Risk stratification in influenza

Abstract

The present invention relates to methods for the identification of clinical risk in patients having, or suspected of having, influenza. The invention also relates to methods for distinguishing between patients having influenza or viral pneumonia from patients having a symptomatically similar condition. The methods of the invention comprise determination of the level of expression of interferon alpha inducible protein 27 (IFI27) in a biological sample from a patient having, or suspected of having, influenza. Kits comprising suitable components for the performance of the methods are also provided by the invention. The invention allows stratification of patients into groups defining clinical risk, for example groups based on the severity of risk to the long-term health of the subject.

1 . A method for identifying clinical risk in a patient having or suspected of having an influenza infection, the method comprising determining the expression level of interferon alpha inducible protein 27 (IFI27) gene in a biological sample from said patient and comparing the determined level of IFI27 gene product to a standard level. 
     
     
         2 . The method of  claim 1 , wherein the standard level of IFI27 gene product is indicative of no clinical risk, and an elevated level of IFI27 gene product in said patient sample compared to said standard level is indicative of clinical risk in said patient. 
     
     
         3 . The method of  claim 2  wherein the standard level is based on the level of IFI27 gene product in healthy subjects, subjects infected with influenza that are asymptomatic or subjects that are infected with influenza that do not develop severe disease. 
     
     
         4 . The method of  claim 1 , wherein the standard level of IFI27 gene product is indicative of clinical risk, and an equal or elevated level of IFI27 gene product in said patient sample compared to said standard level is indicative of clinical risk in said patient. 
     
     
         5 . The method of  claim 1  wherein the standard level is based on the level of IFI27 gene product in healthy subjects and an elevated level of IFI27 gene product in said patient sample compared to said standard level is indicative of clinical risk in said patient. 
     
     
         6 . The method of  claim 1  wherein the standard level is based on the level of IFI27 gene product in subjects infected with influenza virus that are asymptomatic and an elevated level of IFI27 gene product in said patient sample compared to said standard level is indicative of clinical risk in said patient. 
     
     
         7 . The method of any one of  claims 1  to  6  wherein the standard level is prepared at the same time as determining the expression level of IFI27 gene in the biological sample from said patient. 
     
     
         8 . The method of  claim 5  or  6 , wherein a level of IFI27 gene product that is at least 40 to 60 times higher than the standard level is indicative of clinical risk. 
     
     
         9 . The method of  claim 8 , wherein a level of IFI27 gene product that is at least 40 times higher than the standard level is indicative of clinical risk. 
     
     
         10 . The method of  claim 8 , wherein a level of IFI27 gene product that is at least 50 times higher than the standard level is indicative of clinical risk. 
     
     
         11 . The method of  claim 8 , wherein a level of IFI27 gene product that is at least 60 times higher than the standard level is indicative of clinical risk. 
     
     
         12 . The method of  claim 1  wherein the standard level is prepared by subjecting one or more known sample(s) of an IFI27 gene product to the same methods for determining IFI27 gene expression level as the biological sample from said patient, wherein the one or more known sample(s) of an IFI27 gene product are of a pre-determined amount or amounts indicative of clinical risk, and an equal or elevated level of IFI27 gene product in said patient sample compared to said standard level is indicative of clinical risk in said patient. 
     
     
         13 . The method of  claim 1  wherein the standard level is prepared by subjecting one or more known sample(s) of an IFI27 gene product to the same methods for determining IFI27 gene expression level as the biological sample from said patient, wherein the one or more known sample(s) of an IFI27 gene product are of a pre-determined amount or amounts indicative of no clinical risk, and an elevated level of IFI27 gene product in said patient sample compared to said standard level is indicative of clinical risk in said patient. 
     
     
         14 . The method of  claim 1  wherein the method provides a method of monitoring the progress of a patient having influenza, the method comprising determining the expression level of IFI27 gene in a first biological sample from said patient and determining the expression level of IFI27 gene in a second biological sample from said patient, wherein the first and second samples are obtained from the patient at different times, and assessing the patient status on the basis of the relative expression levels of IFI27 in the first and second samples. 
     
     
         15 . The method of  claim 14  wherein an increase in the expression level of IFI27 gene in said second biological sample compared to said first biological sample is indicative of an increased clinical risk in said patient. 
     
     
         16 . The method of  claim 14  wherein a decrease in the expression level of IFI27 gene in said second biological sample compared to said first biological sample is indicative of a decreased clinical risk in said patient. 
     
