated with a Flavivirus infection, or for modulating vascular leakage that is associated with a Flavivirus infection, or for inhibiting sNSl-mediated disease symptoms {e.g., production of a proinflammatory mediator, vascular leakage, etc. associated with a Flavivirus infection, or for treating or preventing a Flaviviridae virus infection. In some embodiments, the CD14 antagonist antibody is manufactured as a medicament for any one or more of those applications.

[0099] Representative non-antibody direct antagonists encompassed by the present invention include non-antibody polypeptide/peptide CD14 antagonists such as, but not limited to, thrombomodulin (TM) polypeptides and TM fragments including TM domains, representative examples of which include TM lectin-like domain and epidermal growth factor-like domain plus serine/threonine-rich domain (rTMD23), as described for example by Ma etai. (2015. J. Immunol. 194: 1905-1915, which is incorporated herein by reference).

[0100] The present invention also contemplates CD14 antagonistic nucleic acid molecules that function to inhibit the transcription or translation of CD14 transcripts.

Representative transcripts of this type include nucleotide sequences corresponding to any one the following sequences: (1) human CD14 nucleotide sequences as set forth for example in GenBank Accession Nos. AF097942.1, BT007331.1, NM_000591.3, M86511.1, NM_001040021.2,

BC010507.2, X13334.1, AK313277.1, AB446505.1, AY044269.1, NM_001174105.1, and

NM_001174104.1; (2) nucleotide sequences that share at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity with any one of the sequences referred to in (1); (3) nucleotide sequences that hybridize under at least medium or high stringency conditions to the sequences referred to in (1); (4) nucleotide sequences that encode any one of the following amino acid sequences: human CD14 amino acid sequences as set forth for example in GenPept Accession Nos. NP_001167576.1, NP_001167575.1, NP_001035110.1, NP_000582.1 and AAH10507; (5) nucleotide sequences that encode an amino acid sequence that shares at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence similarity with any one of the sequences referred to in (4); and nucleotide sequences that encode an amino acid sequence that shares at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity with any one of the sequences referred to in (4).

[OlOl] Illustrative antagonist nucleic acid molecules include antisense molecules, aptamers, ribozymes and triplex forming molecules, RNAi and external guide sequences. The nucleic acid molecules can act as effectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules can possess a de novo activity independent of any other molecules.

[0102] Antagonist nucleic acid molecules can interact with any macromolecule, such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, antagonist nucleic acid molecules can interact with CD14 mRNA or the genomic DNA of CD14 or they can interact with a CD14 polypeptide. Often antagonist nucleic acid molecules are designed to interact with other nucleic acids based on sequence homology between the target molecule and the antagonist nucleic acid molecule. In other situations, the specific recognition between the antagonist nucleic acid molecule and the target molecule is not based on sequence homology between the antagonist nucleic acid molecule and the target molecule, but rather is based on the formation of tertiary structure that allows specific recognition to take place.

[0103] In some embodiments, anti-sense RNA or DNA molecules are used to directly block the translation of CD14 by binding to targeted mRNA and preventing protein translation. Antisense molecules are designed to interact with a target nucleic acid molecule through either canonical or non-canonical base pairing. The interaction of the antisense molecule and the target molecule may be designed to promote the destruction of the target molecule through, for example, RNAse H mediated RNA-DNA hybrid degradation. Alternatively the antisense molecule may be designed to interrupt a processing function that normally would take place on the target molecule, such as transcription. Antisense molecules can be designed based on the sequence of the target molecule. Numerous methods for optimization of antisense efficiency by finding the most accessible regions of the target molecule exist. Non-limiting methods include in vitro selection experiments and DNA modification studies using DMS and DEPC. In specific examples, the antisense molecules bind the target molecule with a dissociation constant (K_d) less than or equal to 10^"5, 10^"8, 10 ¹⁰, or 10 ¹². In specific embodiments, antisense oligodeoxyribonucleotides derived from the translation initiation site, e.g., between -10 and +10 regions are employed. Illustrative CD14 antisense compounds are disclosed for example in Furusako et al. (2001. Acta. Med. Okayama 55(2) : 105- 15) and Amano et al. (1997. J. Cell. Physio/.173{3) : 301-9).

[0104] In other embodiments, RNA molecules that mediate RNA interference (RNAi) of the CD14 gene or transcript thereof can be used to reduce or abrogate gene expression. RNAi refers to interference with or destruction of the product of a target gene by introducing a single- stranded or usually a double-stranded RNA (dsRNA) that is homologous to the transcript of a target gene. RNAi methods, including double-stranded RNA interference (dsRNAi) or small interfering RNA (siRNA), have been extensively documented in a number of organisms (Fire etai, 1998. Nature 391 : 806-811). In mammalian cells, RNAi can be triggered by 21- to 23-nucleotide (nt) duplexes of small interfering RNA (siRNA) (Chiu etai, 2002 Mol. Cell. 10 : 549-561; Elbashir etai, 2001. Nature 411 : 494-498), or by microRNAs (miRNA), functional small-hairpin RNA (shRNA), or other dsRNAs which are expressed in vivo using DNA templates with RNA polymerase III promoters

(Zeng etai, 2002. Mol. Cell^ \ >TJ-\ > ; Paddison etai, 2002. Genes Dev. 16: 948-958; Lee et ai, 2002. Nature Biotechnol. 20 : 500-505; Paul etai, 2002. Nature Biotechnol. 20 : 505-508;

Tuschl, T., 2002. Nature Biotechnol. 20 :440-448; Yu etai, 2002. Proc. Natl. Acad. Sci. USA 99(9) : 6047-6052; McManus etai, 2002. RNA 8: 842-850; Sui etai, 2002. Proc. Natl. Acad. Sci. USA 99(6) : 5515-5520).

[0105] In specific embodiments, dsRNA per se and especially dsRNA-producing constructs corresponding to at least a portion of the CD14 gene are used to reduce or abrogate its expression. RNAi-mediated inhibition of gene expression may be accomplished using any of the techniques reported in the art, for instance by introducing a nucleic acid construct encoding a stem-loop or hairpin RNA structure into the genome of the target cell, or by expressing a transfected nucleic acid construct having homology for a CDJ4 gene from between convergent promoters, or as a head-to-head or tail-to-tail duplication from behind a single promoter. Any similar construct may be used so long as it produces a single RNA having the ability to fold back on itself and produce a dsRNA, or so long as it produces two separate RNA transcripts, which then anneal to form a dsRNA having homology to a target gene.

[0106] Absolute homology is not required for RNAi, with a lower threshold being described at about 85% homology for a dsRNA of about 200 base pairs (Plasterk and Ketting, 2000, Current Opinion in Genetics and Dev. 10 : 562-67). Therefore, depending on the length of the dsRNA, the RNAi-encoding nucleic acids can vary in the level of homology they contain toward the target gene transcript, i.e., with dsRNAs of 100 to 200 base pairs having at least about 85% homology with the target gene, and longer dsRNAs, i.e., 300 to 1000 base pairs, having at least about 75% homology to the target gene. RNA-encoding constructs that express a single RNA transcript designed to anneal to a separately expressed RNA, or single constructs expressing separate transcripts from convergent promoters, are suitably at least about 100 nucleotides in length. RNA-encoding constructs that express a single RNA designed to form a dsRNA via internal folding are usually at least about 200 nucleotides in length.

[0107] The promoter used to express the dsRNA-forming construct may be any type of promoter, provided it is operable in the cell in which a target transcript is expressed.

