WO2011098806A1 - Proteins of the jak, stat, socs and pias families for use in the treatment hypercytokinemia or viral infection - Google Patents

Proteins of the jak, stat, socs and pias families for use in the treatment hypercytokinemia or viral infection Download PDF

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WO2011098806A1
WO2011098806A1 PCT/GB2011/050233 GB2011050233W WO2011098806A1 WO 2011098806 A1 WO2011098806 A1 WO 2011098806A1 GB 2011050233 W GB2011050233 W GB 2011050233W WO 2011098806 A1 WO2011098806 A1 WO 2011098806A1
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cytokine expression
expression regulator
infection
family
cell
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PCT/GB2011/050233
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French (fr)
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Kin Chow Chang
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The University Of Nottingham
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/11—Orthomyxoviridae, e.g. influenza virus

Definitions

  • the present invention relates to an assay of an agent, methods of screening an agent for use in a treatment, a cytokine expression regulator, or a vector encoding a cytokine expression regulator, for use in a treatment for hypercytokinemia.
  • Influenza A viruses have a wide host range for birds and mammals, posing a major threat to animal health as well as a zoonotic threat to humans. Although conventional swine and human influenza viruses are usually not life threatening in their respective host, the outcomes of highly pathogenic avian influenza (HPAI) H5N1 infections in humans and pigs are very different. The mortality rate of human cases of H5N1 infections is in excess of 60% (303 deaths out of 510 official WHO cases, Dec 2010) whereas the clinical effects of H5N1 in experimentally infected pigs are almost absent (Choi, Y. K. et al. (2005) J.Virol. 79: 10821- 10825 , Lipatov, A. S.et al. (2008) Plos Pathogens 4: 1-10) .
  • HPAI highly pathogenic avian influenza
  • the two broad approaches adopted to combat a virulent influenza infection are to target the influenza virus to reduce its pathogenicity, and to target the host to improve resistance or immunity against the virus.
  • anti- viral drugs are of limited efficacy and suffer from the growing emergence of drug resistant influenza strains.
  • Effective vaccination against seasonal influenza infections requires recurring vaccine formulation and, in the face of a pandemic, the significant lag period of 6 months or more between the identification of a specific vaccine subtype and vaccine distribution makes the vaccination route much too slow to stem the rapid spread of the virus.
  • One treatment option to improve patient survival is to restore or maintain an appropriate host innate response. Host cytokine dysregulation as a consequence of virulent influenza infection is a major cause of severe disease and mortality in mammals.
  • Pronounced activation of proinflammatory cytokines is a feature of HPAI H5N1 infections in humans .
  • Human H5N1 infections are characterised by severe pneumonia (often presented as acute respiratory distress syndrome, ARDS) , leucopaenia and even multi-organ failure which are largely explained by an inordinate hyperacute host proinflammatory response to the virus (Wong and Yuen (2006) . Chest 129: 156-168; Peiris et al. , (2009) Trends Immunol. 30: 574-584)) .
  • Tumour necrosis factor a (TNFa) , interleukin 6 (IL6) , interferon ⁇ (IFNP) , and various chemokines, including CXCL10, are some of the cytokines that are highly up-regulated in human patients (Korteweg, C. and J. Gu. (2008) Am. J. Pathol. 172: 1155-1170, Peiris, J. S. M. et al. (2009) Trends Immunol. 30: 574-584) and in H5N1 infected cells (respiratory epithelial cells and macrophages) (Chan, M. C. W. et al. (2005) Resp.Res . 6: 135 , Lee, S. M. Y. et al. (2008) J. Infect. Dis . 198: 525-535) .
  • the chemokine CXCL10 (IP10) is another possible target for the control of excessive proinflammatory response to H5N1 infection.
  • the use of an antagonist (AMG487) to CXCR3 (a receptor of CXCL10) has been shown to reduce H5N1 morbidity in ferrets (Cameron, C . M. et al. (2008) J.Virol. 82: 11308-11317) .
  • a method of treatment or prevention of hypercytokinemia comprising the administration of a cytokine expression regulator, or a vector encoding a cytokine expression regulator,
  • cytokine expression regulator is selected from any one of the group comprising members of the Janus Kinase (JAK) family, members of the signal transducer and activator of transcription (STAT) family, members of the suppressor of cytokine signalling (SOCS) family, and members of the protein inhibitor of activated STAT (PI AS) family, or analogues thereof.
  • JAK Janus Kinase
  • STAT signal transducer and activator of transcription
  • SOCS suppressor of cytokine signalling
  • PI AS protein inhibitor of activated STAT
  • the cytokine expression regulator is selected from any one of the group comprising members of the signal transducer and activator of transcription (STAT) family, members of the suppressor of cytokine signalling (SOCS) family, and members of the protein inhibitor of activated STAT (PI AS) family, or analogues thereof.
  • STAT signal transducer and activator of transcription
  • SOCS suppressor of cytokine signalling
  • PI AS protein inhibitor of activated STAT
  • proteins of the JAK, STAT, SOCS and PIAS families are key regulators of cytokine expression.
  • Members of these cytokine expression regulators are found to be strongly down-regulated in for example human respiratory epithelial cells and macrophages during H5N1 infection.
  • An advantage of treating or preventing hypercytokinemia by the provision/administration of a cytokine expression regulator is that it may restore normal levels of the cytokine expression regulator in a patient. Restoring normal levels of the cytokine expression regulator would provide regulation of the dysregulated cytokine pathways , for example in a cytokine storm.
  • the restoration of an appropriate host cytokine response or the prevention of its dysregulation is relatively unexplored as a treatment option to reduce clinical severity and improve patient survival.
  • This approach strategically targets the over-production of proinflammatory cytokines rather than the effects of excess cytokines .
  • Such an intervention approach could have broad applications for a range of acute viruses , for example, highly virulent influenza infections .
  • the administration may be topically, to the airways or systemically.
  • a method of treatment of a viral infection comprising the administration of a cytokine expression regulator, or a vector encoding a cytokine expression regulator,
  • cytokine expression regulator is selected from any one of the group comprising members of the Janus Kinase (JAK) family, members of the signal transducer and activator of transcription (STAT) family, members of the suppressor of cytokine signalling (SOCS) family, and members of the protein inhibitor of activated STAT (PIAS) family, or analogues thereof.
  • JAK Janus Kinase
  • STAT signal transducer and activator of transcription
  • SOCS suppressor of cytokine signalling
  • PIAS protein inhibitor of activated STAT
  • cytokine expression regulator is selected from the group consisting of members of JAK, STAT, SOCS and PIAS proteins , or analogues thereof.
  • Prevention of hypercytokinemia in a mammalian subject may comprise treatment for a viral infection.
  • the cytokine expression regulator is selected from the group consisting of members of STAT, SOCS and PIAS proteins , or analogues thereof.
  • the vector may be arranged to provide over-expression of JAK, STAT, SOCS or PIAS proteins, or analogues thereof.
  • agent or cytokine expression regulator discussed herein may act as an agonist to at least one member of JAK family and/or STAT family and/or SOCS family, and/or PIAS family.
  • the treatment may comprise prevention or reduction of hyperacute proinflammatory effects, such as hypercytokinemia, from hyperacute viral infection, such as highly pathogenic influenza or severe acute respiratory syndrome (SARS) .
  • the treatment may comprise a reduction in the level of primary proinflammatory cytokines , such as TNF-alpha.
  • the hypercytokinemia may be a result of infection, such as virus infection or bacterial infection.
  • the hypercytokinemia may be a result of pneumonia.
  • the virus or bacterium may be implicated in pneumonia infection and/or acute respiratory distress syndrome (ARDS) .
  • ARDS acute respiratory distress syndrome
  • the hypercytokinemia may be a result of an infection of any of the group selected from influenza virus, SARS virus, respiratory syncytial virus (RSV) , human parainfluenza virus, adenoviruses, metapneumovirus, herpes simplex virus (HSV) , varicella-zoster virus (VZV) and cytomegalovirus (CMV) .
  • the treatment may be for H5N1 viral infection.
  • the prevention may comprise treatment for infection, such as viral infection, preferably H5N1 infection.
  • JAK family members may be selected from JAKl , JAK2 , JAK3 , and TYK2, or combinations thereof.
  • JAK family members may be JAKl .
  • JAKl may comprise the polypeptide sequence of SEQ ID NO: 27.
  • JAK2 may comprise the polypeptide sequence of SEQ ID NO: 28.
  • JAK3 may comprise the polypeptide sequence of SEQ ID NO : 29.
  • TYK2 may comprise the polypeptide sequence of SEQ ID NO: 30.
  • STAT family members may be selected from STAT1 , STAT2, STAT3 , STAT4, STAT5 (STAT5A and STAT5B) , and STAT6, or combinations thereof.
  • STAT family members may be selected from STAT1 , STAT2, STAT3 , or combinations thereof.
  • STAT1 (isoform alpha) may comprise the polypeptide sequence of SEQ ID NO: 1.
  • STAT1 (isoform beta) may comprise the polypeptide sequence of SEQ ID NO: 2.
  • STAT2 may comprise the polypeptide sequence of SEQ ID NO: 3.
  • STAT3 (isoform 1) may comprise the polypeptide sequence of SEQ ID NO: 4.
  • STAT3 (isoform 2) may comprise the polypeptide sequence of SEQ ID NO: 5.
  • STAT3 (isoform 3) may comprise the polypeptide sequence of SEQ ID NO: 6.
  • STAT4 may comprise the polypeptide sequence of SEQ ID NO: 7.
  • STAT5 (signal transducer and activator of transcription 5A) may comprise the polypeptide sequence of SEQ ID NO: 8.
  • STAT5 (signal transducer and activator of transcription 5B) may comprise the polypeptide sequence of SEQ ID NO: 9.
  • STAT6 may comprise the polypeptide sequence of SEQ ID NO: 10.
  • SOCS family members may be selected from SOCS1 , SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, SOCS7 and CIS, or combinations thereof.
  • SOCS family members may be selected from SOCS1 , SOCS2, SOCS3 , or combinations thereof.
  • SOCS1 may comprise the polypeptide sequence of SEQ ID NO: 11.
  • SOCS2 may comprise the polypeptide sequence of SEQ ID NO: 12.
  • SOCS3 may comprise the polypeptide sequence of SEQ ID NO: 13.
  • SOCS4 may comprise the polypeptide sequence of SEQ ID NO: 14 or SEQ ID NO: 15.
  • SOCS5 may comprise the polypeptide sequence of SEQ ID NO: 16 or SEQ ID NO: 17.
  • SOCS6 may comprise the polypeptide sequence of SEQ ID NO: 18.
  • SOCS7 may comprise the polypeptide sequence of SEQ ID NO: 19.
  • CIS cytokine-inducible SH2-containing protein isoform 1
  • CISH cytokine-inducible SH2-containing protein isoform 2
  • CISH cytokine-inducible SH2-containing protein isoform 2
  • PIAS family members may be selected from PIAS1 , PIAS2, PIAS3, PIAS4, or combinations thereof.
  • PIAS family members may be selected from PIAS1 , and PIAS2, or combinations thereof.
  • PIAS1 protein inhibitor of activated STAT 1
  • PIAS2 protein inhibitor of activated STAT X isoform alpha
  • PIAS2 protein inhibitor of activated STAT X isoform beta
  • PIAS3 protein inhibitor of activated STAT 3
  • PIAS4 protein inhibitor of activated STAT 4
  • An analogue may be a natural or synthetic molecule that has the same 3- dimensional molecular structure to the regulator of cytokine expression, and optionally functions like the cytokine expression regulator.
  • An analogue of STAT family members may include proteins having at least 60% sequence identity, alternatively at least 70%, 80%, 90%, 95% or 99% sequence identity, to any one of the sequences selected from the group consisting of SEQ ID NO: 1 ; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; and SEQ ID NO: 10.
  • An analogue may be a molecule, synthetic or otherwise, that has the same 3-dimensional molecular structure as a STAT family member, and optionally functions like a STAT family member.
  • An analogue of SOCS family members may include proteins having at least 60% sequence identity, alternatively at least 70%, 80%, 90%, 95% or 99% sequence identity to any one of the sequences selected from the group consisting of SEQ ID NO: 11 ; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; and SEQ ID NO: 21.
  • An analogue may be a molecule, synthetic or otherwise, that has the same 3-dimensional molecular structure as a SOCS family member, and optionally functions like a SOCS family member.
  • An analogue of PIAS family members may include proteins having at least 60% sequence identity, alternatively at least 70%, 80% , 90% , 95% or 99% sequence identity to any one of the sequences selected from the group consisting of SEQ ID NO: 22; SEQ ID NO: 23 ; SEQ ID NO: 24; SEQ ID NO : 25 ; and SEQ ID NO: 26.
  • An analogue may be a molecule, synthetic or otherwise, that has the same 3-dimensional molecular structure as a PIAS family member, and optionally functions like a PIAS family member.
  • An analogue of JAK family members may include proteins having at least 60% sequence identity, alternatively at least 70% , 80%, 90% , 95% or 99% sequence identity to any one of the sequences selected from the group consisting of SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; and SEQ ID NO: 30.
  • An analogue may be a molecule, synthetic or otherwise, that has the same 3-dimensional molecular structure as a JAK family member, and optionally functions like a JAK family member.
  • cytokine expression regulator or analogue thereof, the protein, or the vector may be provided in a pharmaceutically acceptable carrier.
  • suitable pharmaceutically acceptable carriers include water, saline solution and other aqueous solutions , which will be known to those skilled in the art.
  • an assay comprising the steps of:
  • cytokine expression regulator is selected from any one of the group comprising members of the Janus kinases (JAK) family, members of the signal transducer and activator of transcription (STAT) family, members of the suppressor of cytokine signalling (SOCS) family, and members of the protein inhibitor of activated STAT (PI AS) family, or combinations thereof.
  • JAK Janus kinases
  • STAT signal transducer and activator of transcription
  • SOCS suppressor of cytokine signalling
  • PI AS protein inhibitor of activated STAT
  • the cytokine expression regulator is selected from any one of the group comprising members of the signal transducer and activator of transcription (STAT) family, members of the suppressor of cytokine signalling (SOCS) family, and members of the protein inhibitor of activated STAT (PIAS) family, or combinations thereof.
  • STAT signal transducer and activator of transcription
  • SOCS suppressor of cytokine signalling
  • PIAS protein inhibitor of activated STAT
  • the assay may be for the identification of the agent as a potential therapeutic agent for use in the treatment of hypercytokinemia.
  • the assay may be for the identification of the agent as useful in the reduction of proinflammatory cytokines, such as TNF-alpha, in the cell, or in a patient.
  • Such an assay would advantageously provide a method of identifying therapeutic drug candidates for restoring regulation of cytokines in an infection.
  • the restoration of an appropriate host cytokine response or the prevention of its dysregulation is relatively unexplored as a treatment option to reduce clinical severity and improve patient survival.
  • This approach strategically targets the over-production of proinflammatory cytokines rather than the effects of excess cytokines.
  • Such an intervention approach could have broad applications for a range of acute viruses , for example, highly virulent influenza infections .
  • the assay may further comprise triggering an infection response in the cell. Triggering an infection response in the cell has the use of inducing cytokine dysregulation, such as suppression of the cytokine expression regulator, for which the agent may have an effect.
  • the infection response in the cell may be triggered by introducing an infectious material to the cell, or by introducing a material to the cell that mimics an infection of the cell.
  • the material may be a virus , a glycopeptide, a protein, a nucleic acid, for example a viral nucleic acid, or a bacterium.
  • the material may also be part of one of the aforementioned materials , such as part of a virus , or part of a bacterium, such as endotoxin/lipopolysacharride.
  • the virus , or part of the virus , or the viral nucleic acid may be derived from influenza virus , such as H5N1 or HINT , or SARS virus .
  • the virus or bacterium may be implicated in pneumonia infection and/or acute respiratory distress syndrome (ARDS) .
  • the virus, or part of the virus , or the viral nucleic acid may be derived from any of the group selected from respiratory syncytial virus (RSV) , human parainfluenza viruses, adenoviruses, metapneumovirus, herpes simplex virus (HSV) , varicella-zoster virus (VZV) and cytomegalovirus (CMV) .