     
         17 . A method for identifying influenza or viral pneumonia in a patient, the method comprising determining the expression level of interferon alpha inducible protein 27 (IFI27) gene in a biological sample from said patient and comparing the determined level of IFI27 gene product to a standard level. 
     
     
         18 . The method of  claim 17  wherein the standard level is based on the level of IFI27 gene product in healthy subjects, and an elevated level of IFI27 in said patient sample compared to said standard level is indicative of influenza or viral pneumonia in said patient. 
     
     
         19 . The method of  claim 17  wherein the patient is suspected of having viral pneumonia or bacterial pneumonia. 
     
     
         20 . The method of  claim 17  wherein the standard level is based on the level of IFI27 gene product in subjects with bacterial pneumonia, and an elevated level of IFI27 gene product in said patient sample compared to said standard level is indicative of a patient with influenza or viral pneumonia. 
     
     
         21 . The method of any one of  claims 18  to  20 , wherein a level of IFI27 gene product that is at least 10 times higher than the standard level is indicative of influenza or viral pneumonia. 
     
     
         22 . The method of any one of  claims 18  to  20 , wherein a level of IFI27 gene product that is at least 40 to 60 times higher than the standard level is indicative of a patient with influenza or viral pneumonia and clinical risk. 
     
     
         23 . The method of  claim 22 , wherein a level of IFI27 gene product that is at least 40 times higher than the standard level is indicative of a patient with influenza or viral pneumonia and clinical risk. 
     
     
         24 . The method of  claim 22  wherein a level of IFI27 gene product that is at least 50 times higher than the standard level is indicative of a patient with influenza or viral pneumonia and clinical risk. 
     
     
         25 . The method of  claim 22 , wherein a level of IFI27 gene product that is at least 60 times higher than the standard level is indicative of a patient with influenza or viral pneumonia and clinical risk. 
     
     
         26 . The method of any one of  claims 1  to  25  wherein the method further comprises determining the expression level of at least one additional gene(s) in said biological sample. 
     
     
         27 . The method of  claim 26  wherein the at least one additional gene(s) is a gene the expression of which is constitutive. 
     
     
         28 . The method of  claim 27  wherein the at least one additional gene(s) is GAPDH gene. 
     
     
         29 . The method of any one of  claims 1  to  28  wherein the biological sample is blood, or a component thereof such as blood cell subsets. 
     
     
         30 . The method of any one of  claims 1  to  29  wherein the method comprises contacting the biological sample with an agent capable of binding to an IFI27 gene product and detecting binding between the agent and the IFI27 gene product. 
     
     
         31 . The method of any one of  claims 1  to  30  wherein the biological sample is RNA, mRNA, cDNA or protein. 
     
     
         32 . The method of any one of  claims 1  to  31  wherein the IFI27 gene product is an IFI27 mRNA or fragment thereof. 
     
     
         33 . The method of any one of  claims 1  to  32  wherein the IFI27 gene product comprises a nucleic acid sequence of SEQ ID NO:1 or a fragment or variant thereof or a nucleic acid sequence of SEQ ID NO:2 or a fragment or variant thereof. 
     
     
         34 . The method of any one of  claims 1  to  31  wherein the IFI27 gene product is an IFI27 polypeptide or a fragment or variant thereof. 
     
     
         35 . The method of  claim 34 , wherein the IFI27 gene product comprises an amino acid sequence comprising the sequence of SEQ ID NO:3 or a fragment or variant thereof or comprises an amino acid sequence comprising the sequence of SEQ ID NO:4 or a fragment or variant thereof. 
     
     
         36 . The method of any one of  claims 1  to  33  wherein the method comprises reverse transcription of mRNA to cDNA. 
     
     
         37 . The method of any one of  claim 1  to  33  or  36 , wherein the method comprises amplification of an IFI27 nucleic acid sequence of the sample and detecting an amplified sequence. 
     
     
         38 . The method of any one of  claim 1  to  33 ,  36  or  37 , wherein the method comprises quantitative polymerase chain reaction (qPCR). 
     
     
         39 . The method of  claim 38  wherein the polymerase chain reaction utilizes one or more primers capable of amplifying a nucleic acid sequence selected from the sequences of (i) SEQ ID NO:1, (ii) SEQ ID NO:2, (iii) SEQ ID NO:5, (iv) SEQ ID NO:6, and (v) or a fragment or variant of any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6. 
     
