WO2013055811A1

WO2013055811A1 - Ex vivo methods of predicting responsiveness of a subject to interferon therapy

Info

Publication number: WO2013055811A1
Application number: PCT/US2012/059585
Authority: WO
Inventors: Masato Mitsuhashi; Satoru Saito
Original assignee: Hitachi Chemical Co., Ltd.; Hitachi Chemical Research Center, Inc.; Yokohama City University
Priority date: 2011-10-12
Filing date: 2012-10-10
Publication date: 2013-04-18

Abstract

Embodiments of the invention relate generally to ex vivo methods of quantifying expression of interferon responsive genes and using that quantified expression information to characterize (e.g., predict) a subject's potential responsiveness to interferon administration. In certain embodiments, the methods provided here are for the characterization of a subject's likelihood of responding to interferon therapy for hepatitis.

Description

EX VIVO METHODS OF PREDICTING RESPONSIVENESS OF A SUBEJCT

TO INTERFERON THERAPY

RELATED CASES

[0001] This application claims the benefit of United States Provisional Application No: 61/546,509, filed on October 12, 2011, which is incorporated in its entirety by reference herein.

BACKGROUND

Field of the Invention

[0002] Embodiments of the present invention relate generally to methods for the characterization of the responsiveness of an individual to certain therapeutic agents. More specifically, embodiments disclosed herein relate to the prediction of the whether an individual will respond or fail to respond to interferon (IFN) therapy in certain disease contexts, such as, for example, hepatitis therapy. Additional embodiments relate to methods of monitoring the effectiveness of ongoing IFN therapy in an individual.

Description of the Related Art

[0003] Interferons are anti-viral drugs used in laboratory research settings, as well as clinically, to combat viral infections and/or in therapies for indications such as cancer. Interferons are also clinically approved for treatment for a variety of cancers, including certain leukemias, myelomas, and melanomas, as well as of hepatitis B and C.

[0004] Hepatitis is a viral liver disease characterized by the presence of inflammatory cells in the liver. While there are many causes of the disease (including exposure to infectious blood or body fluids, unprotected sexual contact, blood transfusions, use of contaminated needles, and vertical transmission from mother to child during childbirth), diagnosis is challenging because viral antigens may not appear until well after initiation of the infection. Hepatitis also exists in many forms, most prominently Hepatitis B (caused by the hepatitis B virus, "HBV") and Hepatitis C (caused by the hepatitis C virus "HCV"). Both of these forms of Hepatitis may either be acute (less than six months to resolution) or chronic (infection greater than six months). [0005] Both HBV and HCV exist in various genetic subtypes, which affect the severity of the disease symptoms, the likelihood of complications, and the possible responsiveness of the host to various available treatments, of which interferon therapy is one.

[0006] IFN has anti-proliferative, pro-apoptotic, and anti-angiogenic effects on cultured cancer cells, but the precise molecular targets set in motion by IFN therapy are not presently known. Despite its current clinical use, there remains a need for assessing the likelihood that a given individual will respond positively to IFN administration, in particular in such settings as hepatitis. There is also a need for monitoring the ongoing efficacy of IFN administration in an individual receiving the IFN on an ongoing basis.

SUMMARY

[0007] In several embodiments, there is provided a method for identifying a subject having hepatitis as likely to respond to interferon therapy, comprising obtaining at least a first and a second sample of whole blood from the subject, exposing the first sample of whole blood from the subject having hepatitis to interferon in a solvent for an amount of time sufficient for the interferon to alter the expression of two or more interferon-responsive markers, exposing the second sample of whole blood from the subject having hepatitis to the solvent without the interferon for the amount of time, quantifying the expression of the two or more interferon- responsive markers by measuring the amount of mRNA encoding the one or more interferon-responsive markers in both the first whole blood sample and the second whole blood sample, evaluating the potential responsiveness of the subject to the interferon therapy by: a) predetermining a parameter by multivariate discriminant analysis for each combination of the two or more interferon-responsive markers and the interferon using a plurality of patients in one or more of a plurality of groups with known clinical responsiveness to interferon therapy for hepatitis, b) calculating a predictor value for the subject for each of the plurality of groups; and c) comparing the predictor values with the predetermined parameters in order to categorize the subject within one of the plurality of groups of known clinical responsiveness, and identifying the subject as likely to respond to interferon therapy when the subject is categorized in any of the plurality of groups of known clinical responsiveness.