  • RSV respiratory syncytial virus
  • HSV herpes simplex virus
  • VZV varicella-zoster virus
  • CMV cytomegalovirus
  • the modulation of expression of the cytokine expression regulator may be upregulation.
  • the modulation of expression of the cytokine expression regulator may be the prevention or reversal of suppression of the regulator of cytokine expression.
  • the modulation of expression of the cytokine expression regulator may lead to the restoration of normal levels (i.e. non-infected levels) of the cytokine expression regulator.
  • Detecting upregulation, prevention of suppression, or dysregulation, of the cytokine expression regulator in the cell has an advantage that it provides a new measurable effect for finding novel therapeutic agents for treatment against hypercytokinemia, for example due to viral infection. This contrasts with previous studies which suggest that induction of SOCS3 (a regulator of cytokine expression) is an unfavourable response of host cells during infection . The infection response may be triggered before, during or after exposure of the cell to the agent.
  • the infection response may be triggered before, exposure of the cell to the agent in order to investigate the ability of the agent to restore function or expression of the cytokine expression regulator.
  • the infection response may be triggered after exposure of the cell to the agent in order to evaluate the agent's ability to prevent dysregulation or inactivation of the regulator of cytokine expression.
  • the cell may be a mammalian primary cell or from an established mammalian cell line.
  • the cell may be a human cell, pig cell or avian cell.
  • the cell may be a macrophage (or monocyte) or a tracheal/bronchial epithelial cell (TEC) .
  • Detecting whether there is modulation of expression or activity of at least one cytokine expression regulator may comprise detecting expression levels of RNA encoding the cytokine expression regulator or relevant cytokine, for example by real-time-PCR, or of protein, for example by enzyme-linked immunosorbent assay (ELISA) .
  • the agent may be a compound/drug, nucleic acid, peptide, or protein.
  • the agent may be an analogue of the cytokine expression regulator.
  • the agent may be a truncated form of the cytokine expression regulator.
  • the agent is not significantly cytotoxic at pharmaceutically useful levels.
  • the agent is permeable to cell membrane.
  • a method of screening an agent for use in a treatment comprising determining whether said agent has affinity against a negative-regulator of at least one protein, wherein the at least one protein is selected from the group consisting of: (a) members of the signal transducer and activator of transcription (STAT) family, (b) members of the suppressor of cytokine signalling (SOCS) family, (c) members of the protein inhibitor of activated STAT (PIAS) family (d) members of the Janus kinases (JAK) family, or combinations of (a) , (b) , (c) or (d) .
  • STAT signal transducer and activator of transcription
  • SOCS suppressor of cytokine signalling
  • PIAS protein inhibitor of activated STAT
  • JOK Janus kinases
  • a cytokine expression regulator pathway in a cell for determining the therapeutic activity of an agent, wherein the cytokine expression regulator pathway is selected from JAK, STAT, SOCS and PIAS.
  • the cytokine expression regulator pathway is selected from STAT, SOCS and PIAS.
  • FIG. 1 Induction of TNFa by avian H5N1 and USSR/77 virus in macrophages and tracheal epithelial cells (TECs) from human and pig. Note that TNFa induction is much greater in human cells than corresponding pig cells, and that TNFa induction is much higher in macrophages than TECs .
  • Cells were incubated with influenza subtype, as indicated, at 1.0 MOI for 3h followed by PBS rinses and further incubation overnight for an overall duration of 24h. Total RNA was extracted for cDNA conversion from which
  • Taqman real-time PCR (Lightcycler 480) was performed. Quantification of expression was based on the relative standard curve method and normalised to 18S RNA. Data bars represent the average of 4 biological replicates. Error bars are the standard error of the means.
  • FIG. 2 Induction of IFNfil and ILlfi by avian H5N1 and USSR/77 virus in human macrophages and TECs . Note that IFNfil induction in human macrophages and TECs is greater with
  • ILlfi is consistently down-regulated by avian H5N1 infection.
  • Cells were incubated with influenza subtype, as indicated, at 1.0 MOI for 3h followed by PBS rinses and further incubation overnight for an overall duration of 24h.
  • Total RNA was extracted for cDNA conversion from which Taqman real-time PCR (Lightcycler 480) was performed. Quantification of expression was based on the relative standard curve method and normalised to 18S RNA. Data bars represent the average of 4 biological replicates. Error bars are the standard error of the means .
  • TECs tracheal epithelial cells
  • USSR human H1N1 A/USSR/90/77.
  • FIG. 3 Induction of IFNfil and ⁇ by avian H5N1 and USSR/77 virus in pig macrophages .
  • IFNfil induction in pig macrophages is comparable between avian H5N1 and USSR/77 virus , and that ⁇ in pig macrophages is not induced by avian H5N1 infection.
  • Cells were incubated with influenza subtype, as indicated, at 1.0 MOI for 3h followed by PBS rinses and further incubation overnight for an overall duration of 24h.
  • Total RNA was extracted for cDNA conversion from which Taqman real-time PCR (Lightcycler 480) was performed. Quantification of expression was based on the relative standard curve method and normalised to 18S RNA. Data bars represent the average of 4 biological replicates . Error bars are the standard error of the means .
  • TECs tracheal epithelial cells
  • USSR human H1N1 A/USSR/90/77.
  • FIG. 4 Down-regulation of JAK1 and STAT1 by avian H5N1 and USSR/77 virus in human macrophages and TECs. Note that JAK1 and ST ATI are more severely suppressed in macrophages and TECs by avian H5N1 than by USSR/77 virus.
  • Cells were incubated with influenza subtype, as indicated, at 1.0 MOI for 3h followed by PBS rinses and further incubation overnight for an overall duration of 24h.
  • Total RNA was extracted for cDNA conversion from which Taqman real-time PCR (Lightcycler 480) was performed. Quantification of expression was based on the relative standard curve method and normalised to 18S RNA. Data bars represent the average of 4 biological replicates . Error bars are the standard error of the means .
  • TECs tracheal epithelial cells
  • USSR human H1N1 A/USSR/90/77.
  • FIG. 5 Down-regulation of SOCS3 by avian H5N1 in human TECs.
  • SOCS3 in human TECs is consistently down- regulated by avian H5N1 but not USSR/77.
  • SOCS3 expression in human macrophages is not affected by avian H5N1 or USSR/77 virus.
  • Cells were incubated with influenza subtype, as indicated, at 1.0 MOI for 3h followed by PBS rinses and further incubation overnight for an overall duration of 24h.
  • Total RNA was extracted for cDNA conversion from which Taqman real-time PCR (Lightcycler 480) was performed. Quantification of expression was based on the relative standard curve method and normalised to 18S RNA. Data bars represent the average of 4 biological replicates. Error bars are the standard error of the means .
  • TECs tracheal epithelial cells ;
  • USSR human H1N1 A/USSR/90/77.
  • FIG. 6 Down-regulation of PIAS1 by avian H5N1 and USSR/77 virus in human macrophages and TECs .
  • PIAS1 in human macrophages and TECs is more severely suppressed by avian H5N1 than by USSR/77 virus .
  • Cells were incubated with influenza subtype, as indicated, at 1.0 MOI for 3h followed by PBS rinses and further incubation overnight for an overall duration of 24h.
  • Total RNA was extracted for cDNA conversion from which Taqman real-time PCR (Lightcycler 480) was performed. Quantification of expression was based on the relative standard curve method and normalised to 18S RNA. Data bars represent the average of 4 biological replicates.
  • TECs tracheal epithelial cells
  • USSR human H1N1 A/USSR/90/77.
  • Figure 7 - Induction of TNFa in USSR/77-infected primary cells is reduced by over-expression of SOCS3 and PIAS2.
  • ELISAs for the detection of TNFa was performed on supernatants collected 24h post-infection.
  • B Human TECs were transfected by electroporation with SOCS3 , PIAS2 and non-coding empty vector.
  • HPAI H5N1 Human cases of highly pathogenic avian influenza (HPAI) H5N1 infection carry a mortality rate in excess of 60% , in which hyperacute host inflammatory response (cytokine storm) is often cited as a major cause of complication and death.
  • cytokine storm hyperacute host inflammatory response
  • pigs are susceptible to HPAI H5N1 infection they show no clinical signs of disease during infection.
  • cytokine regulatory factors By comparing host innate response to low and high pathogenicity influenza viruses between the resistant and susceptible species (pig and human respectively) in primary macrophages and tracheal epithelial cells, several cytokine regulatory factors have been identified that are prospective targets for therapeutic intervention to prevent or ameliorate the damaging effects of hypercytokinemia.
  • Identifying specific signalling drivers that are potential host triggers of cytokine dysfunction is based on the inherent contrast in disease resistance and innate response to HPAI H5N1 infections between the host species of human (susceptible) and pig (resistant) .
  • Extensive comparative infection studies show molecular differences in host innate response between human and pig primary macrophages and tracheal epithelial cells (TECs) , the two most important cell types in the pathogenesis of influenza infection.
  • TECs tracheal epithelial cells
  • STAT signal transducer and activator of transcription
  • SOCS suppressor of cytokine signalling
  • PI AS protein inhibitor of activated STAT
  • TNFa is a key proinflammatory cytokine that is highly up-regulated in human patients and cells infected with HPAI H5N1 virus (Peiris, J. S. M. et al. (2009) Trends Immunol. 30: 574-584) .
  • low pathogenicity influenza subtypes such as PR8 strain and seasonal subtypes , typically show little or no TNF induction in human cells (Mok, K. P. et al. (2009) J. Infect. Dis. 200: 1104-1112, Pauli, E. -K. et al. (2008) Plos Pathogens 4:el000196) .
  • the induction of TNFa by the same avian H5N1 is small in pig macrophages, and is virtually none in pig TECs (Fig. l) .
  • the human H1N1 virus (denoted as USSR/77) induces the most TNFa expression in both human macrophages and epithelial cells, at levels that are higher or comparable with those induced by the avian H5N1 virus (Fig. l) .
  • Swine H1N1 and avian H2N3 in human TECs did not produce detectable TNFa as determined by ELISA.
  • the USSR/77 virus was responsible for the 1977/78 epidemic and has been known to cause severe respiratory illness with widespread lung pathology in humans and experimental ferrets (Svitek, N. et al. (2008) Virology 376:53-59) .
  • Systemic acute release of TNFa is detrimental to the host.
  • Mice deficient in TNF receptor 1 (TNFR1) or treated with anti-TNFa antibody show reduce morbidity and less weight loss when challenged with avian H5N1 virus (Szretter, K. J. J. (2007) Virology 81 :2736-2744) . Therefore controlling the excessive production of TNFa caused by virulent influenza infection could ameliorate the damaging effects of hypercytokinemia.
  • TNFa signalling One of the pleiotropic targets of TNFa signalling is the degradation of STAT5 proteins reported in 3T3-L1 adipocytes (Floyd, Z. E. et al. (2007) Am. J. Physiol. Endocrinol. Metab. 292: E461-E468) .
  • sustained elevated levels of TNFa could lead to a major disruption of the JAK-STAT signalling pathway, a major cascade activated by type I and II IFNs, and members of several interleukin families that culminates in the transcriptional activation of further proinflammatory cytokines and the innate anti-viral state (Schindler, C. and Plumlee, C.
  • the avian H5N1 virus was found to consistently down-regulate the JAK- STAT pathway in human macrophages and TECs (Fig.4) .
  • Several members of the JAK and STAT families are transcriptionally suppressed in the presence of the avian H5N1 virus.
  • the USSR/77 virus also exhibits similar inhibitory effect on the JAK-STAT pathway but the degree of suppression is not as severe as the avian H5N1.
  • the JAK-STAT cascade among its multiple roles, is a key innate immunity pathway activated during early infection. Its down-regulation by a highly virulent virus could compromise the host's ability to mount an effective innate response. A further complication to JAK-STAT inhibition could be an increase of inflammation.
  • STAT3 is one of several STAT members that is down- regulated in avian H5N1 infected human TECs . It has been shown that STAT3 activation elicits an anti-inflammatory effect (Schindler, C. and Plumlee, C . (2008) Seminars in Cell and Developmental Biology 19: 311- 318) . Constitutively activated STAT3 is able to suppress bacterial lipopolysaccharides (LPS) -induced TNFa and IL6 production in macrophages (Yoshimura, A. (2009) Keido J. Med. 58:73-83) .
  • LPS lipopolysaccharides
  • SOCS proteins inhibit JAK-STAT signalling through their high binding affinity for cytokine receptors , JAK and STAT proteins.
  • Many factors endocrine factors and cytokines are able to induce SOCS family proteins, including insulin, IL1 , IL6, IL10, IFNa/ ⁇ , IFNy and TNFa (Palmer, D. C. and Restifo, N. P. (2009) Trends Immunol. 30 :592-602. 2009) .
  • H1N1 H1N1 (PR8) and H3N2 subtypes
  • H1N1 H1N1 (PR8) and H3N2 subtypes
  • the invention provides that the restoration or over-expression of SOCS can prevent or reduce the excessive production of proinflammatory cytokines, such as TNFa, in human cases of highly pathogenic influenza infection. This is supported by the present findings (Fig.5) together with the documented protective anti-inflammatory properties of SOCS
  • PIAS1 , PIAS2/x, PIAS3 and PIAS4/y possess SUMO (small ubiquitin-like modifier) E3 ligase activity and can interact with over 60 proteins , many of which are transcription factors, to regulate their transcriptional activity (Shuai, K. (2006) Cell Res . 16: 196-202, Shuai, K. and B . Liu. (2005) Nature Rev. Immunol. 5 : 593-605) .
  • PIAS proteins are not usually inducible (O ' Shea, J. J. and W. Watford. (2004) Nature Immunol. 5 : 875-876) .
  • PIAS proteins regulate transcription factors through blocking the DNA-binding ability of transcription factors , recruiting transcriptional co-repressors or co-activators , or promoting sumoylation of its binding partner (Shuai, K. (2006) Cell Res . 16: 196-202) .
  • PIAS proteins are inhibitors of STAT, IRF and NF- ⁇ p65 transcription factors which are responsible for the induction of primary cytokines (type I and II IFNPs, IL1 , IL6 and TNFa) and cytokine-inducible genes (Shuai, K. and B . Liu. (2005) Nature Rev. Immunol. 5 : 593-605) .
  • mice deficient in PIAS1 or PIAS4 which show raised serum levels of TNFa and ILip, and heightened sensitivity to LPS-induced endotoxic shock (Liu, B. et al. (2005) Mol. Cell. Biol. 25 : 1113-1123 , Tahk, S. et al. (2007) Proc. Natl. Acad Sci U.S.A. 104: 11643-11648) .
  • avian H5N1 strongly down-regulates members of the PIAS family. Although USSR/77 is able to suppress the expression of PIAS1 , avian H5N1 is more potent in the suppression of PIAS1 in human TECs and macrophages (Fig.6) .
  • the invention provides that the restoration or over-expression of PIAS can prevent or reduce the excessive production of proinflammatory cytokines, such as TNFa, in human cases of highly pathogenic influenza infection.
  • SOCS or PIAS over-expression reduces TNFa induction in influenza infected human cells
  • RNA detected in USSR/77 infected human macrophages correlate with high TNFa protein detection by ELISAs (Fig.7 A) .
  • Human TECs and macrophages were transfected (NHBE Nucleofector Kit, Lonza) by electroporation (Amaxa electroporator) in accordance with the supplier' s instructions .
  • Expression plasmids used to evaluate the effects of SOCS and PIAS on TNFa induction by USSR/77 virus were: pCMV-SOCS3 (plasmid 11486, Addgene) , pCMV-PIASxa (plasmid 15209, Addgene, also known as PIAS2) , and empty vector control pBK-CMV. Transfection efficiency was typically around 40% . Forty-eight hours post-transfection one set of wells were infected with USSR/77 at 1.0 MOI, while the remaining duplicate set was left uninfected.