[0108] In some embodiments, RNA molecules of about 21 to about 23 nucleotides, which direct cleavage of specific mRNA to which they correspond, as for example described by Tuschl eta/, in U.S. Pat. App. Pub. No. 2002/0086356, can be utilized for mediating RNAi . Such 21- to 23-nt RNA molecules can comprise a 3' hydroxyl group, can be single-stranded or double stranded (as two 21- to 23-nt RNAs) wherein the dsRNA molecules can be blunt ended or comprise overhanging ends {e.g., 5', 3')·

[0109] In some embodiments, the antagonist nucleic acid molecule is a siRNA. siRNAs can be prepared by any suitable method. For example, reference may be made to International Publication WO 02/44321, which discloses siRNAs capable of sequence-specific degradation of target mRNAs when base-paired with 3' overhanging ends, which is incorporated by reference herein. Sequence specific gene silencing can be achieved in mammalian cells using synthetic, short double-stranded RNAs that mimic the siRNAs produced by the enzyme Dicer. siRNA can be chemically or In iz/frr'-synthesized or can be the result of short double-stranded hairpin-like RNAs (shRNAs) that are processed into siRNAs inside the cell . Synthetic siRNAs are generally designed using algorithms and a conventional DNA/RNA synthesizer. Suppliers include Ambion (Austin,

Tex.), ChemGenes (Ashland, Mass.), Dharmacon (Lafayette, Colo.), Glen Research (Sterling, Va.), MWB Biotech (Esbersberg, Germany), Proligo (Boulder, Colo.), and Qiagen (Vento, The

Netherlands). siRNA can also be synthesized in vitro using kits such as Ambion's SILENCER™ siRNA Construction Kit.

[OllO] The production of siRNA from a vector is more commonly done through the transcription of a short hairpin RNAs (shRNAs). Kits for the production of vectors comprising shRNA are available, such as, for example, Imgenex's GEN ESUPPRESSOR™ Construction Kits and

Invitrogen's BLOCK-IT™ inducible RNAi plasmid and lentivirus vectors. In addition, methods for formulation and delivery of siRNAs to a subject are also well known in the art. See, e.g., U.S. Pat. App. Pub. Nos. 2005/0282188; 2005/0239731; 2005/0234232; 2005/0176018; 2005/0059817; 2005/0020525; 2004/0192626; 2003/0073640; 2002/0150936; 2002/0142980; and

2002/0120129, each of which is incorporated herein by reference. [0111] Illustrative RNAi molecules {e.g., CD14 siRNA or shRNA) are available commercially from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), and OriGene

Technologies, Inc. (Rockville, MD, USA).

3.1 Screening Methods

[0112] The present invention also provides methods for the identification of direct antagonists of CD14 suitable for use in the treatment or prevention of Flavivirus infection or symptom thereof. Antagonists identified by these methods may be antagonists of CD14 having any of the characteristics or effects described herein. For example, antagonists identified by the methods described herein may be suitable for use in the treatment or prevention of Flavivirus infection, or in the treatment or prevention of any of the conditions or symptoms described herein, such as acute febrile illness, malaise, headache, flushing, diarrhea, nausea, vomiting, abdominal pain, myalgias and, in severe disease, production of pro-inflammatory mediators, including proinflammatory cytokines and vascular leakage.

[0113] Accordingly, the invention provides methods of identifying an agent for use in treating or preventing a Flavivirus infection or symptom thereof. These methods generally comprise determining whether a test agent is capable of directly antagonizing CD14. For example, the methods may involve determining whether a test agent is capable of decreasing the amount or agonist activity of CD14, wherein the ability to decrease the amount or agonist activity of CD14 indicates that the test agent may be suitable for use in treating or preventing Flavivirus infection or symptom thereof as described herein. In some embodiments, the test agent is contacted with

CD14, or a cell that expresses CD14 on its surface, or a nucleic acid sequence from which CD14 is expressed, suitably in the presence of a CD14 agonist such as sNSl, wherein a decrease in the amount or agonist activity of CD14 in the presence of the agonist, when compared to a control, indicates that the test agent binds to CD14 and directly antagonizes CD14. A reduction or inhibition of CD14 agonist activity, includes for example inhibiting, reducing or abrogating activation of downstream pathways such as TLR signaling pathways {e.g., TLR4 signaling pathway) and the TRIF pathway, or elicitation of a cellular response {e.g., production of pro-inflammatory mediators including pro-inflammatory cytokines).

[0114] A test agent for use in a screening method of the invention refers to any compound, molecule or agent that may potentially bind to and antagonize CD14. The test agent may be, or may comprise, for example, a peptide, polypeptide, protein, antibody, polynucleotide, small molecule or other compound that may be designed through rational drug design starting from known antagonists of CD14.

[0115] The test agent may be any agent having one or more characteristics of an antagonist of CD14 as described above.

[0116] The test agent to be screened could be derived or synthesized from chemical compositions or man-made compounds. Candidate agents may be obtained from a wide variety of sources including libraries of synthetic or natural compounds. Suitable test agents which can be tested in the above assays include compounds derived from combinatorial libraries, small molecule libraries and natural product libraries, such as display {e.g., phage display) libraries. Multiple test agents may be screened using a method of the invention in order to identify one or more agents having a suitable effect on CD14, such as inhibition of CD14 activity or expression. [0117] The screening methods of the invention may be carried out in vivo, ex vivo or in vitro. In particular, the step of contacting a test agent with CD14 or with a cell that expresses CD14 on its surface {e.g., immune cells, endothelial cells, etc.) may be carried out in vivo, ex vivo or in vitro. The screening methods of the invention may be carried out in a cell-based or a cell-free system. For example, the screening method of the invention may comprise a step of contacting a cell expressing CD14 on its surface with a test agent and determining whether the contacting of the cell with the test agent leads to a decrease in the amount or agonist activity of CD14.

[0118] In such a cell-based assay, the CD14 and/or the test agent may be endogenous to the host cell, may be introduced into a host cell or tissue, may be introduced into the host cell or tissue by causing or allowing the expression of an expression construct or vector or may be introduced into the host cell by stimulating or activating expression from an endogenous gene in the cell. In such a cell-based method, the amount of activity of CD14 may be assessed in the presence or absence of a test agent in order to determine whether the agent is altering the amount of CD14 in the cell, such as through regulation of CD14 expression in the cell or through destabilization of CD14 protein within the cell, or altering the CD14 agonist activity of the cell . The presence of a lower CD14 agonist activity or a decreased amount of CD14 on the cell surface in the presence of the test agent indicates that the test agent may be a suitable antagonist of CD14 for use in accordance with the present invention in the treatment of an individual with a Flavivirus infection or symptom thereof.

[0119] In one embodiment, such a cell-based assay may be carried out in vitro or ex vivo on cells or tissue deriving from the patient to be treated. It may therefore be determined whether or not the test agent is capable of decreasing the activity or amount of CD14 in the cells of that subject.

[0120] In preferred embodiments, the methods further comprise determining whether the test agent lacks substantial or detectable bind to another cellular component, suitably a binding partner of CD14, such as a CD14 binding partner that is either secreted {e.g., MD2) or located on the cell membrane {e.g., TLR4), to thereby determine that the test agent is a specific antagonist of CD14. In a non-limiting example of this type, the test agent is contacted in the presence of a CD14 agonist such as sNSl (1) with a wild-type cell that expresses CD14 on its surface {e.g., an immune cell such a macrophage), and (2) with a CD14 negative cell {e.g., an immune cell that is the same as in (1) but has a loss of function in the CD14 gene). If the test agent inhibits a CD14 agonist activity of the wild-type cell but not of the CD14 negative cell, this indicates that the test agent is a CD14 specific antagonist. Cells of this type may be constructed using routine procedures or animals {e.g., the homozygous knockout mouse B6.129S4-Cdl4tmlFrm/J) having such cells can be purchased from a commercial supplier {e.g., The Jackson Laboratory (Bar Harbor, ME USA)).