[0008] In several embodiments, the two or more interferon-responsive markers are selected from the group consisting of IFI6, IFI27, IFIT2, TNFRSF5, SOCS1, CXCL10, AIM2, SOCS1, CXCL9, IL15RA, LGALS9, CXCL11, GADD45B, TNFSF10, CXCL2, VEGF, MAP3KB, DUSP5, APOBEC3G.

[0009] There is also provided a method for identifying a subject having hepatitis as likely to respond to interferon therapy, comprising obtaining at least a first and a second sample of whole blood from the subject, exposing the first sample of whole blood from the individual to interferon in a solvent for an amount of time sufficient for the interferon to alter the expression of an interferon-responsive marker, wherein the interferon-responsive marker is one or more of IFIT2, IFI27, and IFI6, exposing the second sample of whole blood from the individual to the solvent without the interferon for the amount of time, quantifying the effect of the interferon as a change in expression of the interferon-responsive marker by measuring the amount of mPvNA encoding the interferon-responsive marker in both the first whole blood sample and the second whole blood sample, comparing the quantified effect of interferon on expression of the interferon-responsive marker with a normal effect of interferon on expression of the interferon responsive marker, wherein the normal effect is calculated as an average of change in expression levels of a panel of control individuals, evaluating the potential responsiveness of the subject by identifying significant differences between the change in expression of the interferon-responsive marker in the subject and the average change in expression of the interferon- responsive marker in the panel of control individuals and a) identifying the subject as potentially responsive to the interferon therapy when the identified significant differences are substantially greater in the subject as compared to the panel of control individuals, or b) identifying the subject as potentially non-responsive to the interferon therapy when the identified significant differences are substantially less in the subject as compared to the panel of control individuals.

[0010] Additionally provided is a method for identifying a patient having hepatitis as likely to respond to an immune based therapy, comprising, obtaining at least a first and a second sample of whole blood from the patient, exposing the first sample of whole blood from the patient to interferon in a solvent for an amount of time sufficient for the interferon to alter the expression one or more interferon- responsive markers, wherein the one or more interferon-responsive markers are selected from the group consisting of TNFRSF5, SOCS1, CXCL10, AIM2, SOCS1, CXCL9, IL15RA, LGALS9, CXCL11, GADD45B, TNFSF10, CXCL2, VEGF, MAP3KB, DUSP5, APOBEC3G, exposing the second sample of whole blood from the individual to the solvent without the interferon for the amount of time, quantifying the effect of the interferon as a change in expression of the one or more interferon- responsive markers by measuring the amount of mRNA encoding the one or more interferon-responsive markers in both the first whole blood sample and the second whole blood sample, wherein at least one of the one or more interferon-responsive markers are associated with reduced immune function, comparing the quantified effect of interferon on expression of the one or more interferon-responsive markers with a normal effect of interferon on expression of the one or more interferon- responsive markers, wherein the normal effect is calculated as an average of change in expression levels of a panel of control individuals, identifying significant differences between the change in expression of the one or more interferon-responsive markers in the subject and the average change in expression of the one or more interferon- responsive markers in the panel of control individuals, characterizing the patient as immuno-compromised if the change in expression in the interferon-responsive markers associated with reduced immune function is significantly different than the change in expression in the panel of control individuals; and identifying the patient as not-likely to respond to an immune-based therapy if the patient is characterized as immuno-compromised.

[0011] In several embodiments, the quantification in the methods disclosed herein is performed by a method selected from the group consisting of reverse-transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, northern blotting, fluorescence activated cell sorting, ELISA, and mass spectrometry.