  • KVLFDKDVNE RNTVKGFRKF NILGTHTKVM NMEESTNGSL AAEFRHLQLK EQKNAGTRTN
  • KVLFDKDVNE RNTVKGFRKF NILGTHTKVM NMEESTNGSL AAEFRHLQLK EQKNAGTRTN
  • NVHMNPPQVK A IISEQQAK SLLKNENTRN ECSGEILNNC CVMEYHQATG TLSAHFRNMS
  • NVHMNPPQVK A IISEQQAK SLLKNENTRN DYSGEILNNC CVMEYHQATG TLSAHFRNMS
  • VLLQDTKPTF EMQQPSPPIP PVHPDVQLKN LPFYDVLDVL IKPTSLVQSS IQRFQEKFFI
  • EEDPPAKRKC IFMSETQSSP TKGVLMYQPS SVRVPSVTSV DPAAIPPSLT DYSVPFHHTP
  • VLLQDTKPTF EMQQPSPPIP PVHPDVQLKN LPFYDVLDVL IKPTSLVQSS IQRFQEKFFI
  • EEDPPAKRKC IFMSETQSSP TKGVLMYQPS SVRVPSVTSV DPAAIPPSLT DYSVPFHHTP
  • KRCCQPKPRE ISNLLVATKK AQEWQPVYPM SQLSFDRILK KDLVQGEHLG RGTRTHIYSG
  • KRCCQPKPRE ISNLLVATKK AQEWQPVYPM SQLSFDRILK KDLVQGEHLG RGTRTHIYSG

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Abstract

The invention relates to a cytokine expression regulator,or a vector encoding a cytokine expression regulator, for use in the treatment or prevention of hypercytokinemia and/or a viral infection in a mammalian subject,wherein the cytokine expression regulator is selected from the group consisting of members of JAK,STAT, SOCS and PIAS proteins, or analogues thereof. The invention also relates to a method of treatmentor prevention of hypercytokinemia and/or viral infection comprising the administration to a subject of a cytokine expression regulator, or a vector encoding a cytokine expression regulator. Furthermore, the invention relates to an assay for identifying therapeutic drug candidates for use in restoring regulation of cytokines in an infection.

Description

PROTEINS OF THE JAK, STAT, SOCS AND PIAS FAMILIES FOR USE IN THE TREATMENT HYPERCYTOKINEMIA OR VIRAL INFECTION
The present invention relates to an assay of an agent, methods of screening an agent for use in a treatment, a cytokine expression regulator, or a vector encoding a cytokine expression regulator, for use in a treatment for hypercytokinemia.
Influenza A viruses have a wide host range for birds and mammals, posing a major threat to animal health as well as a zoonotic threat to humans. Although conventional swine and human influenza viruses are usually not life threatening in their respective host, the outcomes of highly pathogenic avian influenza (HPAI) H5N1 infections in humans and pigs are very different. The mortality rate of human cases of H5N1 infections is in excess of 60% (303 deaths out of 510 official WHO cases, Dec 2010) whereas the clinical effects of H5N1 in experimentally infected pigs are almost absent (Choi, Y. K. et al. (2005) J.Virol. 79: 10821- 10825 , Lipatov, A. S.et al. (2008) Plos Pathogens 4: 1-10) .
Presently, the two broad approaches adopted to combat a virulent influenza infection are to target the influenza virus to reduce its pathogenicity, and to target the host to improve resistance or immunity against the virus. Unfortunately, anti- viral drugs are of limited efficacy and suffer from the growing emergence of drug resistant influenza strains. Effective vaccination against seasonal influenza infections requires recurring vaccine formulation and, in the face of a pandemic, the significant lag period of 6 months or more between the identification of a specific vaccine subtype and vaccine distribution makes the vaccination route much too slow to stem the rapid spread of the virus. One treatment option to improve patient survival is to restore or maintain an appropriate host innate response. Host cytokine dysregulation as a consequence of virulent influenza infection is a major cause of severe disease and mortality in mammals. Pronounced activation of proinflammatory cytokines , described as cytokine storm, is a feature of HPAI H5N1 infections in humans . Human H5N1 infections are characterised by severe pneumonia (often presented as acute respiratory distress syndrome, ARDS) , leucopaenia and even multi-organ failure which are largely explained by an inordinate hyperacute host proinflammatory response to the virus (Wong and Yuen (2006) . Chest 129: 156-168; Peiris et al. , (2009) Trends Immunol. 30: 574-584)) . Tumour necrosis factor a (TNFa) , interleukin 6 (IL6) , interferon β (IFNP) , and various chemokines, including CXCL10, are some of the cytokines that are highly up-regulated in human patients (Korteweg, C. and J. Gu. (2008) Am. J. Pathol. 172: 1155-1170, Peiris, J. S. M. et al. (2009) Trends Immunol. 30: 574-584) and in H5N1 infected cells (respiratory epithelial cells and macrophages) (Chan, M. C. W. et al. (2005) Resp.Res . 6: 135 , Lee, S. M. Y. et al. (2008) J. Infect. Dis . 198: 525-535) .
There is no licensed product for the specific treatment of cytokine dysregulation in human H5N1 infections. The few anti-inflammatory drugs, including corticosteroids (Salomon, R. et al. (2007) Proc.Natl.Acad.Sci.U.S.A. 104: 12479-12481) , that had been cited for possible use were empirically chosen and appeared to be ineffective or not fully tested. There is much interest in targeting the transcription factor NFKB to suppress its activation to prevent hypercytokinemia. NFKB deficient mice do not exhibit hyperacute cytokine dysregulation in response to H5N1 infection but with no improvement in survival rate (Droebner, K. , et al. (2008) J.Virol. 82: 11461-11466) . However, high doses of aspirin (salicylate) , known to inhibit both NFKB activation and cyclooxygenase activity improved the survival of H5N1 infected mice (Ludwig, S. (2009) J. Antimicrob. Chemother. 64: 1-4) . Another prospective treatment to reduce lung inflammation is the use of a fusion immunoglobulin to block the activation of the receptor Ox40 (TNFRSF4/CD134) ; Ox40 signals to activate NFKB in T-cells leading to inflammation (Humphreys , I. R. et al. (2003) J. Exp. Med. 198: 1237- 1242) . However the clinical efficacy or safety of the Ox40 inhibitor is not known. The chemokine CXCL10 (IP10) is another possible target for the control of excessive proinflammatory response to H5N1 infection. The use of an antagonist (AMG487) to CXCR3 (a receptor of CXCL10) has been shown to reduce H5N1 morbidity in ferrets (Cameron, C . M. et al. (2008) J.Virol. 82: 11308-11317) .
There is a need to improve treatment of acute and severe inflammatory infections , such as from virulent strains of influenza. Potential new therapies and identification of new therapeutic agents is required.
According to a first aspect of the invention there is provided a method of treatment or prevention of hypercytokinemia comprising the administration of a cytokine expression regulator, or a vector encoding a cytokine expression regulator,
wherein the cytokine expression regulator is selected from any one of the group comprising members of the Janus Kinase (JAK) family, members of the signal transducer and activator of transcription (STAT) family, members of the suppressor of cytokine signalling (SOCS) family, and members of the protein inhibitor of activated STAT (PI AS) family, or analogues thereof.
In one embodiment the cytokine expression regulator is selected from any one of the group comprising members of the signal transducer and activator of transcription (STAT) family, members of the suppressor of cytokine signalling (SOCS) family, and members of the protein inhibitor of activated STAT (PI AS) family, or analogues thereof. Proteins of the JAK, STAT, SOCS and PIAS families are key regulators of cytokine expression. Members of these cytokine expression regulators are found to be strongly down-regulated in for example human respiratory epithelial cells and macrophages during H5N1 infection. An advantage of treating or preventing hypercytokinemia by the provision/administration of a cytokine expression regulator is that it may restore normal levels of the cytokine expression regulator in a patient. Restoring normal levels of the cytokine expression regulator would provide regulation of the dysregulated cytokine pathways , for example in a cytokine storm. The restoration of an appropriate host cytokine response or the prevention of its dysregulation is relatively unexplored as a treatment option to reduce clinical severity and improve patient survival. This approach strategically targets the over-production of proinflammatory cytokines rather than the effects of excess cytokines . Such an intervention approach could have broad applications for a range of acute viruses , for example, highly virulent influenza infections . The administration may be topically, to the airways or systemically.
According to another aspect of the invention, there is provided a method of treatment of a viral infection comprising the administration of a cytokine expression regulator, or a vector encoding a cytokine expression regulator,
wherein the cytokine expression regulator is selected from any one of the group comprising members of the Janus Kinase (JAK) family, members of the signal transducer and activator of transcription (STAT) family, members of the suppressor of cytokine signalling (SOCS) family, and members of the protein inhibitor of activated STAT (PIAS) family, or analogues thereof. According to another aspect of the invention there is provided a cytokine expression regulator, or a vector encoding a cytokine expression regulator, for use in a treatment or prevention of hypercytokinemia in a mammalian subject,
wherein the cytokine expression regulator is selected from the group consisting of members of JAK, STAT, SOCS and PIAS proteins , or analogues thereof.
Prevention of hypercytokinemia in a mammalian subject, may comprise treatment for a viral infection.
In one embodiment the cytokine expression regulator is selected from the group consisting of members of STAT, SOCS and PIAS proteins , or analogues thereof.
The vector may be arranged to provide over-expression of JAK, STAT, SOCS or PIAS proteins, or analogues thereof.
The agent or cytokine expression regulator discussed herein may act as an agonist to at least one member of JAK family and/or STAT family and/or SOCS family, and/or PIAS family.
The treatment may comprise prevention or reduction of hyperacute proinflammatory effects, such as hypercytokinemia, from hyperacute viral infection, such as highly pathogenic influenza or severe acute respiratory syndrome (SARS) . The treatment may comprise a reduction in the level of primary proinflammatory cytokines , such as TNF-alpha. The hypercytokinemia may be a result of infection, such as virus infection or bacterial infection. The hypercytokinemia may be a result of pneumonia. The virus or bacterium may be implicated in pneumonia infection and/or acute respiratory distress syndrome (ARDS) . The hypercytokinemia may be a result of an infection of any of the group selected from influenza virus, SARS virus, respiratory syncytial virus (RSV) , human parainfluenza virus, adenoviruses, metapneumovirus, herpes simplex virus (HSV) , varicella-zoster virus (VZV) and cytomegalovirus (CMV) . The treatment may be for H5N1 viral infection. The prevention may comprise treatment for infection, such as viral infection, preferably H5N1 infection.
JAK family members may be selected from JAKl , JAK2 , JAK3 , and TYK2, or combinations thereof. In one embodiment, JAK family members may be JAKl . JAKl may comprise the polypeptide sequence of SEQ ID NO: 27. JAK2 may comprise the polypeptide sequence of SEQ ID NO: 28. JAK3 may comprise the polypeptide sequence of SEQ ID NO : 29. TYK2 may comprise the polypeptide sequence of SEQ ID NO: 30. STAT family members may be selected from STAT1 , STAT2, STAT3 , STAT4, STAT5 (STAT5A and STAT5B) , and STAT6, or combinations thereof. In one embodiment, STAT family members may be selected from STAT1 , STAT2, STAT3 , or combinations thereof. STAT1 (isoform alpha) may comprise the polypeptide sequence of SEQ ID NO: 1. STAT1 (isoform beta) may comprise the polypeptide sequence of SEQ ID NO: 2. STAT2 may comprise the polypeptide sequence of SEQ ID NO: 3. STAT3 (isoform 1) may comprise the polypeptide sequence of SEQ ID NO: 4. STAT3 (isoform 2) may comprise the polypeptide sequence of SEQ ID NO: 5. STAT3 (isoform 3) may comprise the polypeptide sequence of SEQ ID NO: 6. STAT4 may comprise the polypeptide sequence of SEQ ID NO: 7. STAT5 (signal transducer and activator of transcription 5A) may comprise the polypeptide sequence of SEQ ID NO: 8. STAT5 (signal transducer and activator of transcription 5B) may comprise the polypeptide sequence of SEQ ID NO: 9. STAT6 may comprise the polypeptide sequence of SEQ ID NO: 10.
SOCS family members may be selected from SOCS1 , SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, SOCS7 and CIS, or combinations thereof. In one embodiment, SOCS family members may be selected from SOCS1 , SOCS2, SOCS3 , or combinations thereof.
SOCS1 may comprise the polypeptide sequence of SEQ ID NO: 11. SOCS2 may comprise the polypeptide sequence of SEQ ID NO: 12. SOCS3 may comprise the polypeptide sequence of SEQ ID NO: 13. SOCS4 may comprise the polypeptide sequence of SEQ ID NO: 14 or SEQ ID NO: 15. SOCS5 may comprise the polypeptide sequence of SEQ ID NO: 16 or SEQ ID NO: 17. SOCS6 may comprise the polypeptide sequence of SEQ ID NO: 18. SOCS7 may comprise the polypeptide sequence of SEQ ID NO: 19. CIS (cytokine-inducible SH2-containing protein isoform 1) may comprise the polypeptide sequence of SEQ ID NO: 20. CISH (cytokine-inducible SH2-containing protein isoform 2) may comprise the polypeptide sequence of SEQ ID NO: 21.
PIAS family members may be selected from PIAS1 , PIAS2, PIAS3, PIAS4, or combinations thereof. PIAS family members may be selected from PIAS1 , and PIAS2, or combinations thereof.
PIAS1 (protein inhibitor of activated STAT 1) may comprise the polypeptide sequence of SEQ ID NO: 22. PIAS2 (protein inhibitor of activated STAT X isoform alpha) may comprise the polypeptide sequence of SEQ ID NO: 23. PIAS2 (protein inhibitor of activated STAT X isoform beta) may comprise the polypeptide sequence of SEQ ID NO: 24. PIAS3 (protein inhibitor of activated STAT 3) may comprise the polypeptide sequence of SEQ ID NO: 25. PIAS4 (protein inhibitor of activated STAT 4) may comprise the polypeptide sequence of SEQ ID NO: 26.
An analogue may be a natural or synthetic molecule that has the same 3- dimensional molecular structure to the regulator of cytokine expression, and optionally functions like the cytokine expression regulator.
An analogue of STAT family members may include proteins having at least 60% sequence identity, alternatively at least 70%, 80%, 90%, 95% or 99% sequence identity, to any one of the sequences selected from the group consisting of SEQ ID NO: 1 ; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; and SEQ ID NO: 10. An analogue may be a molecule, synthetic or otherwise, that has the same 3-dimensional molecular structure as a STAT family member, and optionally functions like a STAT family member.
An analogue of SOCS family members may include proteins having at least 60% sequence identity, alternatively at least 70%, 80%, 90%, 95% or 99% sequence identity to any one of the sequences selected from the group consisting of SEQ ID NO: 11 ; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; and SEQ ID NO: 21. An analogue may be a molecule, synthetic or otherwise, that has the same 3-dimensional molecular structure as a SOCS family member, and optionally functions like a SOCS family member. An analogue of PIAS family members may include proteins having at least 60% sequence identity, alternatively at least 70%, 80% , 90% , 95% or 99% sequence identity to any one of the sequences selected from the group consisting of SEQ ID NO: 22; SEQ ID NO: 23 ; SEQ ID NO: 24; SEQ ID NO : 25 ; and SEQ ID NO: 26. An analogue may be a molecule, synthetic or otherwise, that has the same 3-dimensional molecular structure as a PIAS family member, and optionally functions like a PIAS family member. An analogue of JAK family members may include proteins having at least 60% sequence identity, alternatively at least 70% , 80%, 90% , 95% or 99% sequence identity to any one of the sequences selected from the group consisting of SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; and SEQ ID NO: 30. An analogue may be a molecule, synthetic or otherwise, that has the same 3-dimensional molecular structure as a JAK family member, and optionally functions like a JAK family member.
Where reference is made to a percentage sequence identity, it is intended to refer to the percentage sequence homology between two aligned sequences using a standard BLAST alignment tool (available at http: //blast.ncbi.nlm.nih.gov/Blast.cgi) under default parameters.
The cytokine expression regulator or analogue thereof, the protein, or the vector may be provided in a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include water, saline solution and other aqueous solutions , which will be known to those skilled in the art.