[0121] In other embodiments, the screening methods of the invention may use a cell- free assay. For example, the CD14 may be present in a cell-free environment. A suitable cell-free assay may be carried out in a cell extract. For example, the contacting steps of the methods of the invention may be carried out in extracts obtained from cells that may express, produce or otherwise contain CD14 and/or a test agent. A cell-free system comprising CD14 may be incubated with the other components of the methods of the invention such a test agent. [0122] The contacting step(s) of the method of the invention may comprise incubation of the various components. Such incubations may be performed at any suitable temperature, typically between 4° C and 40° C. Incubation periods may be selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening. Following the contact and optional incubation steps, the subject methods may further include a washing step to remove unbound components, where such a washing step is generally employed when required to remove label that would give rise to a background signal during detection, such as radioactive or fluorescently labeled non-specifically bound components.

[0123] Incubation in cell-based or cell-free assay systems may be performed in a microtiter plate e.g., a 96-well plate or other microwell plate). Further, incubation may be performed in an automated fashion {e.g., for high-throughput screening).

[0124] A screening method of the invention may be carried out in vivo. For example, a screening method may be carried out in an animal model. In such an in vivo model, the effects of a test agent may be assessed in the circulation {e.g., blood), or in other organs such as the liver, kidney or heart. Suitably, the animal is a non-human animal such as a mouse or rat. Such a model may be used to assess the in vivo effects of a test agent. For example, such a model may be used to assess whether the test agent is capable of decreasing the activity or amount of CD14 in vivo. In such a method, the amount and/or agonist activity of CD14 may be assessed.

[0125] An in vivo model may also be used to determine whether the test agent has any unwanted side effects. For example, a method of the invention may compare the effects of a test agent on CD14 with its effects on other receptors or cellular components {e.g., CD14 binding partners such as MD2 and TLR4) in order to determine whether the test agent is specific.

[0126] In an in vivo model as described herein, or an in vitro model such as a cell- based or cell-free assay model as described herein, the effects of a test agent on CD14 may be compared with the effects of the same agent on cellular components including CD14 binding partners such as MD2 and TLR4. As discussed above, a desirable CD14 antagonist for use in a method of treatment and prophylaxis as described herein may be an agent that specifically antagonizes CD14. The screening methods of the invention may thus include an additional step of assessing whether the test agent has any effect on the activity or amount of one or more other such cellular components. In such a method, a test agent may be identified as a suitable CD14 antagonist if it is found to decrease the activity or amount of CD14, but not to decrease, not to significantly decrease, not to significantly decrease, not to alter, or not to significantly alter, the activity or amount of one or more other cellular components, including CD14 binding partners such as MD2 and TLR4.

[0127] In the screening methods described herein, the presence of a lower CD14 agonist activity or a decreased amount of CD14 in the presence of the test agent indicates that the test agent may be a suitable antagonist of CD14 for use in accordance with the present invention to treat an individual with a Flavivirus infection or symptom thereof.

[0128] A test agent that is an antagonist of CD14 may result in a decrease in CD14 activity or levels of at least 5%, at least 10%, at least 25%, at least 50%, at least 60%, at least 75%, or at least 85% or more in the presence of the test agent compared to in the absence of the test agent. A test agent that is an antagonist of CD14 may result in a decrease in CD14 agonist activity or levels such that the agonist activity or level of CD14 is no longer detectable in the presence of the test agent. Such a decrease may be seen in the sample being tested or, for example where the method is carried out in an animal model, in particular tissue from the animal such as in the circulation or other organs such as the liver, kidney or heart.

[0129] A test agent that is an antagonist of CD14 is preferably a specific antagonist of

CD14 as described above. However, this does not mean that a specific antagonist of CD14 has a complete absence of off-target antagonistic activity. In this regard, the specific antagonist of CD14 may have negligible or a minor direct binding and effect on other cellular components, such that the antagonism of the activity, signaling or expression of a non-CD14 cellular component, is less than less than 15%, less than 10%, less than 5%, less than 1%, or less than 0.1% of the direct binding and effect of that agent on the activity, signaling or expression of CD14.

[0130] Levels or amounts of CD14 may be measured by assessing expression of the CD14 gene. Gene expression may be assessed by looking at mRNA production or levels or at protein production or levels. Expression products such as mRNA and proteins may be identified or quantified by methods known in the art. Such methods may utilize hybridization to specifically identify the mRNA of interest. For example such methods may involve PCR or real-time PCR approaches. Methods to identify or quantify a protein of interest may involve the use of antibodies that bind that protein. For example, such methods may involve western blotting. Regulation of CD14 gene expression may be compared in the presence and absence of a test agent. Thus test agents can be identified that decrease CD14 gene expression compared to the level seen in the absence of the test agent. Such test agents may be suitable antagonists of CD14 in accordance with the invention.

[0131] The screening methods may assess the agonist activity of CD14. For example, such a method may be carried out using peripheral blood mononuclear cells. Such cells will produce cytokines such as IL-6, TN F-a, IFN-β, IL-Ιβ and IL-8 on response to stimulation with, for example, sNSl. A screening method may therefore comprise combining peripheral blood mononuclear cells with the test agent or a vehicle and adding sNSl. The cells may then be incubated for an amount of time {e.g., 24 hours) to allow the production of pro-inflammatory mediators such as cytokines. The level of cytokines such as IL-6, TNF-a, IFN-β, IL-Ιβ and IL-8 produced by the cells in that time period can then be assessed. If the test agent has anti-CD14 properties, then the production of such cytokines should be reduced compared to the vehicle-treated cells.

[0132] Further tests may also be carried out in order to confirm that the test agent is suitable for use in the claimed methods. For example, as explained above, a suitable antagonist of CD14 should be capable of reducing the deleterious consequences of pro-inflammatory mediator production (also commonly referred to as a cytokine storm) and vascular leakage. The screening methods of the invention may therefore incorporate further steps, such as those discussed above, which involve assessing the effect of the test agent in an animal with such production of proinflammatory mediator and vascular leakage {e.g., one infected with a Flavivirus) and comparing that effect with that seen in the absence the test agent. A suitable CD14 antagonist will be capable of ameliorating at least some of the effects of the Flavivirus infection in the test animal. 3.2 Ancillary Flavivirus agents

[0133] As indicated, compounds according to the present invention may be

administered alone or in combination with other agents (also referred to herein as "ancillary agents"), especially including other compounds of the present invention or compounds which are not direct CD14 antagonists and are otherwise disclosed as being useful for the treatment of

Flavivirus infections including Dengue virus, Japanese encephalitis virus, Yellow fever virus, Murray Valley encephalitis virus, West Nile virus, Tick-borne encephalitis virus, St Louis encephalitis virus, Alfuy virus, Koutango virus, Kunjin virus, Cacipacore virus, Yaounde virus, Zika virus and related Flavivirus infections, such as those relevant compounds and compositions which are disclosed in the following United States patents, which are incorporated by reference herein: U.S. Pat. Nos. 5,922,757; 5,830,894; 5,821,242; 5,610,054; 5,532,215; 5,491,135; 5,179,084; 4,902,720; 4,898,888; 4,880,784; 5,929,038; 5,922,857; 5,914,400; 5,922,711; 5,922,694; 5,916,589; 5,912,356; 5,912,265; 5,905,070; 5,892,060; 5,892,052; 5,892,025; 5,883,116; 5,883,113; 5,883,098; 5,880,141; 5,880,106; 5,876,984; 5,874,413; 5,869,522; 5,863,921; 5,863,918; 5,863,905; 5,861,403; 5,852,027; 5,849,800; 5,849,696; 5,847,172; 5,627,160; 5,561,120; 5,631,239; 5,830,898; 5,827,727; 5,830,881 and 5,837,871, among others as well as U.S. Pat. App. Pub. Nos. 2009/0130123, 2014/0170186 and 2014/0248336. Alternatively, reference may be made to various compounds disclosed in the scientific literature, including anti- Flavivirus antibodies, as disclosed for example by Robinson etai (2015. Ceil 162 :493-504), and anti- Flavivirus small molecule compounds, as disclosed for example by Low etai (2014. Lancet Infect. Dis. 14: 706-715) and Whitehorn etai (2014. PLOS Neglected Tropical Diseases 8(8) : e3205), which are also incorporated by reference herein . In specific embodiments, ancillary anti- Flavivirus agents that are useful in combination with CD14 antagonists include antivirals and vaccines as well as agents that alleviate the symptoms of Flavivirus infection or prevent secondary infections such as antibiotics used to prevent pneumonia and urinary tract infections, anticonvulsants for seizure control, anti-nausea medicaments, mannitol, interferon alpha {e.g., interferon alpha 2a and interferon alpha 2b), interferon beta {e.g., interferon beta la and interferon beta lb), as well as ant\- Flavivirus antibodies and small molecules, nucleic acid constructs from which any of these are expressible, antibody therapy or any combination thereof.

