WO2009025743A2

WO2009025743A2 - Use of trail compositions as antiviral agents

Info

Publication number: WO2009025743A2
Application number: PCT/US2008/009666
Authority: WO
Inventors: Irene Bosch; Rajas V. Warke; Katherine J. Martin
Original assignee: University Of Massachusetts Medical School
Priority date: 2007-08-17
Filing date: 2008-08-13
Publication date: 2009-02-26
Also published as: WO2009025743A3

Abstract

Dengue fever is an important tropical illness for which there is currently no virus-specific treatment. Common gene expression changes, using Affymetrix GeneChips (HG-Ul 33A), were seen using several types of infected primary human cells (human umbilical vein endothelial cells (HUVECs), dendritic cells (DCs), monocytes and B cells). Tissue necrosis factor-related apoptosis inducing ligand (TRAIL), one of the common response genes, may provide additional immune modulator functions present in virus infected cells, and additional interactions between type-I and II IFN response genes and TRAIL that will increase the innate immunity to the virus or even to other pathogens like bacteria.. Dengue virus induces TRAIL expression in immune cells and HUVECs at the mRNA and protein level and was found to be dependent on an intact IFN type I signaling pathway. Anti-TRAIL antibody incubation with primary cells showed an increase in DV accumulation and conversely, a decrease in DV RNA was seen in presence of recombinant TRAIL (i.e., for example, human rTRAIL). These data suggest that TRAIL may play a role in the anti-viral response to DV infection and is a candidate for anti-viral interventions against DV. Further, TRAIL antiviral function does not promote apoptosis. The role of exogenous TRAIL in dendritic cells confirmed a strong anti-inflammatory response due to the lowering of production of mediators of inflammation present in dengue infection.

Description

Use Of TRAIL Compositions As Antiviral Agents

Statement Of Government Interest

This invention was made with government support under U19-AI057319, POl Al 34533, UOl AI070484, UOl AI45440, and Ul 9 AI057319 awarded by the National Institutes of Health. The government has certain rights in the invention.

Field Of The Invention

This invention relates to the field anti-viral compositions and methods. Compositions comprising tumor necrosis factor-related apoptotic ligand (TRAIL) have been observed to •control flavivirus infections (i.e., for example, dengue fever or West Nile virus). Methods of administering TRAIL compositions comprise mediating interferon anti-viral pathways. Further, TRAIL-mediated control of dengue fever virus infections has been shown to be apoptosis- independent and inhibits mediators of inflammation.

Background

Dengue virus (DV) has reemerged as a major global health problem in the tropics, particularly among children Gubler, D. J. 2001. "Human arbovirus infections worldwide" Ann N Y Acad Sd 951:13-24; and Mairuhu et al., 2004. "Dengue: an arthropod-borne disease of global importance" Eur J Clin Microbiol Infect Dis 23:425-33. This mosquito-borne flavivirus, for which there is no vaccine or anti-viral treatment, causes an estimated 50 million infections annually. 2003. "Joint WHO HQ/SEAROP/WPRO meeting on DengueNet implementation in South-East Asia and the Western Pacific, Kuala Lumpur, 11-13 December 2003" WkIy Epidemiol Rec 78:346-7; and Petersen et al., 2005. "Shifting epidemiology of Flaviviridae" J Travel Med 12 Suppl 1 :S3-11. Most dengue infections result in a self limited febrile illness (i.e., for example, dengue fever, DF). Less frequently, infections can cause dengue hemorrhagic fever (DHF), a potentially fatal plasma leakage syndrome.

Effective control of DV replication is possible after a short period of viremia in most individuals. It is unclear, however, what host factors induced by DV infection are involved in regulating the virus. For example, Type-I and type- II interferons have been suggested as possible mediators as increased serum levels are observed during DV infection. Kurane et al., 1993. "High levels of interferon alpha in the sera of children with dengue virus infection" Am J Trop Med Hyg 48:222-9; and Libraty et al., 2002 "Differing influences of virus burden and immune activation on disease severity in secondary dengue-3 virus infections" J Infect Dis 185:1213-21. On one hand, Type-I interferon pre-treatment of cells was shown to block DV- infection of cells by a protein kinase receptor (PKR) and 2-5 oligo adenylate synthase (2-5 OAS) independent mechanism. Diamond et al., 2001. "Interferon inhibits dengue virus infection by preventing translation of viral RNA through a PKR-independent mechanism" Virology 289:297- 311. On the other hand, it has also been shown that DV infection inhibits type-I interferon (EFN- I) signaling within infected cells. Munoz- Jordan et al., 2003. "Inhibition of interferon signaling by dengue virus" Proc Natl Acad Sci U S A 100: 14333-8.

Further, the in vivo tropism and cellular response to DV is only partially understood. Current research has identified that, in vivo; i) macrophages (Halstead, S. B. 1989. "Antibody, macrophages, dengue virus infection, shock, and hemorrhage: a pathogenetic cascade" Rev Infect Dis 11 Suppl 4:S830-9); ii) B-cells (King et al., 1999. "B cells are the principal circulating mononuclear cells infected by dengue virus" Southeast Asian J Trop Med Public Health 30:718- 28; and Lin et al., 2002. "Virus replication and cytokine production in dengue virus-infected human B lymphocytes" J Virol 76:12242-9; and iii) dendritic cells (27, 45) are known sites for in vivo DV replication. Similarly, in vitro dengue fever virus infection has been reported in primary endothelial cell cultures. Huang et al., 2000. "Dengue virus infects human endothelial cells and induces IL-6 and IL-8 production" Am J Trop Med Hyg 63:71-5; Jessie et al., 2004.

"Localization of dengue virus in naturally infected human tissues, by immunohistochemistry and in situ hybridization" J Infect Dis 189:1411-8; and Warke et al., 2003. "Dengue virus induces novel changes in gene expression of human umbilical vein endothelial cells" J Virol 77:11822- 32. For example, in human umbilical vein endothelium cells (HUVEC) and monocytes, DV infections resulted in an upregulation of different sets of genes. Warke et al., (supra); and Moreno-Altamirano et al., 2004. "Gene expression in human macrophages infected with dengue virus serotype-2" Scand J Immunol 60:631-8, respectively. However, in human patients using whole blood cells, gene expression profiles have shown attenuation of the IFN-induced gene response was found in dengue shock syndrome patients compared to dengue hemorrhagic fever patients. Simmons et al., 2007. "Patterns of host genome-wide gene transcript abundance in the peripheral blood of patients with acute dengue hemorrhagic fever" J Infect Dis 195:1097-107.

What is needed are compositions and methods to effectively control DV infection based upon gene regulation.

Summary Of The Invention

This invention relates to the field anti-viral compositions and methods. Compositions comprising tumor necrosis factor-related apoptotic ligand (TRAIL) have been observed to control flavivirus infections (i.e., for example, dengue fever or West Nile virus). Methods of administering TRAIL compositions comprise mediating interferon anti- viral pathways. Further, TRAIL-mediated control of dengue fever virus infections has been shown to be apoptosis- independent and inhibits mediators of inflammation.

In one embodiment, the present invention contemplates a method comprising: a) providing; i) a patient exhibiting at least one symptom of a virus infection, wherein said virus includes, but is not limited to, flaviviruses and bunyaviruses; and ii) a composition comprising a TRAIL protein or a fragment thereof; and b) administering the TRAIL protein under conditions such that the at least one symptom of said infection is reduced. In one embodiment, the flavivirus includes, but is not limited to, a dengue virus, a yellow fever virus, a West Nile virus, and/or an encephalitis virus. In one embodiment, the bunyavirus includes, but is not limited to, a Hantaan virus and/or a Sin Nombre virus. In one embodiment, the TRAIL protein is part of a fusion protein. In one embodiment, the administering comprises a topical administration. In one embodiment, the topical administration is selected from the group consisting of transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. In one embodiment, the administering comprises parenteral administration. In one embodiment, the parenteral administration is selected from the group consisting of intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, intrathecal or intraventricular, administration.

In one embodiment, the present invention contemplates a method comprising: a) providing; i) a patient exhibiting at least one symptom of a virus infection, wherein the virus includes, but is not limited to, flaviviruses and/or bunyaviruses; and ii) a composition comprising a nucleic acid, wherein said nucleic acid encodes a TRAIL protein or a fragment thereof; and b) administering the nucleic acid under conditions such that the at least one symptom of the infection is reduced. In one embodiment, the flavivirus includes, but is not limited to, a dengue virus, a yellow fever virus, a West Nile virus, and/or an encephalitis virus. In one embodiment, the bunyavirus includes, but is not limited to, a Hantaan virus and/or a Sin Nombre virus. In one embodiment, the TRAIL nucleic acid comprises mRNA. hi one embodiment, the nucleic acid is encapsulated in a liposome, hi one embodiment, the administering comprises a topical administration. In one embodiment, the topical administration is selected from the group consisting of transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. In one embodiment, the administering comprises parenteral administration. In one embodiment, the parenteral administration is selected from the group consisting of intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, intrathecal or intraventricular, administration. hi some embodiments, the present invention contemplates a method comprising: a) providing; i) a subject exibiting at least one symptom of an inflammation in a subject; and ii) a composition comprising a TRAIL protein or a fragment thereof; and b) administering the protein under conditions such that the at least one symptom of the inflammation is reduced. In one embodiment, the inflammation is derived from a virus infection. In one embodiment, the inflammation is derived from a disease. In one embodiment, the inflammation is derived from a wound. In one embodiment, the inflammation is derived from surgery. In one embodiment, the virus comprises a flavivirus. In one embodiment, the virus comprises a bunyavirus. In one embodiment, the flavivirus includes, but is not limited to, a dengue virus, a yellow fever virus, a West Nile virus, and/or an encephalitis virus. In one embodiment, the bunyavirus includes, but is not limited to, a Hantaan virus and/or a Sin Nombre virus. In one embodiment, the protein is encapsulated in a liposome. In one embodiment, the administering comprises a topical administration. In one embodiment, the topical administration is selected from the group consisting of transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. In one embodiment, the administering comprises parenteral administration. In one embodiment, the parenteral administration is selected from the group consisting of intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, intrathecal or intraventricular, administration. In some embodiments, the present invention contemplates a method comprising: a) providing; i) a subject exibiting at least one symptom of an inflammation in a subject; and ii) a composition comprising a nucleic acid, wherein said nucleic acid encodes a TRAIL protein or a fragment thereof; and b) administering the nucleic acid under conditions such that the at least one symptom of the inflammation is reduced. In one embodiment, the inflammation is derived from a virus infection. In one embodiment, the inflammation is derived from a disease. In one embodiment, the inflammation is derived from a wound. In one embodiment, the inflammation is derived from surgery. In one embodiment, the virus comprises a flavivirus. In one embodiment, the virus comprises a bunyavirus. In one embodiment, the flavivirus includes, but is not limited to, a dengue virus, a yellow fever virus, a West Nile virus, and/or an encephalitis virus. In one embodiment, the bunyavirus includes, but is not limited to, a Hantaan virus and/or a Sin Nombre virus. In one embodiment, the TRAIL nucleic acid comprises mRNA. In one embodiment, the nucleic acid is encapsulated in a liposome. In one embodiment, the administering comprises a topical administration. In one embodiment, the topical administration is selected from the group consisting of transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. In one embodiment, the administering comprises parenteral administration. In one embodiment, the parenteral administration is selected from the group consisting of intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, intrathecal or intraventricular, administration. In some embodiments, the present invention provides a method for characterizing virus infected tissue in a subject, comprising: providing a virus infected tissue sample from a subject; and detecting the presence or absence of expression of a TRAIL composition in the sample, thereby characterizing the virus infected tissue sample. In one embodiment, the virus includes, but is not limited to, flaviviruses and bunyaviruses. In one embodiment, the flavivirus includes, but is not limited to, a dengue virus, a yellow fever virus, a West Nile virus, and/or an encephalitis virus. In one embodiment, the bunyavirus includes, but is not limited to, a Hantaan virus and/or a Sin Nombre virus. In some embodiments, detecting the presence of expression of a TRAIL composition comprises detecting the presence of TRAIL mRNA. In other embodiments, detecting the presence of expression of TRAIL mRNA comprises exposing the TRAIL mRNA to a nucleic acid probe complementary to the TRAIL mRNA. In yet other embodiments, detecting the presence of expression of TRAIL comprises detecting the presence of a TRAIL polypeptide. In some embodiments, detecting the presence of a TRAIL polypeptide comprises exposing the TRAIL polypeptide to an antibody specific to the TRAIL polypeptide and detecting the binding of the antibody to the TRAIL polypeptide. In some embodiments, the subject comprises a human subject. In some embodiments, the sample comprises a blood sample. In some embodiments, the blood sample is a serum sample. In some embodiments the blood sample is a plasma sample. In some embodiments, the blood sample comprises monocytes.

In some embodiments, the present invention provides a kit for characterizing a virus infection in a subject, comprising: a reagent capable of specifically detecting the presence of absence of expression of a TRAIL composition; and instructions for using the kit for characterizing virus infection in the subject. In one embodiment, the virus includes, but is not limited to, flaviviruses and bunyaviruses. In one embodiment, the flavivirus includes, but is not limited to, a dengue virus, a yellow fever virus, a West Nile virus, and/or an encephalitis virus. In one embodiment, the bunyavirus includes, but is not limited to, a Hantaan virus and/or a Sin Nombre virus. In some embodiments, the reagent comprises a nucleic acid probe complementary to a TRAIL mRNA. In other embodiments, the reagent comprises an antibody that specifically binds to a TRAIL polypeptide. In some embodiments, the instructions comprise instructions in compliance with the United States Food and Drug Administration recommendations for use in in vitro diagnostic products. The present invention also provides a method of screening compounds, comprising providing a virus infected sample; and one or more test compounds; and contacting the virus infected sample with the test compound; and detecting a change in TRAIL composition expression in the virus infected sample in the presence of the test compound relative to the absence of the test compound. In one embodiment, the virus includes, but is not limited to, flaviviruses and bunyaviruses. In one embodiment, the flavivirus includes, but is not limited to, a dengue virus, a yellow fever virus, a West Nile virus, and/or an encephalitis virus. In one embodiment, the bunyavirus includes, but is not limited to, a Hantaan virus and/or a Sin Nombre virus. In some embodiments, the detecting comprises detecting TRAIL mRNA. In other embodiments, the detecting comprises detecting TRAIL polypeptide. In some embodiments, the sample comprises an in vitro cell. In other embodiments, the sample comprises an in vivo cell. In some embodiments, the test compound comprises a peptide. In other embodiments, the test compound comprises a drug.

The present invention provides a virus expression profile map comprising gene expression level information for at least one marker selected from the group consisting of: G1P2, IRF7, ISG20, OAS3, OASL, RSAD2, TRIM5, HSXIAPAFl, TRAIL, CD38, HERC5, IFI44, IFI44L, IFITMl, LGALS3BP, USP18, FLJ20035, FLJ38348, HERC6, IFITl, IFIT3, LY6E, and SAMD9. In one embodiment, the virus includes, but is not limited to, flaviviruses and bunyaviruses. In one embodiment, the fiavivirus includes, but is not limited to, a dengue virus, a yellow fever virus, a West Nile virus, and/or an encephalitis virus. In one embodiment, the bunyavirus includes, but is not limited to, a Hantaan virus and/or a Sin Nombre virus. In some embodiments, the map is digital information stored in computer memory. In some embodiments, the map comprises information for two or more markers. In some embodiments, the map comprises information for three or more markers. In other embodiments, the map comprises information for five or more markers. In still further embodiments, the map comprises information for ten or more markers.

Definitions

The term "fusion protein" as used herein refers to a protein formed by expression of a hybrid gene made by combining two gene sequences. Typically this is accomplished by cloning a cDNA into an expression vector in frame with an existing gene. The fusion partner may act as a reporter (e.g., β-gal) or may provide a tool for isolation purposes (e.g., GST).

The term "gene" refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide or precursor or RNA (e.g., tRNA, siRNA, rRNA, etc.). The polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, etc.) of the full-length or fragment are retained. The term also encompasses the coding region of a structural gene and the sequences located adjacent to the coding region on both the 5' and 3' ends, such that the gene corresponds to the length of the full-length mRNA. The sequences that are located 5' of the coding region and which are present on the mRNA are referred to as 5' untranslated sequences. The sequences that are located 3' or downstream of the coding region and that are present on the mRNA are referred to as 3' untranslated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region, which may be interrupted with non-coding sequences termed "introns" or "intervening regions" or "intervening sequences." Introns are removed or "spliced out" from the nuclear or primary transcript, and are therefore absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

As used herein, the term "purified" refers to molecules (polynucleotides or polypeptides) that are removed from their natural environment, isolated or separated. "Substantially purified" molecules are at least 50% free, preferably at least 75% free, and more preferably at least 90% free from other components with which they are naturally associated.

The term "recombinant DNA" refers to a DNA molecule that is comprised of segments of DNA joined together by means of molecular biology techniques. Similarly, the term "recombinant protein" refers to a protein molecule that is expressed from recombinant DNA. As used herein, the term "coding region" refers to the nucleotide sequences that encode the amino acid sequences found in the nascent polypeptide as a result of translation of an mRNA molecule. The coding region is bounded in eukaryotes, on the 5' side by the nucleotide triplet "ATG" that encodes the initiator methionine and on the 3' side by one of the three triplets which specify stop codons (i.e., TAA, TAG, and TGA).

Where amino acid sequence is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms, such as

"polypeptide" or "protein," are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule. The term "wild-type" refers to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild type gene is that which is most frequently observed in a population and is thus arbitrarily designed the "normal" or "wild-type" form of the gene.

In contrast, the terms "modified," "mutant," and "variant" refer to a gene or gene product that displays modifications in sequence and or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product. The term "fragment" when used in reference to a nucleotide sequence refers to that sequence, which ranges in size from 10 nucleotides to the entire nucleotide sequence minus one nucleotide. When used in reference to an amino acid sequence the term "fragment" refers to that sequence, which ranges in size from 3 amino acids to the entire amino acid sequence minus one amino acid.

The terms "patient" and "subject" refer to a mammal or an animal who is a candidate for receiving medical treatment.

As used herein, the term "effective amount" refers to the amount of a compound (e.g., a TRAIL composition) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not limited intended to be limited to a particular formulation or administration route.

As used herein, the term "pharmaceutical composition" refers to the combination of an active agent (i.e., for example, a TRAIL composition) with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo, in vivo or ex vivo. The term, "TRAIL composition" as used herein, refers to any composition having a molecule derived from a TRAIL gene. Such a composition may include, but not be limited to, a nucleic acid sequence (i.e., for example, TRAIL mRNA) or an amino acid sequence (i.e., for example, TRAIL peptide, polypeptide, and/or protein and fragments thereof).

As used herein, the term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil/water or water/oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants. (See e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa. (1975)). As used herein, the term "pharmaceutically acceptable salt" refers to any pharmaceutically acceptable salt (e.g., acid or base) of a compound of the present invention which, upon administration to a subject, is capable of providing a compound of this invention or an active metabolite or residue thereof. "Salts" of the compounds of the present invention may be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic acid, and the like. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts. Examples of bases include, but are not limited to, alkali metals (e.g., sodium) hydroxides, alkaline earth metals (e.g., magnesium), hydroxides, ammonia, and compounds of formula NW₄ ⁺, wherein W is C^ alkyl, and the like.

Examples of salts include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include anions of the compounds of the present invention compounded with a suitable cation such as Na⁺, NH₄ ⁺, and NW₄₊ (wherein W is a C_1-4 alkyl group), and the like. For therapeutic use, salts of the compounds of the present invention are contemplated as being pharmaceutically acceptable. However, salts of acids and bases that are non- pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.

As used herein, the terms "solid phase supports" or "solid supports," are used in their broadest sense to refer to a number of supports that are available and known to those of ordinary skill in the art. Solid phase supports include, but are not limited to, silica gels, resins, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels, and the like. As used herein, "solid supports" also include synthetic antigen-presenting matrices, cells, liposomes, and the like. A suitable solid phase support may be selected on the basis of desired end use and suitability for various protocols. For example, for peptide synthesis, solid phase supports may refer to resins such as polystyrene (e.g., PAM-resin obtained from Bachem, Inc., Peninsula Laboratories, etc.), POLYHIPE) resin (obtained from Aminotech, Canada), polyamide resin (obtained from Peninsula Laboratories), polystyrene resin grafted with polyethylene glycol (TENTAGEL, Rapp Polymere, Tubingen, Germany) or polydimethylacrylamide resin (obtained from Milligen/Biosearch, California).

As used herein, the term "virus" refers to minute infectious agents, which with certain exceptions, are not observable by light microscopy, lack independent metabolism, and are able to replicate only within a living host cell. The individual particles (i.e., virions) typically consist of nucleic acid and a protein shell or coat; some virions also have a lipid containing membrane. The term "virus" encompasses all types of viruses, including animal, plant, phage, and other viruses. In particular, a virus may refer to a flavivirus (i.e., for example, a dengue fever virus).

The term "symptoms" as used herein, refers to any subjective evidence of a disease or physical disturbance observed by a patient.

The term "infection" as used herein, refers to any invasion and/or multiplication of microorganisms in biological cell or body tissue. For example, an infection may be a virus infection, wherein the virus invades a biological cell or body tissue and undergoes replication using the cell's biochemical sources. Such a virus infection may also result in inflammation. The term "inflammation" as used herein, refers to any local or systemic response to cellular injury that is marked by symptoms of capillary dilatation, leukocytic infiltration, redness, heat, pain, swelling, and often loss of function and that serves as a mechanism initiating the elimination of noxious agents and of damaged tissue. Symptoms of inflammation may also be identified by elevated cytokine levels including, but not limited to, IL6, MCP2, TNFa, IPlO, MIP-Ib, IL8, mda5, or IRF7. Reduction of these cytokine levels would normally be expected to have an antiinflammatory effect (i.e., for example, by the adminsitration of TRAIL compositions). Inflammation may result from an infection (i.e., for example, microorganisms including but not limited to, viruses, bacteria, fungi etc) or from a disease (i.e., for example, cardiovascular disease, pulmonary disease, renal disease, kidney disease etc), or from non- biological sources (i.e., for example, wounds including but not limited to, lacerations, cuts, scrapes, abrasions, punctures or surgical procedures).

The term "administering" or "administer" as used herein, refers to providing a patient with a composition intended for therapeutic benefit. Such an administration may be parenteral or non-parenteral, acute, chronic, or under conditions such that a controlled release of a therapeutic composition takes place. The term "topical" as used herein, refers to an administration to, or action on, any surface of a part of the body.

The term "parenteral" as used herein, refers to any administration of a therapeutic composition to a part of the body that does not involve the gastrointestinal system. The term "map" as used herein, refers to any data complication reflecting the relative gene expression profiles of at least one gene marker. A map may comprises many gene markers such a 1 - 1,000 markers, preferably 100 - 500 markers, more preferably 200 - 300 markers.

The term "marker" as used herein, refers to any quantifiable biological measurement that determines gene expression. For example, a marker may be mRNA resulting from transcription of a gene. Alternatively, a marker may be a protein resulting from translation of an mRNA.

Brief Description Of The Figures

Figure 1 presents exemplary data demonstrating gene expression profiles in HUVECs for three flavivirus species and two bunyavirus species. D: dengue, W: West Nile, HN: Hantaan, SN: Sin Nombre, and YF: Yellow Fever. Poly IC is shown as non-virus control. EB: Epstein- Barr; and VC: vaccinia, both non-RNA viruses, are added for comparison.

Figure 2 presents an exemplary gene expression analysis using Affymetrix GeneChips HG-Ul 33 A. The data presents expression levels of twenty-three (23) dengue virus response genes in cells exposed for 48 hours; HUVEC cells (n=2), Monocyte cells (n=2), and B-cells (n=l). In vitro infection gene expression profiles were normalized to C6/36 insect cell supernatant treated samples for each cell type. A hierarchical cluster analysis using a Pearson correlation is represented by increased color density as the fold induction increases; Maximum Color Density: ~ 20-fold up; White Color: No change. Affymetrix microarray analysis was performed using GeneSpring software (Agilent) to identified a common response gene set (ANOVA) with p<0.05.

Figure 3 presents exemplary data showing TRAIL mRNA and protein induction by dengue virus.

Figure 3A: TRAIL mRNA levels were measured by qRT-PCR. Monocytes were infected with DV, at MOI of 0.1 and 1 PFU/ml, dendritic cells were infected with DV at MOI of 0.1 PFU/ml and B cells were infected with DV at MOI of 1 PFU/ml, for 48 hours. TRAIL mRNA expression was quantified by qRT-PCR analysis on total RNA extracts, β-actin mRNA, a constitutively expressed protein, was used as a control probe. Data shown are representative of several different number of experiments. The mean and standard deviation (SD) was calculated by Excel software. Histograms represent means and SD.

Figure 3B: TRAIL protein levels were measured in cell lysates by ELISA. Monocytes were infected with DV at MOI of 1 PFU/cell and then cultured for 48 hours. Levels of

TRAIL protein were quantified in cell lysates using a TRAIL ELISA (R&D Systems).