According to another aspect of the invention there is provided an assay comprising the steps of:
-exposing a cell to an agent -detecting whether there is modulation of expression or activity of at least one cytokine expression regulator in the cell after exposure of the cell to the agent,
wherein the cytokine expression regulator is selected from any one of the group comprising members of the Janus kinases (JAK) family, members of the signal transducer and activator of transcription (STAT) family, members of the suppressor of cytokine signalling (SOCS) family, and members of the protein inhibitor of activated STAT (PI AS) family, or combinations thereof.
In one embodiment the cytokine expression regulator is selected from any one of the group comprising members of the signal transducer and activator of transcription (STAT) family, members of the suppressor of cytokine signalling (SOCS) family, and members of the protein inhibitor of activated STAT (PIAS) family, or combinations thereof.
The assay may be for the identification of the agent as a potential therapeutic agent for use in the treatment of hypercytokinemia. The assay may be for the identification of the agent as useful in the reduction of proinflammatory cytokines, such as TNF-alpha, in the cell, or in a patient.
Such an assay would advantageously provide a method of identifying therapeutic drug candidates for restoring regulation of cytokines in an infection. The restoration of an appropriate host cytokine response or the prevention of its dysregulation is relatively unexplored as a treatment option to reduce clinical severity and improve patient survival. This approach strategically targets the over-production of proinflammatory cytokines rather than the effects of excess cytokines. Such an intervention approach could have broad applications for a range of acute viruses , for example, highly virulent influenza infections . The assay may further comprise triggering an infection response in the cell. Triggering an infection response in the cell has the use of inducing cytokine dysregulation, such as suppression of the cytokine expression regulator, for which the agent may have an effect.
The infection response in the cell may be triggered by introducing an infectious material to the cell, or by introducing a material to the cell that mimics an infection of the cell. The material may be a virus , a glycopeptide, a protein, a nucleic acid, for example a viral nucleic acid, or a bacterium. The material may also be part of one of the aforementioned materials , such as part of a virus , or part of a bacterium, such as endotoxin/lipopolysacharride. The virus , or part of the virus , or the viral nucleic acid, may be derived from influenza virus , such as H5N1 or HINT , or SARS virus . The virus or bacterium may be implicated in pneumonia infection and/or acute respiratory distress syndrome (ARDS) . The virus, or part of the virus , or the viral nucleic acid, may be derived from any of the group selected from respiratory syncytial virus (RSV) , human parainfluenza viruses, adenoviruses, metapneumovirus, herpes simplex virus (HSV) , varicella-zoster virus (VZV) and cytomegalovirus (CMV) . The modulation of expression of the cytokine expression regulator may be upregulation. The modulation of expression of the cytokine expression regulator may be the prevention or reversal of suppression of the regulator of cytokine expression. Where the cytokine expression regulator has become dysregulated, the modulation of expression of the cytokine expression regulator may lead to the restoration of normal levels (i.e. non-infected levels) of the cytokine expression regulator. Detecting upregulation, prevention of suppression, or dysregulation, of the cytokine expression regulator in the cell has an advantage that it provides a new measurable effect for finding novel therapeutic agents for treatment against hypercytokinemia, for example due to viral infection. This contrasts with previous studies which suggest that induction of SOCS3 (a regulator of cytokine expression) is an unfavourable response of host cells during infection . The infection response may be triggered before, during or after exposure of the cell to the agent. The infection response may be triggered before, exposure of the cell to the agent in order to investigate the ability of the agent to restore function or expression of the cytokine expression regulator. In an alternative embodiment the infection response may be triggered after exposure of the cell to the agent in order to evaluate the agent's ability to prevent dysregulation or inactivation of the regulator of cytokine expression.
The cell may be a mammalian primary cell or from an established mammalian cell line. The cell may be a human cell, pig cell or avian cell. The cell may be a macrophage (or monocyte) or a tracheal/bronchial epithelial cell (TEC) .
An advantage of the cell being a primary macrophage or a tracheal epithelial cell is that these are important/primary cell types involved in the pathogenesis of influenza infection. Pig cells advantageously have similar receptors to human cells , which provide a good model for human disease studies. Detecting whether there is modulation of expression or activity of at least one cytokine expression regulator may comprise detecting expression levels of RNA encoding the cytokine expression regulator or relevant cytokine, for example by real-time-PCR, or of protein, for example by enzyme-linked immunosorbent assay (ELISA) . The agent may be a compound/drug, nucleic acid, peptide, or protein. The agent may be an analogue of the cytokine expression regulator. The agent may be a truncated form of the cytokine expression regulator. Preferably the agent is not significantly cytotoxic at pharmaceutically useful levels. Preferably the agent is permeable to cell membrane.
According to another aspect of the present invention, there is provided a method of screening an agent for use in a treatment, comprising determining whether said agent has affinity against a negative-regulator of at least one protein, wherein the at least one protein is selected from the group consisting of: (a) members of the signal transducer and activator of transcription (STAT) family, (b) members of the suppressor of cytokine signalling (SOCS) family, (c) members of the protein inhibitor of activated STAT (PIAS) family (d) members of the Janus kinases (JAK) family, or combinations of (a) , (b) , (c) or (d) .
According to another aspect of the invention there is provided the use of a cytokine expression regulator pathway in a cell for determining the therapeutic activity of an agent, wherein the cytokine expression regulator pathway is selected from JAK, STAT, SOCS and PIAS.
In another embodiment the cytokine expression regulator pathway is selected from STAT, SOCS and PIAS.
It will be appreciated that optional features applicable to one aspect of the invention can be used in any combination, and in any number. Moreover, they can also be used with any of the other aspects of the invention in any combination and in any number. This includes , but is not limited to, the dependent claims from any claim being used as dependent claims for any other claim in the claims of this application. The present invention will now be described herein, by way of example only, with reference to the following figures .
Figure 1 - Induction of TNFa by avian H5N1 and USSR/77 virus in macrophages and tracheal epithelial cells (TECs) from human and pig. Note that TNFa induction is much greater in human cells than corresponding pig cells, and that TNFa induction is much higher in macrophages than TECs . Cells were incubated with influenza subtype, as indicated, at 1.0 MOI for 3h followed by PBS rinses and further incubation overnight for an overall duration of 24h. Total RNA was extracted for cDNA conversion from which
Taqman real-time PCR (Lightcycler 480) was performed. Quantification of expression was based on the relative standard curve method and normalised to 18S RNA. Data bars represent the average of 4 biological replicates. Error bars are the standard error of the means. TECs = tracheal epithelial cells; USSR = human
H1N1 A/USSR/90/77.
Figure 2 - Induction of IFNfil and ILlfi by avian H5N1 and USSR/77 virus in human macrophages and TECs . Note that IFNfil induction in human macrophages and TECs is greater with
USSR/77 than avian H5N1 virus , and that in human TECs, ILlfi is consistently down-regulated by avian H5N1 infection. Cells were incubated with influenza subtype, as indicated, at 1.0 MOI for 3h followed by PBS rinses and further incubation overnight for an overall duration of 24h. Total RNA was extracted for cDNA conversion from which Taqman real-time PCR (Lightcycler 480) was performed. Quantification of expression was based on the relative standard curve method and normalised to 18S RNA. Data bars represent the average of 4 biological replicates. Error bars are the standard error of the means . TECs = tracheal epithelial cells; USSR = human H1N1 A/USSR/90/77.
Figure 3 - Induction of IFNfil and ΙΤΙβ by avian H5N1 and USSR/77 virus in pig macrophages . Note that IFNfil induction in pig macrophages is comparable between avian H5N1 and USSR/77 virus , and that ΙΤΙβ in pig macrophages is not induced by avian H5N1 infection. Cells were incubated with influenza subtype, as indicated, at 1.0 MOI for 3h followed by PBS rinses and further incubation overnight for an overall duration of 24h. Total RNA was extracted for cDNA conversion from which Taqman real-time PCR (Lightcycler 480) was performed. Quantification of expression was based on the relative standard curve method and normalised to 18S RNA. Data bars represent the average of 4 biological replicates . Error bars are the standard error of the means . TECs = tracheal epithelial cells; USSR = human H1N1 A/USSR/90/77.
Figure 4 - Down-regulation of JAK1 and STAT1 by avian H5N1 and USSR/77 virus in human macrophages and TECs. Note that JAK1 and ST ATI are more severely suppressed in macrophages and TECs by avian H5N1 than by USSR/77 virus. Cells were incubated with influenza subtype, as indicated, at 1.0 MOI for 3h followed by PBS rinses and further incubation overnight for an overall duration of 24h. Total RNA was extracted for cDNA conversion from which Taqman real-time PCR (Lightcycler 480) was performed. Quantification of expression was based on the relative standard curve method and normalised to 18S RNA. Data bars represent the average of 4 biological replicates . Error bars are the standard error of the means . TECs = tracheal epithelial cells; USSR = human H1N1 A/USSR/90/77.
Figure 5 - Down-regulation of SOCS3 by avian H5N1 in human TECs. Note that SOCS3 in human TECs is consistently down- regulated by avian H5N1 but not USSR/77. SOCS3 expression in human macrophages is not affected by avian H5N1 or USSR/77 virus. Cells were incubated with influenza subtype, as indicated, at 1.0 MOI for 3h followed by PBS rinses and further incubation overnight for an overall duration of 24h. Total RNA was extracted for cDNA conversion from which Taqman real-time PCR (Lightcycler 480) was performed. Quantification of expression was based on the relative standard curve method and normalised to 18S RNA. Data bars represent the average of 4 biological replicates. Error bars are the standard error of the means . TECs = tracheal epithelial cells ; USSR = human H1N1 A/USSR/90/77.
Figure 6 - Down-regulation of PIAS1 by avian H5N1 and USSR/77 virus in human macrophages and TECs . Note that PIAS1 in human macrophages and TECs is more severely suppressed by avian H5N1 than by USSR/77 virus . Cells were incubated with influenza subtype, as indicated, at 1.0 MOI for 3h followed by PBS rinses and further incubation overnight for an overall duration of 24h. Total RNA was extracted for cDNA conversion from which Taqman real-time PCR (Lightcycler 480) was performed. Quantification of expression was based on the relative standard curve method and normalised to 18S RNA. Data bars represent the average of 4 biological replicates. Error bars are the standard error of the means. TECs = tracheal epithelial cells; USSR = human H1N1 A/USSR/90/77. Figure 7 - Induction of TNFa in USSR/77-infected primary cells is reduced by over-expression of SOCS3 and PIAS2. ELISAs for the detection of TNFa was performed on supernatants collected 24h post-infection. (A) Human and pig macrophages , as described earlier, were infected in duplicates at 1.0 and 0.1 MOI. Note that there is a virus-dose dependent response of TNFa induction and that human macrophages show a much stronger TNFa induction than pig macrophages. (B) Human TECs were transfected by electroporation with SOCS3 , PIAS2 and non-coding empty vector.
After 48h, the cells were infected with USSR/77 at 1.0 MOI, and 24h post-infection were supernatants collected for TNFa detection by ELISA. Reduction in TNFa induction was evident post-infection in the presence of SOCS3 or PIAS2 over-expression. (C) Similar results were obtained with the use of human macrophages . Human macrophages were transfected by electroporation with SOCS3 , PIAS2 and non-coding empty vector. After 48h, the cells were infected with USSR/77 at 1.0 MOI, and 24h post-infection were supernatants collected for TNFa detection by ELISA. There was reduced induction of TNFa post-infection in the presence of
SOCS3 or PIAS2 over-expression.
Examples Human cases of highly pathogenic avian influenza (HPAI) H5N1 infection carry a mortality rate in excess of 60% , in which hyperacute host inflammatory response (cytokine storm) is often cited as a major cause of complication and death. By contrast, although pigs are susceptible to HPAI H5N1 infection they show no clinical signs of disease during infection. The ability to restore or maintain an appropriate host innate response in the face of virulent influenza or other viral infection, which triggers the hyperacute proinflammatory response, is likely to improve human survival. By comparing host innate response to low and high pathogenicity influenza viruses between the resistant and susceptible species (pig and human respectively) in primary macrophages and tracheal epithelial cells, several cytokine regulatory factors have been identified that are prospective targets for therapeutic intervention to prevent or ameliorate the damaging effects of hypercytokinemia.
Strategy to identify targets that can restore or maintain an appropriate host innate cytokine response
Identifying specific signalling drivers that are potential host triggers of cytokine dysfunction is based on the inherent contrast in disease resistance and innate response to HPAI H5N1 infections between the host species of human (susceptible) and pig (resistant) . Extensive comparative infection studies show molecular differences in host innate response between human and pig primary macrophages and tracheal epithelial cells (TECs) , the two most important cell types in the pathogenesis of influenza infection. The consistent finding from mammalian work is that relative innate resistance in the pig to avian H5N1 influenza infections correlates with more rapid cell death and reduced proinflammatory response to the virus. Conversely, susceptible host cells (from human) show delayed cell death and more vigorous inflammatory changes.
Summary of candidate targets for the control of hypercytokinemia Low and high pathogenicity influenza subtypes were used to infect human and pig primary TECs and macrophages to characterise the molecular differences in host innate response. The generation of RNA (Affymetrix microarray) and protein (Kinex antibody array) profiles of influenza infected human and pig TECs, and validation work by real-time PCR and ELISAs show that the gene and protein expression profile in pig cells is substantially different from the human profile. A notable finding is the marked difference in TNFa response between human and pig cells (Fig. 1) . The elevated TNFa expression in H5Nl-infected human macrophages and TECs closely mirrors the in vivo finding of human H5N1 cases. The analysis indicates that the hyperactive proinflammatory response evident in H5Nl-infected human cells could be connected to the dysregulation of a number of key regulators of cytokine expression. They are (1) members of the signal transducer and activator of transcription (STAT) family, (2) members of the suppressor of cytokine signalling (SOCS) family, and (3) members of the protein inhibitor of activated STAT (PI AS) family. Notably members of these 3 families of regulators are strongly down-regulated in H5N1 infected human respiratory epithelial cells and macrophages. Restoring the expression of SOCS, PIAS and/or STAT family members in H5N1 infection could be pivotal in the correction or maintenance of an appropriate host proinflammatory response. Significant insights have been gained into the pathogenesis of H5Nl-induced hypercytokinemia and the early host events post-infection to facilitate the development of an intervention strategy to prevent or alter the intensity of a cytokine storm, thereby reducing morbidity and mortality. This approach strategically targets the over-production of proinflammatory cytokines rather than the effects of excess cytokines .
Findings of innate response to influenza infection
TNFa induction in human and pig cells
TNFa is a key proinflammatory cytokine that is highly up-regulated in human patients and cells infected with HPAI H5N1 virus (Peiris, J. S. M. et al. (2009) Trends Immunol. 30: 574-584) . In contrast, low pathogenicity influenza subtypes, such as PR8 strain and seasonal subtypes , typically show little or no TNF induction in human cells (Mok, K. P. et al. (2009) J. Infect. Dis. 200: 1104-1112, Pauli, E. -K. et al. (2008) Plos Pathogens 4:el000196) . The innate response of primary macrophages and macrophages of human and pig to classical swine H1N1 (A/sw/Iowa/15/30) , avian H2N3 (A/mallard duck/England/7277/06) , human H1N1 (A/USSR/90/77) and a high pathogenicity avian H5N1 virus (A/turkey/Turkey/1/05) was examined. A strong induction of TNFa expression by avian H5N1 in human macrophages was found, which is substantially higher than in human TECs (Fig. l) . By comparison, the induction of TNFa by the same avian H5N1 is small in pig macrophages, and is virtually none in pig TECs (Fig. l) . Among the 3 non-H5Nl subtypes evaluated in infection studies, the human H1N1 virus (denoted as USSR/77) induces the most TNFa expression in both human macrophages and epithelial cells, at levels that are higher or comparable with those induced by the avian H5N1 virus (Fig. l) . Swine H1N1 and avian H2N3 in human TECs did not produce detectable TNFa as determined by ELISA. The USSR/77 virus was responsible for the 1977/78 epidemic and has been known to cause severe respiratory illness with widespread lung pathology in humans and experimental ferrets (Svitek, N. et al. (2008) Virology 376:53-59) . Systemic acute release of TNFa is detrimental to the host. Mice deficient in TNF receptor 1 (TNFR1) or treated with anti-TNFa antibody show reduce morbidity and less weight loss when challenged with avian H5N1 virus (Szretter, K. J. J. (2007) Virology 81 :2736-2744) . Therefore controlling the excessive production of TNFa caused by virulent influenza infection could ameliorate the damaging effects of hypercytokinemia.