[0134] Ancillary anti -Flavivirus agents may be used in combination with CD14 antagonists for their additive activity or treatment profile Flavivirus infections and, in certain instances, for their synergistic effects in combination with compounds of the present invention.

[0135] When combination therapy is desired, the CD14 antagonist is administered separately, simultaneously or sequentially with ancillary agent. In some embodiments, this may be achieved by administering a single composition or pharmacological formulation that includes both types of agent, or by administering two separate compositions or formulations at the same time, wherein one composition includes the CD14 antagonist and the ancillary agent. In other embodiments, the treatment with the CD14 antagonist may precede or follow the treatment with the ancillary agent by intervals ranging from minutes to days. In embodiments where the CD14 antagonist is applied separately to the ancillary agent, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the CD14 antagonist would still be able to exert an advantageously combined effect on inhibiting a CD14- mediated effect including inhibiting production of pro-inflammatory mediators by cells (e.g., an immune cell such as but not limited to a macrophage or monocyte, or an endothelial cell) with the ancillary agent, and in particular, to maintain or enhance a subject's capacity to reverse or inhibit the development of disease or symptoms associated with Flavivirus infection. In some situations, one may administer both modalities within about 1-12 hours of each other and, more suitably, within about 2-6 hours of each other. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several hours (2, 3, 4, 5, 6 or 7) to several days (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations. In specific embodiments, the antiviral agent is administered prior to the administration of the CD14 antagonist.

[0136] It is conceivable that more than one administration of either the CD14 antagonist or the ancillary agent will be desired. Various combinations may be employed, where the CD14 antagonist is "A" and the ancillary agent is "B", as exemplified below:

[0137] A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B.

[0138] Where two or more therapeutic agents are administered to a subject "in conjunction" or "concurrently" they may be administered in a single composition at the same time, or in separate compositions at the same time, or in separate compositions separated in time.

3.3 Compositions

[0139] In accordance with the present invention, it is proposed that CD14 antagonists, whether alone or in combination with ancillary anW- Flavivirus agents, can be used to inhibit production of pro-inflammatory mediators, including pro-inflammatory cytokines, and reducing the sequelae of that production including inhibiting or ameliorating vascular leakage, and more particularly, to treat or prevent Flavivirus infections and their symptoms, including in severe disease.

[0140] CD14 antagonists and optionally the ancillary anW- Flavivirus agents can be administered either by themselves or with a pharmaceutically acceptable carrier. Thus, in some embodiments, the compositions of the invention are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients. Depending on the specific conditions being treated, the compositions may be administered systemically or locally. Techniques for formulation and administration may be found in

"Remington's Pharmaceutical Sciences", Mack Publishing Co. , Easton, Pa., latest edition. The compositions may be administered orally, topically, transdermally, parenterally, subcutaneously, intravenously {e.g., hepatic vein), intramuscularly, intraperitoneal^, intracavitary, by intravesical instillation, intranasally, intraocularly, intraarterially, intralesionally, by intranasal instillation, or by application to mucous membranes, such as, that of the nose, throat, and bronchial tubes. They may be administered alone or with suitable pharmaceutical carriers, and can be in solid or liquid form such as, tablets, capsules, powders, solutions, suspensions, or emulsions.

[0141] CD14 antagonists and optionally ancillary anW- Flavivirus agents may be orally administered, for example, with an inert diluent, or with an assimilable edible carrier, or they may be enclosed in hard or soft shell capsules, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet. For oral therapeutic administration, these active compounds may be incorporated with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compound in these compositions may, of course, be varied and may conveniently be between about 2% to about 60% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.

[0142] The tablets, capsules, and the like may also contain a binder such as gum tragacanth, acacia, corn starch, or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose, or saccharin. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a fatty oil .

[0143] Various other materials may be present as coatings or to modify the physical form of the dosage unit. For instance, tablets may be coated with shellac, sugar, or both. A syrup may contain, in addition to active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and flavoring such as cherry or orange flavor.

[0144] CD14 antagonists and optionally the ancillary anW-F/avMrus agents may also be administered parenterally. Solutions or suspensions of these active compounds can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil . In general, water, saline, aqueous dextrose and related sugar solution, and glycols such as, propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0145] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi . The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol {e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

[0146] CD14 antagonists and optionally the ancillary ax\W- Flavivirus agents may also be administered directly to the airways in the form of an aerosol . For use as aerosols, the inhibitors of the present invention in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. The materials of the present invention also may be administered in a non-pressurized form such as in a nebulizer or atomizer.

[0147] Persons of skill in the art are readily able to test and assess optimal dosage schedules based on the balance of efficacy and any undesirable side effects. The optimal dosage of each type of inhibitor will vary, of course, and the minimal effective dose will be administered for therapeutic regimen. 4. Methods of Treatment

[0148] The invention provides for both prophylactic and therapeutic methods of treating a subject that has or is at risk of (or susceptible to) of developing a /¾i//V/ z/s--related disease. These methods include within their scope the treatment of Flavivirus infections in humans, e.g., including pediatric and geriatric populations, and ani mals, e.g. , veterinary applications, as well as diseases associated with such infections . Such diseases include for example, fever, meningitis, encephalitis, yellow fever, dengue fever.

4.1 Prophylactic Methods

[0149] The present invention contemplates methods for preventing a Flavivlrus-re\ate0 disease in a subject by administering to the subject a CD14 antagonist of the invention, and optionally an ancillary anW- Flavivirus agent. The CD14 antagonist, and optionally the ancillary anti- Flavivirus agent (also referred to herein as "therapeutic agents"), may be administered in an "effective amount(s)", to achieve an intended purpose in a subject. The dose of therapeutic agents(s) administered to a patient should be sufficient to achieve a beneficial response in the subject over ti me such as a reduction in at least one symptom associated with a Flavivirus infection . The quantity or dose frequency of the therapeutic agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof. In this regard, precise amounts of the therapeutic agent(s) for administration will depend on the judgment of the practitioner. One skilled in the art would be able, by routine

experimentation, to determine an effective, non-toxic amount of a CD14 antagonist, and optionally an ancillary anW- Flavivirus agent described herein, to include in a pharmaceutical composition of the present invention for the desired therapeutic outcome.

[0150] In some embodi ments, an "effective amount" of a therapeutic agent is an amount sufficient to reduce a symptom associated with infection, and/or to reduce the number of infectious agents in the individual . In these embodiments, an effective amount reduces a symptom associated with infection and/or reduces the number of infectious agents in an individual by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, or more, when compared to the symptom or number of infectious agents in an individual not treated with the therapeutic agent. Symptoms of infection by a Flavivirus, as well as methods for measuring such symptoms, are known in the art. Methods for measuring the number of Flaviviruses in an individual are standard in the art.

[0151] Subjects at risk for a /¾i//V/ z/s--related diseases include patients who have been exposed to the Flavivirus from an infected arthropod (i. e. , mosquito or tick), or via a blood transfusion or sexual intercourse with a Flavivirus-infec eti individual . For example, the subjects have traveled to regions or countries of the world in which other flavivirus infections have been reported and confirmed . Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the Flavivirus-relateti disease, such that a disease is prevented or, alternatively, delayed in its progression .