Data shown are the average of three experiments and their mean and SD.

Figure 2C: Intracellular TRAIL protein levels were determined in dendritic cells infected with DV for 12, 24 and 48 hours at M.O.I, of 0.1. Cells were treated with Brefeldin A for 8 hours. Levels of TRAIL protein were quantified by flow cytometry using TRAIL-PE

(BD Biosciences). Data shown is one representation of two experiments.

Figure 4 presents exemplary data showing TRAIL regulation of dengue virus levels in primary human monocytes. Beta-actin mRNA was used as a control. DV-infected Monocytes (N=5), control mouse antibody (N=3). For B cells and HUVECs two independent experiments were done. The mRNA of dengue was normalized by beta actin mRNA and fold inductions calculated with the mean of triplicate reactions.

Figure 5 presents exemplary data following FACS analysis to test TRAIL inhibition of dengue virus infection in dendritic cells (DC).

Figure 5A: Recombinant TRAIL ( rTRAIL) treatment inhibits DV infection in DV- infected DCs. DC were pre-treated with rTRAIL for 24 hours followed by infection with 0.1 M.O.I, of DV. DV infection was stopped at the 48 hour timepoint and cells were stained intracellularly for DV antigen using 2H2 anti-complex dengue antibody (Upstate New York, NY). Data presented from one experiment is a representation of seven experiments.

Figure 5B: Summary of results from FACS analysis in 4A. Mean ± SD for both, the rTRAIL (N=7) and IFN-gamma (N=6) treatments are shown. Data is presented as percent inhibition of DV antigen staining using dengue monoclonal 2H2 antibody coupled to FITC (Upstate New York) following rTRAIL or IFN-gamma treatment.

Figure 6 presents exemplary data from plaque assays showing rTRAIL reduction of DV in infected dendritic cells supernatants. DCs were pre-treated with rTRAIL for 24 hours and infected with DV for 48hr at M.O.I, of 0.1. LLCMK2 cell monolayers were exposed to the cell culture supernatants to determine the DV titers (PFU/ml) in triplicate wells and five serial dilutions (data not shown). Results for four independent experiments are shown for each line with viral titers in the absence and presence of rTRAIL. The log transform of the data set gave a p=0.034 (T-test) for 6 degrees of freedom between the two groups. The mean±SD were 3.05±1.00 and 2.54±0.74 for log virus increase in the untreated groups and rTRAIL treated groups respectively.

Figure 7 presents exemplary data showing that TRAIL induction in response to DV- infection is IFN-α dependent. 2fTGH (Wild type fibroblast human cell line) and UlA, U3A, U4A and U5 A (IFN-α and signaling mutants human fibroblasts) were infected with DV at MOI of 1 PFU/cell and then cultured for 48 hours. TRAIL mRNA levels were quantified by qRT-PCR analysis. B-actin mRNA was used as a control probe. Results shown are one out of two independent experiments. Means of triplicate of each point of the qRT-PCR are used to obtain the fold inductions reported. SD is less than 1% within triplicate results.

Figure 8 presents exemplary data showing that antibodies for TRAIL interfere with IFN- α antiviral effect. Monocytes were infected with DV at MOI of 0.1 for 48 hours. Monocytes were pre-treated with IFN-α (5000U/ml) for 6 hours or a combination of IFN-α (5000LVmI) (6 hours) and anti-TRAIL antibody (50 ng/μl) 24 hours prior to infection with DV. Isotype mouse antibodies were used a negative control. Dengue virus copy number was quantified by qRT- PCR. Beta actin mRNA was used to normalize dengue virus mRNA in duplicate independent experiments. A mouse isotype control IgGl antibody was used at 50 ng/μl. The values of the two experiments are shown in white and black bars.

Figure 9 presents exemplary data demonstrating cell death quantification in rTRAIL treated DV-infected cells. Monocytes were pretreated with rTRAIL followed by infection with DV for 48 hr at an MOI of 0.1. Live/Dead Aqua fluorescence was used to identify apoptotic dendritic cells at various times after rTRAIL treatment and DV infected. Campothecin B (2mM) treated THP-I cells were used as positive controls for Live/Dead Aqua fluorescence.

Figure 9A: 12 hours after infection and treatment (N = 2).

Figure 9B: 24 hours after infection and treatment (N = 2).

Figure 9C: 48 hours after infection and treatment (N = 5).

Figure 1OA presents one embodiment of a TRAIL amino acid sequence (NM_003810)(SEQ ID NO: 10). Figure 1OB presents one embodiment of a TRAIL nucleic acid sequence (mRNA). (NM_003810)(SEQ ID NO: 11).

Figure 11 illustrates dengue fever virus-induced gene expression in apoptosis-related proteins (see boxes) showing data from four cell types as shaded bands (see insert). A stronger intensity shade indicates a higher level of expression.

Figure 12 presents one embodiment of a human TRAIL nucleic acid full length sequence (SEQ ID NO: 1).

Figure 13 present one embodiment of a human TRAIL amino acid full length sequence (SEQ ID NO:2). Figure 14 presents one embodiment of a human TRAIL nucleic acid fragment sequence

(SEQ ID NO: 3).

Figure 15 presents one embodiment of a human TRAIL amino acid fragment sequence (SEQ ID NO:4).

Figure 16 presents one embodiment of a murine TRAIL nucleic acid full length sequence (SEQ ID NO:5).

Figure 17 presents one embodiment of a murine TRAIL amino acid full length sequence (SEQ ID NO:6).

Figure 18 presents one embodiment of an expression vector comprising:

Figure 18A: A FLAG peptide (SEQ ID NO:7); Figure 18B: A TRAIL fragment (SEQ ID NO:8); and

Figure 18C: A CMV leader sequence (SEQ ID NO:9)

Figure 19 presents exemplary data showing: MCP-2, IP-10 and IL-6 protein levels in DC culture supernatants. White Bars: Supernatants from uninfected DCs. Black Bars: Supernatants from DV-infected DCs (N=3) at 12, 24 and 48 hours post-infection. Mean values ± standard error of mean are shown. Mann- Whitney statistical analysis between uninfected and DV- infected: MCP-2 (48 hours, p=0.001); IP-10 (48 hours, p-0.022).

Figure 20 presents exemplary data showing serum levels of MCP-2 (A), IP-10 (B), MIP- 1" (C) and TRAIL (D) in DV-infected patients (black bars) and OFI (white bars). Results are expressed as a mean value ± standard error of mean, for each patient group and each disease day. Mean levels for healthy donors: 33.7 ± 8.8 (N=8) pg/ml for MCP-2; 0.25 ± 0.08 (N=5) ng/ml for IP-10; 105.2 ± 10.4 (N=I 1) pg/ml for MIP-I "; 33.7 ± 8.8 (N=8) pg/ml for TRAIL. Mann- Whitney statistical analysis between OFI and dengue at each disease day: MCP-2 (febrile p=0.015; post-febrile p=0.001); IP- 10 (febrile pO.OOl; post-febrile pO.OOl); MlP-lβ (febrile p=0.024); TRAIL (febrile p=0.009). Conv.: convalescence.

Figure 21 presents exemplary data showing the effect of rTRAIL pre-treatment on chemokines and cytokines levels in culture supernatants from DV-infected DCs.

Figure 21 A: MCP-2, IP-IO, and IL-6 levels in culture supernatants from untreated (white bars) or rTRAIL pre-treated (black bars) DV-infected DC at 48 hours post-infection (N=3-5). Mean values ± standard error of mean are shown. Mann- Whitney analysis showed significantdifferences for MCP-2 (p=0.050); IP-IO (p=0.021) when comparing untreated DENV- infected DC versus rTRAIL pre-treated DV-infected DC.

Figure 2 IB: Levels of IFN-! in supernatants from DV-infected (white bars) and DV- infected + rTRAIL (black bars) at 48 hours, determined by commercial ELISA (N=7). Mean values ± standard error of mean are shown. Mann- Whitney analysis showed significant differences (p=0.025) when comparing untreated versus rTRAIL pretreated DENV-infected DC. Figure 21C: Percentage of infection in DV-infected (white bars) and DV-infected + rTRAIL (black bars) DC, determined by flow cytometry (N=7). Mean values ± standard error of mean are shown. Paired t-test analysis showed significant differences (p=0.034).

Figure 22 presents exemplary data showing the in vitro effect of rTRAIL on DV levels and various proinflammatory cytokines induced by dengue virus infection of dendritic cells (MOI=O-I). X Axis: Proinflammatory cytokines: IL6; MCP2, TNFα, IPlO, MlP-lβ, IL8, mda5, IRF7; and dengue virus (i.e., DENV or DV). Y Axis: Percent Inhibition relative to the absence of rTRAIL.

Figure 23 presents exemplary data showing gene expression analysis of the effect of rTRAIL and/or DV infection on in vitro expression of various TNF receptors in human cells. Figure 24 presents exemplary data showing gene expression analysis of the effect of rTRAIL and/or DV infection on in vitro expression of IL- 15 and IL- 15 receptors in human cells.

Figure 25 presents illustrative data showing gene expression analysis of TRAIL exposure on thrombospondin 1 related genes.

Figure 26 presents an representative list of the 30 highest selectively expressed genes in HUVEC, DC, and LSEC cells. Figure 27 presents an illustrative gene pathway analysis for genes having an at least 2- fold induction by TRAIL in DCs.

Figure 28 presents an illustrative gene pathway analysis for genes having an at least 2- fold induction by TRAIL in HUVECs. Figure 29 presents exemplary data showing the common selective expression of IL- 15 and OAS-I in both HUVECs DCs, and LESCs following exogenous TRAIL exposure.

Figure 30 presents an illustrative Venn diagram showing the relative distributions of selectively expressed genes between HUVECs and DCs in response to exogenous TRAIL.

Figure 31 presents exemplary data comparing TRAIL-induced gene expression between DCs and HUVECs showing selectivity for wounding and apoptotic genes.

Figure 32 presents a specific gene categorization of the selective gene expression profile in Figure 27.

Detailed Description Of The Invention This invention relates to the field anti-viral compositions and methods. Compositions comprising tumor necrosis factor-related apoptotic ligand (TRAIL) have been observed to control flavivirus infections (i.e., for example, dengue fever or West Nile virus). Methods of administering TRAIL compositions comprise mediating interferon anti-viral pathways. Further, TRAIL-mediated control of dengue fever virus infections has been shown to be apoptosis- independent and inhibits mediators of inflammation.

Many viruses are relatively inncocous, such as the common cold. However, serious diseases may result from some virus infections. For example, twelve distinct viruses associated with hemorrhagic fever in humans are classified among four families: Arenaviridae, which includes Lassa, Junin, and Machupo viruses; Bunyaviridae, which includes Rift Valley fever, Crimean-Congo hemorrhagic fever, and Hantaan viruses; Filoviridae, which includes Marburg and Ebola viruses; and Flaviviridae, which includes yellow fever, dengue, Kyasanur Forest disease, and Omsk viruses. Most hemorrhagic fever viruses are zoonoses, with the possible exception of the four dengue viruses, which may continually circulate among humans. Hemorrhagic fever viruses are found in both temperate and tropical habitats and generally infect both sexes and all ages, although the age and sex of those infected are frequently influenced by the possibility of occupational exposure. Transmission to humans is frequently by bite of an infected tick or mosquito or via aerosol from infected rodent hosts. Aerosol and nosocomial transmission are especially important with Lassa, Junin, Machupo, Crimean-Congo hemorrhagic fever, Marburg, and Ebola viruses. Seasonality of hemorrhagic fever among humans is influenced for the most part by the dynamics of infected arthropod or vertebrate hosts. Mammals, especially rodents, appear to be important natural hosts for many hemorrhagic fever viruses. The transmission cycle for each hemorrhagic fever virus is distinct and is dependent upon the characteristics of the primary vector species and the possibility for its contact with humans. LeDuc JW, "Epidemiology of hemorrhagic fever viruses" Rev Infect Dis. 1 1 :S730-S735 (1989). The present invention contemplates that two of these families, Flavivirus and Bunyavirus, may be susceptible to treatment by a composition comprising TRIAL.

In one embodiment, the present invention contemplates a method identifying a common response profile of twenty-three (23) genes induced by a virus infection (i.e., for example a Flavivirus and/or Bunyavirus infection). In one embodiment, the common response profile is identified in primary human cells. In one embodiment, the primary human cells include but are not limited to, human umbilical vein endothelial cells (HUVECs), monocyte cells, dendritic cells (DCs), and B cells. Although it is not necessary to understand the mechanism of an invention, it is believed that a signaling pathway analysis identified Tumor Necrosis Factor-Related Tumor Necrosis Ligand (TRAIL) as a potential common linker between the IFNα and IFNγ pathways. It has been commonly believed that TRAIL (a member of the TNF family) may promote apoptosis in virus induced cells by binding to and activating the death receptors DR4 and DR5. thereby resulting in recruitment of adaptor protein Fas associated death domain (FADD). Huang et al., December 15, 2006. "TRAIL death receptors and virus induced therapeutics" Toxicol Appl Pharmacol. E-Publication. It has been further believed that FADD recruits procaspase-8 into the death receptor complex, thereby causing autoproteo lytic cleavage of procaspase-8, which in turn leads to activation of the apoptosis signaling pathway. Thorburn et al., 2003. "Caspase- and serine protease-dependent apoptosis by the death domain of FADD in normal epithelial cells" MoI Biol Cell 14:67-77. In contrast, TRAIL has been also shown to negatively regulate innate immune response independent of apoptosis. Dieh et al., 2004. "TRAIL-R as a negative regulator of innate immune cell responses" Immunity 21 : 877-89. The data presented herein provides a basis for an apoptosis-independent effect of TRAIL consequent to a virus infection. Specifically, TRAIL is observed to increase gene expression of: i) TRAIL-R; ii) FADD; iii) CASP8; and iv) CASP7. All these proteins are sequential players in the traditional apoptosis pathway. However, a dengue fever virus infection has not been observed to increase the gene expression of DFFA, a necessary link between CASP7 and apoptosis. See, Figure 11. Previous studies have also indicated that TRAIL can function as an anti-viral and anti-tumor protein. Kemp et al., 2003. "Plasmacytoid dendritic cell-derived IFN-alpha induces TNF-related apoptosis-inducing ligand/Apo-2L-mediated antitumor activity by human monocytes following CpG oligodeoxynucleotide stimulation" J Immunol 171:212-8; Ma et al., 2005. "Recombinant adeno-associated virus-mediated TRAIL gene therapy suppresses liver metastatic tumors" Int J Cancer 116:314-21; and Sato et al., 2001. "Antiviral response by natural killer cells through TRAIL gene induction by IFN-alpl^eta" Eur J Immunol 31 :3138-46. Further, TRAIL may regulate viral replication in DV-infected monocytes at a concentration which is much lower than the amount used to induce cell death in vitro. Abdollahi et al., 2003. "Identification of interleukin 8 as an inhibitor of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in the ovarian carcinoma cell line OVCAR3" Cancer Res 63:4521-6; and Clarke et al., 2000. "Reovirus-induced apoptosis is mediated by TRAIL" J Virol 74:8135-9.

Although it is not necessary to understand the mechanism of an invention, it is believed that TRAIL regulates the anti-viral function mediated by IFNα against DV. Cellular immune responses to DV infection were identified by using global gene expression profiling. The data shown herein suggest that IFNα and IFNγ signaling pathway inducible genes have similar changes in expression profiles (i.e., are commonly regulated) in several DV-susceptible cell types.

TRAIL, one of these common response genes, was identified as a novel anti-viral molecule against DV. The data presented herein further demonstrate that TRAIL functions to mediate an IFNα anti-viral response to DV infection. Furthermore, these data suggest that rTRAIL-mediated decreases in DV titer is mediated by an apoptosis-independent mechanism.

I. TRAIL Compositions

TRAIL amino acid sequences disclosed herein reveal that TRAIL is a member of the TNF family of ligands (Smith et al. Cell, 73:1349, 1993; Suda et al., Cell, 75:1169, 1993; Smith et al., Cell, 76:959, 1994). The percent identities between the human TRAIL extracellular domain amino acid sequence and the amino acid sequence of the extracellular domain of other proteins of this family are as follows: 28.4% with Fas ligand, 22.4% with lymphotoxin-β, 22.9% with TNF-β, 23.1% with TNF-β, 22.1% with CD30 ligand, and 23.4% with CD40 ligand.

The TRAIL DNA of the present invention includes cDNA, chemically synthesized DNA, DNA isolated by PCR, genomic DNA, and combinations thereof. Genomic TRAIL DNA may be isolated by hybridization to the TRAIL cDNA disclosed herein using standard techniques. RNA transcribed from the TRAIL DNA is also encompassed by the present invention.

Due to degeneracy of the genetic code, two DNA sequences may differ, yet encode the same amino acid sequence. The present invention thus provides isolated DNA sequences encoding biologically active TRAIL, selected from DNA comprising the coding region of a native human or murine TRAIL cDNA, or fragments thereof, and DNA which is degenerate as a result of the genetic code to the native TRAIL DNA sequence.

Also provided herein are purified TRAIL polypeptides, both recombinant and non- recombinant. Variants and derivatives of native TRAIL proteins that retain a desired biological activity are also within the scope of the present invention. In one embodiment, the biological activity of an TRAIL variant is essentially equivalent to the biological activity of a native TRAIL protein. One desired biological activity of TRAIL is the ability to inhibit a virus infection as described herein.

A. Full Length TRAIL Compositions In one embodiment, an isolated human TRAIL nucleotide sequence comprises SEQ ID

NO:1, and the amino acid sequence encoded thereby comprises SEQ ID NO:2. In one embodiment, the protein comprises an N-terminal cytoplasmic domain (i.e., for example, amino acids 1-18). In one embodiment, the protein comprises a transmembrane region (i.e., for example, amino acids 19-38). In one embodiment, the protein comprises an extracellular domain (i.e., for example, amino acids 39-281). In one embodiment, the extracellular domain comprises a receptor-binding region.

In one embodiment, an isolated human TRAIL nucleotide sequence comprises SEQ ID NO:3, and an amino acid sequence encoded thereby comprises SEQ ID NO:4. hi one embodiment, the protein comprises an N-terminal cytoplasmic domain (amino acids 1-18). In one embodiment, the protein comprises a transmembrane region (amino acids 19-38). In one embodiment, the protein comprises an extracellular domain (amino acids 39-101). TRAIL polypeptides of the present invention may include, but are not limited to, polypeptides having amino acid sequences that differ from, but are highly homologous to, those presented in SEQ ID NOs: 2 and 6. Examples include, but are not limited to, homologs derived from other mammalian species, variants (i.e., for example, both naturally occurring variants and those generated by recombinant DNA technology), and TRAIL fragments that retain a desired biological activity. Such polypeptides exhibit a biological activity of the TRAIL proteins of SEQ ID NOS :2 and 6, and preferably comprise an amino acid sequence that is at least 80% identical (most preferably at least 90% identical) to the amino acid sequence presented in SEQ ID NO:2, SEQ ID NO:6 and/or SEQ ID NO: 10. In one embodiment, a TRAIL composition comprises a TRAIL amino acid sequence (i.e., for example, a full-length TRAIL protein; SEQ ID NO: 10). See, Figure 1OA. Further, the present invention contemplates fragments of TRAIL protein, wherein the TRAIL protein retains antiviral activity exhibited by the full length TRAIL protein. In one embodiment, a TRAIL protein fragment comprises an extracellular domain of human TRAIL with N-terminal His(6) tag (His-TRAIL, amino acids 95-281). Plasilova M, "TRAIL (Apo2L) suppresses growth of primary human leukemia and myelodysplasia progenitors" Leukemia 16(l):67-73 (2002).

In one embodiment, a TRAIL composition comprises a nucleic acid sequence (i.e., for example, a full-length TRAIL gene coding sequence (i.e, for example, a full-length TRAIL mRNA; SEQ ID NO: 11). See, Figure 1OB. Further, the present invention contemplates fragments of a TRAIL nucleic acid, wherein the nucleic acid fragment is capable of producing an active TRAIL peptide fragment. In one embodiment, a TRAIL nucleic acid fragment comprises a human TRAIL cDNA fragment corresponding to amino acids 114 to 281. Yao et al., "Intraarticular injection of recombinant TRAIL induces synovial apoptosis and reduces inflammation in a rabbit knee model of arthritis" Arthritis Res Ther. 8(1):R16 (2006). B. TRAIL Composition Fragments

In one embodiment, the present invention contemplates TRAIL fragments comprising either nucleic acid sequences and/or amino acid sequences. In one embodiment, a DNA fragment of SEQ ID NO:3 lacks a portion of the DNA of SEQ ID NO:1, and may be designated a human TRAIL deletion variant and/or truncated protein (huTRAILdv). In one embodiment, nucleotides 18 through 358 of SEQ ID NO:1 are identical to nucleotides 8 through 348 of the huTRAILdv DNA of SEQ ID NO:3. In one embodiment, nucleotides 359 through 506 of SEQ ID NO:1 are missing from the cloned DNA of SEQ ID NO:3.

In one embodiment, an isolated murine DNA TRAIL nucleotide sequence comprises SEQ ID NO:5 and the amino acid sequence encoded thereby comprises SEQ ID NO:6. In one embodiment, the protein comprises an N-terminal cytoplasmic domain (amino acids 1-17). In one embodiment, the protein a transmembrane region (amino acids 18-38). In one embodiment, the protein comprises an extracellular domain (amino acids 39-291). In one embodiment, SEQ ID NO: 6 is 64% identical to SEQ ID NO:2. In one embodiment, SEQ ID NO:5 is 75% identical to SEQ ID NO: 1. In one embodiment, human TRAIL nucleic acid sequence fragments incldude, but are not limited to NCBI accession numbers T90422, T82085, T10524, R31020, or Z36726. Alternatively, other isolated TRAIL nucleic acid sequence fragment embodiments comprise a nucleotide sequence selected from the group consisting of nucleotides 88 to 933 of SEQ ID NO:1 (i.e., for example, a human TRAIL coding region fragment); nucleotides 202 to 933 of SEQ ID NO:1 (i.e., for example, a human TRAIL extracellular domain fragment); nucleotides 47 to 922 of SEQ ID NO:5 (i.e., for example, a mouse TRAIL coding region fragment); and nucleotides 261 to 922 of SEQ ID NO:5 (i.e, for example, a mouse TRAIL extracellular domain fragment). In other embodiments, DNAs encoding biologically active fragments of the proteins of SEQ ID NOs:2 and 6 are also provided. Further embodiments include, but are not limited to, sequences comprising nucleotides 370 to 930 of SEQ ID NO:1 and nucleotides 341 to 919 of SEQ ID NO:5, which encode human and murine soluble TRAIL polypeptide fragments, respectively.

In one embodiment, the present invention comprises a human TRAIL protein fragment comprising an N-terminal amino acid sequence comprising Met-Ala-Met-Met-Glu-Val-Gln-Gly- Gly-Pro-Ser-Leu-Gly-Gln-Thr (i.e., for example, amino acids 1-15 of SEQ ID NOS:2 and 4). In one embodiment, the present invention contemplates a murine TRAIL protein fragment comprising an N-terminal amino acid sequence comprising Met-Pro-Ser-Ser-Gly-Ala-Leu-Lys- Asp-Leu- Ser-Phe-Ser-Gln-His (i.e., for example, amino acids 1-15 of SEQ DD NO:6). A search of the NCBI databank identified five expressed sequence tags (ESTs) having regions of identity with TRAIL DNA. Although it is not necessary to understand the mechanism of an invention, it is believed that the deletion causes a shift in the reading frame, which results in an in-frame stop codon after amino acid 101 of SEQ ID NO:4. In one embodiment, amino acids 1 through 90 of SEQ ID NO:2 are identical to amino acids 1 through 90 of SEQ ID NO:4. However, due to the deletion, the C-terminal portion of the huTRAILdv protein (amino acids 91 through 101 of SEQ ID NO:4) differs from the residues in the corresponding positions in SEQ ID NO:2.

It is believed that some amino acids in the human TRAIL protein are conserved in positions 124-125 (AH), 136 (L), 154 (W), 169 (L), 174 (L), 180 (G), 182 (Y), 187 (Q), 190 (F), 193 (Q), and 275-276 (FG) of SEQ ID NO:2. Another structural feature of TRAIL is a spacer region between the C-terminus of the trans-membrane region and the portion of the extracellular domain that is believed to be most important for biological activity. This spacer region, located at the N-terminus of the extracellular domain, consists of amino acids 39 through 94 of SEQ ID NO:2. Analogous spacers are found in other family members, e.g., CD40 ligand. Amino acids 138 through 153 correspond to a loop between the b-sheets of the folded (three dimensional) human TRAIL protein.