[0012] In several embodiments, the two or more interferon-responsive markers are selected from the group consisting of IFI6, IFI27, and IFIT2. In several embodiments, the interferon is chosen from the group consisting of a type I interferon, a type II interferon, and a type III interferon, or agonists or analogs thereof. In several embodiments, the interferon is chosen from the group consisting of interferon alpha, interferon beta, interferon omega, interferon gamma, combinations thereof, and subtypes thereof. In one embodiment, the interferon is interferon alpha 2b. In several embodiments, the interferon alpha 2b is present in a concentration from about 1 to about 1 x 10 units per mL. In several embodiments, the interferon alpha 2b is lyophilized in a blood collection tube prior to the exposing. [0013] In several embodiments, the solvent is phosphate buffered saline. In alternative embodiments, depending on the stimulant, the solvent is another composition (e.g.,. DMSO).

[0014] In several embodiments, the exposing is for a time between about one hour and about seven hours. In some embodiments, the exposing is for about four hours.

[0015] In several embodiments, the exposing occurs at about thirty-seven (37) degrees Celsius.

[0016] In several embodiments, the whole blood is heparinized.

[0017] In several embodiments, the hepatitis is hepatitis C, while in other embodiments the hepatitis is hepatitis B

[0018] There are also provided herein, ex vivo methods for characterizing the potential responsiveness of a subject having hepatitis to interferon therapy comprising exposing a first sample of whole blood from the subject having hepatitis to interferon in a solvent for an amount of time sufficient for the interferon to alter the expression of two or more interferon-responsive markers, exposing a second sample of whole blood from the subject having hepatitis to the solvent without the interferon for the amount of time, quantifying the expression of the two or more interferon- responsive markers by measuring the amount of mRNA encoding the one or more interferon-responsive markers in both the first whole blood sample and the second whole blood sample evaluating the potential responsiveness of the subject to the interferon therapy by predetermining a parameter by multivariate discriminant analysis for each combination of the two or more interferon-responsive markers and the interferon using a plurality of patients in one or more of a plurality of groups with known clinical responsiveness to interferon therapy for hepatitis, calculating a predictor value for the subject for each of the plurality of groups; and comparing the predictor values with the predetermined parameters in order to categorize the subject within one of the plurality of groups of known clinical responsiveness; and then characterizing the subject as a potential responder to interferon therapy when the subject is categorized in any of the plurality of groups of known clinical responsiveness.

[0019] In several embodiments, the two or more interferon-responsive markers are selected from the group consisting of IFI6, IFI27, IFIT2, TNFRSF5, SOCS1, CXCL10, AIM2, SOCS1, CXCL9, IL15RA, LGALS9, CXCL11, GADD45B, TNFSF10, CXCL2, VEGF, MAP3KB, DUSP5, and APOBEC3G.

[0020] An ex vivo method for characterizing the potential responsiveness of a subject having hepatitis to interferon therapy comprising exposing a first sample of whole blood from the individual to interferon in a solvent for an amount of time sufficient for the interferon to alter the expression of an interferon-responsive marker, wherein the interferon-responsive marker is one or more of IFIT2, IFI27, and IFI6, exposing a second sample of whole blood from the individual to the solvent without the interferon for the amount of time, quantifying the effect of the interferon as a change in expression of the interferon-responsive marker by measuring the amount of mPvNA encoding the interferon-responsive marker in both the first whole blood sample and the second whole blood sample, comparing the quantified effect of interferon on expression of the interferon-responsive marker with a normal effect of interferon on expression of the interferon responsive marker, wherein the normal effect is calculated as an average of change in expression levels of a panel of control individuals, evaluating the potential responsiveness of the subject by identifying significant differences between the change in expression of the interferon-responsive marker in the subject and the average change in expression of the interferon- responsive marker in the panel of control individuals; and a) characterizing the subject as potentially responsive to the interferon therapy when the identified significant differences are substantially greater in the subject as compared to the panel of control individuals, or b) characterizing the subject as potentially non-responsive to the interferon therapy when the identified significant differences are substantially less in the subject as compared to the panel of control individuals.