4.2 Therapeutic Methods

[0152] Also encompassed by the present invention are methods of treating a Flavivirus infection and related disease in a patient. Suitably, these methods involve administering a CD14 antagonist of the invention, and optionally an ancillary anW-F/avMrus agent, in an "effective amount" to a patient with a Flavivirus infection and/or related disease.

[0153] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting example.

EXAMPLES

EXAMPLE 1

CD14 ANTAGONIST ANTIBODY, IC14, INHIBITS DENGUE VIRUS NSI-MEDIATED ACTIVATION

[0154] Human PBMCs were incubated at 37° C with IC14 antibody or with a negative control human IgG4 antibody in media at various concentrations. After 1 h incubation, CD14 agonist (sNSl or LPS) was added and the cells were incubated for 24 h at 37° C in a humidified incubator. At 24 h post agonist addition the media of treated cells was harvested and centrifuged at 1000 x g for 5 min. The levels of IL-6 production were quantified by ELISA (# DY206, R&D Systems).

[0155] The results shown in Figure 1 demonstrate that IC14 specifically inhibits DENV2

NS1 mediated production of the pro-inflammatory cytokine IL-6.

EXAMPLE 2

IC14 BLOCKS NSl- AND LPS-DEPENDENT IL-6 PRODUCTION BY HUMAN PBMCS

[0156] Human PBMCs were treated with IC14, or a human IgG4 control antibody (both at 100 ng/mL) for 1 h or left untreated. Then LPS or DENV2 NSl were added at the indicated concentrations and incubated overnight. The medium was diluted 1 : 5 with PBS and then 50 pL was assayed for secreted IL-6 using ELISA.

[0157] The results presented in Figure 2 demonstrate that IC14 specifically blocks IL-6 secretion by human myeloid cells over a range of ligand concentrations, revealing that CD14 is essential for NSl activation.

EXAMPLE 3

CD14 IS ESSENTIAL FOR NSl ACTIVATION OF MOUSE BMMS

Cytokine expression

[0158] C57 WT or CD14-deficient murine BMMs were incubated with LPS or NSl at the indicated concentrations for 3 h. Cells were harvested, RNA isolated and RT-qPCR performed with primers to interleukin-6 (IL-6), interferon-β (IFN-β), interleukin-ΐβ (IL-Ιβ), tumor necrosis factor-a (TNF-a), and hypoxanthine phosphorylribosyl transferase (HPRT). The calculated cytokine Ct values were normalized to the housekeeping gene HPRT.

[0159] The results presented in Figure 3 show that mCD14 is required for both NS1- and low dose LPS-mediated induction of cytokine mRNA expression in murine BMMs.

CSF-1 receptor down-regulation

[0160] C57 WT or CD14-deficient BMMs were grown overnight without CSF-1 to upregulate CSF-1 receptor expression. Cells were then treated with LPS or NSl at varying concentrations for 1 h and then stained with a PE-conjugated CSFl receptor antibody and analyzed by flow cytometry.

[0161] The results presented in Figure 4 reveal that CD14 is required for NS1 and LPS to induce CSF-1R down-regulation in murine BMMs.

Materials and Methods for Examples 1 to 3

Cell Culture

[0162] PBMCs were obtained from healthy volunteers under approval from the

University of Queensland Medical Research Ethics Committee. Human PBMCs were isolated from whole blood (Australian Red Cross) by a Ficoll gradient and then cultured in RPMI media containing 10% fetal calf serum, in a 37° C humidified incubator.

[0163] BMMs were obtained by differentiation of mouse bone marrow progenitors in the presence of CSFl ( 10⁴ U/mL) for 7 days (Sester et a/. , 1999. J. Immunol. 163 : 6541-6550) .

Generation and purification of DENV2 sNSl

[0164] Recombinant DENV2 NS1 was expressed by stably transfected Drosophila S2 cells. The protein was affinity-purified from culture medium using a column coupled with the 2A5.1 anti-NSl monoclonal antibody. Transfection complex was washed away after 4 hours. The medium was harvested at day 2 after transfection and concentrated using a 100-kD cutoff spin column (Millipore) . Recombinant NS1 preparations were tested for LPS contamination by the LAL assay using the LAL Chromogenic Endotoxin Quantitation Kit (Pierce) and shown to be endotoxin-free (background readings representing <0.1 EU/mL or < 10 pg of endotoxin/mL equivalent were found for purified DENV2 NS1 at 40 mg/ml and for the working stocks of transiently expressed and concentrated NS1 preparations) . Immunodepletions of NS1 were performed using mouse monoclonal anti-NSl antibody ( 1H7 or 2A5) and control anti-E antibody (3H5), purified from ascites, and bound to protein G Dynabeads (Life Technologies) .

Determination of protein concentration

[0165] Purified protein concentrations were determined using a BCA assay kit (Pierce) . NS1 protein expression in the CHO expression system was determined by capture-ELISA as described previously (Avirutnan et a/., 2006. J. Infect. Dis. 193 : 1078-1088), with the following modifications: the inclusion of NS1 standards (purified recombinant DENV2 sNSl (0.3 ng/mL to 1250 ng/mL) and 100 pg/mL TM B and sulfuric acid were used for color detection.

Detection of IL-6 production using ELISA

[0166] Human PBMCs from healthy donors were plated at 50,000 cells per well in 12 well plates in the presence of IC14 or a human IgG4 control antibody for 1 hour prior to incubation with NS1 or LPS (Ultrapure LPS from E. coli strain 0111 : B4, InvivoGen) . After a 24 h incubation at 37° C in a humidified incubator, the media of treated cells were harvested and centrifuged at 1,000 x g for 5 min. The level of IL-6 production was quantitated by ELISA (# DY206, R & D Systems) according to the protocol recommended by the manufacturer. CSF1R down-modulation assay

[0167] Mouse BMMs were cultured overnight in complete RPMI 1640 medium without CSF1. Cells were subsequently treated with NS1 or LPS for 1 h in a microcentrifuge tube, then stained for CSF1R, and analyzed by flow cytometry as described (Sester eta/., 1999, supra).

Analysis of mRNA by quantitative real-time reverse transcription PCR

[0168] Mouse BMMs (with CSF1) were treated with NS1 or LPS for indicated times for 3 hours before harvest for RNA extraction. Complementary DNA was prepared using random hexamer primers, and quantitative PCR was carried out using gene-specific primers for IL-6, TNF- a, IL-Ιβ and IFN-β, HPRT, and SYBR Green PCR MasterMix (Life Technologies). HPRTqene expression was used as the reference to normalize the expression of mouse cytokine transcripts.

EXAMPLE 4

DOSE RESPONSE OF IC14 FOR INHIBITION OF DENVl, DENV2 AND DENV3 NS1 RESPONSES

[0169] Human PBMCs were treated with IC14, or a human IgG4 control antibody at concentrations ranging from 0.001 to 10 pg/mL for 1 h. Then NS1 from DENVl, DENV2 or DENV3 was added and incubated overnight. The medium was diluted 1 : 5 with PBS and then 50 pL was assayed for secreted IL-6 using ELISA.

[0170] The results presented in Figure 5 demonstrate that IC14 inhibits IL-6 secretion by human myeloid cells in response to activation by NS1 from each of DENVl, DENV2 and DENV3.

EXAMPLE 5

IC14-MEDIATED INHIBITION OF CYTOKINE MRNA INDUCTION BY NSl

[0171] In order to check the inhibition of induction of a number of different cytokines by IC14, PBMCs from two donors were pretreated with IC14 or isotype control antibody, and then treated with NSl. Analogous treatment with LPS was performed for a single donor. RNA was extracted after 2 hours, and cDNA prepared for analysis of gene expression by real time PCR. The results presented in Figure 6 show that IL-Ιβ, IL-6, TNF-a, IFN-γ and IFN-β mRNA were all induced by NSl and inhibited by IC14, but not the control antibody.