TRAIL fragments, including soluble polypeptides, may be prepared by any of a number of conventional techniques. A DNA sequence encoding a desired TRAIL fragment may be subcloned into an expression vector for production of the TRAIL fragment. The TRAIL- encoding DNA sequence advantageously is fused to a sequence encoding a suitable leader or signal peptide. The desired TRAIL-encoding DNA fragment may be chemically synthesized using known techniques. DNA fragments also may be produced by restriction endonuclease digestion of a full length cloned DNA sequence, and isolated by electrophoresis on agarose gels. If necessary, oligonucleotides that reconstruct the 5¹ or 3' terminus to a desired point may be ligated to a DNA fragment generated by restriction enzyme digestion. Such oligonucleotides may additionally contain a restriction endonuclease cleavage site upstream of the desired coding sequence, and position an initiation codon (ATG) at the N-terminus of the coding sequence. A polymerase chain reaction (PCR) procedure also may be employed to isolate and amplify a DNA sequence encoding a desired protein fragment. Oligonucleotides that define the desired termini of the DNA fragment are employed as 5' and 3' primers. The oligonucleotides may additionally contain recognition sites for restriction endonucleases, to faciliate insertion of the amplified DNA fragment into an expression vector. PCR techniques are described in Saiki et al., Science 239:487 (1988); Recombinant DNA Methodology, Wu et al., eds., Academic Press, Inc., San Diego (1989), pp. 189-196; and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds., Academic Press, Inc. (1990).

A transmembrane region of each TRAIL protein may be identified in accordance with conventional criteria for identifying a hydrophobic domain. The exact boundaries of a transmembrane region may vary slightly (i.e., for example, most likely by no more than five amino acids on either end). Computer programs are commercially available for identifying such hydrophobic regions in proteins are.

C. Soluble TRAIL Compositions Provided herein are membrane-bound TRAIL proteins (comprising a cytoplasmic domain, a transmembrane region, and an extracellular domain) as well as TRAIL fragments that retain a desired biological property of the full length TRAIL protein. In one embodiment, TRAIL fragments are soluble TRAIL polypeptides comprising all or part of the extracellular domain, but lacking the transmembrane region that would cause retention of the polypeptide on a cell membrane. Soluble TRAIL proteins are capable of being secreted from the cells in which they are expressed. Advantageously, a heterologous signal peptide is fused to the N-terminus such that the soluble TRAIL is secreted upon expression.

Soluble TRAIL may be identified (and distinguished from its non-soluble membrane- bound counterparts) by separating intact cells which express the desired protein from the culture medium, e.g., by centrifugation, and assaying the medium (supernatant) for the presence of the desired protein. The presence of TRAIL in the medium indicates that the protein was secreted from the cells and thus is a soluble form of the TRAIL protein. Naturally-occurring soluble forms of TRAIL are encompassed by the present invention.

The use of soluble forms of TRAIL is advantageous for certain applications. Purification of the proteins from recombinant host cells is facilitated, since the soluble proteins are secreted from the cells. Further, soluble proteins are generally more suitable for intravenous administration.

Examples of soluble TRAIL polypeptides are those containing the entire extracellular domain (e.g., amino acids 39 to 281 of SEQ ID NO:2 or amino acids 39 to 91 of SEQ ID NO:6). Fragments of the extracellular domain that retain a desired biological activity are also provided. Additional examples of soluble TRAIL polypeptides are those lacking not only the cytoplasmic domain and transmembrane region, but also all or part of the above-described spacer region. Soluble human TRAIL polypeptides thus include, but are not limited to, polypeptides comprising amino acids x to 281, wherein x represents any of the amino acids in positions 39 through 95 of SEQ ED NO:2. In the embodiment in which residue 95 is the N-terminal amino acid, the entire spacer region has been deleted.

D. TRAIL Variants

TRAIL variants may be obtained by mutations of native TRAIL nucleotide sequences, for example. A TRAIL variant, as referred to herein, is a polypeptide substantially homologous to a native TRAIL, but which has an amino acid sequence different from that of native TRAIL because of one or a plurality of deletions, insertions or substitutions. TRAIL-encoding DNA sequences of the present invention encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native TRAIL DNA sequence, but that encode an TRAIL protein that is essentially biologically equivalent to a native TRAIL protein.

A variant amino acid or DNA sequence preferably is at least 80% identical to a native TRAIL sequence, most preferably at least 90% identical. The degree of homology (percent identity) between a native and a mutant sequence may be determined, for example, by comparing the two sequences using computer programs commonly employed for this purpose. One suitable program is the GAP computer program, version 6.0, described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (J. MoI. Biol. 48:443, 1970), as revised by Smith and Waterman (Adv. Appl. Math 2:482, 1981). Briefly, the GAP program defines identity as the number of aligned symbols (i.e., nucleotides or amino acids) which are identical, divided by the total number of symbols in the shorter of the two sequences. The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.

Alterations of the native amino acid sequence may be accomplished by any of a number of known techniques. Mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered gene having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations include those disclosed by Walder et al. (Gene 42: 133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are incorporated by reference herein. Variants may comprise conservatively substituted sequences, meaning that one or more amino acid residues of a native TRAIL polypeptide are replaced by different residues, but that the conservatively substituted TRAIL polypeptide retains a desired biological activity that is essentially equivalent to that of a native TRAIL polypeptide. Examples of conservative substitutions include substitution of amino acids that do not alter the secondary and/or tertiary structure of TRAIL. Other examples involve substitution of amino acids outside of the receptor- binding domain, when the desired biological activity is the ability to bind to a receptor on target cells and induce apoptosis of the target cells. A given amino acid may be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, VaI, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; GIu and Asp; or GIn and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. TRAIL polypeptides comprising conservative amino acid substitutions may be tested in one of the assays described herein to confirm that a desired biological activity of a native TRAIL is retained. DNA sequences encoding TRAIL polypeptides that contain such conservative amino acid substitutions are encompassed by the present invention. Conserved amino acids located in the C-terminal portion of proteins in the TNF family, and believed to be important for biological activity, have been identified. These conserved sequences are discussed in Smith et al. (Cell, 73:1349, 1993, see page 1353 and Figure 6); Suda et al. (Cell, 75:1169, 1993, see figure 7); Smith et al. (Cell, 6:959, 1994, see figure 3); and Goodwin et al. (Eur. J. Immunol., 23:2631, 1993, see figure 7 and pages 2638-39).

Advantageously, the conserved amino acids are not altered when generating conservatively substituted sequences. If altered, amino acids found at equivalent positions in other members of the TNF family are substituted.

TRAIL also may be modified to create TRAIL derivatives by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like. Covalent derivatives of TRAIL may be prepared by inking the chemical moieties to functional groups on TRAIL amino acid side chains or at the N-terminus or C-terminus of a TRAIL polypeptide or the extracellular domain thereof. Other derivatives of TRAIL within the scope of this invention include covalent or aggregative conjugates of TRAIL or its fragments with other proteins or polypeptides, such as by synthesis in recombinant culture as N-terminal or C-terminal fusions. For example, the conjugate may comprise a signal or leader polypeptide sequence (e.g. the .alpha.-factor leader of Saccharomyces) at the N-terminus of a TRAIL polypeptide. The signal or leader peptide co-translationally or post-translationally directs transfer of the conjugate from its site of synthesis to a site inside or outside of the cell membrane or cell wall.

In another example, sequences encoding Cys residues that are not essential for biological activity can be altered to cause the Cys residues to be deleted or replaced with other amino acids, preventing formation of incorrect intramolecular disulfide bridges upon renaturation. Other variants are prepared by modification of adjacent dibasic amino acid residues to enhance expression in yeast systems in which KEX2 protease activity is present. EP 212,914 discloses the use of site-specific mutagenesis to inactivate KEX2 protease processing sites in a protein. KEX2 protease processing sites are inactivated by deleting, adding or substituting residues to alter Arg— Arg, Arg-Lys, and Lys-Arg pairs to eliminate the occurrence of these adjacent basic residues. Lys— Lys pairings are considerably less susceptible to KEX2 cleavage, and conversion of Arg- Lys or Lys-Arg to Lys— Lys represents a conservative and preferred approach to inactivating KEX2 sites. Potential KEX2 protease processing sites are found at positions 89-90 and 149-150 in the protein of SEQ ID NO:2, and at positions 85-86, 135-136, and 162-163 in the protein of SEQ ED NO:6.

Naturally occurring TRAIL variants are also encompassed by the present invention. Examples of such variants are proteins that result from alternative mRNA splicing events (since TRAIL is encoded by a multi-exon gene) or from proteolytic cleavage of the TRAIL protein, wherein a desired biological activity is retained. Alternative splicing of mRNA may yield a truncated but biologically active TRAIL protein, such as a naturally occurring soluble form of the protein, for example. Variations attributable to proteolysis include, for example, differences in the N- or C-termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the TRAIL protein. In addition, proteolytic cleavage may release a soluble form of TRAIL from a membrane-bound form of the protein. Allelic variants are also encompassed by the present invention. E. TRAIL Oligomers The present invention encompasses TRAIL polypeptides in the form of oligomers, such as dimers, trimers, or higher oligomers. Oligomers may be formed by disulfide bonds between cysteine residues on different TRAIL polypeptides, or by non-covalent interactions between TRAIL polypeptide chains, for example. In other embodiments, oligomers comprise from two to four TRAIL polypeptides joined via covalent or non-covalent interactions between peptide moieties fused to the TRAIL polypeptides. Such peptides may be peptide linkers (spacers), or peptides that have the property of promoting oligomerization. Leucine zippers and certain polypeptides derived from antibodies are among the peptides that can promote oligomerization of TRAIL polypeptides attached thereto, as described in more detail below. The TRAIL polypeptides preferably are soluble.

Alternatively, oligomeric TRAIL may comprise two or more soluble TRAIL polypeptides joined through peptide linkers. Examples include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference). Fusion proteins comprising multiple TRAIL polypeptides separated by peptide linkers may be produced using conventional recombinant DNA technology.

Another method for preparing oligomeric TRAIL polypeptides involves use of a leucine zipper. Leucine zipper domains are peptides that promote oligomerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, 1988), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble oligomeric TRAIL proteins are those described in PCT application WO 94/10308, hereby incorporated by reference. Recombinant fusion proteins comprising a soluble TRAIL polypeptide fused to a peptide that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble oligomeric TRAIL is recovered from the culture supernatant.

Certain members of the TNF family of proteins are believed to exist in trimeric form (Beutler and Huffel, Science 264:667, 1994; Banner et al., Cell 73:431, 1993). Thus, trimeric TRAIL may offer the advantage of enhanced biological activity. Preferred leucine zipper moieties are those that preferentially form trimers. One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, 1994) and in U.S. patent application Ser. No. 08/446,922, hereby incorporated by reference. Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric TRAIL.

For example, a soluble Flag.RTM.-TRAIL polypeptide expressed in CV-1/EBNA cells spontaneously forms oligomers believed to be a mixture of dimers and trimers. The activity of this soluble Flag-TRAIL is believed to be enhanced by including an anti-Flag→ antibody, possibly because the antibody facilitates cross-linking of TRAIL/receptor complexes. In one embodiment of the invention, biological activity of TRAIL is enhanced by employing TRAIL in conjunction with an antibody that is capable of cross-linking TRAIL. Cells that are to be killed may be contacted with both a soluble TRAIL polypeptide and such an antibody.

As one example, a virally infected cells (i.e., for example, flavivirus and/or bunyavirus) are contacted with an anti-Flag^® antibody and a soluble Flag^®.-TRAIL polypeptide. Preferably, an antibody fragment lacking the Fc region is employed. Bivalent forms of the antibody may bind the Flag^® moieties of two soluble Flag^®.-TRAIL polypeptides that are found in separate dimers or trimers. The antibody may be mixed or incubated with a Flag^®.-TRAIL polypeptide prior to administration in vivo. II. Global Gene Expression Profiling

Three (3) flaviviruses (i.e., for example, dengue virus, West Nile virus, and Yellow Fever virus) and two (2) bunyaviruses (i.e., for example, Hantaan virus, and Sin Nombre virus) were tested for their ability to regulate gene expression in HUVECs. In particular, seventy-nine individual genes were selected for study. The data show that all five (5) viruses from both Flaviridiae and Bunyaviridae induced gene expression in a common fashion. See, Figure 1.

Twenty-three (23) of these genes showing a common induced expression profile by the three (3) flaviviruses and two (2) bunyaviruses were selected for further study using a standard microchip library screening technology (GeneChip). As dengue virus showed the most intense gene induction, this virus was selected for this detailed study. However, it is believed that the data shown herein is representative for all flaviviruses and bunyaviruses.

Common gene expression profiles were seen in DV-infected HUVECs and PBMC subsets (i.e., for example, B cells, monocytes). Expression profiles of each indicated gene is represented by a shaded horizontal bar. Changes in expression levels (i.e., for example 0.0 - 20.0) are reflected in the intensity of the shaded bar. For example, if a gene increased expression by 20- fold the horizontal bar would a deep red. On the contrary, if gene expression is absent (i.e., O.O-fold) the horizontal bar would be a deep blue. Otherwise, if gene expression does not change (i.e, 1.0 -fold) the horizontal bar would be white. See, Figure 2.

These findings identified genes also observed in previous studies of: i) gene expression in DV-infected HUVECs (Warke et al., 2003. "Dengue virus induces novel changes in gene expression of human umbilical vein endothelial cells" J Virol 77:11822-32; and ii) cytokine gene expression in macrophages (Moreno-Altamirano et al., 2004. "Gene expression in human macrophages infected with dengue virus serotype-2" Scand J Immunol 60:631-8. Moreno- Altamirano et al., however, did not identify a common gene expression profile as contemplated herein in a DV-upregulated cytokine gene array.

Of the 23 genes that comprised the common dengue response profile, functions and/or inducers of 19 genes have been described in the literature (Table 2). Table 2: Biological function of the 23 dengue virus response genes common to primary cells from human (Monocytes, B, DC and HUVEC).

These include, but are not limited to, classical anti-viral response genes (OAS3 and IRF7), more recently identified anti-viral genes (ISGl 5, HERC5, RSAD2, TRIM5, TRAIL, OASL, ISG20), genes regulating ubiquitination (USP 18), cell adhesion and cyclic ADP-ribose (cADPR) metabolism (CD38), apoptosis (XAFl), immune suppression (IFITMl), immune activation (LGALs3BP) and 9 other genes (FLJ20035, FLJ38348, HERC6, IFI44, IFI44L, IFITl, IFIT3, LY6E and SAMD9) with unknown function. Although it is not necessary to understand the mechanism of an invention, it is believed that functions of these identified genes may play a role of the cellular responses to dengue virus infection. Of these common response genes, G1P2, MxI and OAS3 were also detected to be associated with dengue shock patients. Simmons et al., 2007. "Patterns of host genome-wide gene transcript abundance in the peripheral blood of patients with acute dengue hemorrhagic fever" J Infect Dis 195:1097-107.

III. TRAIL Mediation Of Interferon Activity

One of the common response genes, TRAIL may have a potential link between EFNa and IFNγ signaling pathways. As indicated above, TRAIL has been identified as having anti-viral and anti-tumor function. TRAIL has been reported as capable of initiating apoptosis through the engagement of its receptors, TRAIL-Rl (DR4) and TRAIL-R2 (DR5). Cretney et al., 2005. "TNF-related apoptosis-inducing ligand (TRAIL)/ Apo2L suppresses experimental autoimmune encephalomyelitis in mice" Immunol Cell Biol 83:511-9. Both in vitro and in vivo studies have demonstrated tumoricidal and anti- viral activity of TRAIL without significant toxicity towards normal cells or tissues. Pitti et al., 1996. "Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family" J Biol Chem 271 : 12687-90; and Sato et al., 2005. "TRAIL-transduced dendritic cells protect mice from acute graft-versus-host disease and leukemia relapse" J Immunol 174:4025-33. TRAIL-mediated killing by activated CD4⁺ T cells, NK cells, and B cells has been shown against influenza virus and others. Kemp et al., 2003. "Plasmacytoid dendritic cell-derived IFN-alpha induces TNF-related apoptosis-inducing ligand/ Apo-2L-mediated antitumor activity by human monocytes following CpG oligodeoxynucleotide stimulation" J Immunol 171:212-8; Sato et al., 2001. "Antiviral response by natural killer cells through TRAIL gene induction by IFN-alpha/beta" Eur J Immunol 31:3138-46; and Wang et al., 2004. "8-Chloro-adenosine sensitizes a human hepatoma cell line to TRAIL-induced apoptosis by caspase-dependent and -independent pathways" Oncol Rep 12:193-9.

EFNs enhance expression of TRAIL, while on the other hand, TRAIL treatment can enhance expression of IFN-inducible genes like BFITMl, IFITl, STATl, LGaBBP, PRKR as well as EFN alpha itself. Kumar-Sinha et al., 2002. "Molecular cross-talk between the TRAIL and interferon signaling pathways" J Biol Chem 277:575-85. Consequently, the molecular crosstalk and functional synergy observed between TRAIL and IFN signaling pathways may not be not limited to the genes involved in apoptosis and may have implications for the physiological role and mechanism of action of TRAIL protein. In support of this speculation, the data provided herein support the idea that TRAIL-mediated antiviral function is apoptosis- independent and contributes to the Type-I EFN response against dengue virus. See, Figure 7.

TV. TRAIL And Apoptosis

In primary monocytes, dendritic cells, B cells and HUVECs infected with DV induced TRAIL mRNA expression TRAIL protein levels were also found to be highly induced (i.e., for example, in DV-infected monocyte cell lysates). TRAIL, however, was not expressed on the monocyte cell surface or secreted in the supernatant from DV-infected primary monocytes. On the contrary, secretion of TRAIL protein by HepG2 cells after DV-infection was reported as partly responsible for apoptosis of uninfected HepG2 cells. Matsuda et al., 2005. "Dengue virus- induced apoptosis in hepatic cells is partly mediated by Apo2 ligand/tumour necrosis factor- related apoptosis-inducing ligand" J Gen Virol 86: 1055-65.

This observation may be explained by global gene expression analysis in DV-infected HepG2 cells and human primary cells (i.e., for example, monocytes, B cells and HUVECs) showing a distinct set of differentially regulated genes in HepG2 versus primary human cells (data not shown). Unlike human primary cells, TRAIL mRNA levels were not found to be up- regulated in DV-infected HepG2 cells by GeneChip analysis (data not shown). One hypothesis suggests that DV-infection of primary cells may better reflect a physiological response. On the other hand, since it has been observed that 80% of DV-infected HepG2 cells stain positive for cell death, this raises the possibility that the TRAIL protein detected in the supernatant of DV- infected cells was not secreted but resulted from cell death and release of intracellular TRAIL. DCs infected with DV have been observed to stain minimally using an apoptosis detection dye (Live/Dead Aqua) and/or apoptotic markers (PARP-I and Caspase-8). These results indicate that DV infection may not induce apoptosis in monocytes and DCs. See, Figures 9A-C. rTRAIL has been shown to inhibit DV replication at about 40 to 100 times lower concentrations than used in studies demonstrating TRAIL induced apoptosis of tumor cells (i.e., for example, 1-20 ng/ml). Sato et al., 2005. "TRAIL-transduced dendritic cells protect mice from acute graft-versus-host disease and leukemia relapse" J Immunol 174:4025-33. Although it is not necessary to understand the mechanism of an invention, it is believed that TRAIL might be inhibiting DV copy number by acting as an antiviral and not as a result of apoptosis induction. It is further believed that one possible mechanism of TRAIL anti-viral function which is apoptosis- independent would be TRAIL-mediated increased expression of known/novel anti-viral cellular protein/s which is/are secreted by cells.

V. TRAIL And Anti- Viral Actions

TRAIL has been shown to mediate anti-viral functions in vivo in mouse models of influenza virus. Ishikawa et al., 2005. "Role of tumor necrosis factor-related apoptosis-inducing ligand in immune response to influenza virus infection in mice" J Virol 79:7658-63. Influenza viral clearance was prolonged in mice injected with anti-TRAIL antibody. Previous studies have shown that both Type-I and Type-II IFNs are involved in controlling different stages of DV infection in mice, although the precise mechanism(s) by which IFNs mediate an anti-viral response is unknown. Shresta et al., 2005. "Critical roles for both STATl -dependent and STATl -independent pathways in the control of primary dengue virus infection in mice" J Immunol 175:3946-54. In one embodiment, the present invention contemplates a method comprising mediating type-I IFN anti-viral function using a TRAIL protein. Induction of TRAIL gene expression by DV was shown to be type-I IFN dependent when using wild type (2fTGH) and type-I IFN mutant (UlA, U3A, U4A and U5A) fibroblast cells. Lutfalla et al., 1995. "Mutant U5A cells are complemented by an interferon-alpha beta receptor subunit generated by alternative processing of a new member of a cytokine receptor gene cluster" Embo J 14:5100-8; McKendry et al., 1991. "High-frequency mutagenesis of human cells and characterization of a mutant unresponsive to both alpha and gamma interferons" Proc Natl Acad Sd USA 88:11455-9; and Pellegrini et al., 1989. "Use of a selectable marker regulated by alpha interferon to obtain mutations in the signaling pathway" MoI Cell Biol 9:4605-12. Further, it has been shown that the anti-viral effect of type-I IFN was independent of IFN-inducible protein kinase, interferon-inducible double stranded RNA dependent (PRKR), 2',5'-oligoadenylate synthetase 1 (OASl) and myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 (mouse) (MxI) anti- viral pathways. Diamond et al., 2001. "Interferon inhibits dengue virus infection by preventing translation of viral RNA through a PKR-independent mechanism" Virology 289:297-311.

In one embodiment, the present invention contemplates a method comprising inhibiting TRAIL function by anti-TRAIL antibody treatment, wherein said inhibiting blocks an EFN-α anti-viral effect. Although it is not necessary to understand the mechanism of an invention, it is believed that TRAIL mediated anti-dengue activity may mediate type-I IFN dependent anti-viral response. It is further believed that TRAIL inhibits DV infection in an apoptosis independent manner.

In one embodiment, the present invention contemplates a method comprising activating a TRAIL signaling pathway, thereby providing an anti-viral therapy. TRAIL may further play a role in providing anti-viral (CMV) and pro-viral (HIV-I and Reovirus) effects. Clarke et al., 2003. "Reovirus-induced apoptosis: A minireview" Apoptosis 8:141-50; and Herbeuval et al., 2005. "TNF-related apoptosis-inducing ligand (TRAIL) in HIV-I -infected patients and its in vitro production by antigen-presenting cells" Blood 105:2458-64.

VI. TRAIL And Chemokine Activity In one embodiment, the present contemplates a gene expression analysis of primary human cells in vitro showing an induction of several genes in response to dengue virus (DV) infection. In one embodiment, these genes are selected from the group comprising chemokines. For example, these chemokines include, but are not limited to, monocyte chemotactic protein 2 (MCP-2), chemokine (C-C motif) ligand 8, CCL8, interferon gamma-induced protein 10 (IP-10), chemokine (C-X-C motif) ligand 10, CXCLlO, or cytokine tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

In one embodiment, in vitro MCP-2 and IP-10 protein expression is higher in DV- infected dendritic cells (DC) as compared to non-infected DC. In another embodiment, in vitro MCP-2 and EP-IO mRNA expression is higher in DV-infected DC as compared to non-infected DC.

In one embodiment, in vivo expression of MCP-2, IP-10, and TRAIL in DV-infected patient serum is higher during the febrile period as compared to healthy donors and patients with other febrile illnesses (OFIs).

In one embodiment, the present invention contemplates in vitro expression of MCP-2 and IP-10 mRNA and/or protein is lower in DV-infected DCs following pre-treatment with recombinant TRAIL (rTRAIL) as compared to un-treated DENV-infected DC.

In one embodiment, the present invention contemplates that in vitro expression of EFN-I decreased following pre-treatment with rTRAIL. In one embodiment, rTRAIL pretretment decreases the percentage of DV-infected DCs, as compare to untreated DC. A. Overview

The results discussed below indicate that DV induces the expression of chemokines including, but not limted to, MCP-2, IP-10/CXCLlO and TRAIL both in vitro and in vivo. Although it is not necessary to understand the mechanism of an invention, it is believed that TRAIL protein and/or mRNA inhibits the production of chemokines and cytokines induced in response to virus infection, an effect that might be related to the decreased infection of DC after rTRAIL treatment. Dengue virus (DV) is a single-stranded RNA mosquito-borne virus that belongs to the Flaviviridae family and exists as four different serotypes: DVl, DV2, DV3, and DV4 (Monath T.P., "Dengue: the risk to developed and developing countries" Proc Natl Acad Sci USA 91 :2395-2400 (1994). All known serotypes can infect humans and produce a disease with a broad spectrum of clinical manifestations that ranges from an acute self-limiting febrile illness (Dengue Fever, DF) to various grades of a severe disease (Dengue Hemorrhagic Fever, DHF and Dengue Shock Syndrome, DSS). Chaturvedi et al., "Dengue and dengue haemorrhagic fever: implications of host genetics" FEMS Immunol Med Microbiol 47:155-166 (2006).