     
         40 . The method of  claim 38  wherein the polymerase chain reaction utilizes one or both of the primer sequences of SEQ ID NO:7 (acctcatcagcagtgaccagt) and SEQ ID NO:8 (acatcatcttggctgctatgg), or a sequence variant thereof capable of amplifying the same target sequence. 
     
     
         41 . The method of  claim 38  wherein the polymerase chain reaction may utilize one or both of the primer sequences of SEQ ID NO:9 (TGCCTCGGGCAGCCT) and SEQ ID NO:10 (TTGGTCAATCCGGAGAGTCC), or a sequence variant thereof capable of amplifying the same target sequence. 
     
     
         42 . The method of  claim 1  or  17  wherein the method comprises contacting the sample with one or more probe(s) capable of specifically binding to an IFI27 gene product or fragment or variant thereof. 
     
     
         43 . The method of  claim 42  wherein the one or more probe(s) is a nucleic acid comprising (i) a sequence complementary to the sequence shown in SEQ ID NO:1 or (ii) a sequence complementary to the sequence shown in SEQ ID NO:2, or a fragment or variant of (i) or (ii). 
     
     
         44 . The method of  claim 42  wherein the one or more probe(s) is a nucleic acid comprising (i) a sequence complementary to the sequence shown in SEQ ID NO:5 or (ii) a sequence complementary to the sequence shown in SEQ ID NO:6 or a fragment or variant of (i) or (ii). 
     
     
         45 . The method of  claim 42  wherein the nucleic acid probe comprises 10 to 50 bases. 
     
     
         46 . The method of  claim 42  wherein the nucleic acid probe comprises 30 to 600 bases. 
     
     
         47 . The method of  claim 1  or  17  wherein the method comprises the steps of (i) obtaining a blood sample from a patient having or suspected of having an influenza infection; (ii) preparing an isolate of total RNA from said blood sample; (iii) preparing cDNA by reverse transcription of said total RNA isolate; (iv) amplifying an IFI27 nucleic acid sequence by polymerase chain reaction; (v) comparing the level of said amplified IFI27 nucleic acid sequence to a standard; (vi) determining if said patient has clinical risk to said patient on the basis of said comparing. 
     
     
         48 . The method of  claim 47  wherein the polymerase chain reaction is quantitative polymerase chain reaction. 
     
     
         49 . The method of  claim 1  or  17  wherein the method comprises determining the level of an IFI27 polypeptide, or fragment thereof. 
     
     
         50 . The method of  claim 49  wherein the method comprises determining the level of an IFI27 polypeptide comprising (i) the amino acid sequence of SEQ ID NO:3 or (ii) the amino acid sequence of SEQ ID NO:4, or an antigenic fragment or variant of (i) or (ii). 
     
     
         51 . The method of  claim 49  wherein the method comprises contacting the sample with an antibody capable of selectively binding to an IFI27 polypeptide comprising an amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:4, or an antigenic fragment or variant thereof. 
     
     
         52 . The method of  claim 1  or  17  wherein the method comprises one or more of gel electrophoresis, nucleic acid sequencing and amino acid sequencing. 
     
     
         53 . The method of  claim 1  or  17  wherein the method is performed entirely ex vivo. 
     
     
         54 . The method of  claim 1  or  17  wherein comparing the level of determined IFI27 gene product in a biological sample to a standard level is done with the assistance of a computer program. 
     
     
         55 . The method of any one of  claims 1  to  30  wherein identifying a clinical risk in a patient on the basis of the comparison is done with the assistance of a computer program. 
     
     
         56 . A kit for determining the level of an interferon alpha inducible protein 27 (IFI27) gene product in a biological sample, the kit comprising at least one agent for detecting the presence of an IFI27 gene product. 
     
     
         57 . The kit of  claim 56  wherein the at least one agent is a primer, antibody or probe. 
     
     
         58 . The kit of  claim 56  wherein the primer or probe is specific for a nucleic acid sequence selected from the sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, or a variant or fragment thereof. 
     
     
         59 . The kit of  claim 56  wherein the kit comprises a forward and a reverse primer capable of selectively amplifying a nucleic acid sequence selected from the sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, or a variant or fragment thereof. 
     
     
         60 . The kit of  claim 56  wherein the kit comprises one or more nucleic acid sequences selected from the group consisting of SEQ ID NO:7 (acctcatcagcagtgaccagt), SEQ ID NO:8 (acatcatcttggctgctatgg), SEQ ID NO:9 (TGCCTCGGGCAGCCT) and SEQ ID NO:10 (TTGGTCAATCCGGAGAGTCC), or a sequence variant thereof capable of specifically binding the same target sequence. 
     