[0172] These results demonstrate that IC14 inhibits expression of IL-Ιβ, IL-6, TNF-a, IFN-γ and IFN-β in human myeloid cells in response to activation by NSl .

EXAMPLE 6

EFFECT OF IC14 ON VIRUS INFECTION AND REPLICATION

[0173] Human monocyte-derived macrophages were prepared and differentiated for 7 days in the presence of macrophage growth factor, M-CSF. These cells were infected with DENV2 in the presence or absence of anti-DENV antibody that mediates antibody-dependent enhancement (ADE) of infection. Prior to infection, cells were pretreated with either IC14 or isotype control human antibody. Assessment of viral titer showed production of virus within the first day of infection, and a slight decline in titer on day 2 (Figure 7). Viral titer was boosted almost 10-fold by ADE by days 1 and 2, but there was no effect of IC14 relative to the isotype control antibody (IgG4).

[0174] The results presented in Figure 7 indicate that prior exposure of human myeloid cells to IC14 does not promote DENV infection and replication. Materials and Methods for Examples 4 to 6

IC14

[0175] IC14 was provided by Implicit Bioscience Ltd (IC14-3, Lot l-FIN-0779). Isotype control human IgG4 antibody was obtained from Biolegend (Clone ET904, #403402). E. coli ultrapure LPS was obtained from Invivogen.

Expression of Dengue virus NS1 protein

[0176] DNA encoding NS1 from serotypes 1, 2, and 3 of dengue virus (DENV1, DENV2, DENV3) were cloned into the pNBF Xpress plasmid (Acyte Biotech, USA). Plasmid DNA was purified using Qiagen maxiprep or midiprep kits. LPS was removed by four extractions with Triton X-114 and the DNA was further purified by phenol chloroform extraction followed by ethanol precipitation.

[0177] CHO-S cells were grown in conical flasks with shaking in a 37°C incubator supplied with 7% C0₂. CHO-S cells were transiently transfected with expression plasmids. Briefly, 30 xlO⁵ CHO-S cells were diluted into 30 mL of CD CHO medium (Life Technologies) supplemented with Glutamax-1 and 50 units/mL penicillin and 50 pg/mL of streptomycin (Life Technologies) and grown overnight. Prior to transfection, cells were first washed with PBS and resuspended in 30 ml of CD CHO medium with Glutamax-1 but without antibiotics. LPS-free plasmid DNA (60 pg) and 240 pL of PEI transfection reagent (lmg/ml) were added to 5 mL of Opti-MEM (Life Technologies) and incubated at room temperature for 10 min. The transfection complexes were added to the CHO cells and the cells incubated at 37°C, 7% C0₂ without shaking for 6 h. The cells were then washed three times with PBS and resuspended in CD CHO medium supplemented with Glutamax-1, penicillin, streptomycin and 5% CHO CD Effi ci entFeed™ A and B (Life Technologies). Cultures were grown at 37°C with shaking for 4 days and then the cells were pelleted and the medium containing recombinant NS1 was filtered through a 0.22 pm filter and frozen at -80°C. The concentration of DENV2 NS1 was determined by ELISA, but this could not be estimated for DENV1 and DENV3 NS1 due to lack of purified standard NS1 for these strains.

Cell culture conditions

[0178] Human PBMCs, and monocyte-derived macrophages were cultured in "complete RPMI 1640 medium" which is RPMI 1640 supplemented with 10% heat inactivated fetal calf serum (FCS), 50 Units/ml penicillin, 50 pg/ml streptomycin and IX Glutamax (Life Technologies). Aedes albopictus C6/36 cells were maintained in RPMI 1640 medium supplemented with 10% FCS, IX Glutamax and 20 mM HEPES. African green monkey kidney cells (Vero) were maintained in Optimem medium supplemented with 3% FCS. Media were from Life Technologies. Except where noted otherwise mammalian cells were grown at 37°C in a humidified incubator with 5% C0₂ and insect cell lines at 28°C.

Preparation of human PBMCs and monocyte-derived macrophages

[0179] Human PBMCs were obtained from blood buffy coat samples obtained from the Australian Red Cross Blood Service, under approval from the University of Queensland Medical Research Ethics Committee. PBMCs were isolated by Ficoll-Paque (GE Healthcare Life Sciences) density centrifugation. Monocytes were isolated from PBMC by negative selection using the human Monocyte Isolation kit II (Miltenyi Biotec) as per manufacturer's instructions. Monocytes were differentiated into macrophages as previously described (Wu eta/., 2013) with 10⁴ U/ml of M-CSF in bacterial petri dishes. Assessment of binding of IC14 to monocytes within PBMC population

[0180] Human PBMCs were resuspended in complete RPMI 1640 and diluted to a density of lxlO⁵ cells/mL. An aliquot of cells (140,000 cells) was placed in each per tube and mixed with 1 ml of ice cold PBS with 0.1% NaN₃. The cells were pelleted and resuspended in 100 μΙ_ of blocking buffer (PBS/0.1% NaN₃/2% FCS) with 5-20% human Fc receptor blocking agent (Miltenyi Biotec Australia Pty Ltd) for 30 min. A further 80 pL of blocking buffer (PBS/0.1%

NaN₃/2% FCS) was added followed by 20 pL of IC14 Ab, which had been diluted in blocking buffer to the following concentrations; 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01 pg/mL. Cells were incubated with the IC14 antibody for 1 h on ice and then 1 ml of cold wash buffer (PBS/0.1% NaN₃/0.2%FCS) was added. The cells were pelleted and resuspended in 100 pL of FITC-labeled anti-human IgG4 (Abeam) at a 1 : 500 dilution in blocking buffer with 5% human Fc receptor blocking agent. Cells were incubated with the secondary antibody for 1 h on ice and then 1 ml of cold wash buffer was added. Cells were pelleted and resuspended in 150 pL of wash buffer and run on a BD Accuri C6 flow cytometer.

Titration of IC14 for inhibition of NS1 and LPS activation of human PBMCs

[0181] Human PBMCs were resuspended in complete RPMI 1640 and diluted to a density of lxlO⁵ cells/mL. An aliquot (70 pL; 70,000 cells) was added to each well of a 96-well plate. IC14 or control IgG4 was diluted to the following concentrations; 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01 pg/mL in RPMI and 10 pL of diluted antibody was added to the cells. The cells were incubated for 1 h at 37°C and then either NS1 (20 pL of medium from NSl-expressing CHO cells) or LPS (20 pL of 50 ng/mL LPS solution in RPMI) was added to each well. After overnight incubation at 37°C, plates were centrifuged and the medium was removed and sequentially diluted 5-fold, first into PBS and then into PBS/BSA. An aliquot (50 pL) of the 25-fold diluted supernatant was used in an IL-6 ELISA assay using the human IL-6 DuoSet ELISA kit (In Vitro Technologies Pty Ltd).

IC14 inhibition of NSl-induced gene expression

[0182] Human PBMCs were resuspended in complete RPMI 1640 and 2 million cells were plated in 500 pL final volume after all additions. Cells were incubated with either no addition or IC14 or control IgG at a final concentration of 3 pg/mL for 1 h at 37°C. Then DENV2 NS1 (100 pL CHO cell expressed, giving final concentration of 1.6 pg/mL) or LPS (10 ng/mL) was added for 2 h. Total RNA was prepared with RNeasy RNA Mini Kit (Qiagen) and cDNA was prepared using reverse transcription of 1.7 pg of total RNA using oligo dT primers. Quantitative PCR was carried out in triplicate for each sample using gene-specific primer sets for IL-Ιβ, IL-6, TNF-a IFNy, ΙΕΝ , and SYBR Green PCR MasterMix (Life Technologies). PRTqene expression was used as the housekeeping gene. Analysis was done by Applied Biosystems ViiA 7 Real-time PCR system. Levels of gene expression relative to HPRT reference gene were quantified using the Delta-C_T method. Primer sequences are shown in Table 2.