Clinical symptoms of DV include, but are not limited to, high fever, headache, myalgias, skin rash, thrombocytopenia, coagulation alterations, hepatic inflammation and hemorrhagic manifestations. Furthermore, increased vascular permeability that results in vascular leakage is the characteristic event that occurs and defines DHF. Rothman et al., "Immunopathogenesis of Dengue hemorrhagic fever" Virology 257:1-6 (1999). Infection with one of the serotypes imparts immunity to the infecting serotype. Multiple infections with different (heterologous) serotypes can occur during one's lifetime and DHF/DSS is usually associated with secondary infections. Halstead et al., "Observations related to pathogenesis of dengue hemorrhagic fever. IV. Relation of disease severity to antibody response and virus recovered" Yale J Biol Med 42:311-328 (1970); and Guzman et al., "Dengue hemorrhagic fever in Cuba, 1981: a retrospective seroepidemiologic study" Am J Trop Med Hyg 42: 179-184 (1990). It has been suggested that the transient vascular leakage observed in vivo could be associated with the response of the endothelium to inflammatory mediators produced in response to DV infection. Avirutnan et al., "Dengue virus infection of human endothelial cells leads to chemokine production, complement activation, and apoptosis" J/wm««o/ 161 : 6338-6346 (1998). Others have discused quantifying the levels of chemokines during the acute phase of DV infected patients: i). elevated levels of interleukin 8 (IL-8) (Raghupathy et al., "Elevated levels of IL-8 in dengue hemorrhagic fever" J Med Virol 56:280-285.(1998); and Juffrie et al., "Inflammatory mediators in dengue virus infection in children: interleukin-8 and its relationship to neutrophil degranulation" Infect Immun 68:702-707 (2000); and ii) monocyte chemotactic protein 1 (MCP-I) (Lee et al., "MCP-I, a highly expressed chemokine in dengue haemorrhagic fever/dengue shock syndrome patients, may cause permeability change, possibly through reduced tight junctions of vascular endothelium cells" J Gen Virol 87:3623-3630 (2006). The expression of macrophage inflammatory protein- 1 alpha (MIP- lα) and macrophage inflammatory protein- 1 beta (MIP- lβ) was reported in peripheral blood mononuclear cells (PBMC) from DV-infected patients. Spain-Santana et al., MIP-I alpha and MIP-I beta induction by dengue virus" J Med Virol 65:324-330 (2001). In vitro DV infection of different cell types, including, but not limited to, PBMC, monocytes, macrophages, mast cells, umbilical vein endothelial cells (HUVEC) and human primary hepatocytes, have been reported to induce the expression of various chemokines, including but not limited to, IL-8, MIP- lα/β, and Regulated upon Activation, Normal T Cell Expressed and Secreted (RANTES). Chen et al., "Activation of terminally differentiated human monocytes/macrophages by dengue virus: productive infection, hierarchical production of innate cytokines and chemokines, and the synergistic effect of lipopolysaccharide" J Virol 76:9877-9887 (2002); Huang et al., "Dengue virus infects human endothelial cells and induces IL-6 and IL-8 production" Am J Trop Med Hyg 63:71-75. (2000); Suksanpaisan et al., "Infection of human primary hepatocytes with dengue virus serotype 2" J Med Virol 79:300-307 (2007); and King et al., "Dengue virus selectively induces human mast cell chemokine production" J Virol 76:8408-8419 (2002).

DV-induced immunopathology is complex and not well known. Potential mediators of disease have been identified in earlier studies, but none of these molecules alone can explain the events that have been documented in DV-infected patients. Hence, the identification of new mediators up-regulated in response to DV infection could contribute to the understanding of the immune response against the virus and the related immune-mediated pathology.

In one embodiment, the present invention contemplates an expression of novel soluble mediators that mediate the inflammatory response to DV. In one embodiment, the DV mediation of the inflammatory response comprises a regulatory feedback loop to control in vivo " inflammation. In one embodiment, a gene expression analysis of primary human cells infected with DENV in vitro identifies selective gene expression. In one embodiment, at least three genes (i.e., for example, two chemokines and one cytokine) showed selective expression in the gene expression analysis, thereby being predicted as possible disease markers present in a patient serum, as compared to both healthy donors and OFI patients. In one embodiment, the chemokine disease marker comprises MCP-2. In one embodiment, the chemokine disease marker comprises IP-10. In one embodiment, the cytokine disease marker comprises TRAIL. A potent antiviral effect of TRAIL in DV infection in vitro has recently been reported. Warke et al., "TRAIL is a novel anti-viral protein against dengue virus" J Virol. 82(l):555-564 (2008).

It is believed thatTRAIL is a member of the TNF family of proteins, originally identified as a promoter of apoptosis in tumor cell lines and some primary tumors. Recent studies have shown that TRAIL can induce proliferation of T lymphocytes and regulate inflammation, as well as negatively regulate the innate immune response through an apoptosis-independent mechanism. Song et al., "Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an inhibitor of autoimmune inflammation and cell cycle progression" J Exp Med 191 :1095-l 104 (2000); Vassina et al., "Increased expression and a potential anti-inflammatory role of TRAIL in atopic dermatitis" J Invest Dermatol 125:746-752. 2005); Diehl et al., "TRAIL-R as a negative regulator of innate immune cell responses" Immunity 21 :877-889 (2004); Secchiero et al., "TRAIL counteracts the proadhesive activity of inflammatory cytokines in endothelial cells by down-modulating CCL8 and CXCLlO chemokine expression and release" Blood 105:3413-3419 (2005). In one embodiment, the present invention contemplates a method using an in vitro dendritic cell (DC) model for DV infection, wherein TRAIL is a negative regulator of DV iinfection -induced chemokines and cytokines expression. Although it is not necessary to understand the mechanism of an invention, it is believed that TRAIL may control both the the virus and the resultant inflammatory response. It is further believed that TRAIL may be involved in the balance between proinflammatory and anti-inflammatory mediators in response to viral infection disease pathology.

B. _ Cytokine Regulation Of Chemokine Expression

1. Gene Expression Analysis In Vitro MCP-2, IP-10, MEP- lβ and TRAIL were selected for gene expression analysis based on level of in vitro expression and possible role in disease. The expression of these four genes are shown below in Table 1. Table 1 : Gene Expression Changes in Primary Human Cells Induced by DV determined using Microarray Analysis.

HUVEC Mo DC

Gene Alias Fold change⁸ N^b Fold change^a N^b Fold change^a N^b

CCL4 MIP- lβ, 0.8 2 2.2 2 3.0 1

CXCLlO IP-IO 29.2 2 3.7 2 122.2 1

TNFSFlO TRAIL 4.1 2 9.4 2 67.1 1

CCL8 MCP-2 47.0 2 16.4 2 11.6 1 a. Gene expression fold-change in DENV-infected cells compared to mockinfected cells. b. N is the number of independent experiments.

These tabulated values indicate the fold-change induced by DV infection in three different cell types. Values were derived from single experiments or are the mean of duplicate experiments as indicated. For TRAIL, which has three different probe sets on HG-Ul 33 A chips (202687_s_at, 202688_at, and 214329_x_at), values are the mean of all three probe sets; values for other genes were obtained from single probe sets (MlP-lβ 204103_at; IP-10 204533_at; MCP-2 214038_at). The chemokines MCP-2 and IP-10 were strongly induced in HUVEC and DC in response to DENV infection after 48 hours of infection. On the other hand, the mRNA expression of the chemokine MIP- lβ showed lower levels of induction. MCP-2 expression has not been previously described in DV infections in vitro cell culture or in vivo patients. IP-10 has been shown to be induced in vitro and recently was reported to be increased in serum of DV-infected patients. Fink et al., "Host gene expression profiling of dengue virus infection in cell lines and patients" PLoS Negl Trop Dis I:e86. (2007). MIP- lβ has been reported using PBMC from DV- infected patients but not in serum. These three chemokines were selected to be tested in serum from patients.

Expression of the TRAIL cytokine was induced in response to DV infection in different primary human cells and which have previously been shown to have a potent antiviral effect in vitro against DV. Warke et al., "TRAIL is a novel anti-viral protein against dengue virus" J Virol. 82(l):555-564 (2008). Induction of MCP-2, IP-10 and MIP-lβ in response to DV, the expression of these genes in DV-infected DC in vitro was determined at 12, 24 and 48 hours post-infection. IL-6, a cytokine that has been previously shown to be increased in DV infections both in patients and in vitro, was also subjected to gene expression profiling, Pinto et al., "Increased pro-inflammatory cytokines (TNF-alpha and IL-6) and anti-inflammatory compounds (sTNFRp55 and sTNFRp75) in Brazilian patients during exanthematic dengue fever" Mem Inst Oswaldo Cruz 94:387-394 (1999); and Deauvieau et al., "Innate immune responses in human dendritic cells upon infection by chimeric yellow-fever dengue vaccine serotypes 1-4" Am J Trop Med Hyg 76: 144-154.2007).

The results demonstrated that MCP-2 mRNA expression was up-regulated 60 times at 48 hours; IP-10 mRNA expression was up-regulated more than 100 times at 24 and 48 hours; and IL-6 expression was up-regulated more than 3 times at 48 hours; MIP-I β mRNA was up- regulated less than 1.6 times at both 24 and 48 hours. Accumulated levels of MCP-2, IP-10 and IL-6 proteins in cell culture supernatants were also determined at each time point where higher levels of MCP-2 (p=0.001), IP-10 (p=0.022) and IL-6 (p=0.083) in supernatants from DENV- infected DC compared to uninfected DC at 48 hours. Figure 19. 2. Characterization of Patients

The characteristics of the patients enrolled in the clincial study are shown below in Table 2.

Table 2: Clinical Profile of Patients Enrolled in the Study Protocol.

a. Patients were classified as OFI or Dengue according to DENV RNA and IgM detection. Dengue patients were positive for both DENV RNA and IgM; OFI patients were negative for both parameters. b. Age in years (average ± standard deviation). c. Average value ± standard error. d. Mann- Whitney test was used to compare maximum or minimum values between OFI and Dengue patients.

The study included a total of 11 OFI and 31 dengue patients. Among the dengue patients, 15 had primary infections and 15 had secondary infections (1 patient was not sub-classified as primary or secondary infection). Out of 31 DENVinfected patients, 24 had hemorrhagic manifestations (petechiae, epistaxis, gum bleeding), 21 had platelet counts lower than 100,000 / μl, two had more than 20% hemoconcentration, and three had evidence of vascular leakage. Two of the dengue patients had DHF according to the 2003 WHO classification (2003). 3. MCP-2, IP-10, MIP-I β, and TRAIL levels in Serum from Patients

Levels of MCP-2, IP-10, MIP- lβ and TRAIL in serum from DV-infected patients and OFI were determined. Figure 20.

MCP-2 levels were measured in serum from 22 DV-infected patients and 8 OFI. Figure 2OA. Increased MCP-2 levels were observed during the febrile days, followed by a progressive decrease at postfebrile days to reach values close to normal by the convalescent visit. MCP-2 levels were significantly higher in DV-infected patients compared to OFI during the febrile (p=0.015) and early post- febrile (p=0.001) days of the disease. Serum levels of MCP-2 in healthy donors were 33.7 ± 8.8 (N=8). There were not significant differences in levels of MCP-2 in serum from OFI and healthy donors. IP-10 serum levels were markedly increased in DENV-infected patients during the febrile and early post-febrile days of the disease. Figure 2OB. Levels of IP-10 had decreased by the convalescent visit, but still remained elevated compared to OFI and healthy donors. Statistical analysis showed that levels of IP-10 were significantly higher in DV-infected patients (N=I 9) compared to OFI (N=7) at febrile (p<0.001), early post-febrile (pO.OOl) and convalescent (p=0.022) days. Serum levels of IP-10 in healthy donors were 0.25 ± 0.08 (N=5) ng/ml.

Significant differences in IP-10 levels were also found in serum from OFI at the febrile days of the disease and healthy donors (p=0.014).

MIP-I β levels were slightly increased in serum from DENV-infected patients (N=23). Figure 2OC. Statistical analysis showed a significant difference during the febrile days of the disease when compared to OFI (p=0.024). Serum levels of MIP-I β in healthy donors were 105.2 ± 10.4 (N=I 1) pg/ml. There were no significant differences between MEP- lβ levels in serum from OFI and healthy donors.

TRAIL levels in serum from DV-infected patients were also increased during the febrile days of the disease and dropped close to normal levels after defervescence. Figure 2)D. Statistically significant differences were found when comparing TRAIL levels in serum from DV-infected patients (N=I 9) and OFI (N=4) during the febrile days of the disease (p=0.009). There were no differences in TRAIL levels in serum from OFI and healthy donors. Serum levels of TRAIL in healthy donors were 33.7 ± 8.8 (N=8) pg/ml.

A comparasion of the levels of MCP-2, LP-IO, MIP- lβ and TRAIL in DV-infected patients sub-classified as primary and secondary infections was performed. Significant differences were not found in levels of MCP-2 (10 primary, 11 secondary infections), EP-IO (8 primary, 11 secondary infections) and MEP-I β (8 primary, 15 secondary infections) at any period of the disease. Higher TRAIL levels were found in primary infections as compared to secondary infections during the febrile period of the disease: 217.7 ± 34.4 pg/ml (N=IO) versus 112.7 ± 20.8 pg/ml (N=8) (mean ± standard error) but the differences were not quite significant (p=0.051).

A comparasion of MCP-2, LP-IO, MLP- lβ and TRAIL levels was performed in DV- infected patients classified according to the absence or presence of hemorrhagic manifestations. A higher LP-IO level was found in patients with hemorrhagic manifestations as compared to patients without hemorrhagic manifestations; the difference was significant during the postfebrile period: 6.0 ± 0.5 ng/ml (N=15) versus 4.2 ± 1.0 ng/ml (N=4) (mean ± standard error), (p=0.037). No differences were found in levels of MCP-2 (4 without, 18 with hemorrhagic manifestations), MEP- lβ (6 without, 17 with hemorrhagic manifestations) and TRAIL (4 without, 15 with hemorrhagic manifestations) at any period of the disease.

4. TRAIL Suppresses the Expression of MCP-2 and IP- 10 Induced in DV infected DC

The effect of rTRAIL pre-treatment on DV-induced expression of MCP-2 and EP-IO, two potential inflammatory mediators that we found increased in DENV-infected patients, was determined. TRAIL-mediated suppression of MCP-2 and LP-IO induced by TNF-α in endothelial cells in vitro has been reported. Secchiero et al., "TRAIL counteracts the proadhesive activity of inflammatory cytokines in endothelial cells by down-modulating CCL8 and CXCLlO chemokine expression and release" Blood 105:3413-3419 (2005).

The expression of MCP-2, IP-10 and IL-6 in untreated or rTRAIL pre-treated DENV- infected DC was determined. A higher expression of MCP-2 and IP-10 mRNA was found in untreated DV-infected DCs as compared to rTRAIL pre-treated DV-infected DCs. Table 3.

Table 3: Gene Expression Changes in untreated relative to rTRAIL pre-treated DENV-infected DC.

a. Relative quantification (Rq) of mRNA using qRT-PCR / 2-""Ct {Livak, 2001 #29} ; #-actin was used endogenous control and rTRAIL pre-treated DENV-infected samples as calibrator. Gene expression in rTRAIL pretreated DENV-infected DC =1. b. Mean values (M) ± standard error of mean (SEM) and number of independent experiments (N) are presented. c. Percentage inhibition of RNA levels in rTRAIL pre-treated DENV-infected DC as compared to untreated DENV-infected DC

The levels of MCP-2, IP-10, and IL-6 were analyzed in cell culture supernatants at 48 hours post-infection from rTRAIL pre-treated or untreated DV-infected DCs. Figure 21 A, 2 IB, and 21C. Significantly lower levels of MCP-2 (p=0.050) and IP-10 (p=0.021) were found in supernatants from rTRAIL pre-treated DENV-infected DC as compared to supernatants from untreated DENV-infected DC. The effect of TRAIL on the expression of other genes at the mRNA level was studied in DV-infected DC. A higher expression for most of the genes tested in untreated DENV-infected DC was observed as compared to rTRAIL pretreated DV-infected DC (Table 3, supra). 5. TRAIL Suppresses IFN-α Production in DENV-infected DC The effect of TRAIL on the expression of MCP-2, IP-IO, and other genes in DCs could be a result of an antiviral effect of TRAIL. Such an effect maybe characterized by a lower percentage of DV-infected cells in rTRAIL pretreated DCs as compared to untreated DENV- infected DCs. Viral infection has been reported to induce the production of type-I EFN which then modulates an antiviral response. Taniguchi et al., "The interferon-alpha/beta system in antiviral responses: a multimodal machinery of gene regulation by the IRF family of transcription factors" Curr Opin Immunol 14:111-116 (2002).

IFN-α levels were determined in DC supernatants from untreated or rTRAIL pre-treated DENV-infected DC. Figure 2 ID. Lower levels of IFN-α (p=0.025) were observed in cell culture supernatants of rTRAIL pre-treated DV-infected DC as compared to untreated DV- infected DC at 48 hours post-infection. We further confirmed a lower percentage of infection in rTRAIL pre-treated DENV-infected DC as compared to untreated DENV-infected DC. Figure 21E. Further, a significant positive correlation was found between IFN-α levels in culture supernatants and the percentage of DV-infected DC (r= 0.73; p<0.003).

Interferon regulatory factor 7 (IRF-7), a well known regulator of the type-I IFN response, was also studied. The expression of IRF-7 mRNA was increased in response to DV infection (11.4 ± 2.5; N=3). rTRAIL pre-treatment slightly increased IRF-7 mRNA expression in untreated DENV-infected DC as compared to rTRAIL pre-treated DENV-infected DCs. The effect of rTRAIL pre-treatment also slightly increased the expression of the melanoma differentiation molecule 5 (MD A-5), a RNA-sensing protein that induces the expression of type-I FN, in untreated DV-infected DC as compared to rTRAIL pre-treated DV-infected DC. Table 3, supra.

C. Discussion Gene expression analyses to identify new proteins involved in the cellular response to DV has been reported. Warke et al., "TRAIL is a novel anti-viral protein against dengue virus" J Virol. 82(1 ):555-564 (2008). These studies identified aa set of genes upregulated in response to DV infection in primary human cells in vitro. The present invention contemplates a method comprising an up-regulation of genes for TRAIL, IP-10,a nd MCP-2 when using DC, monocytes and HUVEC as in vitro cell models of infection. These same genes were screened in patient samples. Several chemokines have been reported to be induced in response to DV infection in vitro and in vivo. It has been suggested that the short-lived vascular leakage observed in DV infections in vivo is most likely mediated by a transiently released/produced soluble mediator(s). The effect of chemokines may not be restricted to the regulation of local trafficking of leukocytes, but also might have an effect on systemic targets. (Farber J. M., "Mig and IP-IO: CXC chemokines that target lymphocytes" J Leukoc Biol 61:246-257 (1997). Although it is not necessary to understand the mechanism of an invention, it is believed that a combination of different chemokines signaling through different receptors in different cells or tissues, rather than an individual molecule, determines the final outcome. Hence, it is believed that the over- expression of different chemokines and cytokines would probably contribute to the DV-induced immune-mediated pathology, including endothelial cell dysfunction.

The data presented herein, suggested that the chemokines MCP-2 and IP-IO might also be involved in these phenomena. Among the genes selected from gene expression analysis, MCP-2 has not previously been associated with DV infections. In one embodiment, the present invention contemplates a method for diagnosing DV-infected pateints, wherein the patients have an increased level of serum MCP-2 as compared to healthy subjects or patients with OFI.

Previous studies, however, have shown higher levels of MCP-I in patients with DHF. Although it is not necessary to understand the mechanism of an invention, it is believed that a mechanism of vascular leakage in an in vitro model using DV2- infected HUVECs was partially dependent on MCP-I. Lee et al., "MCP-I, a highly expressed chemokine in dengue haemorrhagic fever/dengue shock syndrome patients, may cause permeability change, possibly through reduced tight junctions of vascular endothelium cells" J Gen Virol 87:3623-3630 (2006).

It has been reported that MCP-I and MCP-2 are co-expressed, although MCP-I may be produced in higher quantities but lower concentrations of MCP-I are required to induce chemotaxis of monocytes and activated T lymphocytes. Proost et al., "Human monocyte chemotactic proteins-2 and -3: structural and functional comparison with MCP-I" J Leukoc Biol 59:61-1 A (1996). MCP-I and MCP-2 could have independent effects during DV infection. For example, MCP-I may bind only to CCR2 while MCP-2 is able to bind to CCRl, CCR2 and CCR5. Interestingly, a recent report showed that the MCP-2 - CCR2 interaction may counteract the activation signaling normally generated by other chemokines. O'Boyle et al., "Chemokine- mediated inflammation: Identification of a possible regulatory role for CCR2" MoI Immunol 44:1944-1953 (2007). Further, MCP-2 might contribute to the immune response to DV infection by engaging with different receptors in various cell types and/or suppressing the effect of other chemokines. IP-10 was observed to be among the highest upregulated genes in DCs and was also up- regulated in other primary cells in response to DV infection. The data presented herein showed increased levels of IP-10 in DV-infected patients. A tendency of higher levels of IP- 10 in DV-infected patients was also seen with hemorrhagic manifestations, but no differences in IP-10 levels were found in serum from patients with primary and secondary DENV infections. Recently, higher serum levels of EP-IO in DV infected patients were reported. Fink et al., Host gene expression profiling of dengue virus infection in cell lines and patients" PLoS Negl Trop Dis 1 :e86.(2007). The data presented herein is consistent with this study. Although it is not necessary to understand the mechanism of an invention, it is believed that IP-10 is induced by IFN-γ and EFN-α/β and is chemoattractant to activated T lymphocytes and NK cells. It is further believed that IP-10 has been found to be induced in viral infections, including DENV infection. Diago et al., "Association of pretreatment serum interferon gamma inducible protein 10 levels with sustained virological response to peginterferon plus ribavirin therapy in genotype 1 infected patients with chronic hepatitis C" Gut 55:374-379 (2006); Roe et al., "Elevated serum levels of interferon- gamma -inducible protein- 10 in patients coinfected with hepatitis C virus and HIV. J" Infect Dis 196: 1053-1057 (2007); and Chen et al., "Dengue virus induces expression of CXC chemokine ligand 10/EFNgamma- inducible protein 10, which competitively inhibits viral binding to cell surface heparan sulfate" J Immunol 177:3185-3192 (2006). It has been suggested that EP-IO may have a role in the protective immune response against DV, competing with the virus for the binding to heparan sulfate on the surface of Hepal-6 cells, and blocking entry and replication. Chen et al., 2006; and Hsieh et al., "Both CXCR3 and CXCLl 0/IFN inducible protein 10 are required for resistance to primary infection by dengue virus" J Immunol 177:1855-1863 (2006). On the other hand, high serum levels of IP-10 have been found in patients with chronic inflammatory conditions. Laine et al., "Palmitic acid induces IP-10 expression in human macrophages via NF-kappaB activation" Biochem Biophys Res Commun 358:150-155 (2007). Thus, EP-IO could have a generalized protective role against inflammation if produced in high amount and in an uncontrolled manner, and is not necessarily specific for DV infection.

DV infection gene expression analysis did not detect a significant increase of MEP- 1 β expression in various DV infected primary human cells. The data presented herein shows a slight increase of MIP-I β in serum from DV-infected patients. Additionally, these results in DV-infected DCs in vitro showed low levels of induction of MEP-I β mRNA in response to DV infection.

MIP- lβ induction has been reported in response to LPS, TNF-α, EFN-γ, and viral infections. Maurer et al., "Macrophage inflammatory protein-1" Int J Biochem Cell Biol 36:1882-1886 (2004) Further, it has been reported that MlPlβ induces chemotaxis of monocytes, T lymphocytes, NK cells and immature DCs. MEP- lβ expression was induced in response to DV infection in an in vitro model using the K562 cell line and also was found in PBMC isolated from DV-infected patients. Spain-Santana et al., "MIP-I alpha and MEP-I beta induction by dengue virus" J Med Virol 65:324-330. (2001). Nevertheless, the induction of MEP-I β in response to DV infection might be limited to certain cell types and to restricted or specialized areas. Hence, the systemic levels or cell-specific levels detected in the present data might underestimate the role of MEP-I β at areas where immune reactions take place.

The data presented herein also demonstrate the up-regulation ofTRAIL expression both in vitro, and in serum from DV-infected patients during the febrile period of the disease. A potent antiviral effect of TRAIL against DV infection in vitro was recently reported. (Warke et al., 2007, supra). The present data demonstrate that TRAIL pre-treatment not only suppresses DV levels, but also DV-mediated induction of inflammatory cytokines including, but not limited to, MCP-2 and EP-IO, as well as IL-6 (supra).