[0021] There is also provided an ex-vivo method for characterizing the present immune status of a hepatitis patient, comprising exposing a first sample of whole blood from the patient to interferon in a solvent for an amount of time sufficient for the interferon to alter the expression one or more interferon-responsive markers, wherein the one or more interferon-responsive markers are selected from the group consisting of TNFRSF5, SOCS1, CXCL10, AIM2, SOCS1, CXCL9, IL15RA, LGALS9, CXCL11, GADD45B, TNFSF10, CXCL2, VEGF, MAP3KB, DUSP5, APOBEC3G, exposing a second sample of whole blood from the individual to the solvent without the interferon for the amount of time, quantifying the effect of the interferon as a change in expression of the one or more interferon-responsive markers by measuring the amount of mRNA encoding the one or more interferon-responsive markers in both the first whole blood sample and the second whole blood sample, wherein at least one of the one or more interferon-responsive markers are associated with reduced immune function, comparing the quantified effect of interferon on expression of the one or more interferon-responsive markers with a normal effect of interferon on expression of the one or more interferon-responsive markers, wherein the normal effect is calculated as an average of change in expression levels of a panel of control individuals, identifying significant differences between the change in expression of the one or more interferon-responsive markers in the subject and the average change in expression of the one or more interferon-responsive markers in the panel of control individuals, and characterizing the subject as immuno-compromised if the change in expression in the interferon-responsive markers associated with reduced immune function is significantly different than the change in expression in the panel of control individuals.

[0022] In several embodiments, the quantification is performed by a method selected from the group consisting of reverse-transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, northern blotting, fluorescence activated cell sorting, ELISA, and mass spectrometry. In one embodiment real-time RT-PCR is used.

[0023] There is also provided a method for advising a subject to undertake an interferon therapy regime for hepatitis comprising ordering a test of the subject's blood, the test comprising obtaining at least a first and a second sample of whole blood from the subject, exposing the first sample of whole blood from the subject having hepatitis to interferon in a solvent for an amount of time sufficient for the interferon to alter the expression of two or more interferon-responsive markers, exposing the second sample of whole blood from the subject having hepatitis to the solvent without the interferon for the amount of time, quantifying the expression of the two or more interferon-responsive markers by measuring the amount of mRNA encoding the one or more interferon-responsive markers in both the first whole blood sample and the second whole blood sample, evaluating the potential responsiveness of the subject to the interferon therapy by a) predetermining a parameter by multivariate discriminant analysis for each combination of the two or more interferon-responsive markers and the interferon using a plurality of patients in one or more of a plurality of groups with known clinical responsiveness to interferon therapy for hepatitis, b) calculating a predictor value for the subject for each of the plurality of groups; and c) comparing the predictor values with the predetermined parameters in order to categorize the subject within one of the plurality of groups of known clinical responsiveness; and characterizing the subject as a potential responder to interferon therapy when the subject is categorized in any of the plurality of groups of known clinical responsiveness; and advising the subject to undergo an interferon therapy regime for treating hepatitis when the subject is categorized in any of the clinical response groups.

[0024] In several embodiments, the two or more interferon-responsive markers are selected from the group consisting of IFI6, IFI27, IFIT2, TNFRSF5, SOCS1, CXCL10, AIM2, SOCS1, CXCL9, IL15RA, LGALS9, CXCL11, GADD45B, TNFSF10, CXCL2, VEGF, MAP3KB, DUSP5, and APOBEC3G.

[0025] Methods are also provided for advising a subject to undertake an interferon therapy regime for hepatitis comprising ordering a test of the subject's blood, the test comprising: obtaining at least a first and a second sample of whole blood from the subject, exposing the first sample of whole blood from the individual to interferon in a solvent for an amount of time sufficient for the interferon to alter the expression of an interferon-responsive marker, wherein the interferon-responsive marker is one or more of IFIT2, IFI27, and IFI6, exposing the second sample of whole blood from the individual to the solvent without the interferon for the amount of time, quantifying the effect of the interferon as a change in expression of the interferon- responsive marker by measuring the amount of mRNA encoding the interferon- responsive marker in both the first whole blood sample and the second whole blood sample, comparing the quantified effect of interferon on expression of the interferon- responsive marker with a normal effect of interferon on expression of the interferon responsive marker, wherein the normal effect is calculated as an average of change in expression levels of a panel of control individuals, evaluating the potential responsiveness of the subject by identifying significant differences between the change in expression of the interferon-responsive marker in the subject and the average change in expression of the interferon-responsive marker in the panel of control individuals and characterizing the subject as potentially responsive to the interferon therapy when the identified significant differences are substantially greater in the subject as compared to the panel of control individuals, or characterizing the subject as potentially non-responsive to the interferon therapy when the identified significant differences are substantially less in the subject as compared to the panel of control individuals and advising the subject to undertake an interferon therapy regime for hepatitis when the subject is characterized as potentially responsive.