TABLE 2

ΙΡΝ CAGTCCTG G AAG AAAAACTG GAGA [SEQ ID TTG G CCTTCAG GTAATG CAG AA [SEQ ID NO: 29] NO: 30]

IFNY G G AG ACCATCAAG G AAG ACATG A [SEQ ID TG G ACATTCAAGTCAGTTACCG AA [SEQ ID N0: 31] NO: 32]

IL-6 CTCAGCCCTGAGAAAGGAGACAT [SEQ ID 1 CAGCCA 1 I 1 1 GAAGG 1 1 CA LSbQ ID

NO: 33] NO: 34]

TNFa G CCTCTTCTCCTTCCTG ATCG [SEQ ID NO: 35] AG AG AG GTCCCTG G G G AACTC [SEQ ID

NO: 36]

HPRT TCAG G CAGTATAATCCAAAG ATG GT [SEQ ID AGTCTG G CTTATATCCAACACTTCG [SEQ ID NO: 37] NO: 38]

Preparation of virus stocks

[0183] DENV2 strain (Timor ET003) (Muller eta/., 2012, J Virol Methods 183(1) : 90-93) was propagated in C6/36 cells. C6/36 were infected at a multiplicity of infection (MOI) of 0.01. Cell supernatants were harvested after 7 days, centrifuged (500^ for 5 minutes) and transferred to ultracentrifuge tubes (344058, Beckman Coulter), then 2.5 ml 20% sucrose in ultrapure water was layered underneath. Virus was pelleted by ultracentrifugation on a SW32 Ti rotor (Beckman Coulter) at 28,000rpm for 2 hours at 4°C, and resuspended in RPMI 1640. Virus was aliquoted, stored at -80 °C, and one aliquot was used to determine the titer.

Quantifying virus titer using immunofluorescent plaque assay

[0184] The infectious titer of the virus was quantified using virus immunofluorescent plaque assay. Confluent Vero cells in 96-well plates were infected with 10-fold serial dilutions of virus for two hours at 37°C. Medium was then removed and replaced with M 199 medium supplemented with 2% FCS and 1.5% carboxymethyl cellulose. After 72 hours, the M 199 medium was removed and cells were fixed with 80% acetone in PBS for 20 minutes at -20°C. Acetone was removed completely by air drying and cells were blocked with 5% skim milk powder in PBS, 0.05% Tween20 (PBS-T) . Cells were then stained for 1 hour with 4E11, human anti-DENV E antibody (1 : 1000). Cells were washed thrice with PBS-T and stained for 1 hour with IRdye 800CW goat anti- human antibody. Viral plaques were visualized and quantified using LI-COR Biosciences Odyssey Infrared Imaging System, giving the virus titer measured in PFU/mL.

Infection of human monocyte-derived macrophages and effect of IC14

[0185] Monocyte-derived macrophages were harvested from the petri dish using ice-old PBS. Prior to the infection of cells, virus sufficient to give MOI 10 in the final incubation was pretreated, in a final volume of 10 μΙ_, with or without 2.5 pg/mL of anti-DENV 4G2 antibodies for 30 mins on ice, to allow the formation of virus-antibody complexes. Human monocyte-derived macrophages, at a concentration of 1 x 10⁵ cell/mL, were pre-treated with IC14 or IgG4 (1 pg) in final volume of 90 μΙ_ for 30 mins in polypropylene tubes. The cells subsequently were inoculated with the virus or virus-antibody complex mixture, and incubated for 90 minutes at 37°C. To ensure that the cells were in suspension, tubes were tapped every 15 minutes. Cells were washed once by addition of 900 μΙ_ of PBS and centrifuged at 600^ for 10 mins. Cells were resuspended in 200 μΙ_ of complete RPMI 1640 medium and were then transferred to a 96-well plate pre-coated with poly- HEMA. After 1 or 2 days, supernatants and cells were harvested for viral titer quantification by immunofluorescent plaque assay, and infected cell number by flow cytometry.

[0186] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0187] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.

[0188] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for modulating production of a pro-inflammatory mediator {e.g., a cytokine such as IL-6, TNF-a, IL-Ιβ and IFN-β) in a subject with a Flavivirus infection, the method comprising, consisting or consisting essentially of contacting CD14 {e.g., mCD14 and/or sCD14) in the subject with a pro-inflammatory mediator-modulating amount of a CD14 antagonist antibody.

2. A method for modulating vascular leakage in a subject with a Flavivirus infection, the method comprising, consisting or consisting essentially of contacting CD14 {e.g., mCD14 and/or sCD14) in the subject with a vascular leakage-modulating amount of a CD14 antagonist antibody.

3. A method for inhibiting sNSl-mediated disease symptoms {e.g., production of a pro-inflammatory mediator, vascular leakage, etc. associated with a Flavivirus infection, the method comprising, consisting or consisting essentially of contacting CD14 {e.g., mCD14 and/or sCD14) with a CD14 antagonist antibody suitably in an amount sufficient to inhibit binding of sNSl to CD14.

4. A method for modulating vascular leakage in a subject with a Flavivirus infection, the method comprising, consisting or consisting essentially of administering to the subject a vascular leakage-modulating amount of a CD14 antagonist antibody.

5. A method for treating or preventing a Flavivirus infection or a symptom thereof in a subject, the method comprising, consisting or consisting essentially of administering an effective amount of a CD14 antagonist antibody to the subject.

6. A method according to any one of claims 1 to 5, further comprising identifying that the subject has or is at risk of developing a Flavivirus infection, suitably prior to administration of the CD14 antagonist antibody.

7. A method according to claim 6, comprising determining the presence of NS1

{e.g., soluble or non-soluble forms of NS1) in the subject.

8. A method according to claim 7, comprising determining the presence of NS1 in a biological sample of the subject.

9. A method according to claim 8, wherein the biological sample is selected from blood, serum, plasma, saliva, cerebrospinal fluid, urine, skin or other tissues, or fractions thereof.

10. Use of a CD14 antagonist antibody for inhibiting the binding of sNSl to CD14 {e.g., mCD14 and/or sCD14) in a subject with a Flavivirus infection.

11. Use of a CD14 antagonist antibody for modulating production of a pro- inflammatory mediator {e.g., a cytokine such as IL-6, TNF-a, IL-Ιβ and IFN-β) that is associated with a Flavivirus infection.

12. Use of a CD14 antagonist antibody for modulating vascular leakage that is associated with a Flavivirus infection.

13. Use of a CD14 antagonist antibody for inhibiting the binding of sNSl to a cell that mediates disease symptoms associated with a Flavivirus infection.

14. Use of a CD14 antagonist antibody for treating or preventing a Flaviviridae virus infection.

15. A use according to any one of claims 10 to 14, wherein the CD14 antagonist antibody is manufactured as a medicament for the said application.

16. A method or use according to any one of claims 1 to 15, wherein the Flavivirus is a virus selected from the group consisting of Dengue virus (DENV), Japanese encephalitis virus (JEV) , Yellow fever virus (YFV), Murray Valley encephalitis virus (MVEV) , West Nile virus (WNV), Tick-borne encephalitis virus (TBEV), St Louis encephalitis virus (SLEV), Alfuy virus (AV), Kou tango virus (KV), Cacipacore virus (CM), Yaounde virus (YV) and Zika virus (ZV).

17. A method or use according to claim 16, wherein the Flavivirus is selected from Dengue virus serotype I, II, III, or IV.