These results suggest a possible role ofTRAIL in the regulation of the inflammatory response triggered by DV, although the mechanism of action is not known. This hypothesis was further studied by examining the in vitro effect of rTRAIL on other cytokines induced by DV infection. For example, rTRAIL inhibited DV-induced mRNA levels of TNFα. EPlO, MEP- lβ, EP8, mda5, ERF7 and DV mRNA levels themselves. Figure 22. Consequently, the present invention contemplates a method of treating inflammation comprising administering rTRAIL. In the disclosed in vitro model for DV infection, the effect ofTRAIL as an antiviral could be responsible for the suppression of DV-induced chemokines and cytokines, as lower levels of virus might account for lower induction of soluble mediators. The antiviral responses to DV infection are reported to have a strong component of type-I IFN. Honda et al., "Type I interferon gene induction by the interferon regulatory factor family of transcription factors" Immunity 25:349-360 (2006). It has been reported that the induction of IFN-α/β in response to viral infection may involve the activation of cellular helicases (i.e., for example, RIG-I and MDA-5) which in turn activate IRF-3; this transcription factor regulates the expression of IFN-β. Then, IFN-β induces the expression of IRF-7, which is believed to be a regulator of the type-I IFN response and expression of IFN-α/β and type-I IFN-induced genes Fitzgerald-Bocarsly et al., "The role of type I interferon production by dendritic cells in host defense" Biochimie 89:843-855 (2007).

Nevertheless, rTRAIL was observed to suppress IFN-α production in response to DV- infection, and levels of IFN-α were strongly correlated with the percentage of infected DC. These observations may suggest that rTRAIL signalling is unique and different from either TNF- α and/or IFN-α. While TRAIL may induce apoptosis by binding to many TNF receptors including, but not limited to, TNFRSF 10 A/TRAILR1, TNFRSF 10B/TRAILR2,

TNFRSF 10C/TRAILR3, TNFRSFl 0D/TRAILR4 and possibly also to TNFRSFl 1B/OPG, its activity may be modulated by binding to decoy TNF receptors including, TNFRSF10C/TRAILR3, TNFRSF 10D/TRAILR4 and TNFRSFl 1B/OPG that cannot induce apoptosis. The data presented herein demonstrate that, as a whole, the expresison of TNF receptors is not largely modulated by the presence of either rTRAIL or DV infection. Figure 23.

In contrast, Interleukin 15 (IL- 15) and the IL- 15 receptors are selectively expressed in gene expression analysis when DCs are exposed to either rTRAIL or DV infection. Figure 24. This selective expression of IL-15 related genes were compared to the expression of thrombospondin receptors, CD36 and CD47 or Interleukin 6 (IL-6). Figure 25. These data show that niether the thrombospondin receptors, nor IL-6 were selectively expressed in response to either rTRAIL or DV infection. Thrombospondin- 1 binds to cell surface receptors including, CD36, CD47, some syndecans, LDL receptor-related protein- 1 (via calreticulin) and the integrins αV/β3. α3/βl, α4/βl and α6/βl. Thrombospondin 1 is believed to be a slow, tight inhibitor of plasmin, cathepsin.G, and neutrophil elastase. Further, it is believed that thrombospondin 1 directly binds and activates latent TGF-βi . Additionally, MDA-5 mRNA expression was suppressed about 60% and the expression of IRF-7 mRNA was suppressed about 40% following TRAIL pretreatment. These results suggest that a lower activation of the type-I IFN antiviral response occurs in TRAIL pre-treated DC, either as a result of an early and more efficient control of the infection or as an alternative antiviral pathway triggered by TRAIL. TRAIL pre-treatment could generate an "antiviral state" that might lower the requirement for IFN-α and type-I IFN-inducible genes for control of the infection. TRAIL could be exerting its effect downstream in the signaling pathways that lead to cytokine and chemokine expression or by activating parallel pathways of viral control.

Selective gene expression data collected herein has allowed the generation of a gene pathway analysis for TRAIL in both DCs and HUVECs. In both instances, the pathway construction was subject to genes that were induced at least two-fold by the presence of TRAIL. For example, a list of the 30 highest selectively expressed genes are presented. Figure 26.

For the DC analysis, 243 2-fold induced genes were evaluated by Pathway Architect^® (Aligent) such that a Relevance Network was generated where the genes are automatically ordered such that upstream genes are placed at the top of the network, and downstream genes are placed at the bottom of the network. Figure 27. The blue outlined genes represent those in the original 243 genes, whereas TNF was added by the software as a relevant gene.

For the FIUVEC analysis, 159 2-fold induced genes were evaluated by Pathway Architect^® (Aligent) such that a Relevance Network was generated. Figure 28. The blue outlined genes represent those in the original 159 genes, whereas TNF, NFKBl, LTA, MAPKl 4 and PRKCA were added by the software as relevant genes.

The differences between DCs and HUVECs were further compared by side-by-side gene expression analysis in response to TRAIL exposure. The comparison used 75 genes from HUVECs and 137 genes from DCs that showed an at least 2-fold TRAIL induction. The two gene lists were merged (i.e., creating 210 genes) and it was identified that IL-15 and 2'-5'- oligoadenylate synthetase 1 (OASl) were present in both lists. A gene profiling comparision confirms that IL — 15 and OASl were selectively expressed in both HUVEC and DCs by exogenous TRAIL. Figure 29 and Figure 30.

The data show that exogenous TRAIL treatment appears to cause tissue damage as there was selective gene expression for wounding response genes and apoptosis genes in both HUVEC and DCs. Figure 31. Their respective patterns explains the observations that a more limited inflammatory response is seen HUVECs, because only the DCs have chemokine and inflammatory response genes. These data are consistent earlier reports. Li et al., "TRAIL Induces Apoptosis and Inflammatory Gene Expression in Human Endothelial Cells" J Immunol 171:1526-1533 (2003). These genes are specifically identified and categorized. Figure 32. Although the individual genes differ, both cell types show a wounding and apoptosis response to exogenous TRAIL treatment.

Recently, the suppression of infection and cytokine production in dexamethasone-treated DV-infected monocytes was reported. Reis et al., "An in vitro model for dengue virus infection that exhibits human monocyte infection, multiple cytokine production and dexamethasone immunomodulation" Mem Inst Oswaldo Cruz 102:983-990 (2007). This study suggested that dexamethasone mediated inhibition of the nuclear factor KB (NF- KB) resulted in the suppression of cytokines, and may involve nitric oxide (NO) production as an antiviral mechanism. TRAIL can induce the production of NO in various in vitro models: i) up-regulation of the inducible nitric oxide synthase. Cantarella et al., "Trail interacts redundantly with nitric oxide in rat astrocytes: potential contribution to neurodegenerative processes" J Neuroimmunol 182:41-47 (2007); and ii) activation of the endothelial nitric oxide synthase (Zauli et al., "Tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL) sequentially upregulates nitric oxide and prostanoid production in primary human endothelial cells" Circ Res 92:732-740 (2003); and Di Pietro et al., "Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) regulates endothelial nitric oxide synthase (eNOS) activity and its localization within the human vein endothelial cells (HUVEC) in culture" J Cell Biochem 97:782-794 (2006).

Severe symptomology of dengue disease often occurs around the time of defervescence. Kalayanarooj et al., "Early clinical and laboratory indicators of acute dengue illness" J Infect Dis 176:313-321 (1997). Although it is not necessary to understand the mechanism of an invention, it is believed that higher levels of MCP-2 and IP-10, as well as other mediators, around the time of defervescence could have a role promoting and increasing the potentially damaging inflammatory response and contributing to the events that lead to endothelial cell dysfunction, vascular leakage and coagulation alterations. The tendency of higher IP-10 levels in patients with hemorrhagic manifestations supports this observation. A specific up-regulation of TRAIL expression in DV-infected patients would suggest a role in the control of DV virus. A tendency of higher serum TRAIL levels in primary infections deserves further investigation, using a larger cohort of patients; additionally, the analysis should be extended to patients with DF and DHF. Additionally, TRAIL-mediated in vitro suppression of some proinflammatory chemokines and cytokines, via decrease of the total virus burden or by directly regulating the synthesis of pro-inflammatory cytokines, suggests a potential therapeutic use for TRAIL in limiting the damaging inflammatory response triggered by DV infection. Overall, TRAIL emerges as a potential regulator not only for virus replication but also for proinflammatory chemokines and cytokines, and TRAIL could be useful as a therapeutic agent in DV infection. Although it is not necessary to understand the mechanism of an invention, it is believed that higher levels of proinflammatory mediators as well as negative regulators of the immune system are present in DV-infected patients and the balance among them would define the final outcome of the infection.

VI. Flavivirus Infections

Flaviriuses are a genus of the family Flaviviridae of single-stranded RNA viruses that are transmitted by arthropod vectors and especially by ticks and mosquitoes. Numerous diseases a caused by such virues including, but not limited to, dengue fever, Japanese B encephalitis, Saint Louis encephalitis, West Nile fever, West Nile encephalitis, West Nile meningitis Hantaan fever, Sin Nombre fever, and yellow fever. Some of these diseases have mild symptoms (i.e., for example, denge fever), others are fatal (i.e., for example, West Nile encephalitis). A. Dengue Fever (DF)

Dengue virus infection is an acute infection cleared approximately within one week (22). Dengue fever is a virus-based disease spread by mosquitoes. DF is caused by four different arboviruses (i.e., for example, Flaiviridae). DF spread by the bite of mosquitoes, most commonly the mosquito Aedes aegypti, which found in tropic and subtropic regions (i.e., for example, Southeast Asia, Indonesian archipelago into northeastern Australia, Sub-Saharan Africa, or South and Central America). Dengue fever, therefore, is also common among world travelers. DF generally lasts a week or more, is uncomfortable, but not deadly and a full recovery is usually expected. DF should not be confused with Dengue hemorrhagic fever, which is a separate disease and frequently deadly.

Dengue fever begins with a sudden high fever, often to 104-105 degrees Fahrenheit. A flat, red rash may appear over most of the body early during the fever. A second rash, measles- like in appearance, appears later in the disease. Infected people may have increased skin sensitivity and are very uncomfortable. Other symptoms of dengue fever include, but are not limited to, headache, joint aches, muscle aches, nausea, swollen lymph nodes, and/or vomiting.

Diagnostic testing that may be performed to diagnose this condition include, but are not limited to, complete blood count (CBC), serology studies to look for antibodies to flaviviruses, and/or antibody titer for flavivirus types (i.e., for example dengue viruses). Until the present invention, there was no specific treatment for dengue fever. Fluids are necessary if there are signs of dehydration. Acetaminophen (i.e., for example, Tylenol^®) is used to treat a high fever but aspirin should be avoided. B. West Nile Fever

West Nile virus was first identified in 1937 in Uganda in eastern Africa. It was first identified in the United States in the summer of 1999 in New York. Since then, the virus has spread throughout the United States. The West Nile virus is a type of organism called a flavivirus. Although it is not necessary to understand the mechanism of an invention, it is believed that West Nile virus is spread when a mosquito bites an infected bird and then bites a person. Mosquitos carry the highest amounts of West Nile virus in the early fall, which is why the rate of the disease increases in late August to early September. The risk of disease decreases as the weather becomes colder and mosquitos die off.

Mild, flu-like illness is often called West Nile fever. More severe forms of disease, which can be life threatening, may be called West Nile encephalitis or West Nile meningitis. Risk factors for developing a more severe form of West Nile virus include, but are not limited to, conditions that weaken the immune system, such as HIV, organ transplants, and recent chemotherapy; pregnancy; or advanced age.

West Nile virus may also be spread through blood transfusions and organ transplantation. It is possible for an infected mother to spread the virus to her child through breast milk. The mildest West Nile disease, is generally called West Nile fever, has some or all of the following symptoms: fever, headache, back pain, muscle aches, lack of appetite, sore throat, nausea, vomiting, abdominal pain, and diarrhea. These symptoms usually last for 3 to 6 days. The more severe West Nile diseases (i.e., for example, encephalitis and/or meningitis), may also have symptoms including, but not limited to: muscle weakness, stiff neck, confusion or change in clarity of thinking, or loss of consciousness. Further, a rash may be present in 20-50% of patients and true muscle weakness in the presence of other related symptoms is suggestive of a West Nile virus infection.

Tests to diagnose West Nile virus may include, but are not limited to, complete blood count, lumbar puncture and cerebrospinal fluid (CSF) testing, or head computer tomography (CT) and multiple resonance intensity (MRI) scanning. However, a definitive diagnosis may be obtained using a serology test, which checks a blood or CSF sample for antibodies against the virus. Alternatively, the virus can also be identified in body fluids using polymerase chain reaction (PCR). Antiviral drug treatments (i.e., for example, ribavirin) are still in the reseach phase, therefore standard care (i.e., bedrest, and/or fluids) are recommended to prevent secondary complications. Complications from mild West Nile virus infection are extremely rare. In contrast, complications from severe West Nile virus infection include permanent brain damage or muscle weakness (sometimes similar to polio), and death. In general, the likely outcome of a mild West Nile virus infection is excellent.

For patients with severe cases of West Nile virus infection, the outlook is more guarded. West Nile encephalitis or meningitis has the potential to lead to brain damage and death. Approximately 10% of patients with brain inflammation do not survive. C. Yellow Fever Yellow fever is a viral infection transmitted by mosquitoe bites that causes fever, jaundice, kidney failure, and bleeding. The responsible virus is believed to be a single-stranded RNA virus of the genus Flavivirus (species Yellow fever virus) transmitted especially by the yellow- fever mosquito - called also the yellow jack mosquito. The disease is most common in South America and in sub-Saharan Africa. Yellow fever ranges in severity. Severe infections with internal bleeding and fever (hemorrhagic fever) are deadly in 25 - 50% of cases. Anyone can get yellow fever, but the elderly have a higher risk of severe infection. If a person is bitten by an infected mosquito, symptoms usually develop 3 to 6 days later. Yellow fever can be divided into at least three stages: 1) Early stage: Headache, muscle aches, fever, loss of appetite, vomiting, and jaundice are common. After approximately 3 to 4 days, victims often experience brief remission; 2) Period of remission: After a few days (3 to 4) fever and other symptoms go away. Most individuals will recover at this stage, but others may move onto the third, most dangerous stage (intoxication stage) within 24 hours; 3) Period of intoxication: Multi- organ dysfunction occurs. This includes, but is not limited to, liver and kidney failure, bleeding disorders/hemorrhage, brain dysfunction including, but not limited to, delirium, seizures, coma, shock, and death.

In general yellow fever symptoms include, but are not limited to, fever, headache, muscle aches (myalgia), vomiting, red eyes, red face, red tongue, jaundice, bleeding and/or hemorrhage, decreased urination, arrhythmias, heart dysfunction, vomiting blood, delirium, seizures, or coma. A person with advanced yellow fever may also show signs of liver failure, renal failure, and shock. A symptmatic diagnosis may be confirmed by blood tests that reveal the virus, viral antigens, or antibodies.

Currently, there is no specific treatment for yellow fever. Treatment for symptoms may include intravenous fluids, blood products for severe bleeding, and dialysis for renal failure. Further secondary complications may occur, including but not limited to, kidney failure, disseminated intravascular coagulation (DIC), secondary bacterial infections, liver failure, parotitis, shock, coma, or death. D. Encephalitis

Encephalitis is an inflammation (irritation and swelling) of the brain, usually caused by infections. Encephalitis is most often caused by a viral infection, and many types of viruses may cause it. Exposure to viruses can occur through insect bites, food or drink contamination, inhalation of respiratory droplets from an infected person, or skin contact. In rural areas, arboviruses (i.e., for example, flaviviruses such as Japanese B virus and/or Saint Louis virus) ~ carried by mosquitoes or ticks, or accidentally ingested, are the most common cause. Encephalitis is relatively uncommon but still affects approximately 1,500 people per year in the U.S. The elderly and infants are more vulnerable and may have a more severe course of the disease. Once an encephalitis virus has entered the bloodstream, it may localize in the brain, causing inflammation of brain tissue and surrounding membranes. White blood cells invade the brain tissue as they try to fight off the infection. The brain tissue swells (cerebral edema), which may cause destruction of nerve cells, bleeding within the brain (intracerebral hemorrhage), and brain damage.

Encephalitis symptoms may include, but are not limited to, fever, headache, vomiting, light-sensitivity, stiff neck and/or back, confusion, disorientation, drowsiness, clumsiness, unsteady gait, irritability, or poor temper control. More serious symptoms can also develop including, but not limited to, loss of consciousness, poor responsiveness, stupor, coma, seizures, muscle weakness and/or paralysis, memory loss (amnesia), impaired short-term memory or impaired long-term memory. Some behavioural symptoms may also be present including, but not limited to, a "flat" mood or lack of discernible mood, or mood inappropriate for the situation, diminished interest in daily activities, inflexibility, extreme self-centeredness, indecisiveness, withdrawal from social interaction, or impaired judgment An examination may show signs of meningeal irritation (especially neck stiffness), increased intracranial pressure, or other neurologic symptoms such as muscle weakness, mental confusion, speech problems, and abnormal reflexes. The patient may have a skin rash, mouth ulcers, and signs of involvement of other organs such as the liver and lungs. A lumbar puncture test and cerebrospinal fluid (CSF) examination may show clear fluid, high pressure, high white blood cell count and high protein levels ~ indications of inflammation. Blood may be present in the CSF.

Sometimes the virus can be detected in CSF, blood, or urine through a laboratory test called viral culture. In some cases, viral PCR (polymerase chain reaction, a test able to detect very tiny amounts of viral DNA) may identify the virus. Serology tests may also provide evidence of viral infection. Alternatively, an electroencephalogram (EEG) may provide indirect clues for the diagnosis of encephalitis. Some EEG wave patterns may suggest a seizure disorder, or point to a specific virus as cause of the infection. Certain EEG wave patterns can suggest encephalitis due to herpes, for instance. A brain MRI, which provides high-quality pictures of the brain, or a CT scan of the head may be used to determine internal bleeding or specific areas of brain inflammation.

Presently, no specific antiviral drugs are available to combat the infection. Consequently, supportive care (i.e., for example, rest, nutrition, and/or fluids) is usually provided to relieve symptoms. Antiviral medications, such as acyclovir (Zovirax) and foscarnet (Foscavir), may be useful but are not clinically effective.

The outcome viral encephalitis infections varies. Some cases are mild, short, and relatively harmless, followed by full recovery. Other cases are severe, and permanent impairment or death is possible. The acute phase normally lasts for 1 to 2 weeks, with gradual or sudden disappearance of fever and neurologic symptoms. Neurologic symptoms may require many months before full recovery.

VII. Bunyavirus Infections

The virus family Bunyaviridae, are rodent-borne negative-stranded RNA viruses. Members of the genus Hantavirus have been identified as etiologic agents of two severe human diseases: hemorrhagic fever with renal syndrome (HFRS), which is caused by the Old World hantaviruses, and hantavirus pulmonary syndrome (HPS), which is caused by the New World hantaviruses. Case fatality is considerably higher for HPS (up to 40%) than for HFRS (between 0.1 and 15%). The major target in human hantavirus infection is the microvascular endothelium, and severe hantavirus disease in humans has been attributed to microvascular leakage. Several Old and New World hantaviruses have not been associated with any human disease. The basis for disease in humans, and differences between pathogenic and nonpathogenic hantaviruses, remains unclear; however, innate immune responses likely plays a role. A. Hantaan Fever

Hantaan fever (also known as Hantavirus disease) characterized by symptoms that resemble the flu, followed by respiratory failure. Hantaan fever is a potentially fatal respiratory illness first identified in the United States Southwest. Since that discovery, hantavirus disease has been reported in every western state, and in many eastern states.

Hantavirus is carried by rodents, particularly deer mice, and is present in their urine and feces. The virus does not cause disease in the carrier animal. Humans are thought to become infected when they are exposed to contaminated dust from the nests or droppings of mice. The disease is not, however, passed between humans. Contaminated dust is often encountered when cleaning long-vacated dwellings, sheds, or other enclosed areas. Initial symptoms of hantavirus disease closely resemble the flu. The disease begins abruptly with fever, chills, muscle aches , headache, nausea and vomiting, and malaise. A dry cough may be present. The fever may be higher in younger people than in older people.

For a very short period, the infected person feels somewhat better, but this is followed within a day or two by an increased respiratory rate caused by a seepage of fluid into the lungs. The initial shortness of breath is subtle and the patient may be unaware of it, but progression is rapid. The patient ultimately develops respiratory failure.

An effective treatment for hantavirus is not yet available. Even with intensive therapy, more than half of the diagnosed cases have been fatal. Hantaan virus symptoms may include, but are not limited to, chills, dry cough, fever, general ill feeling (malaise), headache, muscle aches, rapid shallow breathing, respiratory failure, or shortness of breath. Other indications of hantaan virus infection may include, but are not limited to, hypoxia, hypotension, or acute respiratory distress syndrome.

Diagnostic tests for Hantaan fever include, but are not limited to, complete blood count (i.e., for example, elevated white blood cells); platelet count (i.e., for example, < 150,000 and decreasing), chest X-ray (i.e., for example, lung tissue invasion/infiltration), liver enzymes (i.e., for example, LDH elevation), decreased serum albumin, increased hematocrit, serological testing for hantavirus presence.

Because the breathing problems progress rapidly, cardiorespiratory failure is common associated with a high death rate (i.e., for example over 50%). Oxygen therapy is used with respiratory support from a breathing tube (i.e., for example, an endotracheal tube) and/or ventilator.

B. Sin Nombre Fever

Sin Nombre virus causes the majority of Hantavirus pulmonary syndrome (HPS cases) in the United States, and the deer mouse (Peromyscus maniculatus) is its predominant reservoir. HPS is a rodentborne viral disease characterized by severe pulmonary illness and a case-fatality ratio of 30%-40%.

HPS is characterized by a febrile illness (i.e., temperature >101.0°F) associated with bilateral diffuse interstitial edema of the lungs developing within 72 hours of hospitalization in a previously healthy person; radiographically, the edema can resemble acute respiratory distress syndrome (1). Annually, the majority of HPS cases occur in spring and summer; however, the seasonality of HPS can vary by elevation, location, and biome, and cases have been identified throughout the winter and early spring. Since recognition of the disease in 1993, CDC has confirmed 438 cases of HPS reported from 30 states among residents of 32 states; 35% (154) of these cases were fatal. HPS typically begins as headache, fever, and myalgia and is soon followed by pulmonary edema, which often leads to severe respiratory compromise; thrombocytopenia, presence of immunoblasts, and hemoconcentration are characteristic laboratory findings (1). Other than supportive care, no treatment exists for hantavirus infection.

VIII. Expression Platforms

The present invention provides recombinant expression vectors for expression of TRAIL, and host cells transformed with the expression vectors. Any suitable expression system may be employed. The vectors include a DNA encoding a TRAIL polypeptide, operably linked to suitable transcriptional or translational regulatory nucleotide sequences, such as those derived from a mammalian, microbial, viral, or insect gene. Examples of regulatory sequences include transcriptional promoters, operators, or enhancers, an mRNA ribosomal binding site, and appropriate sequences which control transcription and translation initiation and termination. Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the TRAIL DNA sequence. Thus, a promoter nucleotide sequence is operably linked to an TRAIL DNA sequence if the promoter nucleotide sequence controls the transcription of the

TRAIL DNA sequence. An origin of replication that confers the ability to replicate in the desired host cells, and a selection gene by which transformants are identified, are generally incorporated into the expression vector.

In addition, a sequence encoding an appropriate signal peptide can be incorporated into expression vectors. A DNA sequence for a signal peptide (secretory leader) may be fused in frame to the TRAIL sequence so that the TRAIL is initially translated as a fusion protein comprising the signal peptide. A signal peptide that is functional in the intended host cells promotes extracellular secretion of the TRAIL polypeptide. The signal peptide is cleaved from the TRAIL polypeptide upon secretion of TRAIL from the cell. Suitable host cells for expression of TRAIL polypeptides include prokaryotes, yeast or higher eukaryotic cells. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described, for example, in Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., (1985). Cell-free translation systems could also be employed to produce TRAIL polypeptides using RNAs derived from DNA constructs disclosed herein. Prokaryotes include gram negative or gram positive organisms, for example, E. coli or

Bacilli. Suitable prokaryotic host cells for transformation include, for example, E. coli, Bacillus subtilis, Salmonella typhimurium, and various other species within the genera Pseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic host cell, such as E. coli, a TRAIL polypeptide may include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in the prokaryotic host cell. The N-terminal Met may be cleaved from the expressed recombinant TRAIL polypeptide.

Expression vectors for use in prokaryotic host cells generally comprise one or more phenotypic selectable marker genes. A phenotypic selectable marker gene is, for example, a gene encoding a protein that confers antibiotic resistance or that supplies an autotrophic requirement. Examples of useful expression vectors for prokaryotic host cells include those derived from commercially available plasmids such as the cloning vector pBR.322 (ATCC 37017). pBR.322 contains genes for ampicillin and tetracycline resistance and thus provides simple means for identifying transformed cells. An appropriate promoter and a TRAIL DNA sequence are inserted into the pBR.322 vector. Other commercially available vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEMl (Promega Biotec, Madison, Wis., USA).