One of the pleiotropic targets of TNFa signalling is the degradation of STAT5 proteins reported in 3T3-L1 adipocytes (Floyd, Z. E. et al. (2007) Am. J. Physiol. Endocrinol. Metab. 292: E461-E468) . Conceivably, sustained elevated levels of TNFa could lead to a major disruption of the JAK-STAT signalling pathway, a major cascade activated by type I and II IFNs, and members of several interleukin families that culminates in the transcriptional activation of further proinflammatory cytokines and the innate anti-viral state (Schindler, C. and Plumlee, C. (2008) Seminars in Cell and Developmental Biology 19: 311-318) . There is evidence to suggest that TNFa can also activate JAK-STAT signalling, but it is uncertain if it is a direct or indirect activation (Guo, D. Q. et al. (1998) J. Immunol. 160:2742-2750) .
Induction oflFNfil and ILi
The induction of ΙΡΝβΙ by USSR/77 is greater than avian H5N1 in both human macrophages and TECs which suggests that the more highly pathogenic H5N1 triggers a weaker IFN-stimulated response (Fig.2) (Zeng, H. et al (2007) J.Virol. 81 : 12439-12449) . In pig macrophages, IFNpi is induced to similar levels by USSR/77 and avian H5N1 (Fig. 3) . ILip is similarly induced by USSR/77 and avian H5N1 in human macrophages (Fig. 2) . Interestingly, in human TECs, ILip is consistently down-regulated by avian H5N1 infection. In pig macrophages, USSR/77 up-regulates ILip expression but avian H5N1 has no effect on ILip production. Neither USSR/77 nor avian H5N1 induces detectable mRNA for IFNpi and ILip in pig TECs, indicating that there are intrinsic innate differences between human and pig, with the latter displaying a less reactive cytokine response.
Down-regulation of JAK and STAT expression
The avian H5N1 virus was found to consistently down-regulate the JAK- STAT pathway in human macrophages and TECs (Fig.4) . Several members of the JAK and STAT families are transcriptionally suppressed in the presence of the avian H5N1 virus. The USSR/77 virus also exhibits similar inhibitory effect on the JAK-STAT pathway but the degree of suppression is not as severe as the avian H5N1. The JAK-STAT cascade, among its multiple roles, is a key innate immunity pathway activated during early infection. Its down-regulation by a highly virulent virus could compromise the host's ability to mount an effective innate response. A further complication to JAK-STAT inhibition could be an increase of inflammation. STAT3 is one of several STAT members that is down- regulated in avian H5N1 infected human TECs . It has been shown that STAT3 activation elicits an anti-inflammatory effect (Schindler, C. and Plumlee, C . (2008) Seminars in Cell and Developmental Biology 19: 311- 318) . Constitutively activated STAT3 is able to suppress bacterial lipopolysaccharides (LPS) -induced TNFa and IL6 production in macrophages (Yoshimura, A. (2009) Keido J. Med. 58:73-83) . Therefore induction of a more responsive JAK-STAT pathway, as seen with the use of low pathogenicity influenza viruses, could confer more effective host innate response and reduce excessive inflammation (Zeng, H. et al (2007) J.Virol. 81 : 12439-12449) .
Down-regulation of SOCS expression
Members of the SOCS family negatively regulate cytokines (including IFNs) that signal through the JAK-STAT pathway (Alexander, W. S. and D. J. Hilton (2004) . Annu.Rev.Immuol. 22: 503-529) . SOCS proteins inhibit JAK-STAT signalling through their high binding affinity for cytokine receptors , JAK and STAT proteins. Many factors (endocrine factors and cytokines) are able to induce SOCS family proteins, including insulin, IL1 , IL6, IL10, IFNa/β, IFNy and TNFa (Palmer, D. C. and Restifo, N. P. (2009) Trends Immunol. 30 :592-602. 2009) . Additionally, low pathogenicity influenza viruses, such as H1N1 (PR8) and H3N2 subtypes , have been shown to induce SOCS1 and 3 in human epithelial cell lines (Palmer, D. C. and Restifo, N. P. (2009) Trends Immunol. 30: 592-602. 2009, Pauli, E. -K. et al. (2008) Plos Pathogens 4:el000196, Pothlichet, J. et al. (2008) Journal of Immunology 180: 2034-2038) . It was reported that over-expression of SOCS3 is associated with raised virus yield, and inhibition of IFNP and STAT1 phosphorylation, which suggest that S0CS3 expression and subsequent dampening of the JAK- STAT cascade benefit virus propagation (Pauli, E. -K. et al. (2008) Plos Pathogens 4:el000196) . However, this observation on A549 human epithelial cells was based on the use of PR8 (A/Puerto-Rico/8/34) , a subtype that is highly pathogenic in mice but does not infect humans. This subtype also does not significantly induce TNF a or ILi p expression in A549 cells.
Contrary to recent findings that low virulence influenza subtypes up- regulate SOCS expression (Pauli, E. -K. et al. (2008) Plos Pathogens 4:el000196, Pothlichet, J. et al. (2008) Journal of Immunology 180: 2034- 2038) , we found that highly pathogenic avian H5N1 consistently down- regulates SOCS3 in infected human TECs (Fig.5) . In human macrophages, avian H5N1 showed little effect on SOCS3 expression. Without being bound by theory, it can be speculated that in hypercytokinemia there is a breakdown in the homeostatic interactions between production of cytokines and their negative regulators, as exemplified by suppressed SOCS expression in the presence of elevated TNFa.
The invention provides that the restoration or over-expression of SOCS can prevent or reduce the excessive production of proinflammatory cytokines, such as TNFa, in human cases of highly pathogenic influenza infection. This is supported by the present findings (Fig.5) together with the documented protective anti-inflammatory properties of SOCS
Down-regulation of PIAS expression
The 4 members of the mammalian PIAS family (PIAS1 , PIAS2/x, PIAS3 and PIAS4/y) possess SUMO (small ubiquitin-like modifier) E3 ligase activity and can interact with over 60 proteins , many of which are transcription factors, to regulate their transcriptional activity (Shuai, K. (2006) Cell Res . 16: 196-202, Shuai, K. and B . Liu. (2005) Nature Rev. Immunol. 5 : 593-605) . Unlike SOCS proteins, PIAS proteins are not usually inducible (O ' Shea, J. J. and W. Watford. (2004) Nature Immunol. 5 : 875-876) . PIAS proteins regulate transcription factors through blocking the DNA-binding ability of transcription factors , recruiting transcriptional co-repressors or co-activators , or promoting sumoylation of its binding partner (Shuai, K. (2006) Cell Res . 16: 196-202) . Importantly, PIAS proteins are inhibitors of STAT, IRF and NF-κΒ p65 transcription factors which are responsible for the induction of primary cytokines (type I and II IFNPs, IL1 , IL6 and TNFa) and cytokine-inducible genes (Shuai, K. and B . Liu. (2005) Nature Rev. Immunol. 5 : 593-605) . The significance of PIAS proteins in the control of inflammation is exemplified in mice deficient in PIAS1 or PIAS4 which show raised serum levels of TNFa and ILip, and heightened sensitivity to LPS-induced endotoxic shock (Liu, B. et al. (2005) Mol. Cell. Biol. 25 : 1113-1123 , Tahk, S. et al. (2007) Proc. Natl. Acad Sci U.S.A. 104: 11643-11648) .
It is found that avian H5N1 strongly down-regulates members of the PIAS family. Although USSR/77 is able to suppress the expression of PIAS1 , avian H5N1 is more potent in the suppression of PIAS1 in human TECs and macrophages (Fig.6) . The invention provides that the restoration or over-expression of PIAS can prevent or reduce the excessive production of proinflammatory cytokines, such as TNFa, in human cases of highly pathogenic influenza infection.
SOCS or PIAS over-expression reduces TNFa induction in influenza infected human cells
High levels of TNFa RNA detected in USSR/77 infected human macrophages (Fig. l) correlate with high TNFa protein detection by ELISAs (Fig.7 A) . Human TECs and macrophages were transfected (NHBE Nucleofector Kit, Lonza) by electroporation (Amaxa electroporator) in accordance with the supplier' s instructions . Expression plasmids used to evaluate the effects of SOCS and PIAS on TNFa induction by USSR/77 virus were: pCMV-SOCS3 (plasmid 11486, Addgene) , pCMV-PIASxa (plasmid 15209, Addgene, also known as PIAS2) , and empty vector control pBK-CMV. Transfection efficiency was typically around 40% . Forty-eight hours post-transfection one set of wells were infected with USSR/77 at 1.0 MOI, while the remaining duplicate set was left uninfected. Supernatants were harvested 24h post-infection for the detection of TNFa by ELISA (Quantikine TNFa immunoassay DTA00C) (Fig.7B and C) . The over-expression of SOCS3 or PIAS2 dampens the induction of TNFa by the USSR/77 virus in human TECs and macrophages.
SEQ ID NO: 1
MSQWYELQQL DSKFLEQVHQ LYDDSFPMEI RQYLAQWLEK QDWEHAANDV SFATIRFHDL
60
LSQLDDQYSR FSLENNFLLQ HNIRKSKRNL QDNFQEDPIQ MSMIIYSCLK EERKILENAQ
120
RFNQAQSGNI QSTVMLDKQK ELDSKVRNVK DKVMCIEHEI KSLEDLQDEY DFKCKTLQNR
180
EHETNGVAKS DQKQEQLLLK KMYLMLDNKR KEWHKIIEL LNVTELTQNA LINDELVEWK
240
RRQQSACIGG PPNACLDQLQ NWFTIVAESL QQVRQQLKKL EELEQKYTYE HDPITKNKQV
300
LWDRTFSLFQ QLIQSSFWE RQPCMPTHPQ RPLVLKTGVQ FTVKLRLLVK LQELNYNLKV
360
KVLFDKDVNE RNTVKGFRKF NILGTHTKVM NMEESTNGSL AAEFRHLQLK EQKNAGTRTN
420
EGPLIVTEEL HSLSFETQLC QPGLVIDLET TSLPVWISN VSQLPSGWAS ILWYNMLVAE
480
PRNLSFFLTP PCARWAQLSE VLSWQFSSVT KRGLNVDQLN MLGEKLLGPN ASPDGLIPWT
540
RFCKENINDK NFPFWLWIES ILELIKKHLL PLWNDGCIMG FISKERERAL LKDQQPGTFL
600
LRFSESSREG AITFTWVERS QNGGEPDFHA VEPYTKKELS AVTFPDI IRN YKVMAAENIP
660
ENPLKYLYPN IDKDHAFGKY YSRPKEAPEP MELDGPKGTG YIKTELISVS EVHPSRLQTT
720
DNLLPMSPEE FDEVSRIVGS VEFDSMMNTV
750
SEQ ID NO: 2
MSQWYELQQL DSKFLEQVHQ LYDDSFPMEI RQYLAQWLEK QDWEHAANDV SFATIRFHDL
60
LSQLDDQYSR FSLENNFLLQ HNIRKSKRNL QDNFQEDPIQ MSMIIYSCLK EERKILENAQ
120
RFNQAQSGNI QSTVMLDKQK ELDSKVRNVK DKVMCIEHEI KSLEDLQDEY DFKCKTLQNR
180
EHETNGVAKS DQKQEQLLLK KMYLMLDNKR KEWHKIIEL LNVTELTQNA LINDELVEWK
240