[0026] There is also provided a method for advising a subject to undergo interferon therapy for hepatitis based on the present immune status of the subject, the method comprising ordering a test of the subject's blood, the test comprising, obtaining at least a first and a second sample of whole blood from the subject, exposing the first sample of whole blood from the patient to interferon in a solvent for an amount of time sufficient for the interferon to alter the expression one or more interferon-responsive markers, wherein the one or more interferon-responsive markers are selected from the group consisting of TNFRSF5, SOCS1, CXCL10, AIM2, SOCS1, CXCL9, IL15RA, LGALS9, CXCL11, GADD45B, TNFSF10, CXCL2, VEGF, MAP3KB, DUSP5, and APOBEC3G, exposing the second sample of whole blood from the individual to the solvent without the interferon for the amount of time, quantifying the effect of the interferon as a change in expression of the one or more interferon-responsive markers by measuring the amount of mRNA encoding the one or more interferon-responsive markers in both the first whole blood sample and the second whole blood sample, wherein at least one of the one or more interferon- responsive markers are associated with reduced immune function, comparing the quantified effect of interferon on expression of the one or more interferon-responsive markers with a normal effect of interferon on expression of the one or more interferon-responsive markers, wherein the normal effect is calculated as an average of change in expression levels of a panel of control individuals, identifying significant differences between the change in expression of the one or more interferon-responsive markers in the subject and the average change in expression of the one or more interferon-responsive markers in the panel of control individuals, characterizing the subject as immuno-compromised if the change in expression in the interferon- responsive markers associated with reduced immune function is significantly different than the change in expression in the panel of control individuals, and advising the subject to undergo interferon therapy for hepatitis only if the subject is not characterized as immuno-compromised.

[0027] In several embodiments, the quantification is performed by a method selected from the group consisting of reverse-transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, northern blotting, fluorescence activated cell sorting, ELISA, and mass spectrometry.

[0028] There are also provided methods of treating a subject having hepatitis based on the subject's diagnostic responsiveness to interferon therapy, comprising ordering a test of the subject's blood, the test comprising obtaining at least a first and a second sample of whole blood from the subject, exposing a first sample of whole blood from the subject having hepatitis to interferon in a solvent for an amount of time sufficient for the interferon to alter the expression of two or more interferon-responsive markers, wherein the two or more interferon-responsive markers are selected from the group consisting of IFI6, IFI27, IFIT2, TNFRSF5, SOCS1, CXCL10, AIM2, SOCS1, CXCL9, IL15RA, LGALS9, CXCL11, GADD45B, TNFSF10, CXCL2, VEGF, MAP3KB, DUSP5, and APOBEC3G, exposing a second sample of whole blood from the subject having hepatitis to the solvent without the interferon for the amount of time, quantifying the expression of the two or more interferon-responsive markers by measuring the amount of mRNA encoding the one or more interferon-responsive markers in both the first whole blood sample and the second whole blood sample by a method selected from the group consisting of reverse-transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, northern blotting, fluorescence activated cell sorting, ELISA, and mass spectrometry; evaluating the potential responsiveness of the subject to the interferon therapy by: a) predetermining a parameter by multivariate discriminant analysis for each combination of the two or more interferon-responsive markers and the interferon using a plurality of patients in one or more of a plurality of groups with known clinical responsiveness to interferon therapy for hepatitis, b) calculating a predictor value for the subject for each of the plurality of groups; and c) comparing the predictor values with the predetermined parameters in order to categorize the subject within one of the plurality of groups of known clinical responsiveness; and characterizing the subject as a potential responder to interferon therapy when the subject is categorized in any of the plurality of groups of known clinical responsiveness; and administering to the subject an interferon therapy when the subject is categorized in any of the plurality of groups of known clinical responsiveness.