Promoter sequences commonly used for recombinant prokaryotic host cell expression vectors include p-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281:544, 1979), tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057, 1980; and EP-A-36776) and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412, 1982). A particularly useful prokaryotic host cell expression system employs a phage λ P_L promoter and a cI857ts thermolabile repressor sequence. Plasmid vectors available from the American Type Culture Collection which incorporate derivatives of the .lambda, λ P_L promoter include plasmid pHUB2 (resident in E. coli strain JMB9, ATCC 37092) and pPLc28 (resident in E. coli RRl, ATCC 53082). TRAIL alternatively may be expressed in yeast host cells, preferably from the Saccharomyces genus (e.g., S. cerevisiae). Other genera of yeast, such as Pichia or Kluyveromyces, may also be employed. Yeast vectors will often contain an origin of replication sequence from a 2μ yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene. Suitable promoter sequences for yeast vectors include, among others, promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900, 1978), such as enolase, glyceraldehyde-3 -phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3- phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other suitable vectors and promoters for use in yeast expression are further described in Hitzeman, EPA-73,657. Another alternative is the glucose-repressible ADH2 promoter described by Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier et al. (Nature 300:724, 1982). Shuttle vectors replicable in both yeast and E. coli may be constructed by inserting DNA sequences from pBR322 for selection and replication in E. coli (Amp^r gene and origin of replication) into the above-described yeast vectors.

The yeast α- factor leader sequence may be employed to direct secretion of the TRAIL polypeptide. The α-factor leader sequence is often inserted between the promoter sequence and the structural gene sequence. See, e.g., Kurjan et al., Cell 30:933, 1982 and Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330, 1984. Other leader sequences suitable for facilitating secretion of recombinant polypeptides from yeast hosts may also be used. Further, a leader sequence may be modified near its 3' end to contain one or more restriction sites. This will facilitate fusion of the leader sequence to the structural gene. Yeast transformation protocols are described. Hinnen et al., Proc. Natl. Acad. Sci. USA

75:1929, 1978. The Hinnen et al. protocol selects for Trp⁺ transformants in a selective medium, wherein the selective medium consists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 μg/ml adenine and 20 μg/ml uracil.

Yeast host cells transformed by vectors containing an ADH2 promoter sequence may be grown for inducing expression in a "rich" medium. An example of a rich medium is one consisting of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 .mu.g/ml adenine and 80 μg/ml uracil. Derepression of the ADH2 promoter occurs when glucose is exhausted from the medium.

Mammalian or insect host cell culture systems could also be employed to express recombinant TRAIL polypeptides. Bacculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988). Established cell lines of mammalian origin also may be employed. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23:175, 1981), L cells, C 127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, and BHK (ATCC CRL 10) cell lines, and the CVI/EBNA cell line derived from the African green monkey kidney cell line CVI (ATCC CCL 70) as described by McMahan et al. (EMBO J. 10: 2821, 1991).

Transcriptional and translational control sequences for mammalian host cell expression vectors may be excised from viral genomes. Commonly used promoter sequences and enhancer sequences are derived from Polyoma virus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites may be used to provide other genetic elements for expression of a structural gene sequence in a mammalian host cell. Viral early and late promoters are particularly useful because both are easily obtained from a viral genome as a fragment which may also contain a viral origin of replication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40 fragments may also be used, provided the approximately 250 bp sequence extending from the Hind III site toward the BgII site located in the SV40 viral origin of replication site is included.

Expression vectors for use in mammalian host cells can be constructed as disclosed by Okayama and Berg (MoI. Cell. Biol. 3:280, 1983), for example. A useful system for stable high level expression of mammalian cDNAs in C 127 murine mammary epithelial cells can be constructed substantially as described by Cosman et al. (MoI. Immunol. 23:935, 1986). A high expression vector, PMLSV N1/N4, described by Cosman et al., Nature 312:768, 1984 has been deposited as ATCC 39890. Additional mammalian expression vectors are described in EP-A- 0367566, and in WO 91/18982. As one alternative, the vector may be derived from a retrovirus. Additional suitable expression systems are described in the examples below. One preferred expression system employs Chinese hamster ovary (CHO) cells and an expression vector designated PG5.7. This expression vector is described in U.S. patent application Ser. No. 08/586,509, filed Jan. 11, 1996, which is hereby incorporated by reference. PG5.7 components include a fragment of CHO cell genomic DNA, followed by a CMV -derived promoter, which is followed by a sequence encoding an adenovirus tripartite leader, which in turn is followed by a sequence encoding dihydrofolate reductase (DHFR). These components were inserted into the plasmid vector pGEMl (Promega, Madison, Wis.). DNA encoding a TRAIL polypeptide (or fusion protein containing TRAIL) may be inserted between the sequences encoding the tripartite leader and DHFR. Methotrexate may be added to the culture medium to increase expression levels.

The fragment of CHO cell genomic DNA in vector PG5.7 enhances expression of TRAIL. A phage lysate containing a fragment of genomic DNA isolated from CHO cells was deposited with the American Type Culture Collection on Jan. 4, 1996, and assigned accession number ATCC 97411. Vector PG5.7 contains nucleotides 8671 through 14507 of the CHO genomic DNA insert in strain deposit ATCC 97411.

For expression of TRAIL, a type II protein lacking a native signal sequence, a heterologous signal sequence or leader functional in mammalian host cells may be added. Examples include the signal sequence for interleukin-7 (IL-7) described in U.S. Pat. No. 4,965,195, the signal sequence for interleukin-2 receptor described in Cosman et al., Nature 312:768 (1984); the interleukin-4 receptor signal peptide described in EP 367,566; the type I interleukin-1 receptor signal peptide described in U.S. Pat. No. 4,968,607; and the type I interleukin-1 receptor signal peptide described in EP 460,846.

A preferred expression system employs a leader sequence derived from cytomegalovirus (CMV). For example, mammalian host cells may be transformed with an expression vector encoding the peptide Met- Ala- Arg-Arg-Leu-Trp-Ile-Leu-Ser- Leu-Leu- Ala- Val-Thr-Leu-Thr- Val-Ala-Leu-Ala-Ala-Pro-Ser-Gln-Lys-Ser-Lys-Arg-Arg-Thr-Ser-Ser (SEQ ID NO:9) fused to the N-terminus of an octapeptide designated FLAG^® (SEQ ID NO:7), which in turn is fused to the N-terminus of a soluble TRAIL polypeptide. Residues 1 through 29 of SEQ ID NO:9 constitute a CMV-derived leader sequence, whereas residues 30 through 32 are encoded by oligonucleotides employed in constructing an expression vector. In one embodiment, DNA encoding a poly-His peptide (e.g., a peptide containing six histidine residues) is positioned between the sequences encoding the CMV leader and the FLAG^® peptide.

Expression systems that employ such CMV-derived leader peptides are useful for expressing proteins other than TRAIL. Expression vectors comprising a DNA sequence that encodes amino acids 1 through 29 of SEQ ID NO:9 are provided herein. In another embodiment, the vector comprises a sequence that encodes amino acids 1 through 28 of SEQ ID NO:9. DNA encoding a desired heterologous protein is positioned downstream of, and in the same reading frame as, DNA encoding the leader. Additional residues (e.g., those encoded by linkers or primers) may be encoded by DNA positioned between the sequences encoding the leader and the desired heterologous protein. Expression vectors may also comprise promoters and any other desired regulatory sequences, operably linked to the sequences encoding the leader and heterologous protein.

The leader peptide presented in SEQ ID NO:9 may be cleaved after the arginine residue at position 29 to yield the mature secreted form of a protein fused thereto. Alternatively or additionally, cleavage may occur between amino acids 20 and 21, or between amino acids 28 and 29, ofSEQ ID NO:9.

Position(s) at which a signal peptide is cleaved may vary according to such factors as the type of host cells employed, whether murine or human TRAIL is expressed by the vector, and the like. Analysis by computer program reveals that the primary cleavage site may be between residues 20 and 21 of SEQ ID NO:9. Cleavage between residues 22 and 23, and between residues 27 and 28, is predicted to be possible, as well. To illustrate, expression and secretion of a soluble murine TRAIL polypeptide resulted in cleavage of a CMV-derived signal peptide at multiple positions. The three most prominent species of secreted protein (in descending order) resulted from cleavage between amino acids 20 and 21 of SEQ ED NO:9, cleavage between amino acids 22 and 23, and cleavage between amino acids 27 and 28.

A method for producing a heterologous recombinant protein involves culturing mammalian host cells transformed with such an expression vector under conditions that promote expression and secretion of the heterologous protein, and recovering the protein from the culture medium. Expression systems employing CMV leaders may be used to produce any desired protein, examples of which include, but are not limited to, colony stimulating factors, interferons, interleukins, other cytokines, and cytokine receptors. E. coli strain DHlOB cells can be transformed with a recombinant vector containing this human TRAIL DNA. (ATCC, Accession No. 69849. Such a recombinant vector may comprise expression vector pDC409. Vectors may be digested with Sail and Notl, and human TRAIL DNA that includes the entire coding region shown in SEQ ID NO:1 was ligated into the vector. The present invention further includes TRAIL polypeptides with or without associated native- pattern glycosylation. TRAIL expressed in yeast or mammalian expression systems may be similar to or significantly different from a native TRAIL polypeptide in molecular weight and glycosylation pattern, depending upon the choice of expression system.

Expression of TRAIL polypeptides in bacterial expression systems, such as E. coli, provides non-glycosylated molecules. Glycosylation sites in the TRAIL extracellular domain can be modified to preclude glycosylation while allowing expression of a homogeneous, reduced carbohydrate analog using yeast or mammalian expression systems. N-glycosylation sites in eukaryotic polypeptides are characterized by an amino acid triplet Asn-X-Y, wherein X is any amino acid except Pro and Y is Ser or Thr. Appropriate modifications to the nucleotide sequence encoding this triplet will result in substitutions, additions or deletions that prevent attachment of carbohydrate residues at the Asn side chain. Known procedures for inactivating N-glycosylation sites in proteins include those described in U.S. Pat. No. 5,071,972 and EP 276,846. A potential N-glycosylation site is found at positions 109-111 in the human protein of SEQ ED NO:2 and at positions 52-54 in the murine protein of SEQ ID NO:6.

IX. Protein Purification

The present invention provides purified TRAIL proteins, which may be produced by recombinant expression systems as described above or purified from naturally occurring cells. The desired degree of purity may depend on the intended use of the protein. A relatively high degree of purity is desired when the protein is to be administered in vivo, for example.

Advantageously, TRAIL polypeptides are purified such that no protein bands corresponding to other proteins are detectable by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). As demonstrated herein, multiple bands corresponding to TRAIL protein may be detected by SDS- PAGE, due to differential glycosylation, variations in post-translational processing. In one embodiment, an SDS-PAGE detection provides purified TRAIL protein when no visual bands corresponding to different (non-TRAIL) proteins are visualized. TRAIL most preferably is purified to substantial homogeneity, as indicated by a single protein band upon analysis by SDS- PAGE. The protein band may be visualized by silver staining, Coomassie blue staining, or (if the protein is radiolabeled) by autoradiography.

One process for producing the TRAIL protein comprises culturing a host cell transformed with an expression vector comprising a DNA sequence that encodes TRAIL under conditions such that TRAIL is expressed. The TRAIL protein is then recovered from the culture (from the culture medium or cell extracts). Procedures for purifying the recombinant TRAIL will vary according to such factors as the type of host cells employed and whether or not the TRAIL is secreted into the culture medium. For example, when expression systems that secrete the recombinant protein are employed, the culture medium first may be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a purification matrix such as a gel filtration medium. Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred. Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, (e.g., silica gel having pendant methyl or other aliphatic groups) can be employed to further purify TRAIL. Some or all of the foregoing purification steps, in various combinations, can be employed to provide a purified TRAIL protein.

Recombinant protein produced in bacterial culture may be isolated by initial disruption of the host cells, centrifugation, extraction from cell pellets if an insoluble polypeptide, or from the supernatant fluid if a soluble polypeptide, followed by one or more concentration, salting-out, ion exchange, affinity purification or size exclusion chromatography steps. Finally, RP-HPLC can be employed for final purification steps. Microbial cells can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Transformed yeast host cells are preferably employed to express TRAIL as a secreted polypeptide. This simplifies purification. Secreted recombinant polypeptide from a yeast host cell fermentation can be purified by methods analogous to those disclosed by Urdal et al. (J. Chromatog. 296:171, 1984). Urdal et al. describe two sequential, reversed-phase HPLC steps for purification of recombinant human IL-2 on a preparative HPLC column.

Alternatively, TRAIL polypeptides can be purified by immunoaffinity chromatography. An affinity column containing an antibody that binds TRAIL may be prepared by conventional procedures and employed in purifying TRAIL.

X. Fusion Proteins

The present invention also provides fusion proteins incorporating all or part of a TRAIL protein. Accordingly, in some embodiments of the present invention, the coding sequences for the polypeptides can be incorporated as a part of a fusion gene including a nucleotide sequence encoding a different polypeptide. TRAIL polypeptide fusions can comprise peptides added to facilitate purification and identification of TRAIL. Such peptides include, for example, poly-His or the antigenic identification peptides described in U.S. Pat. No. 5,011,912 and in Hopp et al., Bio/Technology 6:1204, 1988. One such peptide is the FLAG^® peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK) (SEQ ID NO:7), which is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody, thus enabling rapid assay and facile purification of expressed recombinant protein. This sequence is also specifically cleaved by bovine mucosal enterokinase at the residue immediately following the Asp-Lys pairing. Fusion proteins capped with this peptide may also be resistant to intracellular degradation in E. coli.

Various techniques for making fusion genes have been reported. Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment of the present invention, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, in other embodiments of the present invention, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed to generate a chimeric gene sequence (See e.g., Current Protocols in Molecular Biology, supra).

The above-described TRAIL proteins that can be used in the present invention, may be produced as fusion proteins, constituting a functional variant of one of the previously described proteins or a functional variant only after the fusion moiety has been eliminated. These fusion proteins include, in particular, fusion proteins that have a content of about 1 - 300 foreign amino acids, preferably about 1 - 200 foreign amino acids, particularly preferably about 1 - 150 foreign amino acids, more preferably about 1 - 100 foreign amino acids, and most preferably about 1 - 50 foreign amino acids. Such foreign amino acid sequences may be prokaryotic peptide sequences that can be derived, for example, from E. CoIi β-galactosidase.

Other examples of peptide sequences for fusion proteins are peptides that facilitate detection of the fusion protein; they include, but are not limited to, green fluorescent protein or variants thereof. It is also possible to add on at least one "affinity tag" or "protein tag" for the purpose of purifying the previously described proteins. For example, suitable affinity tags enable the fusion protein to be absorbed with high specificity and selectivity to a matrix. This attachment step is then followed by stringent washing with suitable buffers without eluting the fusion protein to any significant extent, and specific elution of the absorbed fusion protein. Examples of the protein tags include, but are not limited to, a (His)₆ tag, a Myc tag, a FLAG tag, a hemagglutinin tag, a glutathione-S-transferase (GST) tag, a tag consisting of an intein flanked by an affinity chitin-binding domain, and a maltose-binding protein (MBP) tag. These protein tags can be located N-terminally, C-terminally and/or internally.

The proteins that can be used in the methods and compositions of the present invention can also be prepared synthetically. Thus, the entire polypeptide, or parts thereof, can, for example, be produced by classical synthesis techniques (e.g., Merrifield technique). Particular preference is given to using polypeptides which have been prepared recombinantly using one of the previously described nucleic acids. Furthermore, proteins of the present invention can be isolated from an organism or from tissue or cells for use in accordance with the present invention. Thus, it is possible, for example, to purify proteins, which can be used in the present invention, from human serum. Abdullah et al., Arch. Biochem. Biophys., 225:306 312 (1983). Furthermore, it is possible to prepare cell lines expressing the proteins of the present invention. These cell lines can then be used for isolating the proteins of interest. Suitable systems for production of recombinant proteins include but are not limited to prokaryotic (e.g., Escherichia coli), yeast (e.g., Saccaromyces cerevisiae), insect (e.g., baculovirus), mammalian (e.g., Chinese hamster ovary), plant (e.g., safflower), and cell-free systems (e.g., rabbit reticulocyte).

XII. Detection Methodologies

A. Detection of RNA

In some embodiments, detection of a virus infection comprises measuring the expression of corresponding mRNA in a biological sample (i.e., for example, a blood sample). mRNA expression may be measured by any suitable method, including but not limited to, those disclosed below.

In some embodiments, RNA is detection by Northern blot analysis. Northern blot analysis involves the separation of RNA and hybridization of a complementary labeled probe.

In other embodiments, RNA expression is detected by enzymatic cleavage of specific structures (INVADER assay, Third Wave Technologies; See e.g., U.S. Pat. Nos. 5,846,717, 6,090,543; 6,001,567; 5,985,557; and 5,994,069; each of which is herein incorporated by reference). The INVADER assay detects specific nucleic acid (e.g., RNA) sequences by using structure-specific enzymes to cleave a complex formed by the hybridization of overlapping oligonucleotide probes. In still further embodiments, RNA (or corresponding cDNA) is detected by hybridization to a oligonucleotide probe. A variety of hybridization assays using a variety of technologies for hybridization and detection are available. For example, in some embodiments, TaqMan assay (PE Biosystems, Foster City, Calif.; See e.g., U.S. Pat. Nos. 5,962,233 and 5,538,848, each of which is herein incorporated by reference) is utilized. The assay is performed during a PCR reaction. The TaqMan assay exploits the 5'-3' exonuclease activity of the AMPLITAQ GOLD DNA polymerase. A probe consisting of an oligonucleotide with a 5'-reporter dye (e.g., a fluorescent dye) and a 3 '-quencher dye is included in the PCR reaction. During PCR, if the probe is bound to its target, the 5'-3' nucleolytic activity of the AMPLITAQ GOLD polymerase cleaves the probe between the reporter and the quencher dye. The separation of the reporter dye from the quencher dye results in an increase of fluorescence. The signal accumulates with each cycle of PCR and can be monitored with a fluorimeter. In yet other embodiments, reverse-transcriptase PCR (RT-PCR) is used to detect the expression of RNA. In RT-PCR, RNA is enzymatically converted to complementary DNA or "cDNA" using a reverse transcriptase enzyme. The cDNA is then used as a template for a PCR reaction. PCR products can be detected by any suitable method, including but not limited to, gel electrophoresis and staining with a DNA specific stain or hybridization to a labeled probe. In some embodiments, the quantitative reverse transcriptase PCR with standardized mixtures of competitive templates method described in U.S. Pat. Nos. 5,639,606, 5,643,765, and 5,876,978 (each of which is herein incorporated by reference) is utilized. B. Detection of Protein In other embodiments, gene expression in virus infected tissues may be detected by measuring the expression of a protein or polypeptide. Protein expression may be detected by any suitable method. In some embodiments, proteins are detected by immunohistochemistry. In other embodiments, proteins are detected by their binding to an antibody raised against the protein. The generation of antibodies is described below. Antibody binding may be detected by many different techniques including, but not limited to, (e.g., radioimmunoassay, ELISA (enzyme- linked immunosorbant assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (e.g., using colloidal gold, enzyme or radioisotope labels, for example), Western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays, etc.), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.

In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled.

In some embodiments, an automated detection assay is utilized. Methods for the automation of immunoassays include those described in U.S. Pat. Nos. 5,885,530, 4,981,785, 6,159,750, and 5,358,691, each of which is herein incorporated by reference. In some embodiments, the analysis and presentation of results is also automated. For example, in some embodiments, software that generates a prognosis based on the presence or absence of a series of proteins corresponding to virus induced markers is utilized. In other embodiments, the immunoassay described in U.S. Pat. Nos. 5,599,677 and 5,672,480; each of which is herein incorporated by reference.

C. Remote Detection Systems

In some embodiments, a computer-based analysis program is used to translate the raw data generated by the detection assay (e.g., the presence, absence, or amount of a given marker or markers) into data of predictive value for a clinician. The clinician can access the predictive data using any suitable means. Thus, in some preferred embodiments, the present invention provides the further benefit that the clinician, who is not likely to be trained in genetics or molecular biology, need not understand the raw data. The data is presented directly to the clinician in its most useful form. The clinician is then able to immediately utilize the information in order to optimize the care of the subject.

The present invention contemplates any method capable of receiving, processing, and transmitting the information to and from laboratories conducting the assays, wherein the information is provided to medical personal and/or subjects. For example, in some embodiments of the present invention, a sample (e.g., a biopsy or a serum or urine sample) is obtained from a subject and submitted to a profiling service (e.g., clinical lab at a medical facility, genomic profiling business, etc.), located in any part of the world (e.g., in a country different than the country where the subject resides or where the information is ultimately used) to generate raw data. Where the sample comprises a tissue or other biological sample, the subject may visit a medical center to have the sample obtained and sent to the profiling center, or subjects may collect the sample themselves (e.g., a urine sample) and directly send it to a profiling center. Where the sample comprises previously determined biological information, the information may be directly sent to the profiling service by the subject (e.g., an information card containing the information may be scanned by a computer and the data transmitted to a computer of the profiling center using an electronic communication systems). Once received by the profiling service, the sample is processed and a profile is produced (i.e., expression data), specific for the diagnostic or prognostic information desired for the subject.

The profile data is then prepared in a format suitable for interpretation by a treating clinician. For example, rather than providing raw expression data, the prepared format may represent a diagnosis or risk assessment (e.g., likelihood of a virus infection) for the subject, along with recommendations for particular treatment options. The data may be displayed to the clinician by any suitable method. For example, in some embodiments, the profiling service generates a report that can be printed for the clinician (e.g., at the point of care) or displayed to the clinician on a computer monitor.

In some embodiments, the information is first analyzed at the point of care or at a regional facility. The raw data is then sent to a central processing facility for further analysis and/or to convert the raw data to information useful for a clinician or patient. The central processing facility provides the advantage of privacy (all data is stored in a central facility with uniform security protocols), speed, and uniformity of data analysis. The central processing facility can then control the fate of the data following treatment of the subject. For example, using an electronic communication system, the central facility can provide data to the clinician, the subject, or researchers.

In some embodiments, the subject is able to directly access the data using the electronic communication system. The subject may chose further intervention or counseling based on the results. In some embodiments, the data is used for research use. For example, the data may be used to further optimize the inclusion or.elimination of markers as useful indicators of a particular condition or stage of disease. D. Detection Kits

In other embodiments, the present invention provides kits for the detection and characterization of virus infections. In some embodiments, the kits contain antibodies specific for a protein expressed as a result of a virus infection, in addition to detection reagents and buffers. In other embodiments, the kits contain reagents specific for the detection of mRNA or cDNA (e.g., oligonucleotide probes or primers). In preferred embodiments, the kits contain all of the components necessary to perform a detection assay, including all controls, directions for performing assays, and any necessary software for analysis and presentation of results.

XII. Antibodies

The present invention provides isolated antibodies (i.e., for example, polyclonal or monoclonal). In one embodiment, the present invention provides monoclonal antibodies that specifically bind to an isolated polypeptide comprised of at least five amino acid residues of the gene expression profile proteins described herein (e.g., TRAIL). These antibodies find use in the detection methods described above. A murine hybridoma designated 4El 1 produces a monoclonal antibody that binds the peptide DYKDDDDK (SEQ ID N0:7) in the presence of certain divalent metal cations (as described in U.S. Pat. No. 5,011,912), and has been deposited with the American Type Culture Collection under Accession No HB 9259. Expression systems useful for producing recombinant proteins fused to the FLAG^® peptide, as well as monoclonal antibodies that bind the peptide and are useful in purifying the recombinant proteins, are available from Eastman Kodak Company, Scientific Imaging Systems, New Haven, Conn.

Preparation of fusion proteins comprising heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, e.g., by Ashkenazi et al. (PNAS USA 88:10535, 1991); Byrn et al. (Nature 344:667, 1990); and Hollenbaugh and Aruffo ("Construction of Immunoglobulin Fusion Proteins", in Current Protocols in Immunology, Supplement 4, pages 10.19.1-10.19.11, 1992), hereby incorporated by reference. In one embodiment of the invention, an TRAIL dimer is created by fusing TRAIL to an Fc region polypeptide derived from an antibody. The term "Fc polypeptide" includes native and mutein forms, as well as truncated Fc polypeptides containing the hinge region that promotes dimerization. The Fc polypeptide preferably is fused to a soluble TRAIL (e.g., comprising only the extracellular domain).