RRQQSACIGG PPNACLDQLQ NWFTIVAESL QQVRQQLKKL EELEQKYTYE HDPITKNKQV
300
LWDRTFSLFQ QLIQSSFWE RQPCMPTHPQ RPLVLKTGVQ FTVKLRLLVK LQELNYNLKV
360
KVLFDKDVNE RNTVKGFRKF NILGTHTKVM NMEESTNGSL AAEFRHLQLK EQKNAGTRTN
420
EGPLIVTEEL HSLSFETQLC QPGLVIDLET TSLPVWISN VSQLPSGWAS ILWYNMLVAE
480
PRNLSFFLTP PCARWAQLSE VLSWQFSSVT KRGLNVDQLN MLGEKLLGPN ASPDGLIPWT
540
RFCKENINDK NFPFWLWIES ILELIKKHLL PLWNDGCIMG FISKERERAL LKDQQPGTFL
600
LRFSESSREG AITFTWVERS QNGGEPDFHA VEPYTKKELS AVTFPDI IRN YKVMAAENIP
660
ENPLKYLYPN IDKDHAFGKY YSRPKEAPEP MELDGPKGTG YIKTELISVS EV
712
SEQ ID NO: 3
MAQWEMLQNL DSPFQDQLHQ LYSHSLLPVD IRQYLAVWIE DQNWQEAALG SDDSKATMLF
60 FHFLDQLNYE CGRCSQDPES LLLQHNLRKF CRDIQPFSQD PTQLAEMIFN LLLEEKRILI 120
QAQRAQLEQG EPVLETPVES QQHEIESRIL DLRAMMEKLV KSISQLKDQQ DVFCFRYKIQ
180
AKGKTPSLDP HQTKEQKILQ ETLNELDKRR KEVLDASKAL LGRLTTLIEL LLPKLEEWKA
240
QQQKACIRAP IDHGLEQLET WFTAGAKLLF HLRQLLKELK GLSCLVSYQD DPLTKGVDLR
300
NAQVTELLQR LLHRAFWET QPCMPQTPHR PLILKTGSKF TVRTRLLVRL QEGNESLTVE
360
VSIDRNPPQL QGFRKFNILT SNQKTLTPEK GQSQGLIWDF GYLTLVEQRS GGSGKGSNKG
420
PLGVTEELHI ISFTVKYTYQ GLKQELKTDT LPWIISNMN QLSIAWASVL WFNLLSPNLQ
480
NQQFFSNPPK APWSLLGPAL SWQFSSYVGR GLNSDQLSML RNKLFGQNCR TEDPLLSWAD
540
FTKRESPPGK LPFWTWLDKI LELVHDHLKD LWNDGRIMGF VSRSQERRLL KKTMSGTFLL
600
RFSESSEGGI TCSWVEHQDD DKVLIYSVQP YTKEVLQSLP LTEI IRHYQL LTEENIPENP
660
LRFLYPRIPR DEAFGCYYQE KVNLQERRKY LKHRLIWSN RQVDELQQPL ELKPEPELES
720
LELELGLVPE PELSLDLEPL LKAGLDLGPE LESVLESTLE PVIEPTLCMV SQTVPEPDQG
780
PVSQPVPEPD LPCDLRHLNT EPMEIFRNCV KIEEIMPNGD PLLAGQNTVD EVYVSRPSHF
840
YTDGPLMPSD F
851
SEQ ID NO: 4
MAQWNQLQQL DTRYLEQLHQ LYSDSFPMEL RQFLAPWIES QDWAYAASKE SHATLVFHNL
60
LGEIDQQYSR FLQESNVLYQ HNLRRIKQFL QSRYLEKPME IARIVARCLW EESRLLQTAA
120
TAAQQGGQAN HPTAAWTEK QQMLEQHLQD VRKRVQDLEQ KMKWENLQD DFDFNYKTLK
180
SQGDMQDLNG NNQSVTRQKM QQLEQMLTAL DQMRRSIVSE LAGLLSAMEY VQKTLTDEEL
240
ADWKRRQQIA CIGGPPNICL DRLENWITSL AESQLQTRQQ IKKLEELQQK VSYKGDPIVQ
300
HRPMLEERIV ELFRNLMKSA FWERQPCMP MHPDRPLVIK TGVQFTTKVR LLVKFPELNY
360
QLKIKVCIDK DSGDVAALRG SRKFNILGTN TKVMNMEESN NGSLSAEFKH LTLREQRCGN
420
GGRANCDASL IVTEELHLIT FETEVYHQGL KIDLETHSLP VWISNICQM PNAWASILWY
480
NMLTNNPKNV NFFTKPPIGT WDQVAEVLSW QFSSTTKRGL SIEQLTTLAE KLLGPGVNYS
540
GCQITWAKFC KENMAGKGFS FWVWLDNIID LVKKYILALW NEGYIMGFIS KERERAILST
600
KPPGTFLLRF SESSKEGGVT FTWVEKDISG KTQIQSVEPY TKQQLNNMSF AEI IMGYKIM
660
DATNILVSPL VYLYPDIPKE EAFGKYCRPE SQEHPEADPG SAAPYLKTKF ICVTPTTCSN
720
TIDLPMSPRT LDSLMQFGNN GEGAEPSAGG QFESLTFDME LTSECATSPM
770 SEQ ID NO: 5
MAQWNQLQQL DTRYLEQLHQ LYSDSFPMEL RQFLAPWIES QDWAYAASKE SHATLVFHNL
60
LGEIDQQYSR FLQESNVLYQ HNLRRIKQFL QSRYLEKPME IARIVARCLW EESRLLQTAA
120
TAAQQGGQAN HPTAAWTEK QQMLEQHLQD VRKRVQDLEQ KMKWENLQD DFDFNYKTLK
180
SQGDMQDLNG NNQSVTRQKM QQLEQMLTAL DQMRRSIVSE LAGLLSAMEY VQKTLTDEEL
240
ADWKRRQQIA CIGGPPNICL DRLENWITSL AESQLQTRQQ IKKLEELQQK VSYKGDPIVQ
300
HRPMLEERIV ELFRNLMKSA FWERQPCMP MHPDRPLVIK TGVQFTTKVR LLVKFPELNY
360
QLKIKVCIDK DSGDVAALRG SRKFNILGTN TKVMNMEESN NGSLSAEFKH LTLREQRCGN
420
GGRANCDASL IVTEELHLIT FETEVYHQGL KIDLETHSLP VWISNICQM PNAWASILWY
480
NMLTNNPKNV NFFTKPPIGT WDQVAEVLSW QFSSTTKRGL SIEQLTTLAE KLLGPGVNYS
540
GCQITWAKFC KENMAGKGFS FWVWLDNIID LVKKYILALW NEGYIMGFIS KERERAILST
600
KPPGTFLLRF SESSKEGGVT FTWVEKDISG KTQIQSVEPY TKQQLNNMSF AEI IMGYKIM
660
DATNILVSPL VYLYPDIPKE EAFGKYCRPE SQEHPEADPG AAPYLKTKFI CVTPTTCSNT
720
IDLPMSPRTL DSLMQFGNNG EGAEPSAGGQ FESLTFDMEL TSECATSPM
769
SEQ ID NO: 6
MAQWNQLQQL DTRYLEQLHQ LYSDSFPMEL RQFLAPWIES QDWAYAASKE SHATLVFHNL
60
LGEIDQQYSR FLQESNVLYQ HNLRRIKQFL QSRYLEKPME IARIVARCLW EESRLLQTAA
120
TAAQQGGQAN HPTAAWTEK QQMLEQHLQD VRKRVQDLEQ KMKWENLQD DFDFNYKTLK
180
SQGDMQDLNG NNQSVTRQKM QQLEQMLTAL DQMRRSIVSE LAGLLSAMEY VQKTLTDEEL
240
ADWKRRQQIA CIGGPPNICL DRLENWITSL AESQLQTRQQ IKKLEELQQK VSYKGDPIVQ
300
HRPMLEERIV ELFRNLMKSA FWERQPCMP MHPDRPLVIK TGVQFTTKVR LLVKFPELNY
360
QLKIKVCIDK DSGDVAALRG SRKFNILGTN TKVMNMEESN NGSLSAEFKH LTLREQRCGN
420
GGRANCDASL IVTEELHLIT FETEVYHQGL KIDLETHSLP VWISNICQM PNAWASILWY
480
NMLTNNPKNV NFFTKPPIGT WDQVAEVLSW QFSSTTKRGL SIEQLTTLAE KLLGPGVNYS
540
GCQITWAKFC KENMAGKGFS FWVWLDNIID LVKKYILALW NEGYIMGFIS KERERAILST
600
KPPGTFLLRF SESSKEGGVT FTWVEKDISG KTQIQSVEPY TKQQLNNMSF AEI IMGYKIM
660
DATNILVSPL VYLYPDIPKE EAFGKYCRPE SQEHPEADPG SAAPYLKTKF ICVTPFIDAV
720
WK
722 SEQ ID NO: 7
MSQWNQVQQL EIKFLEQVDQ FYDDNFPMEI RHLLAQWIEN QDWEAASNNE TMATILLQNL
60
LIQLDEQLGR VSKEKNLLLI HNLKRIRKVL QGKFHGNPMH VAWISNCLR EERRILAAAN
120
MPVQGPLEKS LQSSSVSERQ RNVEHKVAAI KNSVQMTEQD TKYLEDLQDE FDYRYKTIQT
180
MDQSDKNSAM VNQEVLTLQE MLNSLDFKRK EALSKMTQI I HETDLLMNTM LIEELQDWKR
240
RQQIACIGGP LHNGLDQLQN CFTLLAESLF QLRRQLEKLE EQSTKMTYEG DPIPMQRTHM
300
LERVTFLIYN LFKNSFWER QPCMPTHPQR PLVLKTLIQF TVKLRLLIKL PELNYQVKVK
360
ASIDKNVSTL SNRRFVLCGT NVKAMSIEES SNGSLSVEFR HLQPKEMKSS AGGKGNEGCH
420
MVTEELHSIT FETQICLYGL TIDLETSSLP WMISNVSQL PNAWASIIWY NVSTNDSQNL
480
VFFNNPPPAT LSQLLEVMSW QFSSYVGRGL NSDQLHMLAE KLTVQSSYSD GHLTWAKFCK
540
EHLPGKSFTF WTWLEAILDL IKKHILPLWI DGYVMGFVSK EKERLLLKDK MPGTFLLRFS
600
ESHLGGITFT WVDHSESGEV RFHSVEPYNK GRLSALPFAD ILRDYKVIMA ENIPENPLKY
660
LYPDIPKDKA FGKHYSSQPC EVSRPTERGD KGYVPSVFIP ISTIRSDSTE PHSPSDLLPM
720
SPSVYAVLRE NLSPTTIETA MKSPYSAE
748
SEQ ID NO: 8
MAGWIQAQQL QGDALRQMQV LYGQHFPIEV RHYLAQWIES QPWDAIDLDN PQDRAQATQL
60
LEGLVQELQK KAEHQVGEDG FLLKIKLGHY ATQLQKTYDR CPLELVRCIR HILYNEQRLV
120
REANNCSSPA GILVDAMSQK HLQINQTFEE LRLVTQDTEN ELKKLQQTQE YFI IQYQESL
180
RIQAQFAQLA QLSPQERLSR ETALQQKQVS LEAWLQREAQ TLQQYRVELA EKHQKTLQLL
240
RKQQ I ILDD ELIQWKRRQQ LAGNGGPPEG SLDVLQSWCE KLAEI IWQNR QQIRRAEHLC
300
QQLPIPGPVE EMLAEVNA I TDIISALVTS TFI IEKQPPQ VLKTQTKFAA TVRLLVGGKL
360
NVHMNPPQVK A IISEQQAK SLLKNENTRN ECSGEILNNC CVMEYHQATG TLSAHFRNMS
420
LKRIKRADRR GAESVTEEKF TVLFESQFSV GSNELVFQVK TLSLPVWIV HGSQDHNATA
480
TVLWDNAFAE PGRVPFAVPD KVLWPQLCEA LNMKFKAEVQ SNRGLTKENL VFLAQKLFNN
540
SSSHLEDYSG LSVSWSQFNR ENLPGWNYTF WQWFDGVMEV LKKHHKPHWN DGAILGFVNK
600
QQAHDLLINK PDGTFLLRFS DSEIGGITIA WKFDSPERNL WNLKPFTTRD FSIRSLADRL
660
GDLSYLIYVF PDRPKDEVFS KYYTPVLAKA VDGYVKPQIK QWPEFVNAS ADAGGSSATY
720
MDQAPSPAVC PQAPYNMYPQ NPDHVLDQDG EFDLDETMDV ARHVEELLRR PMDSLDSRLS
780
PPAGLFTSAR GSLS
794 SEQ ID NO: 9
MAVWIQAQQL QGEALHQMQA LYGQHFPIEV RHYLSQWIES QAWDSVDLDN PQENIKATQL
60
LEGLVQELQK KAEHQVGEDG FLLKIKLGHY ATQLQNTYDR CPMELVRCIR HILYNEQRLV
120
REANNGSSPA GSLADAMSQK HLQINQTFEE LRLVTQDTEN ELKKLQQTQE YFI IQYQESL
180
RIQAQFGPLA QLSPQERLSR ETALQQKQVS LEAWLQREAQ TLQQYRVELA EKHQKTLQLL
240
RKQQ I ILDD ELIQWKRRQQ LAGNGGPPEG SLDVLQSWCE KLAEI IWQNR QQIRRAEHLC
300
QQLPIPGPVE EMLAEVNA I TDIISALVTS TFI IEKQPPQ VLKTQTKFAA TVRLLVGGKL
360
NVHMNPPQVK A IISEQQAK SLLKNENTRN DYSGEILNNC CVMEYHQATG TLSAHFRNMS
420
LKRIKRSDRR GAESVTEEKF TILFESQFSV GGNELVFQVK TLSLPVWIV HGSQDNNATA
480
TVLWDNAFAE PGRVPFAVPD KVLWPQLCEA LNMKFKAEVQ SNRGLTKENL VFLAQKLFNN
540
SSSHLEDYSG LSVSWSQFNR ENLPGRNYTF WQWFDGVMEV LKKHLKPHWN DGAILGFVNK
600
QQAHDLLINK PDGTFLLRFS DSEIGGITIA WKFDSQERMF WNLMPFTTRD FSIRSLADRL
660
GDLNYLIYVF PDRPKDEVYS KYYTPVPCES ATAKAVDGYV KPQIKQWPE FVNASADAGG
720
GSATYMDQAP SPAVCPQAHY NMYPQNPDSV LDTDGDFDLE DTMDVARRVE ELLGRPMDSQ
780
WIPHAQS
787
SEQ ID NO: 10
MSLWGLVSKM PPEKVQRLYV DFPQHLRHLL GDWLESQPWE FLVGSDAFCC NLASALLSDT
60
VQHLQASVGE QGEGSTILQH ISTLESIYQR DPLKLVATFR QILQGEKKAV MEQFRHLPMP
120
FHWKQEELKF KTGLRRLQHR VGEIHLLREA LQKGAEAGQV SLHSLIETPA NGTGPSEALA
180
MLLQETTGEL EAAKALVLKR IQIWKRQQQL AGNGAPFEES LAPLQERCES LVDIYSQLQQ
240
EVGAAGGELE PKTRASLTGR LDEVLRTLVT SCFLVEKQPP QVLKTQTKFQ AGVRFLLGLR
300
FLGAPAKPPL VRADMVTEKQ ARELSVPQGP GAGAESTGEI INNTVPLENS IPGNCCSALF
360
KNLLLKKIKR CERKGTESVT EEKCAVLFSA SFTLGPGKLP IQLQALSLPL WIVHGNQDN
420
NAKA ILWDN AFSEMDRVPF WAERVPWEK MCETLNLKFM AEVGTNRGLL PEHFLFLAQK
480
IFNDNSLSME AFQHRSVSWS QFNKEILLGR GFTFWQWFDG VLDLTKRCLR SYWSDRLIIG
540
FISKQYVTSL LLNEPDGTFL LRFSDSEIGG ITIAHVIRGQ DGSPQIENIQ PFSAKDLSIR
600
SLGDRIRDLA QLKNLYPKKP KDEAFRSHYK PEQMGKDGRG YVPATIKMTV ERDQPLPTPE
660
LQMPTMVPSY DLGMAPDSSM SMQLGPDMVP QVYPPHSHSI PPYQGLSPEE SVNVLSAFQE
720 PHLQMPPSLG QMSLPFDQPH PQGLLPCQPQ EHAVSSPDPL LCSDVTMVED SCLSQPVTAF 780
PQGTWIGEDI FPPLLPPTEQ DLTKLLLEGQ GESGGGSLGA QPLLQPSHYG QSGISMSHMD
840
LRANPSW
847
SEQ ID NO: 11
MVAHNQVAAD NAVSTAAEPR RRPEPSSSSS SSPAAPARPR PCPAVPAPAP GDTHFRTFRS
60
HADYRRITRA SALLDACGFY WGPLSVHGAH ERLRAEPVGT FLVRDSRQRN CFFALSVKMA
120
SGPTSIRVHF QAGRFHLDGS RESFDCLFEL LEHYVAAPRR MLGAPLRQRR VRPLQELCRQ
180
RIVATVGREN LARIPLNPVL RDYLSSFPFQ I
211
SEQ ID NO: 12
MTLRCLEPSG NGGEGTRSQW GTAGSAEEPS PQAARLAKAL RELGQTGWYW GSMTVNEAKE
60
KLKEAPEGTF LIRDSSHSDY LLTISVKTSA GPTNLRIEYQ DGKFRLDSI I CVKSKLKQFD
120
SWHLIDYYV QMCKDKRTGP EAPRNGTVHL YLTKPLYTSA PSLQHLCRLT INKCTGAIWG
180
LPLPTRLKDY LEEYKFQV
198
SEQ ID NO: 13
MVTHSKFPAA GMSRPLDTSL RLKTFSSKSE YQLWNAVRK LQESGFYWSA VTGGEANLLL
60
SAEPAGTFLI RDSSDQRHFF TLSVKTQSGT KNLRIQCEGG SFSLQSDPRS TQPVPRFDCV
120
LKLVHHYMPP PGAPSFPSPP TEPSSEVPEQ PSAQPLPGSP PRRAYYIYSG GEKIPLVLSR
180
PLSSNVATLQ HLCRKTVNGH LDSYEKVTQL PGPIREFLDQ YDAPL
225
SEQ ID NO: 14
MAENNENISK NVDVRPKTSR SRSADRKDGY VWSGKKLSWS KKSESYSDAE TVNGIEKTEV
60
SLRNQERKHS CSSIELDLDH SCGHRFLGRS LKQKLQDAVG QCFPIKNCSS RHSSGLPSKR
120
KIHISELMLD KCPFPPRSDL AFRWHFIKRH TAPINSKSDE WVSTDLSQTE LRDGQLKRRN
180
MEENINCFSH TNVQPCVITT DNALCREGPM TGSVMNLVSN NSIEDSDMDS DDEILTLCTS
240
SRKRNKPKWD LDDEILQLET PPKYHTQIDY VHCLVPDLLQ INNNPCYWGV MDKYAAEALL
300
EGKPEGTFLL RDSAQEDYLF SVSFRRYSRS LHARIEQWNH NFSFDAHDPC VFHSPDITGL
360
LEHYKDPSAC MFFEPLLSTP LIRTFPFSLQ HICRTVICNC TTYDGIDALP IPSSMKLYLK
420
EYHYKSKVRV LRIDAPEQQC
440
SEQ ID NO: 15
MAENNENISK NVDVRPKTSR SRSADRKDGY VWSGKKLSWS KKSESYSDAE TVNGIEKTEV
60 SLRNQERKHS CSSIELDLDH SCGHRFLGRS LKQKLQDAVG QCFPIKNCSS RHSSGLPSKR 120
KIHISELMLD KCPFPPRSDL AFRWHFIKRH TAPINSKSDE WVSTDLSQTE LRDGQLKRRN
180
MEENINCFSH TNVQPCVITT DNALCREGPM TGSVMNLVSN NSIEDSDMDS DDEILTLCTS
240
SRKRNKPKWD LDDEILQLET PPKYHTQIDY VHCLVPDLLQ INNNPCYWGV MDKYAAEALL
300
EGKPEGTFLL RDSAQEDYLF SVSFRRYSRS LHARIEQWNH NFSFDAHDPC VFHSPDITGL
360
LEHYKDPSAC MFFEPLLSTP LIRTFPFSLQ HICRTVICNC TTYDGIDALP IPSSMKLYLK
420
EYHYKSKVRV LRIDAPEQQC
440
SEQ ID NO: 16
MDKVGKMWNN FKYRCQNLFG HEGGSRSENV DMNSNRCLSV KEKNISIGDS TPQQQSSPLR