     
         61 . The kit of  claim 56  wherein the kit comprises multiple agents capable of detecting the presence of an interferon alpha inducible protein 27 (IFI27) gene product in a biological sample. 
     
     
         62 . The kit of  claim 56  wherein the kit comprises an antibody capable of specifically binding to a polypeptide, or fragment or variant thereof, encoded by an IFI27 gene sequence. 
     
     
         63 . The kit of  claim 62  wherein the antibody is a conjugated antibody specific for IFI27. 
     
     
         64 . The kit of  claim 62  wherein the antibody is a polyclonal antibody. 
     
     
         65 . The kit of  claim 64  wherein the polyclonal antibody is a rabbit polyclonal antibody. 
     
     
         66 . The kit of  claim 62  wherein the antibody is a monoclonal antibody. 
     
     
         67 . The kit of  claim 62  wherein the antibody binds to the epitope sequence: 
       
         
           
                 
               
                   MEASLATSSAVTSVAKVVRVASGSAVVLPLARIATVVIGGVVAVPMVLSA 
                 
                     
                 
                   MGFTAAGIASSSIAAKMMSAAAIANGGGVASGSLVATLQSLGATGLSGLT 
                 
                     
                 
                   KFILGSIGSAIAAVIARFY). 
                 
             
                
                
                
                
                
               
            
           
         
       
     
     
         68 . The kit of  claim 62  wherein the antibody is capable of selectively binding to (i) an IFI27 polypeptide comprising an amino acid sequence shown in SEQ ID NO:3 or (ii) an IFI27 polypeptide comprising an amino acid sequence shown in SEQ ID NO:4, or (iii) an antigenic fragment or variant of (i) or (ii). 
     
     
         69 . The kit of  claim 56  wherein the kit comprises one or more agents for normalisation of said method. 
     
     
         70 . The kit of  claim 56  wherein the agent(s) for normalisation are selected from the group consisting of an agent or agents for the detection of a constitutively expressed gene product. 
     
     
         71 . The kit of  claim 56  wherein the constitutively expressed gene product is GAPDH. 
     
     
         72 . The kit of  claim 56  wherein the kit comprises one or more calibrated standards wherein the standard comprises a known concentration of IFI27 gene product. 
     
     
         73 . The kit of  claim 56  wherein the kit comprises one or more additional components selected from the group consisting of (i) one or more reference sample(s); (ii) one or more detectable moieties; (iii) one or more substance(s) for immobilising an agent for detecting an IFI27 gene product on a solid support; (iv) a solid support material; (v) one or more container(s) for collection and/or storage of a biological sample; (vi) one or more reagent(s) for use in preparation of a biological sample; (vii) one or more agents for the amplification of a nucleic acid sequence; and (viii) instructions for use of the kit or a component(s) thereof in a method for determining the level of an IFI27 gene product in a biological sample. 
     
     
         74 . A method for assessing the efficacy of an agent for treatment of influenza, the method comprising administering said agent to an individual having an influenza infection and determining the level of an interferon alpha inducible protein 27 (IFI27) gene product in a biological sample from said individual and comparing said determined level of IFI27 gene product to a standard level. 
     
     
         75 . A method for assessing the efficacy of an agent for reducing disease severity in an individual exposed to an influenza virus, the method comprising administering said agent to said individual, determining the level of an interferon alpha inducible protein 27 (IFI27) gene product in a biological sample from said individual and comparing said determined level to a standard level. 
     
     
         76 . The method of  claim 74  or  75  wherein the individual is exposed to an influenza virus prior to administration of said agent. 
     
     
         77 . The method of  claim 74  or  75  wherein the individual has an influenza infection when administered said agent. 
     
     
         78 . The method of  claim 74  or  75  wherein the individual is exposed to an influenza virus after said administration of said agent. 
     
     
         79 . The method of  claim 74  or  75  wherein the method further comprises determining the level of IFI27 gene product in a biological sample obtained from said individual prior to administration of said agent. 
     
     
         80 . The method of  claim 74  or  75  wherein a decrease in the level of an IFI27 gene product in a biological sample from said individual after administration of said agent compared to the level prior to said administration is indicative of an agent capable of reducing disease severity in a patient exposed to an influenza virus or having an influenza infection. 
     
     
         81 . The method of  claim 74  or  75  wherein the method is conducted as part of a research trail or clinical trial of a candidate anti-viral agent for the prevention of treatment of influenza.