A gene fusion encoding the TRAIL/Fc fusion protein may be inserted into an appropriate expression vector. The TRAIL/Fc fusion proteins are allowed to assemble much like antibody molecules, whereupon interchain disulfide bonds form between the Fc polypeptides, yielding divalent TRAIL. In other embodiments, TRAIL may be substituted for the variable portion of an antibody heavy or light chain. If fusion proteins are made with both heavy and light chains of an antibody, it is possible to form an TRAIL oligomer with as many as four TRAIL extracellular regions. One suitable Fc polypeptide is the native Fc region polypeptide derived from a human

IgGl, which is described in PCT application WO 93/10151, hereby incorporated by reference. Another useful Fc polypeptide is the Fc mutein described in U.S. Pat. No. 5,457,035. The amino acid sequence of the mutein is identical to that of the native Fc sequence presented in WO 93/10151, except that amino acid 19 has been changed from Leu to Ala, amino acid 20 has been changed from Leu to GIu, and amino acid 22 has been changed from GIy to Ala. This mutein Fc exhibits reduced affinity for immunoglobulin receptors. An antibody against a protein of the present invention may be any monoclonal or polyclonal antibody, as long as it can recognize the protein. Antibodies can be produced by using a protein of the present invention as the antigen according to a conventional antibody or antiserum preparation process. The present invention contemplates the use of both monoclonal and polyclonal antibodies. Any suitable method may be used to generate the antibodies used in the methods and compositions of the present invention, including but not limited to, those disclosed herein. For example, for preparation of a monoclonal antibody, protein, as such, or together with a suitable carrier or diluent is administered to an animal (e.g., a mammal) under conditions that permit the production of antibodies. For enhancing the antibody production capability, complete or incomplete Freund's adjuvant may be administered. Normally, the protein is administered once every 2 weeks to 6 weeks, in total, about 2 times to about 10 times. Animals suitable for use in such methods include, but are not limited to, primates, rabbits, dogs, guinea pigs, mice, rats, ^"~ sheep, goats, etc. For preparing monoclonal antibody-producing cells, an individual animal whose antibody titer has been confirmed (e.g., a mouse) is selected, and 2 days to 5 days after the final immunization, its spleen or lymph node is harvested and antibody-producing cells contained therein are fused with myeloma cells to prepare the desired monoclonal antibody producer hybridoma. Measurement of the antibody titer in antiserum can be carried out, for example, by reacting the labeled protein, as described hereinafter and antiserum and then measuring the activity of the labeling agent bound to the antibody. The cell fusion can be carried out according to known methods, for example, the method described by Koehler and Milstein (Nature 256:495 (1975)). As a fusion promoter, for example, polyethylene glycol (PEG) or Sendai virus (HVJ), preferably PEG is used. Examples of myeloma cells include NS-I, P3U1, SP2/0, AP-I and the like. The proportion of the number of antibody producer cells (spleen cells) and the number of myeloma cells to be used is preferably about 1 :1 to about 20:1. PEG (preferably PEG 1000-PEG 6000) is preferably added in concentration of about 10% to about 80%. Cell fusion can be carried out efficiently by incubating a mixture of both cells at about 20°C to about 4O⁰C, preferably about 30°C to about 37⁰C for about 1 minute to 10 minutes. Various methods may be used for screening for a hybridoma producing the antibody (e.g., against a tumor antigen or autoantibody of the present invention). For example, where a supernatant of the hybridoma is added to a solid phase (e.g., microplate) to which antibody is adsorbed directly or together with a carrier and then an antiimmunoglobulin antibody (if mouse cells are used in cell fusion, anti-mouse immunoglobulin antibody is used) or Protein A labeled with a radioactive substance or an enzyme is added to detect the monoclonal antibody against the protein bound to the solid phase. Alternately, a supernatant of the hybridoma is added to a solid phase to which an antiimmunoglobulin antibody or Protein A is adsorbed and then the protein labeled with a radioactive substance or an enzyme is added to detect the monoclonal antibody against the protein bound to the solid phase.

Selection of the monoclonal antibody can be carried out according to any known method or its modification. Normally, a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) are added is employed. Any selection and growth medium can be employed as long as the hybridoma can grow. For example, RPMI 1640 medium containing 1 % to 20%, preferably 10% to 20% fetal bovine serum, GIT medium containing 1% to 10% fetal bovine serum, a serum free medium for cultivation of a hybridoma (SFM-101, Nissui Seiyaku) and the like can be used. Normally, the cultivation is carried out at 20°C to 40°C, preferably 37°C for about 5 days to 3 weeks, preferably 1 week to 2 weeks under about 5% CO₂ gas. The antibody titer of the supernatant of a hybridoma culture can be measured according to the same manner as described above with respect to the antibody titer of the anti-protein in the antiserum.

Separation and purification of a monoclonal antibody (e.g., against a virus induced marker of the present invention) can be carried out according to the same manner as those of conventional polyclonal antibodies such as separation and purification of immunoglobulins, for example, salting-out, alcoholic precipitation, isoelectric point precipitation, electrophoresis, adsorption and desorption with ion exchangers (e.g., DEAE), ultracentrifugation, gel filtration, or a specific purification method wherein only an antibody is collected with an active adsorbent such as an antigen-binding solid phase, Protein A or Protein G and dissociating the binding to obtain the antibody.

Polyclonal antibodies may be prepared by any known method or modifications of these methods including obtaining antibodies from patients. For example, a complex of an immunogen (an antigen against the protein) and a carrier protein is prepared and an animal is immunized by the complex according to the same manner as that described with respect to the above monoclonal antibody preparation. A material containing the antibody against is recovered from the immunized animal and the antibody is separated and purified.

As to the complex of the immunogen and the carrier protein to be used for immunization of an animal, any carrier protein and any mixing proportion of the carrier and a hapten can be employed as long as an antibody against the hapten, which is crosslinked on the carrier and used for immunization, is produced efficiently. For example, bovine serum albumin, bovine cycloglobulin, keyhole limpet hemocyanin, etc. may be coupled to an hapten in a weight ratio of about 0.1 part to about 20 parts, preferably, about 1 part to about 5 parts per 1 part of the hapten. In addition, various condensing agents can be used for coupling of a hapten and a carrier.

For example, glutaraldehyde, carbodiimide, maleimide activated ester, activated ester reagents containing thiol group or dithiopyridyl group, and the like find use with the present invention. The condensation product as such or together with a suitable carrier or diluent is administered to a site of an animal that permits the antibody production. For enhancing the antibody production capability, complete or incomplete Freund's adjuvant may be administered. Normally, the protein is administered once every 2 weeks to 6 weeks, in total, about 3 times to about 10 times.

The polyclonal antibody is recovered from blood, ascites and the like, of an animal immunized by the above method. The antibody titer in the antiserum can be measured according to the same manner as that described above with respect to the supernatant of the hybridoma culture. Separation and purification of the antibody can be carried out according to the same separation and purification method of immunoglobulin as that described with respect to the above monoclonal antibody.

The protein used herein as the immunogen is not limited to any particular type of immunogen. For example, a protein expressed resulting from a virus infection (further including a gene having a nucleotide sequence partly altered) can be used as the immunogen. Further, fragments of the protein may be used. Fragments may be obtained by any methods including, but not limited to expressing a fragment of the gene, enzymatic processing of the protein, chemical synthesis, and the like. XIII. Pharmaceutical Compositions

The present invention further provides pharmaceutical compositions (e.g., comprising the protein or nucleic acid compounds described herein). The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (i.e., for example, ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes, but is not limited to, intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.

Pharmaceutical compositions and formulations for topical administration may include, but is not limited to, transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

Compositions and formulations for oral administration may include, but is not limited to, powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Compositions and formulations for parenteral, intrathecal or intraventricular administration may comprise sterile aqueous solutions that may also include, but are not limited to, buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients. Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers. In one embodiment of the present invention the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product.

Agents that enhance uptake of nucleic acids at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (WO 97/30731), also enhance the cellular uptake of oligonucleotides (both publications herein incorporated by reference).

The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation. Certain embodiments of the invention provide pharmaceutical compositions containing (a) one or more TRAIL protein compounds; or (b) one or more TRAIL nucleic acids. Alternatively, other drugs such as an anti-inflammatory drug, may include but not be limited to, nonsteroidal anti-inflammatory drugs and corticosteroids, and other antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. Two or more combined compounds may be used together or sequentially.

Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC₅o's found to be effective in in vitro and in vivo animal models or based on the examples described herein. In general, dosage is from 0.01 μg to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly. The treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the subject undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight, once or more daily, to once every 20 years.

rVX. Drug Screening In some embodiments, the present invention provides drug screening assays (e.g., to screen for anti-viral drugs). The screening methods of the present invention utilize gene expression maps identified using the methods of the present invention (e.g., including but not limited to, TRAIL). For example, in some embodiments, the present invention provides methods of screening for compound that alter (e.g., increase or decrease) the expression of virus-induced gene expression maps. In some embodiments, candidate compounds are antibodies that specifically bind to a protein encoded by a virus-induced gene of the present invention. In one screening method, candidate compounds are evaluated for their ability to alter virus-induced gene expression by contacting a compound with a cell expressing a virus induced protein and then assaying for the effect of the candidate compounds on expression. In some embodiments, the effect of candidate compounds on expression of a virus induced gene is assayed for by detecting the level of mRNA expressed by the cell. mRNA expression can be detected by any suitable method. In other embodiments, the effect of candidate compounds on expression of virus induced genes is assayed by measuring the level of polypeptide encoded by the virus induced genes. The level of polypeptide expressed can be measured using any suitable method, including but not limited to, those disclosed herein. Specifically, the present invention provides screening methods for identifying modulators, i.e., candidate or test compounds or agents (e.g., proteins, peptides, peptidomimetics, peptoids, small molecules or other drugs) which bind to virus induced gene products of the present invention, have an inhibitory (or stimulatory) effect on, for example, gene expression or gene product activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of a virus induced gene substrate. Compounds thus identified can be used to modulate the activity of target gene products (e.g., virus induced genes) either directly or indirectly in a therapeutic protocol, to elaborate the biological function of the target gene product, or to identify compounds that disrupt normal target gene interactions. Compounds which inhibit the activity or expression of virus induced genes are useful in the treatment of virus infections.

In one embodiment, the invention provides assays for screening candidate or test compounds that are substrates of a virus induced protein or polypeptide or a biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds that bind to or modulate the activity of a virus induced protein or polypeptide or a biologically active portion thereof.

The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods, including biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone, which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckennann et al., J. Med. Chem. 37: 2678 85 (1994)); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are preferred for use with peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Antivirus induced Drug Des. 12:145).

Numerous examples of methods for the synthesis of molecular libraries have been reported, for example in: DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90:6909 (1993); Erb et al., Proc. Nad. Acad. Sci. USA 91 :11422 (1994); Zuckermann et al., J. Med. Chem. 37:2678 (1994); Cho et al., Science 261 :1303 (1993); Carrell et al., Angew. Chem. Int. Ed. Engl. 33.2059 (1994); Carell et al., Angew. Chem. Int. Ed. Engl. 33:2061 (1994); and Gallop et al., J. Med. Chem. 37:1233 (1994).

Libraries of compounds may be presented in solution (e.g., Houghten, Biotechniques 13:412 421 (1992)), or on beads (Lam, Nature 354:82 84 (1991)), chips (Fodor, Nature 364:555 556 (1993)), bacteria or spores (U.S. Pat. No. 5,223,409; herein incorporated by reference), plasmids (Cull et al., Proc. Nad. Acad. Sci. USA 89:18651869 (1992)) or on phage (Scott and Smith, Science 249:386 390 (1990); Devlin Science 249:404 406 (1990); Cwirla et al., Proc. Natl. Acad. Sci. 87:6378 6382 (1990); Felici, J. MoI. Biol. 222:301 (1991)).

In one embodiment, an assay is a cell-based assay in which a cell that expresses a virus induced protein or biologically active portion thereof is contacted with a test compound, and the ability of the test compound to the modulate virus induced protein activity is determined.

Determining the ability of the test compound to modulate virus induced protein activity can be accomplished by monitoring, for example, changes in enzymatic activity. The cell, for example, can be of mammalian origin.

The ability of the test compound to modulate a virus induced protein binding to a compound, e.g., a virus induced substrate, can also be evaluated. This can be accomplished, for example, by coupling the compound, e.g., the substrate, with a radioisotope or enzymatic label such that binding of the compound, e.g., the substrate, can be determined by detecting the labeled compound, e.g., substrate, in a complex.

Alternatively, the virus induced protein is coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate virus induced protein binding to a substrate in a complex. For example, compounds (e.g., substrates) can be labeled with ¹²⁵1, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. The ability of a compound to interact with a virus induced protein with or without the labeling of any of the interactants can be evaluated. For example, a microphysiometer can be used to detect the interaction of a compound with a virus induced marker without the labeling of either the compound or the virus induced marker (McConnell et al. Science 257:1906 1912 (1992)). As used herein, a "microphysiometer" (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a compound and markers.

In yet another embodiment, a cell-free assay is provided in which a virus induced marker protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the virus induced marker protein or biologically active portion thereof is evaluated. Preferred biologically active portions of the virus induced marker proteins to be used in assays of the present invention include fragments that participate in interactions with substrates or other proteins, e.g., fragments with high surface probability scores.

Cell- free assays involve preparing a reaction mixture of the target gene protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected.

The interaction between two molecules can also be detected, e.g., using fluorescence energy transfer (FRET) (see, for example, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos et al., U.S. Pat. No. 4,968,103; each of which is herein incorporated by reference). A fluorophore label is selected such that a first donor molecule's emitted fluorescent energy will be absorbed by a fluorescent label on a second, 'acceptor' molecule, which in turn is able to fluoresce due to the absorbed energy.

Alternately, the 'donor' protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the 'acceptor' molecule label may be differentiated from that of the 'donor'. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed, hi a situation in which binding occurs between the molecules, the fluorescent emission of the 'acceptor" molecule label in the assay should be maximal. An FRET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter). In another embodiment, determining the ability of the virus induced marker protein to bind to a target molecule can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander and Urbaniczky, Anal. Chem. 63:2338 2345 (1991) and Szabo et al. Curr. Opin. Struct. Biol. 5:699 705 (1995)). "Surface plasmon resonance" or "BLA" detects biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal that can be used as an indication of real-time reactions between biological molecules.

In one embodiment, the target gene product or the test substance is anchored onto a solid phase. The target gene product/test compound complexes anchored on the solid phase can be detected at the end of the reaction. Preferably, the target gene product can be anchored onto a solid surface, and the test compound, (which is not anchored), can be labeled, either directly or indirectly, with detectable labels discussed herein.

It may be desirable to immobilize virus induced markers, an anti- virus induced marker antibody or its target molecule to facilitate separation of complexed from non-complex ed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to a virus induced marker protein, or interaction of a virus induced marker protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase-virus induced marker fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione Sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione-derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or virus induced marker protein, and the mixture incubated under conditions conducive for complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of virus induced markers binding or activity determined using standard techniques. Other techniques for immobilizing either virus induced marker proteins or a target molecule on matrices include using conjugation of biotin and streptavidin. Biotinylated virus induced marker protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, EL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).

In order to conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non- immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-IgG antibody).

This assay is performed utilizing antibodies reactive with virus induced marker protein or target molecules but which do not interfere with binding of the virus induced markers protein to its target molecule. Such antibodies can be derivatized to the wells of the plate, and unbound target or virus induced markers protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the virus induced marker protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the virus induced marker protein or target molecule. Alternatively, cell free assays can be conducted in a liquid phase. In such an assay, the reaction products are separated from unreacted components, by any of a number of standard techniques, including, but not limited to: differential centrifugation (see, for example, Rivas and Minton, Trends Biochem Sci 18:284 7 (1993)); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g., Ausubel et al., eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York.); and immunoprecipitation (see, for example, Ausubel et al., eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York). Such resins and chromatographic techniques are known to one skilled in the art (See e.g., Heegaard J. MoI. Recognit 11 : 141 8 (1998); Hageand Tweed J. Chromatogr. Biomed. Sci. Appl 699:499 525 (1997)). Further, fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution.

The assay can include contacting the virus induced marker proteins or biologically active portion thereof with a known compound that binds the virus induced marker to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a virus induced marker protein, wherein determining the ability of the test compound to interact with a virus induced marker protein includes determining the ability of the test compound to preferentially bind to virus induced markers or biologically active portion thereof, or to modulate the activity of a target molecule, as compared to the known compound. To the extent that virus induced markers can, in vivo, interact with one or more cellular or extracellular macromolecules, such as proteins, inhibitors of such an interaction are useful. A homogeneous assay can be used can be used to identify inhibitors.

For example, a preformed complex of the target gene product and the interactive cellular or extracellular binding partner product is prepared such that either the target gene products or their binding partners are labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Pat. No. 4,109,496, herein incorporated by reference, that utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt target gene product-binding partner interaction can be identified. Alternatively, virus induced markers protein can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al., Cell 72:223 232 (1993); Madura et al., J. Biol. Chem. 268.12046 12054 (1993); Bartel et al., Biotechniques 14:920 924 (1993); Iwabuchi et al., Oncogene 8:1693 1696 (1993); and Brent WO 94/10300; each of which is herein incorporated by reference), to identify other proteins, that bind to or interact with virus induced markers ("virus induced marker-binding proteins" or "virus induced marker-bp") and are involved in virus induced marker activity. Such virus induced marker-bps can be activators or inhibitors of signals by the virus induced marker proteins or targets as, for example, downstream elements of a virus induced markers-mediated signaling pathway.

Modulators of virus induced markers expression can also be identified. For example, a cell or cell free mixture is contacted with a candidate compound and the expression of virus induced marker mRNA or protein evaluated relative to the level of expression of virus induced marker mRNA or protein in the absence of the candidate compound. When expression of virus induced marker mRNA or protein is greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of virus induced marker mRNA or protein expression. Alternatively, when expression of virus induced marker mRNA or protein is less (i.e., statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of virus induced marker mRNA or protein expression. The level of virus induced marker mRNA or protein expression can be determined by methods described herein for detecting virus induced marker mRNA or protein. A modulating agent can be identified using a cell-based or a cell free assay, and the ability of the agent to modulate the activity of a virus induced marker protein can be confirmed in vivo, e.g., in an animal such as an animal model for a disease (e.g., an animal with dengue fever), or cells from a dengue fever virus induced cell line.

This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein (e.g., a virus induced marker modulating agent, a virus induced marker specific antibody, or a virus induced marker-binding partner) in an appropriate animal model (such as those described herein) to determine the efficacy, toxicity, side effects, or mechanism of action, of treatment with such an agent. Furthermore, novel agents identified by the above-described screening assays can be, e.g., used for treatments as described herein. Experimental

The following examples are only presented to illustrate specific embodiments of the present invention and are not intended to be limiting. The following example were performed with Wild type (2fTGH) and IFN-α pathway mutant (UlA, U3A, U4A and U5A) fibroblast cells obtained from Dr. Paul Fisher (Univ. of Columbia, NY) and cultured in DMEM medium supplemented with 10% FCS. The fibroblast cells were infected with DV for 48 hours. Lutfalla et al., 1995. "Mutant U5A cells are complemented by an interferon-alpha beta receptor subunit generated by alternative processing of a new member of a cytokine receptor gene cluster" Embo J 14:5100-8; McKendry et al., 1991. "High-frequency mutagenesis of human cells and characterization of a mutant unresponsive to both alpha and gamma interferons" Proc Natl Acad Sd USA 88:11455-9; and Pellegrini et al., 1989. "Use of a selectable marker regulated by alpha interferon to obtain mutations in the signaling pathway" MoI Cell Biol 9:4605-12.

Example I

Blood Sample Preparation And Cell Culture

Blood samples were obtained from healthy U.S. volunteers at The University of Massachusetts Medical School. Monocytes and B cells were negatively selected from heparin- anti-coagulated blood using a rosetting antibody precipitation kit (StemCell) and maintained in RPMI 1640 medium supplemented with 10% FCS and antibiotics. Sample purity was determined by cell surface staining of freshly isolated monocytes and B cells. Primary HUVECs were obtained from pooled umbilical cords (two to five donor pools per culture) from Brigham and Women's Hospital (Boston) and maintained in M 199 supplemented with 10% FCS, 1 mM glutamine, endothelial cell growth stimulant, porcine intestinal heparin, and antibiotics. HUVEC cultures were split at a ratio of 1 :3 or 1 :4 for up to two passages. DV (dengue 2 virus, strain New Guinea C) was cultured using standard methods in the C6/36 insect cell line. HUVECs, monocytes and B cells were infected in vitro with DV as previously described. Cells were treated with uninfected C6/36 cell supernatant as a negative control (mock infection). Cells were collected for gene expression analysis at 48 h post-infection based on previous results CD14 microbeads (Cat # 120-000-305, Miltenyi Biotec) were used to positively select CD14 positive cells (monocytes) from ficoll isolated PBMCs from healthy donors. Warke et al., 2003. "Dengue virus induces novel changes in gene expression of human umbilical vein endothelial cells" J Virol 77:11822-32.

Monocytes were cultured for 7 days in RPMI 1640 medium supplemented with 800U/ml of granulocyte-macrophage colony-stimulating factor (GM_CSF), 500U/ml of interleukin-4 (IL- 4) and 10% FCS. Cells were stained for CDIa, CD14, HLA-DR and CD83 on day 6 to determine monocyte to immature dendritic cell (iDC) conversion.

Example II Affvmetrix GeneChip Hybridization And Analysis Total cellular RNA isolated using RNeasy kit (Qiagen) was biotin-labeled and hybridized to human oligonucleotide microarrays (Affymetrix HG-Ul 33A) as previously described (44). Experiments displaying Affymetrix present (P)-call rates of > 30% were included in the analysis. Signal values from each of the 22,283 probe sets were calculated using robust multi-array analysis (RMA). Irizarry et al., 2003. "Exploration, normalization, and summaries of high density oligonucleotide array probe level data" Biostatistics 4:249-64. Signal values were transformed using inverse nlog. For repeated experiments, inverse nlog transformed RMA array results were normalized based on the median and geometric means and were calculated prior to ' importing data into GeneSpring Software (Agilent). Each GeneChip was independently normalized to the median expression level of all genes on the chip. Each gene was then normalized to the median expression levels of that gene. Low expressing signals were excluded, and expression of statistical filters was applied as indicated. Data was analyzed by hierarchical cluster analysis using Pearson correlation. Reproducibility was assessed as previously described (Warke et al, supra).

For the analysis of the common response the following analysis was done: Expression signals for 22,283 genes were filtered to exclude those with very low signals (-2000 genes) and the remaining genes were analyzed by 1-way ANOVA to identify genes with statistically significant differences between samples infected with DV (5 samples) and uninfected samples (5 samples). Variances were calculated using the cross-gene error model, parametric test, p-value cutoff = 0.05, multiple testing correction Benjamin and Hochberg False Discovery Rate. Hierarchical cluster analysis used a Pearson correlation. Example III Quantitative RT-PCR

TaqMan quantification of DV RNA was performed as previously described. Warke et al. {supra). TRAIL mRNA was quantified using the same reaction conditions with TaqMan primer and probes obtained from Applied Biosystems. Results were calculated using the standard curve method or relative quantification method (RQ) using qPCR software (Applied Biosystems). For microfluidic card analysis or qRT-PCR, total RNA was extracted from cells using the Qiagen RNeasy kit. RNA was subjected to 384- well microfluidic card analysis as described by the manufacturer (Applied Biosystems). 100 ng of total cellular RNA was reverse transcribed using TaqMan reverse transcription reagents in the presence of random hexamers as primers. Reverse transcription was performed at 25⁰C for 10 min, 48⁰C for 30 min, followed by 95⁰C for 5 min. For PCR, a 100 μl reaction mix through a single port provided 2 μl of total reaction mix per sample. The 100 μl PCR-reaction mix included 5 μl of cDNA, 45 μl of RNase/DNase-free water and 50 μl of TaqMan Universal PCR Master Mix (2X). PCR reactions were cycled at 50⁰C for 2 min, 94.5⁰C for 10 min, followed by 40 cycles of 97⁰C for 30 sec and 59.7⁰C for 1 min in the PCR signal detection system 7900 (Applied Biosystems). β-actin was used as an endogenous control to equalize loading of total RNA between samples. Each data point was measured in quadruplicate and the standard error was determined.

Example IV

TRAIL protein detection by ELISA and FACs

TRAIL ELISA (R&D Systems) and TRAIL Intracellular Staining (ICS) techniques were utilized to measure TRAIL protein levels in culture supernatants and cell lysates. The minimum detectable amount of TRAIL for this ELISA was 2.86 pg/ml. Surface TRAIL protein was detected in CD14⁺ CD4^" cells (monocytes) by flow cytometry using label monoclonal anti- TRAIL (PE) antibody (BD Biosciences). Intracellular TRAIL protein levels were also determined by flow cytometry in CDIa⁺ CD14^"ve cells (dendritic cells) infected for 12, 24 and 48 hours with DV at M.O.I, of 0.1. Example V

TRAIL Antibody and IFN-α Treatment

Monocytes, B cells and HUVECs were pre-treated for 24 hours with TRAIL blocking monoclonal antibody (50 ng/μl, R&D Systems) or purified IgGl isotype control antibody followed by infection with DV for 48hr at a multiplicity of infection (M.O.I.) of 1. The antibodies were left in the culture during the 48hr DV-infection.

Monocytes were pre-treated with 140 ng/ml of IFN-α or 140 ng/ml of IFN-α and 50 ng/μl of anti-TRAIL antibody or purified IgGl isotype control antibody followed by infection with DV, for 48 hr at a M.O.I, of 0.1. Cells were collected by centrifugation at 500 x g, washed twice in PBS, and cell pellets were stored at -70⁰C until analysis. RNA was extracted from the cell pellet using RNAeasy (Qiagen) and subjected to TaqMan qRT-PCR for detection of TRAIL and dengue virus RNA using β-actin as an endogenous control. A standard curve was run using a pre-calibrated DV sample for absolute quantification of gene expression.