60
ENIALQLGLS PSKNSSRRNQ NCATEIPQIV EISIEKDNDS CVTPGTRLAR RDSYSRHAPW
120
GGKKKHSCST KTQSSLDADK KFGRTRSGLQ RRERRYGVSS VHDMDSVSSR TVGSRSLRQR
180
LQDTVGLCFP MRTYSKQSKP LFSNKRKIHL SELMLEKCPF PAGSDLAQKW HLIKQHTAPV
240
SPHSTFFDTF DPSLVSTEDE EDRLRERRRL SIEEGVDPPP NAQIHTFEAT AQVNPLYKLG
300
PKLAPGMTEI SGDSSAIPQA NCDSEEDTTT LCLQSRRQKQ RQISGDSHTH VSRQGAWKVH
360
TQIDYIHCLV PDLLQITGNP CYWGVMDRYE AEALLEGKPE GTFLLRDSAQ EDYLFSVSFR
420
RYNRSLHARI EQWNHNFSFD AHDPCVFHSS TVTGLLEHYK DPSSCMFFEP LLTISLNRTF
480
PFSLQYICRA VICRCTTYDG IDGLPLPSML QDFLKEYHYK QKVRVRWLER EPVKAK
536
SEQ ID NO: 17
MDKVGKMWNN FKYRCQNLFG HEGGSRSENV DMNSNRCLSV KEKNISIGDS TPQQQSSPLR
60
ENIALQLGLS PSKNSSRRNQ NCATEIPQIV EISIEKDNDS CVTPGTRLAR RDSYSRHAPW
120
GGKKKHSCST KTQSSLDADK KFGRTRSGLQ RRERRYGVSS VHDMDSVSSR TVGSRSLRQR
180
LQDTVGLCFP MRTYSKQSKP LFSNKRKIHL SELMLEKCPF PAGSDLAQKW HLIKQHTAPV
240
SPHSTFFDTF DPSLVSTEDE EDRLRERRRL SIEEGVDPPP NAQIHTFEAT AQVNPLYKLG
300
PKLAPGMTEI SGDSSAIPQA NCDSEEDTTT LCLQSRRQKQ RQISGDSHTH VSRQGAWKVH
360
TQIDYIHCLV PDLLQITGNP CYWGVMDRYE AEALLEGKPE GTFLLRDSAQ EDYLFSVSFR
420
RYNRSLHARI EQWNHNFSFD AHDPCVFHSS TVTGLLEHYK DPSSCMFFEP LLTISLNRTF
480
PFSLQYICRA VICRCTTYDG IDGLPLPSML QDFLKEYHYK QKVRVRWLER EPVKAK
536 SEQ ID NO: 18
MKKISLKTLR KSFNLNKSKE ETDFMWQQP SLASDFGKDD SLFGSCYGKD MASCDINGED
60
EKGGKNRSKS ESLMGTLKRR LSAKQKSKGK AGTPSGSSAD EDTFSSSSAP IVFKDVRAQR
120
PIRSTSLRSH HYSPAPWPLR PTNSEETCIK MEVRVKALVH SSSPSPALNG VRKDFHDLQS
180
ETTCQEQANS LKSSASHNGD LHLHLDEHVP WIGLMPQDY IQYTVPLDEG MYPLEGSRSY
240
CLDSSSPMEV SAVPPQVGGR AFPEDESQVD QDLWAPEIF VDQSVNGLLI GTTGVMLQSP
300
RAGHDDVPPL SPLLPPMQNN QIQRNFSGLT GTEAHVAESM RCHLNFDPNS APGVARVYDS
360
VQSSGPMWT SLTEELKKLA KQGWYWGPIT RWEAEGKLAN VPDGSFLVRD SSDDRYLLSL
420
SFRSHGKTLH TRIEHSNGRF SFYEQPDVEG HTSIVDLIEH SIRDSENGAF CYSRSRLPGS
480
ATYPVRLTNP VSRFMQVRSL QYLCRFVIRQ YTRIDLIQKL PLPNKMKDYL QEKHY
535
SEQ ID NO: 19
MVFRNVGRPP EEEDVEAAPE PGPSELLCPR HRCALDPKAL PPGLALERTW GPAAGLEAQL
60
AALGLGQPAG PGVKTVGGGC CPCPCPPQPP PPQPQPPAAA PQAGEDPTET SDALLVLEGL
120
ESEAESLETN SCSEEELSSP GRGGGGGGRL LLQPPGPELP PVPFPLQDLV PLGRLSRGEQ
180
QQQQQQQPPP PPPPPGPLRP LAGPSRKGSF KIRLSRLFRT KSCNGGSGGG DGTGKRPSGE
240
LAASAASLTD MGGSAGRELD AGRKPKLTRT QSAFSPVSFS PLFTGETVSL VDVDISQRGL
300
TSPHPPTPPP PPRRSLSLLD DISGTLPTSV LVAPMGSSLQ SFPLPPPPPP HAPDAFPRIA
360
PIRAAESLHS QPPQHLQCPL YRPDSSSFAA SLRELEKCGW YWGPMNWEDA EMKLKGKPDG
420
SFLVRDSSDP RYILSLSFRS QGITHHTRME HYRGTFSLWC HPKFEDRCQS WEFIKRAIM
480
HSKNGKFLYF LRSRVPGLPP TPVQLLYPVS RFSNVKSLQH LCRFRIRQLV RIDHIPDLPL
540
PKPLISYIRK FYYYDPQEEV YLSLKEAQLI SKQKQEVEPS T
581
SEQ ID NO: 20
MYLEHTSHCP HHDDDTAMDT PLPRPRPLLA VERTGQRPLW APSLELPKPV MQPLPAGAFL
60
EEVAEGTPAQ TESEPKVLDP EEDLLCIAKT FSYLRESGWY WGSITASEAR QHLQKMPEGT
120
FLVRDSTHPS YLFTLSVKTT RGPTNVRIEY ADSSFRLDSN CLSRPRILAF PDWSLVQHY
180
VASCTADTRS DSPDPAPTPA LPMPKEDAPS DPALPAPPPA TAVHLKLVQP FVRRSSARSL
240
QHLCRLVINR LVADVDCLPL PRRMADYLRQ YPFQL
275
SEQ ID NO: 21
MVLCVQGPRP LLAVERTGQR PLWAPSLELP KPVMQPLPAG AFLEEVAEGT PAQTESEPKV
60 LDPEEDLLCI AKTFSYLRES GWYWGSITAS EARQHLQKMP EGTFLVRDST HPSYLFTLSV 120
KTTRGPTNVR IEYADSSFRL DSNCLSRPRI LAFPDWSLV QHYVASCTAD TRSDSPDPAP
180
TPALPMPKED APSDPALPAP PPATAVHLKL VQPFVRRSSA RSLQHLCRLV INRLVADVDC
240
LPLPRRMADY LRQYPFQL
258
SEQ ID NO: 22
MADSAELKQM VMSLRVSELQ VLLGYAGRNK HGRKHELLTK ALHLLKAGCS PAVQMKIKEL
60
YRRRFPQKIM TPADLSIPNV HSSPMPATLS PSTIPQLTYD GHPASSPLLP VSLLGPKHEL
120
ELPHLTSALH PVHPDIKLQK LPFYDLLDEL IKPTSLASDN SQRFRETCFA FALTPQQVQQ
180
ISSSMDISGT KCDFTVQVQL RFCLSETSCP QEDHFPPNLC VKVNTKPCSL PGYLPPTKNG
240
VEPKRPSRPI NITSLVRLST TVPNTIWSW TAEIGRNYSM AVYLVKQLSS TVLLQRLRAK
300
GIRNPDHSRA LIKEKLTADP DSEIATTSLR VSLLCPLGKM RLTIPCRALT CSHLQCFDAT
360
LYIQMNEKKP TWVCPVCDKK APYEHLI IDG LFMEILKYCT DCDEIQFKED GTWAPMRSKK
420
EVQEVSASYN GVDGCLSSTL EHQVASHHQS SNKNKKVEVI DLTIDSSSDE EEEEPSAKRT
480
CPSLSPTSPL NNKGILSLPH QASPVSRTPS LPAVDTSYIN TSLIQDYRHP FHMTPMPYDL
540
QGLDFFPFLS GDNQHYNTSL LAAAAAAVSD DQDLLHSSRF FPYTSSQMFL DQLSAGGSTS
600
LPTTNGSSSG SNSSLVSSNS LRESHSHTVT NRSSTDTASI FGIIPDIISL D
651
SEQ ID NO: 23
MADFEELRNM VSSFRVSELQ VLLGFAGRNK SGRKHDLLMR ALHLLKSGCS PAVQIKIREL
60
YRRRYPRTLE GLSDLSTIKS SVFSLDGGSS PVEPDLAVAG IHSLPSTSVT PHSPSSPVGS
120
VLLQDTKPTF EMQQPSPPIP PVHPDVQLKN LPFYDVLDVL IKPTSLVQSS IQRFQEKFFI
180
FALTPQQVRE ICISRDFLPG GRRDYTVQVQ LRLCLAETSC PQEDNYPNSL CIKVNGKLFP
240
LPGYAPPPKN GIEQKRPGRP LNITSLVRLS SAVPNQISIS WASEIGKNYS MSVYLVRQLT
300
SAMLLQRLKM KGIRNPDHSR ALIKEKLTAD PDSEIATTSL RVSLMCPLGK MRLTIPCRAV
360
TCTHLQCFDA ALYLQMNEKK PTWICPVCDK KAAYESLILD GLFMEILNDC SDVDEIKFQE
420
DGSWCPMRPK KEAMKVSSQP CTKIESSSVL SKPCSVTVAS EASKKKVDVI DLTIESSSDE
480
EEDPPAKRKC IFMSETQSSP TKGVLMYQPS SVRVPSVTSV DPAAIPPSLT DYSVPFHHTP
540
ISSMSSDLPG EQRRNDINNE LKLGTSSDTV QQ
572
SEQ ID NO: 24
MADFEELRNM VSSFRVSELQ VLLGFAGRNK SGRKHDLLMR ALHLLKSGCS PAVQIKIREL
60 YRRRYPRTLE GLSDLSTIKS SVFSLDGGSS PVEPDLAVAG IHSLPSTSVT PHSPSSPVGS 120
VLLQDTKPTF EMQQPSPPIP PVHPDVQLKN LPFYDVLDVL IKPTSLVQSS IQRFQEKFFI
180
FALTPQQVRE ICISRDFLPG GRRDYTVQVQ LRLCLAETSC PQEDNYPNSL CIKVNGKLFP
240
LPGYAPPPKN GIEQKRPGRP LNITSLVRLS SAVPNQISIS WASEIGKNYS MSVYLVRQLT
300
SAMLLQRLKM KGIRNPDHSR ALIKEKLTAD PDSEIATTSL RVSLMCPLGK MRLTIPCRAV
360
TCTHLQCFDA ALYLQMNEKK PTWICPVCDK KAAYESLILD GLFMEILNDC SDVDEIKFQE
420
DGSWCPMRPK KEAMKVSSQP CTKIESSSVL SKPCSVTVAS EASKKKVDVI DLTIESSSDE
480
EEDPPAKRKC IFMSETQSSP TKGVLMYQPS SVRVPSVTSV DPAAIPPSLT DYSVPFHHTP
540
ISSMSSDLPG LDFLSLIPVD PQYCPPMFLD SLTSPLTASS TSVTTTSSHE SSTHVSSSSS
600
RSETGVITSS GSNIPDIISL D
621
SEQ ID NO: 25
MAELGELKHM VMSFRVSELQ VLLGFAGRNK SGRKHELLAK ALHLLKSSCA PSVQMKIKEL
60
YRRRFPRKTL GPSDLSLLSL PPGTSPVGSP GPLAPIPPTL LAPGTLLGPK REVDMHPPLP
120
QPVHPDVTMK PLPFYEVYGE LIRPTTLAST SSQRFEEAHF TFALTPQQVQ QILTSREVLP
180
GAKCDYTIQV QLRFCLCETS CPQEDYFPPN LFVKVNGKLC PLPGYLPPTK NGAEPKRPSR
240
PINITPLARL SATVPNTIW NWSSEFGRNY SLSVYLVRQL TAGTLLQKLR AKGIRNPDHS
300
RALIKEKLTA DPDSEVATTS LRVSLMCPLG KMRLTVPCRA LTCAHLQSFD AALYLQMNEK
360
KPTWTCPVCD KKAPYESLI I DGLFMEILSS CSDCDEIQFM EDGSWCPMKP KKEASEVCPP
420
PGYGLDGLQY SPVQGGDPSE NKKKVEVIDL TIESSSDEED LPPTKKHCSV TSAAIPALPG
480
SKGVLTSGHQ PSSVLRSPAM GTLGGDFLSS LPLHEYPPAF PLGADIQGLD LFSFLQTESQ
540
HYGPSVITSL DEQDALGHFF QYRGTPSHFL GPLAPTLGSS HCSATPAPPP GRVSSIVAPG
600
GALREGHGGP LPSGPSLTGC RSDIISLD
628
SEQ ID NO: 26
MAAELVEAKN MVMSFRVSDL QMLLGFVGRS KSGLKHELVT RALQLVQFDC SPELFKKIKE
60
LYETRYAKKN SEPAPQPHRP LDPLTMHSTY DRAGAVPRTP LAGPNIDYPV LYGKYLNGLG
120
RLPAKTLKPE VRLVKLPFFN MLDELLKPTE LVPQNNEKLQ ESPCIFALTP RQVELIRNSR
180
ELQPGVKAVQ WLRICYSDT SCPQEDQYPP NIAVKVNHSY CSVPGYYPSN KPGVEPKRPC
240
RPINLTHLMY LSSATNRITV TWGNYGKSYS VALYLVRQLT SSELLQRLKT IGVKHPELCK
300
ALVKEKLRLD PDSEIATTGV RVSLICPLVK MRLSVPCRAE TCAHLQCFDA VFYLQMNEKK PTWMCPVCDK PAPYDQLI ID GLLSKILSEC EDADEIEYLV DGSWCPIRAE KERSCSPQGA 420
ILVLGPSDAN GLLPAPSVNG SGALGSTGGG GPVGSMENGK PGADWDLTL DSSSSSEDEE
480
EEEEEEEDED EEGPRPKRRC PFQKGLVPAC
510
SEQ ID NO: 27
MQYLNIKEDC NAMAFCAKMR SSKKTEVNLE APEPGVEVIF YLSDREPLRL GSGEYTAEEL
60
CIRAAQACRI SPLCHNLFAL YDENTKLWYA PNRTITVDDK MSLRLHYRMR FYFTNWHGTN
120
DNEQSVWRHS PKKQKNGYEK KKIPDATPLL DASSLEYLFA QGQYDLVKCL APIRDPKTEQ
180
DGHDIENECL GMAVLAISHY AMMKKMQLPE LPKDISYKRY IPETLNKSIR QRNLLTRMRI
240
NNVFKDFLKE FNNK ICDSS VSTHDLKVKY LATLETLTKH YGAEIFETSM LLISSENEMN
300
WFHSNDGGNV LYYEVMVTGN LGIQWRHKPN WSVEKEKNK LKRKKLENKH KKDEEKNKIR
360
EEWNNFSYFP EITHIVIKES WSINKQDNK KMELKLSSHE EALSFVSLVD GYFRLTADAH
420
HYLCTDVAPP LIVHNIQNGC HGPICTEYAI NKLRQEGSEE GMYVLRWSCT DFDNILMTVT
480
CFEKSEQVQG AQKQFKNFQI EVQKGRYSLH GSDRSFPSLG DLMSHLKKQI LRTDNISFML
540
KRCCQPKPRE ISNLLVATKK AQEWQPVYPM SQLSFDRILK KDLVQGEHLG RGTRTHIYSG
600
TLMDYKDDEG TSEEKKIKVI LKVLDPSHRD ISLAFFEAAS MMRQVSHKHI VYLYGVCVRD
660
VENIMVEEFV EGGPLDLFMH RKSDVLTTPW KFKVAKQLAS ALSYLEDKDL VHGNVCTKNL
720
LLAREGIDSE CGPFIKLSDP GIPITVLSRQ ECIERIPWIA PECVEDSKNL SVAADKWSFG
780
TTLWEICYNG EIPLKDKTLI EKERFYESRC RPVTPSCKEL ADLMTRCMNY DPNQRPFFRA
840
IMRDINKLEE QNPDIVSEKK PATEVDPTHF EKRFLKRIRD LGEGHFGKVE LCRYDPEGDN
900
TGEQVAVKSL KPESGGNHIA DLKKEIEILR NLYHENIVKY KGICTEDGGN GIKLIMEFLP
960
SGSLKEYLPK NKNKINLKQQ LKYAVQICKG MDYLGSRQYV HRDLAARNVL VESEHQVKIG
1020
DFGLTKAIET DKEYYTVKDD RDSPVFWYAP ECLMQSKFYI ASDVWSFGVT LHELLTYCDS
1080
DSSPMALFLK MIGPTHGQMT VTRLVNTLKE GKRLPCPPNC PDEVYQLMRK CWEFQPSNRT
1140
SFQNLIEGFE ALLK
1154
SEQ ID NO: 28
MGMACLTMTE MEGTSTSSIY QNGDISGNAN SMKQIDPVLQ VYLYHSLGKS EADYLTFPSG
60
EYVAEEICIA ASKACGITPV YHNMFALMSE TERIWYPPNH VFHIDESTRH NVLYRIRFYF
120
PRWYCSGSNR AYRHGISRGA EAPLLDDFVM SYLFAQWRHD FVHGWIKVPV THETQEECLG
180
MAVLDMMRIA KENDQTPLAI YNSISYKTFL PKCIRAKIQD YHILTRKRIR YRFRRFIQQF
240 SQCKATARNL KLKYLINLET LQSAFYTEKF EVKEPGSGPS GEEIFATI I I TGNGGIQWSR 300
GKHKESETLT EQDLQLYCDF PNIIDVSIKQ ANQEGSNESR WTIHKQDGK NLEIELSSLR
360
EALSFVSLID GYYRLTADAH HYLCKEVAPP AVLENIQSNC HGPISMDFAI SKLKKAGNQT
420
GLYVLRCSPK DFNKYFLTFA VERENVIEYK HCLITKNENE EYNLSGTKKN FSSLKDLLNC
480
YQMETVRSDN IIFQFTKCCP PKPKDKSNLL VFRTNGVSDV PTSPTLQRPT HMNQMVFHKI
540
RNEDLIFNES LGQGTFTKIF KGVRREVGDY GQLHETEVLL KVLDKAHRNY SESFFEAASM
600
MSKLSHKHLV LNYGVCVCGD ENILVQEFVK FGSLDTYLKK NKNCINILWK LEVAKQLAWA
660
MHFLEENTLI HGNVCAKNIL LIREEDRKTG NPPFIKLSDP GISITVLPKD ILQERIPWVP
720
PECIENPKNL NLATDKWSFG TTLWEICSGG DKPLSALDSQ RKLQFYEDRH QLPAPKWAEL
780
ANLINNCMDY EPDFRPSFRA I IRDLNSLFT PDYELLTEND MLPNMRIGAL GFSGAFEDRD
840
PTQFEERHLK FLQQLGKGNF GSVEMCRYDP LQDNTGEWA VKKLQHSTEE HLRDFEREIE
900
ILKSLQHDNI VKYKGVCYSA GRRNLKLIME YLPYGSLRDY LQKHKERIDH IKLLQYTSQI
960
CKGMEYLGTK RYIHRDLATR NILVENENRV KIGDFGLTKV LPQDKEYYKV KEPGESPIFW
1020
YAPESLTESK FSVASDVWSF GWLYELFTY IEKSKSPPAE FMRMIGNDKQ GQMIVFHLIE
1080
LLKNNGRLPR PDGCPDEIYM IMTECWNNNV NQRPSFRDLA LRVDQIRDNM AG
1132
SEQ ID NO: 29
MAPPSEETPL IPQRSCSLLS TEAGALHVLL PARGPGPPQR LSFSFGDHLA EDLCVQAAKA
60
SGILPVYHSL FALATEDLSC WFPPSHIFSV EDASTQVLLY RIRFYFPNWF GLEKCHRFGL
120
RKDLASAILD LPVLEHLFAQ HRSDLVSGRL PVGLSLKEQG ECLSLAVLDL ARMAREQAQR
180
PGELLKTVSY KACLPPSLRD LIQGLSFVTR RRIRRTVRRA LRRVAACQAD RHSLMAKYIM
240
DLERLDPAGA AETFHVGLPG ALGGHDGLGL LRVAGDGGIA WTQGEQEVLQ PFCDFPEIVD
300
ISIKQAPRVG PAGEHRLVTV TRTDNQILEA EFPGLPEALS FVALVDGYFR LTTDSQHFFC
360