18. A method or use according to any one of claims 1 to 17, wherein the CD14 antagonist antibody is selected from:

(1) an antibody comprising :

a VL domain that comprises, consists or consists essentially of the sequence:

QS PAS LAVS LG Q RATIS C RASESVDSFGNSFMH WYQQKAGQPPKSSIY RAANLES GIPARFSGSGSRTDFTLTINPVEADDVATYFC QQSYEDPWT FGGGTKLGNQ [SEQ ID NO: 1] (3C10 VL); and

a VH domain that comprises, consists or consists essentially of the sequence:

LVKPGGSLKLSCVASGFTFS SYAMS WVRQTPEKRLEWVA SISSGGTTYYPDNVKG RFTISRDNARNILYLQMSSLRSEDTAMYYCAR GYYDYHY WGQGTTLTVSS [SEQ ID NO: 2] (3C10 VH);

(2) an antibody comprising :

a VL domain that comprises, consists or consists essentially of the sequence:

QS PAS LAVS LG Q RATIS C RASESVDSYVNSFLH WYQQKPGQPPKLLIY RASNLQS GIPARFSGSGSRTDFTLTINPVEADDVATYCC QQSNEDPTT FGGGTKLEIK [SEQ ID NO: 3] (28C5 VL); and

a VH domain that comprises, consists or consists essentially of the sequence:

LQQSG PG LVKPSQSLS LTCTVTGYSIT SDSAWN WIRQFPGNRLEWMG YISYSGSTSYNPSLKS

RISITRDTSKNQFFLQLNSVTTEDTATYYCVR GLRFAY WGQGTLVTVSA [SEQ ID NO: 4] (28C5 VH); and

(3) an antibody comprising :

a VL domain that comprises, consists or consists essentially of the sequence:

QTPSSLSASLGDRVTISC RASQDIKNYLN WYQQPGGTVKVLIY YTSRLHS

GVPSRFSGSGSGTDYSLTISNLEQEDFATYFC QRGDTLPWT FGGGTKLEIK [SEQ ID NO: 5] (18E12 VL); and

a VH domain that comprises, consists or consists essentially of the sequence:

LESG PG LVAPSQS LSITCTVSG FS LT NYDIS WIRQPPGKGLEWLG VIWTSGGTN YN SAFM S RLSITKDNSESQVFLKMNGLQTDDTGIYYCVR GDGN FYLYNFDY WGQGTTLTVSS [SEQ ID NO: 6] (18E12 VH);

19. A method or use according to claim 18, wherein the CD14 antagonist antibody that comprises, consists or consists essentially of the VL and VH CDR sequences of the above antibodies defined in claim 18.

20. A method or use according to claim 19, wherein the CD14 antagonist antibody is selected from:

(1) an antibody that comprises: a) an antibody VL domain, or antigen binding fragment thereof, comprising L-CDR1, L-CDR2 and L-CDR3, wherein : L-CDR1 comprises the sequence RASESVDSFGNSFMH [SEQ ID NO: 7] (3C10 L-CDR1); L-CDR2 comprises the sequence RAANLES [SEQ ID NO: 8] (3C10 L-CDR2); and L-CDR3 comprises the sequence QQSYEDPWT [SEQ ID NO: 9] (3C10 L-CDR3); and b) an antibody VH domain, or antigen binding fragment thereof, comprising H-CDR1, H-CDR2 and H-CDR3, wherein : H-CDR1 comprises the sequence SYAMS [SEQ ID NO: 10] (3C10 H-CDR1); H-CDR2 comprises the sequence SISSGGTTYYPD N VKG [SEQ ID NO: 11] (3C10 H-CDR2); and H-CDR3 comprises the sequence GYYDYHY [SEQ ID NO: 12] (3C10 H-CDR3);

(2) an antibody that comprises: a) an antibody VL domain, or antigen binding fragment thereof, comprising L-CDR1, L-CDR2 and L-CDR3, wherein : L-CDR1 comprises the sequence RASESVDSYVNSFLH [SEQ ID NO: 13] (28C5 L-CDR1); L-CDR2 comprises the sequence RASNLQS [SEQ ID NO: 14] (28C5 L-CDR2); and L-CDR3 comprises the sequence QQSNEDPTT [SEQ ID NO: 15] (28C5 L-CDR3); and b) an antibody VH domain, or antigen binding fragment thereof, comprising H-CDR1, H-CDR2 and H-CDR3, wherein : H-CDR1 comprises the sequence SDSAWN [SEQ ID NO: 16] (28C5 H-CDR1); H-CDR2 comprises the sequence YISYSGSTSYNPSLKS [SEQ ID NO: 17] (28C5 H-CDR2); and H-CDR3 comprises the sequence GLRFAY [SEQ ID NO: 18] (28C5 H-CDR3); and

(3) an antibody that comprises: a) an antibody VL domain, or antigen binding fragment thereof, comprising L-CDR1, L-CDR2 and L-CDR3, wherein : L-CDR1 comprises the sequence RASQDIKNYLN [SEQ ID NO: 19] (18E12 L-CDR1); L-CDR2 comprises the sequence YTSRLHS [SEQ ID NO: 20] (18E12 L-CDR2); and L-CDR3 comprises the sequence

QRGDTLPWT [SEQ ID NO: 21] (18E12 L-CDR3); and b) an antibody VH domain, or antigen binding fragment thereof, comprising H-CDR1, H-CDR2 and H-CDR3, wherein : H-CDR1 comprises the sequence NYDIS [SEQ ID NO: 22] (18E12 H-CDR1); H-CDR2 comprises the sequence VIWTSGGTNYNSAFMS [SEQ ID NO: 23] (18E12 H-CDR2); and H-CDR3 comprises the sequence GDGNFYLYN FDY [SEQ ID NO: 24] (18E12 H-CDR3).

21. A method or use according to claim 20, wherein the CD14 antagonist antibody is humanized.

22. A method or use according to claim 21, wherein the CD14 antagonist antibody comprises a VL domain and a VH domain, wherein :

the VL domain comprises the amino acid sequence:

METDTILLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASESVDSYVNSFLHWYQQKPGQPPKL LIYRASNLQSGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPYTFGGGTKLEIKRTVAAPSVFIF PPSDEQLKSGTASWCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO: 25]; and

the VH domain comprises the amino acid sequence:

MKVLSLLYLLTAIPGILSDVQLQQSGPGLVKPSQSLSLTCTVTGYSITSDSAWNWIRQFPGNRLEWMGYI SYSGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCVRGLRFAYWGQGTLVTVSSASTKGPS VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT KTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVD KS RWQEG N VFSCSVM H EALH N HYTQKSLS LSLG K [SEQ ID NO: 26] .

23. A method or use according to claim 22, wherein the CD14 antagonist antibody is IC14, or an antigen-binding fragment thereof.

24. A method or use according to any one of claims 1 to 23, wherein the CD14 antagonist antibody is a direct antagonist of CD14.

25. A method or use according to any one of claims 1 to 24, wherein the CD14 antagonist antibody is a specific antagonist of CD14.

26. A method or use according to any one of claims 1 to 25, wherein the CD14 antagonist is administered in combination with one or more ancillary agents that treat or ameliorate the symptoms of a Flavivirus infection.

27. A pharmaceutical composition, suitably for treating a Flavivirus infection or symptom thereof, comprising, consisting or consisting essentially of a CD14 antagonist antibody and an ancillary ax\W- Flaviviridae virus agent, optionally together with a

pharmaceutically acceptable carrier or diluent.

28. A method for treating or preventing a Flavivirus infection or associated disease or symptom thereof in a subject, the method comprising, consisting or consisting essentially of administering concurrently to the subject an effective amount of a CD14 antagonist antibody and an effective amount of an ancillary anW- Flavivirus agent.

29. A method according to claim 28, wherein the CD14 antagonist antibody and the ancillary ant -Flavivirus agent are administered in synergistically effective amounts.

30. A method according to claim 28 or claim 29, wherein the ancillary ant -Flavivirus agent is selected from interferons, illustrative examples of which include interferon alpha {e.g., interferon alpha 2a and interferon alpha 2b) and interferon beta {e.g., interferon beta la and interferon beta lb), as well as anW- Flavivirus antibodies and small molecules, or nucleic acid constructs from which an ancillary anW- Flavivirus agent is expressible.