Example VI

Recombinant TRAIL Treatment

Monocytes were treated 24 hours with rTRAIL (Merck, DR or BIOMOL, PA) and infected with DV at a multiplicity of infection (M.O.I.) of 0.1. At the end of 24 and 48 hours cells were washed with PBS two times and cell pellets were stored at -7O⁰C. RNA was extracted and DV copy number was determined using TaqMan qRT-PCR and actin was used as endogenous control to normalize mRNA levels of other genes.

Dendritic cells were treated 24 hours with rTRAIL (BIOMOL, PA) and infected with DV at a M.O.I, of 0.1 for 12, 24 or 48 hours. Dendritic cells were washed two hours post-infection to remove residual virus used for infection. Cells were maintained in fresh RPMI 1640 media containing 20 ng/ml rTRAIL, 10% FCS, IL-4 (500 U/ml) and GM-CSF (800 U/ml) for 12, 24 or 48 hours. At each time point cells were stained for dengue antigen (DV-FITC from Upstate New York, NY), according to previously published procedures (44), CDIa-APC, HLA-DR- PerCpCy5.5 and CD83-PE. Plaque assay was also performed on supernatant collected at the 48 hour timepoint. Example VII

Detection of Apoptosis in Dendritic Cells

Live/Dead Aqua Stain: Live/Dead Aqua dye is an early marker for cells undergoing apoptosis. DV-infected DCs treated with / without TRAIL were stained for apoptosis using Live/Dead Aqua dye at 12, 24 and 48 hour post infection. DCs incubated in 65⁰C water bath for 20 min were used as a positive control for Live/Dead Aqua dye stain at each time point.

Example VIII Plaque Assay Culture supernatants were incubated with a monolayer of Vero cells for 2 hours. Cell monolayers were washed with PBS and overlaid with medium viscosity (mix of high and low) carboxy methylcellulose (CMC) (Sigma-Aldrich, St.Louis, USA). CMC was removed 7 days later, and cells were fixed and stained with 0.2% crystal violet in ethanol. Dengue virus plaques were counted visually. Russell et al., 1967. "A plaque reduction test for dengue virus neutralizing antibodies" J Immunol 99:285-90.

Example IX Statistical calculations

Analysis of variance (ANOVA) was used to identified a set of twenty-three (23) transcripts that demonstrated a common and significant change in expression between the various cell types studied by gene expression analysis (GeneSpring). Means, Standard Deviations (SD) and Student T test were done by Excel software (Microsoft).

Example X Identification Of Common Gene Expression Response To DV Infection

To identify common gene expression changes in host cells in response to DV infection, a global gene expression profile was analyzed in HUVECs, monocytes, DCs and B cells using Affymetrix GeneChip arrays. Monocytes and B cells isolated from healthy donor blood were -80% CD14⁺ and -90% CD19⁺, respectively (data not shown). Monocyte derived DCs (iDCs) were -91% CDIa⁺ See, Figure 5 A. All the cells tested were infected with DV propagated in

C6/36 mosquito cells. Control samples were mock-infected with uninfected C6/36 supernatants. ANOVA identified a set of twenty-three (23) transcripts that demonstrated a common and significant change in expression (p<0.05) in all cell types studied. See, Figure 2. To confirm the gene expression changes detected by Affymetrix GeneChip, eleven (11) genes for which primers and probes were available were selected for validation by qRT-PCR. Quantitative RT-PCR confirmed up regulation (> 3 fold) of all the 11 genes in HUVECs (Table 1).

Table 1. Quantitative RT-PCR validation of eleven (11) exemplary dengue response genes. In vitro dengue virus infections in HUVECs were analyzed by quantitative RT-PCR (qRT-PCR) using microfluidic cards (Applied Biosystems). Relative fold induction was calculated with software from Applied Biosystems.

To identify signaling pathways associated with the host cell response to DV infection, the twenty-three common response genes were analyzed using Pathway Architect software (Stratagene). This analysis found the commonly regulated genes to be predominantly regulated by EFNα and IFNγ signaling pathways. Further, TRAIL/TNFSFIO (one of the 23 common response genes) was identified as a potential linker of these two signaling pathways (data not shown).

Example XI

DV Infection Induces TRAIL/TNFSFIO mRNA And Protein Production Using real-time PCR, TRAIL/TNFSFIO induction in vitro was determined during DV infection at the mRNA level. The data showed that TRAIL mRNA was induced in DV-infected monocytes, B cells and dendritic cells. See, Figure 3 A. TRAIL was not detected on the surface of DV-infected monocytes by flow cytometry or in the supernatant of DV-infected monocytes by ELISA. Higher amounts of TRAIL protein was detected in DV-infected monocyte cell lysates as compared to uninfected monocytes at 48 hours after infection with DV in vitro. See, Figure 3B. hi DV-infected DCs, higher intracellular TRAIL protein levels were detected at the 12 hour but not at the 24 or 48 hour timepoints. See, Figure 3C.

Example XII

TRAIL Inhibits DV Replication

This example determines whether TRAIL was regulating levels of DV. TRAIL neutralizing antibodies were thereby used to block TRAIL function in DV-infected monocytes, B cells, and HUVECs.

The data show that TRAIL blocking monoclonal antibody increases dengue virus progeny. Monocytes (Mo), B cells, HUVECs were infected with DV at MOI of 1 PFU/cell and then cultured for 48 hours. TRAIL blocking antibody (50 ng/μl) was added 24 hr prior to infection with DV. Dengue virus copy number was quantified by qRT-PCR analysis. The concentration of anti-TRAIL antibody (R&D Systems) was titrated to block cell surface TRAIL protein in primary monocytes treated with IFN- β (100U/ml) confirming previous studies (data not shown). Ehrlich et al., 2003. "Regulation of soluble and surface-bound TRAIL in human T cells, B cells, and monocytes" Cytokine 24:244-53. At 48 hours post-infection, a 5 to 12 fold increase in DV RNA was detected by qRT-PCR in the anti-TRAIL treated monocytes, B cells and HUVECs. See, Figure 4A. Monocytes pre- treated with 5 - 20 ng/ml of rTRAIL (Merck) showed a 80% to 90.9% fold decrease in DV RNA copy number after 24 and 48 hours respectively when infection was 0.1 MOI (data not shown). Since DCs were more efficiently infected by DV, flow cytometry was used to calculate the percent of infected cells and plaque assay was used to measure progeny virus in culture supernatants. Dendritic cells treated with rTRAIL showed approximately 60% decrease in DV antigen when stained with the 2H2 antibody. See, Figure 5A and Figure 5B. Further, DV infectious progeny was reduced by more than a log in the supernatant of DV-infected DCs treated with rTRAIL. See, Figure 6.

Example XUI

Tvpe-I IFN Regulates TRAIL mRNA Induction

Tyk2, STATl, JAKl and IFNARα2c mutants (UlA, U3A, U4A and U5A) were used to identify whether the IFN-α signaling pathway regulates TRAIL mRNA induction during DV- infection. TaqMan qRT-PCR was used to quantify TRAIL mRNA levels. None of the mutant cell lines showed upregulation of TRAIL in response to DV-infection. See, Figure 7. These data show that type-I IFN signaling may be necessary for TRAIL mRNA induction.

Example XIV TRAIL Antibodies Can Block IFN-α Mediated -Viral Inhibition

This example using qRT-PCR of dengue virus infected cells to test TRAIL involvement in IFN-α mediated anti-viral function.

Monocytes were pre-treated with IFN-α, with IFN-α and anti-TRAIL antibody, or with IFN-α and a control mouse antibody IgGl . DV RNA copy number was measured after 48 hours of DV-infection. IFN-α treatment inhibited DV RNA copy number, but the IFN-α mediated effect in DV copy number was inhibited by pre-treatment with TRAIL blocking antibodies. The interference of the antiviral function of IFN-α was only seen with TRAIL antibody but not with isotype control mouse antibody. See, Figure 8. Hence, TRAIL is required for the IFN-α mediated anti-viral activity in two independent sources of monocytes. Each mRNA measure represents triplicate PCR reactions, and the means of dengue mRNA were corrected by beta actin levels. Example XV

TRAIL Inhibition OfDV Is Apoptosis-Independent

This example determines whether rTRAIL mediated inhibition of DV was dependent on apoptosis of DV-infected cells.

DV-infected DCs treated with or without rTRAIL were stained for DV antigen and Live/Dead Aqua dye and also for early (Caspase-8) and late (PARP-I) markers of apoptosis (data not shown). The decrease in DV infection of rTRAIL treated DCs did not correlate with an increase in staining with Live Dead Aqua, a dye which detects a cell in its early stage of apoptosis. See, Figure 9A, Figure 9B, and Figure 9C. Similar results for absence of apoptosis were observed when DCs were stained for active caspase-8 and cleaved PARP-I proteins (data not shown). These results suggest that TRAIL is regulating DV by an apoptosis-independent mechanism.

Example VI Detection Of Chemokine And Cytokine Expression In Human DV Infection

I. MATERIALS AND METHODS A. Patients, Clinical Record and Laboratory Analyses

Patients were enrolled in a study protocol conducted by the University of Massachusetts Medical School (UMMS), Worcester, MA, USA, and Banco Municipal de Sangre del Distrito Capital (BMS), Caracas, Venezuela. Written informed consent was obtained from all subjects. Febrile patients with no evidence of other defined infections were enrolled. Patients attended the clinic daily until 2 days after the fever resolved. A convalescent visit was performed at least 2 weeks after the onset of symptoms. Complete clinical exam and routine laboratory tests were performed as described previously. Becerra et al., "Elevated levels of soluble ST2 protein in dengue virus infected patients" Cytokine 41:114-20 (2008). Based on corporal temperature we defined "fever day zero (0)" as the day of defervescence. The febrile period included days before defervescence and the post-febrile period included defervescence and days after defervescence. Healthy donors from BMS and UMMS were used as controls. B. Dengue Case Definition

Dengue cases were defined following the 2003 World Health Organization (WHO) guidelines. DV genomic RNA was isolated from febrile serum samples using the QIAmp Viral RNA kit (QIAGEN). DV serotype-specific reverse transcription and polymerase chain reaction (RT-PCR) was performed using the One-step PCR kit (QIAGEN) and primers, adapted to a one- step RT-PCR, using reverse primer and serotype specific forward primers. Lanciotti et al., "Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction" J CHn Microbiol 30:545-551 (1992). DV-specific antibodies were measured in paired serum samples (enrollment and convalescence samples, Sl and S2), using IgM-ELISA and hemagglutination inhibition assay (HI) at the Instituto Nacional de Higiene "Rafael Rangel", Caracas, Venezuela. Patients were classified as Dengue or as Other Febrile Illness (OFI) based on the detection of DV RNA, presence of IgM antibodies and/or at least a four- fold increase in HI titers in S2 compared to Sl. The HI levels were used to further classify dengue patients as a primary infection (HI titer < 1 : 1280) or secondary infection (HI titer > 1 :1280).

C. Cell Preparation and Culture

Blood was obtained from healthy U.S. volunteers at The University of Massachusetts Medical School. DCs were prepared from peripheral blood-derived CD14-positive cells.

Briefly, PBMC were isolated using a density gradient centrifugation over Ficoll-Paque™ Plus, 1.077g/dl (GE Healthcare). Positive selection of CD 14+ cells was performed using the CD14-positive selection magnetic cell sorting kit (Miltenyi Biotec, Inc.). Monocytes were incubated in RPMI 1640 supplemented with 10% FCS, 500 U/ml of IL-4 (Pepro-Tech) and 800 U/ml of granulocyte-macrophage colony stimulation factor (GM-CSF) (Pepro-Tech). The percentage of immature DC (iDC) conversion (CDIa+ / CD 14-) was determined after 6 days in culture, using flow cytometry. Monocytes for GeneChip hybridization experiments were negatively selected from blood using a rosetting antibody precipitation kit (StemCell) and were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS). The percentage of purity (CD14-positive cells) was determined using flow cytometry. Primary HUVEC for GeneChip hybridization experiments were obtained and cultured. D. In Vito DV Infection

Cells for gene expression analysis were infected with DV (DV2 - New Guinea C, DV2- NGC) at a multiplicity of infection (M.O.I.) of 1 for HUVEC and monocytes and M.O.I, of 0.1 for DC. Cells were incubated with virus for 2 hours at 37°C and then washed thoroughly, resuspended in RPMI 1640 supplemented with 10% FCS (monocytes), RPMI 1640 supplemented with 10% FCS plus 500 U/ml IL-4 and 800 U/ml GM-CSF (DC) or M199 supplemented with 10% FCS, 1 mM glutamine, endothelial cell growth stimulant and porcine intestinal heparine (HUVEC) and incubated for 48 hours.

For the time-course experiment, DCs were infected with DV2-NGC at M.O.I, of 0.1 and incubated for 12, 24 or 48 hours; cells and supernatant were collected at each time point. For TRAIL pre-treatment experiments, DC were treated with 20 μg/ml of recombinant TRAIL (rTRAIL, Biomol International LP) for 24 hours, and then infected with DENV2-NGC at M.O.I of 0.1; cells and supernatants were collected at 48 hours post- infection.

E. Flow Cytometry Analysis Cells were surface-stained using monoclonal antibodies anti-CD 14-pacific blue, anti-

CDIa-APC and/or anti-CD83-PE (all from BD). Intracellular detection of DV antigen was performed in fixed and permeabilized cells, using the Cytofix/Cytoperm kit (BD) and stained with anti-DV complex antibody conjugated to FITC (Chemicon). Cells were analyzed using the BD FACSAria™ and Flow- Jo software. F. RNA Preparation: Affymetrix GeneChip Hybridization and Quantitative RT-PCR

Total cellular RNA was prepared using the RNeasy kit (Qiagen). Affymetrix genechip hybridization was performed, using biotin-labeled total cellular RNA, hybridized to human oligonucleotide microarrays (Affymetrix HG-U133A). Warke et al., "Dengue virus induces novel changes in gene expression of human umbilical vein endothelial cells" J Virol 77:11822- 11832 (2003). Normalized signal values were computed from Affymetrix HG-Ul 33 A chips using Robust Multichip Average (RMA). Irizarry et al., "Summaries of Affymetrix GeneChip probe level data" Nucleic Acids Res 31 :el5 (2003). Genes were normalized to expression levels in controls for each cell type.

Quantitative real time RT-PCR (qRT-PCR) was performed from total cellular RNA using Taqman Reverse Transcription kit, universal PCR Master Mix 2X and specific primers and probes (all from Applied Biosystems). The PCR reaction was performed in the 7300 Taqman PCR System (Applied Biosystems). "-actin was used as an endogenous control and relative quantification (Rq) was done using the 2^"ΔΔ ' method. Livak et al., Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method" Methods 25:402-408 (2001). G. Quantification of Proteins in Serum or Culture Supernatants

Levels of MCP-2, IP-10, MlP-lβ, and TRAIL in serum from patients and MCP-2, IP-10, IL-6, and IFN-α in DC culture supernatants were measured by enzyme linked immunosorbent assay (ELISA) (Ray Biotech Inc., R&D Systems) following the manufacturer's instructions.

H. Statistical Analysis The Mann- Whitney U test was used for comparisons between two groups for continuous variables not normally distributed. The paired t-test was used for comparisons between percentages of infection. Spearman r test was used to test correlations. We used the software SPSS 15.0 for Windows (Copyright SPSS Inc. 1989-2005) for the statistical analysis.

Claims

ClaimsWe claim:

1. A method comprising: a) providing; i) a patient exhibiting at least one symptom of a virus infection, wherein said virus is selected from the group consisting of flaviviruses and bunyaviruses; and ii) a composition comprising a TRAIL protein and fragments thereof; and b) administering said TRAIL protein under conditions such that the at least one symptom of said infection is reduced.

2. The method of Claim 1, wherein said flavivirus is selected from the group consisting of a dengue virus, a yellow fever virus, a West Nile virus, and an encephalitis virus.

3. The method of Claim 1, wherein said bunyavirus is selected from the group consisting of a Hantaan virus and a Sin Nombre virus.

4. The method of Claim 1 , wherein said TRAIL protein is part of a fusion protein.

5. The method of Claim 1, wherein sad administering comprises a topical administration.

6. The method of Claim 5, wherein said topical administration is selected from the group consisting of transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.

7. The method of Claim 1, wherein said administering comprises parenteral administration.

8. The method of Claim 8, wherein said parenteral administration is selected from the group consisting of intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular injection or infusion; and intracranial, intrathecal and intraventricular administration.

9. A method comprising: a) providing; i) a patient exhibiting at least one symptom of a virus infection, wherein said virus is selected from the group consisting of flaviviruses and bunyaviruses; and ii) a composition comprising a nucleic acid, wherein said nucleic acid encodes a TRAIL protein or fragment thereof; and b) administering said nucleic acid under conditions such that the at least one symptom of said infection is reduced.

10. The method of Claim 9, wherein said flavivirus is selected from the group consisting of a dengue virus, a yellow fever virus, a West Nile virus, and an encephalitis virus.

11. The method of Claim 9, wherein said bunyavirus is selected from the group consisting of a Hantaan virus and a Sin Nombre virus.

12. The method of Claim 9, wherein said TRAIL nucleic acid comprises mRNA.

13. The method of Claim 12, wherein said nucleic acid is encapsulated in a liposome.

14. The method of Claim 9, wherein said administering comprises a topical administration.

15. The method of Claim 14, wherein said topical administration is selected from the group consisting of transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.

16. The method of Claim 9, wherein said administering comprises parenteral administration.

17. The method of Claim 16, wherein said parenteral administration is selected from the group consisting of intravenous, intraarterial, subcutaneous, intraperitoneal and intramuscular injection or infusion; and intracranial, intrathecal and intraventricular administration.

18. A method for characterizing virus infected tissue in a subject, comprising: a) providing a virus infected tissue sample from a subject; and b) detecting the presence or absence of expression of a TRAIL composition in said sample, thereby characterizing said virus infected tissue sample.

19. The method of Claim 18, wherein said virus is selected from the group consisting of flaviviruses and bunyaviruses.

20. The method of Claim 19, wherein flavivirus is selected from the group consisting of a dengue virus, a yellow fever virus, a West Nile virus, and an encephalitis virus.

21. The method of Claim 19, wherein said bunyavirus is selected from the group consisting of a Hantaan virus and/or a Sin Nombre virus.

22. The method of Claim 19, wherein said detecting the presence of expression of said TRAIL composition comprises detecting the presence of TRAIL mRNA.

23. The method of Claim 22 wherein said detecting the presence of expression of said TRAIL mRNA comprises exposing said TRAIL mRNA to a nucleic acid probe complementary to said TRAIL mRNA.

24. The method of Claim 19, wherein said detecting the presence of expression of TRAIL comprises detecting the presence of a TRAIL polypeptide.

25. The method of Claim 24, wherein said TRAIL polypeptide is detected by an antibody specific to said TRAIL polypeptide.

26. The method of Claim 19, said subject comprises a human subject.

27. The method of Claim 19, wherein said sample comprises a blood sample.

28. The method of Claim 27, wherein said blood sample is a serum sample.

29. The method of Claim 27, wherein said blood sample is a plasma sample.

30. The method of Claim 19, wherein said blood sample comprises monocytes.

31. A kit for characterizing a virus infection in a subject, comprising: a) a reagent capable of specifically detecting the presence of absence of expression of a TRAIL composition; and b) instructions for using said kit for characterizing said virus infection in said subject.

32. The kit of Claim 31, wherein said virus is selected from the group consisting of flavi viruses and bunyaviruses.

33. The kit of Claim 32, wherein said flavivirus is selected from the group consisting of a dengue virus, a yellow fever virus, a West Nile virus, and an encephalitis virus.

34. The kit of Claim 32, wherein said bunyavirus is selected from the group consisting of a Hantaan virus and a Sin Nombre virus.

35. The kit of Claim 31, wherein said reagent comprises a nucleic acid probe complementary to a TRAIL mRNA.

36. The kit of Claim 31 , wherein said reagent comprises an antibody that specifically binds to a TRAIL polypeptide.

37. The kit of Claim 31, wherein said instructions comprise instructions in compliance with the United States Food and Drug Administration recommendations for use in in vitro diagnostic products.

38. A method of screening compounds, comprising: a) providing; i) a virus infected sample; and ii) at least one test compound; b) contacting said virus infected sample with said at least one test compound; and c) detecting a change in TRAIL composition expression in said virus infected sample in the presence of said at least one test compound relative to the absence of said at least one test compound.

39. The method of Claim 38, wherein said virus is selected from the group consisting of flaviviruses and bunyaviruses.

40. The method of Claim 39, wherein said flavivirus is selected from the group consisting of a dengue virus, a yellow fever virus, a West Nile virus, and an encephalitis virus.

41. The method of Claim 38, wherein said bunyavirus is selected from the group consisting of a Hantaan virus and a Sin Nombre virus.

42. The method of Claim 38, wherein said detecting comprises detecting TRAIL mRNA.

43. The method of Claim 38, wherein said detecting comprises detecting TRAIL polypeptide.

44. The method of Claim 38, wherein said sample comprises an in vitro cell.

45. The method of Claim 38, wherein said sample comprises an in vivo cell.

46. The method of Claim 38, wherein said test compound comprises a peptide.

47. The method of Claim 38, wherein said test compound comprises a drug.

48. A virus expression profile map comprising gene expression level information for at least one marker selected from the group consisting of: G1P2, IRF7, ISG20, OAS3, OASL, RSAD2, TRIM5, HSXIAPAFl, TRAIL, CD38, HERC5, IFI44, IFI44L, IFITMl, LGALS3BP, USPl 8, FLJ20035, FLJ38348, HERC6, IFITl, IFIT3, LY6E, and SAMD9.

49. The map of Claim 48, wherein said virus is selected from the group consisting of flaviviruses and bunyaviruses.

50. The map of Claim 49, wherein said flavivirus is selected from the group consisting of a dengue virus, a yellow fever virus, a West Nile virus, and an encephalitis virus.

51. The map of Claim 49, wherein said bunyavirus is selected from the group consisting of a Hantaan virus and a Sin Nombre virus.

52. The map of Claim 48, wherein said map comprises digital information stored in computer memory.

53. The map of Claim 48, wherein said map comprises information for two or more markers.

54. The map of Claim 48, wherein said map comprises information for three or more markers.

55. The map of Claim 48, wherein said map comprises information for five or more markers.

56. The map of Claim 48, wherein said map comprises information for ten or more markers.

57. A method comprising: a) providing; i) a subject exibiting at least one symptom of an inflammation in a subject; and ii) a composition comprising a TRAIL protein or a fragment thereof; and b) administering the protein under conditions such that the at least one symptom of the inflammation is reduced.

58. The method of Claim 57, wherein said inflammation is derived from a virus infection.

59. The method of Claim 57, wherein said inflammation is derived from a disease.

,

60. The method of Claim 57, wherein said inflammation is derived from a wound.

61. The method of Claim 57, wherein said inflammation is derived from surgery.

62. The method of Claim 58, wherein said virus infection comprises a flavivirus.

63. The method of Claim 58, wherein said virus infection comprises a bunyavirus.

64. The method of Claim 62, wherein said flavivirus includes, but is not limited to, a dengue virus, a yellow fever virus, a West Nile virus, and/or an encephalitis virus.

65. The method of Claim 63, wherein said bunyavirus includes, but is not limited to, a Hantaan virus and/or a Sin Nombre virus.

66. The method of Claim 57, wherein said protein is encapsulated in a liposome.

67. The method of Claim 57, wherein said administering comprises a topical administration.

68. The method of Claim 67, wherein said topical administration is selected from the group consisting of transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.

69. The method of Claim 57, wherein said administering comprises parenteral administration.

70. The method of Claim 69, wherein said parenteral administration is selected from the group consisting of intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, intrathecal or intraventricular, administration.

71. A method comprising: a) providing; i) a subject exibiting at least one symptom of an inflammation in a subject; and ii) a composition comprising a nucleic acid, wherein said nucleic acid encodes a TRAIL protein or a fragment thereof; and b) administering the protein under conditions such that the at least one symptom of the inflammation is reduced.

72. The method of Claim 71, wherein said inflammation is derived from a virus infection.

73. The method of Claim 71, wherein said inflammation is derived from a disease.

74. The method of Claim 71, wherein said inflammation is derived from a wound.

75. The method of Claim 71, wherein said inflammation is derived from surgery.

76. The method of Claim 72, wherein said virus infection comprises a flavivirus.

77. The method of Claim 72, wherein said virus infection comprises a bunyavirus.

78. The method of Claim 76, wherein said flavivirus includes, but is not limited to, a dengue virus, a yellow fever virus, a West Nile virus, and/or an encephalitis virus.

79. The method of Claim 77, wherein said bunyavirus includes, but is not limited to, a Hantaan virus and/or a Sin Nombre virus.

80. The method of Claim 71, wherein said protein is encapsulated in a liposome.

81. The method of Claim 71 , wherein said administering comprises a topical administration.

82. The method of Claim 81, wherein said topical administration is selected from the group consisting of transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.

83. The method of Claim 57, wherein said administering comprises parenteral administration.

84. The method of Claim 83, wherein said parenteral administration is selected from the group consisting of intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, intrathecal or intraventricular, administration.