KEVAPPRLLE EVAEQCHGPI TLDFAINKLK TGGSRPGSYV LRRSPQDFDS FLLTVCVQNP
420
LGPDYKGCLI RRSPTGTFLL VGLSRPHSSL RELLATCWDG GLHVDGVAVT LTSCCIPRPK
480
EKSNLIWQR GHSPPTSSLV QPQSQYQLSQ MTFHKIPADS LEWHENLGHG SFTKIYRGCR
540
HEWDGEARK TEVLLKVMDA KHKNCMESFL EAASLMSQVS YRHLVLLHGV CMAGDSTMVQ
600
EFVHLGAIDM YLRKRGHLVP ASWKLQWKQ LAYALNYLED KGLPHGNVSA RKVLLAREGA
660
DGSPPFIKLS DPGVSPAVLS LEMLTDRIPW VAPECLREAQ TLSLEADKWG FGATVWEVFS
720
GVTMPISALD PAKKLQFYED RQQLPAPKWT ELALLIQQCM AYEPVQRPSF RAVIRDLNSL
780 ISSDYELLSD PTPGALAPRD GLWNGAQLYA CQDPTIFEER HLKYISQLGK GNFGSVELCR 840
YDPLGDNTGA LVAVKQLQHS GPDQQRDFQR EIQILKALHS DFIVKYRGVS YGPGRQSLRL
900
VMEYLPSGCL RDFLQRHRAR LDASRLLLYS SQICKGMEYL GSRRCVHRDL AARNILVESE
960
AHVKIADFGL AKLLPLDKDY YWREPGQSP IFWYAPESLS DNIFSRQSDV WSFGWLYEL
1020
FTYCDKSCSP SAEFLRMMGC ERDVPALCRL LELLEEGQRL PAPPACPAEV HELMKLCWAP
1080
SPQDRPSFSA LGPQLDMLWS GSRGCETHAF TAHPEGKHHS LSFS
1124
SEQ ID NO: 30
MQYLNIKEDC NAMAFCAKMR SSKKTEVNLE APEPGVEVIF YLSDREPLRL GSGEYTAEEL
60
CIRAAQACRI SPLCHNLFAL YDENTKLWYA PNRTITVDDK MSLRLHYRMR FYFTNWHGTN
120
DNEQSVWRHS PKKQKNGYEK KKIPDATPLL DASSLEYLFA QGQYDLVKCL APIRDPKTEQ
180
DGHDIENECL GMAVLAISHY AMMKKMQLPE LPKDISYKRY IPETLNKSIR QRNLLTRMRI
240
NNVFKDFLKE FNNKTICDSS VSTHDLKVKY LATLETLTKH YGAEIFETSM LLISSENEMN
300
WFHSNDGGNV LYYEVMVTGN LGIQWRHKPN WSVEKEKNK LKRKKLENKH KKDEEKNKIR
360
EEWNNFSYFP EITHIVIKES WSINKQDNK KMELKLSSHE EALSFVSLVD GYFRLTADAH
420
HYLCTDVAPP LIVHNIQNGC HGPICTEYAI NKLRQEGSEE GMYVLRWSCT DFDNILMTVT
480
CFEKSEQVQG AQKQFKNFQI EVQKGRYSLH GSDRSFPSLG DLMSHLKKQI LRTDNISFML
540
KRCCQPKPRE ISNLLVATKK AQEWQPVYPM SQLSFDRILK KDLVQGEHLG RGTRTHIYSG
600
TLMDYKDDEG TSEEKKIKVI LKVLDPSHRD ISLAFFEAAS MMRQVSHKHI VYLYGVCVRD
660
VENIMVEEFV EGGPLDLFMH RKSDVLTTPW KFKVAKQLAS ALSYLEDKDL VHGNVCTKNL
720
LLAREGIDSE CGPFIKLSDP GIPITVLSRQ ECIERIPWIA PECVEDSKNL SVAADKWSFG
780
TTLWEICYNG EIPLKDKTLI EKERFYESRC RPVTPSCKEL ADLMTRCMNY DPNQRPFFRA
840
IMRDINKLEE QNPDIVSEKK PATEVDPTHF EKRFLKRIRD LGEGHFGKVE LCRYDPEGDN
900
TGEQVAVKSL KPESGGNHIA DLKKEIEILR NLYHENIVKY KGICTEDGGN GIKLIMEFLP
960
SGSLKEYLPK NKNKINLKQQ LKYAVQICKG MDYLGSRQYV HRDLAARNVL VESEHQVKIG
1020
DFGLTKAIET DKEYYTVKDD RDSPVFWYAP ECLMQSKFYI ASDVWSFGVT LHELLTYCDS
1080
DSSPMALFLK MIGPTHGQMT VTRLVNTLKE GKRLPCPPNC PDEVYQLMRK CWEFQPSNRT
1140
SFQNLIEGFE ALLK 1154

Claims

1. A method of treatment or prevention of hypercytokinemia and/or viral infection comprising the administration of a cytokine expression regulator, or a vector encoding a cytokine expression regulator,
wherein the cytokine expression regulator is selected from any one of the group comprising members of the Janus Kinase (JAK) family, members of the signal transducer and activator of transcription (STAT) family, members of the suppressor of cytokine signalling (SOCS) family, and members of the protein inhibitor of activated STAT (PIAS) family, or analogues thereof.
2. A cytokine expression regulator, or a vector encoding a cytokine expression regulator, for use in a treatment or prevention of hypercytokinemia and/or viral infection in a mammalian subject,
wherein the cytokine expression regulator is selected from the group consisting of members of JAK, STAT, SOCS and PIAS proteins , or analogues thereof 3. The method of claim 1 , or the cytokine expression regulator or vector of claim 2, wherein the hypercytokinemia is a result of infection.
4. The method or the cytokine expression regulator or vector of claim 3 , wherein the hypercytokinemia is a result of viral infection, optionally influenza viral infection.
5. The method of claim 1 or the cytokine expression regulator or vector of claim 2, wherein the prevention comprises treatment for viral infection, optionally influenza viral infection.
6. The method or the cytokine expression regulator or vector of claim 5 , wherein the prevention comprises treatment for H5N1 , HlNl , or SARS infection; or
the method or the cytokine expression regulator or vector of claim 5 , wherein the prevention comprises treatment for respiratory syncytial virus (RSV) infection, human parainfluenza virus infection, adenovirus infection, metapneumovirus infection, herpes simplex virus (HSV) infection, varicella-zoster virus (VZV) infection or cytomegalovirus (CMV) infection.
7. The method of any of claims 1 , or 3 to 6, or the cytokine expression regulator or vector of any of claims 2 to 6, wherein the JAK family member is JAK1.
8. The method of any of claims 1 , or 3 to 7, or the cytokine expression regulator or vector of any of claims 2 to 7, wherein the STAT family member is selected from STAT1 , STAT2, STAT3 , or combinations thereof.
9. The method of any of claims 1 , or 3 to 8, or the cytokine expression regulator or vector of claims 2 to 8, wherein the SOCS family member is selected from SOCS1 , SOCS2, SOCS3 , or combinations thereof.
10. The method of any of claims 1 , or 3 to 9, or the cytokine expression regulator or vector of any of claims 2 to 9, wherein the PI AS family members is selected from PIAS1 , and PIAS2, or combinations thereof.
11. An assay comprising the steps of:
-exposing a cell to an agent -detecting whether there is modulation of expression or activity of at least one cytokine expression regulator in the cell after exposure of the cell to the agent,
wherein the cytokine expression regulator is selected from any one of the group comprising members of the Janus kinases (JAK) family, members of the signal transducer and activator of transcription (STAT) family, members of the suppressor of cytokine signalling (SOCS) family, and members of the protein inhibitor of activated STAT (PIAS) family, or combinations thereof.
12. The assay according to claim 11 , wherein the assay is for the identification of the agent as a potential therapeutic agent for use in the treatment of hypercytokinemia.
13. The assay according to claim 11 , wherein the assay is for the identification of the agent as useful in the reduction of proinflammatory cytokines, such as TNF-alpha, in the cell, or in a patient.
14. The assay according to any of claims 11 to 13, where the assay further comprises triggering an infection response in the cell.
15. The assay according to claim 14, wherein the infection response in the cell is triggered by introducing an infectious material to the cell, or by introducing a material to the cell that mimics an infection of the cell.
16. The assay according to claim 15 , wherein the material is a virus, a glycopeptide, a protein, a nucleic acid, or a bacterium, or a part thereof.
17. The assay according to claim 16, wherein the virus, or part of the virus, or the nucleic acid, may be derived from influenza virus, such as
H5N1 or H1N1 , or SARS virus.
18. The assay according to claim 16, wherein the virus , or part of the virus, or the viral nucleic acid, is derived from any of the group selected from respiratory syncytial virus (RSV) , human parainfluenza viruses, adenoviruses, metapneumovirus, herpes simplex virus (HSV) , varicella- zoster virus (VZV) and cytomegalovirus (CMV) .
19. The assay according to any of claims 11 to 18, wherein the modulation of expression of the cytokine expression regulator is upregulation.
20. The assay according to any of claims 11 to 19, wherein the modulation of expression of the cytokine expression regulator is the prevention or reversal of suppression of the cytokine expression regulator.
21. The assay according to any of claims 11 to 20, wherein the cell is a human cell, pig cell or avian cell.
22. The assay according to any of claims 11 to 21 , wherein the cell is a macrophage, monocyte, or a tracheal epithelial cell (TEC) .
23. The assay according to any of claims 11 to 22, wherein the agent is a compound/drug, nucleic acid, peptide, or protein.
24. The assay according to any of claims 11 to 23 , wherein the agent is an analogue of the cytokine expression regulator.
25. The assay according to any of claims 11 to 24, wherein the agent is a truncated form of the cytokine expression regulator.
26. A method of screening an agent for use in a treatment, comprising determining whether said agent has affinity against a negative-regulator of at least one protein, wherein the at least one protein is selected from the group consisting of: (a) members of the signal transducer and activator of transcription (STAT) family, (b) members of the suppressor of cytokine signalling (SOCS) family, (c) members of the protein inhibitor of activated STAT (PIAS) family (d) members of the Janus kinases (JAK) family, or combinations of (a) , (b) , (c) or (d) .
27. Use of a cytokine expression regulator pathway in a cell for determining the therapeutic activity of an agent, wherein the cytokine expression regulator pathway is selected from JAK, STAT, SOCS and PIAS.
28. Use of a cytokine expression regulator substantially as described herein with reference to the description and drawings .
29. An assay substantially as described herein with reference to the description and drawings .
30. A method of screening substantially as described herein with reference to the description and drawings .
PCT/GB2011/050233 2010-02-12 2011-02-09 Proteins of the jak, stat, socs and pias families for use in the treatment hypercytokinemia or viral infection WO2011098806A1 (en)

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WO2013059559A3 (en) * 2011-10-21 2013-11-14 Glaxosmithkline Llc Compounds and methods for enhancing innate immune responses
EP3199181A4 (en) * 2014-09-22 2018-06-20 Japan Science and Technology Agency Anti-influenza virus agent, and screening method for anti-influenza virus agent
US11253559B2 (en) * 2016-07-19 2022-02-22 University of Pittsburgh—of the Commonwealth System of Higher Education Oncolytic viruses targeting STAT3
CN112639084A (en) * 2018-08-16 2021-04-09 深圳市亦诺微医药科技有限公司 Methods and compositions for treating solid cancers and microbial infections

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