[0029] Also provided are methods for treating a subject having hepatitis based on the subject's diagnostic responsiveness to interferon therapy, comprising ordering a test of the subject's blood, the test comprising obtaining at least a first and a second sample of whole blood from the subject, exposing the first sample of whole blood from the individual to interferon in a solvent for an amount of time sufficient for the interferon to alter the expression of an interferon-responsive marker, exposing the second sample of whole blood from the individual to the solvent without the interferon for the amount of time, quantifying the effect of the interferon as a change in expression of the interferon-responsive marker by measuring the amount of mRNA encoding the interferon-responsive marker in both the first whole blood sample and the second whole blood sample, comparing the quantified effect of interferon on expression of the interferon-responsive marker with a normal effect of interferon on expression of the interferon responsive marker, wherein the normal effect is calculated as an average of change in expression levels of a panel of control individuals, evaluating the potential responsiveness of the subject by identifying significant differences between the change in expression of the interferon-responsive marker in the subject and the average change in expression of the interferon- responsive marker in the panel of control individuals; and a) characterizing the subject as potentially responsive to the interferon therapy when the identified significant differences are substantially greater in the subject as compared to the panel of control individuals, or b) characterizing the subject as potentially non-responsive to the interferon therapy when the identified significant differences are substantially less in the subject as compared to the panel of control individuals; and administering to the subject an interferon therapy when the subject is characterized as potentially responsive to the interferon therapy.

[0030] In several embodiments, the interferon-responsive marker is one or more of IFIT2, IFI27, and IFI6. In several embodiments, the quantification is by a method selected from the group consisting of reverse-transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, northern blotting, fluorescence activated cell sorting, ELISA, and mass spectrometry.

[0031] Moreover, there is provided a method for treating a subject having hepatitis based on the subject's present immune status, comprising ordering a test of the subject's blood, the test comprising obtaining at least a first and a second sample of whole blood from the subject, exposing the first sample of whole blood from the patient to interferon in a solvent for an amount of time sufficient for the interferon to alter the expression one or more interferon-responsive markers, exposing the second sample of whole blood from the individual to the solvent without the interferon for the amount of time, quantifying the effect of the interferon as a change in expression of the one or more interferon-responsive markers by measuring the amount of mRNA encoding the one or more interferon-responsive markers in both the first whole blood sample and the second whole blood sample wherein at least one of the one or more interferon-responsive markers are associated with reduced immune function, comparing the quantified effect of interferon on expression of the one or more interferon-responsive markers with a normal effect of interferon on expression of the one or more interferon-responsive markers, wherein the normal effect is calculated as an average of change in expression levels of a panel of control individuals, identifying significant differences between the change in expression of the one or more interferon-responsive markers in the subject and the average change in expression of the one or more interferon-responsive markers in the panel of control individuals, characterizing the subject as immuno-compromised if the change in expression in the interferon-responsive markers associated with reduced immune function is significantly different than the change in expression in the panel of control individuals; and administering to the subject an interferon therapy for hepatitis only if the subject is not characterized as immuno-compromised.

[0032] In several embodiments, there is provided an ex vivo method for characterizing the potential responsiveness of a subject having interferon, the method comprising exposing a first sample of blood from said subject interferon in a solvent for an amount of time sufficient for said interferon to alter the expression of two or more interferon-responsive markers, exposing a second sample of blood from said subject to the solvent without the interferon for said amount of time, quantifying the expression of said two or more interferon-responsive markers by measuring the amount of mRNA encoding said one or more interferon-responsive markers in both said first blood sample and said second blood sample; and characterizing the potential responsiveness of said subject to said interferon, wherein said characterization comprises: predetermining a parameter by multivariate discriminant analysis for each combination of said two or more interferon-responsive markers and said interferon using a plurality of patients in one or more of a plurality of groups with known clinical responsiveness to interferon, calculating a predictor value for each patient for each of said plurality of groups; and comparing said predictor values with said predetermined parameters in order to categorize said subject within one of said plurality of groups of known clinical responsiveness, wherein categorization of said subject in a group having patients that responded to interferon results in said subject being characterized as a responder to interferon.

[0033] In several embodiments, said two or more interferon-responsive markers are two or more of IFI6, IFI27, IFIT2, TNFRSF5, SOCS1, CXCL10, AIM2, SOCS1, CXCL9, IL15RA, LGALS9, CXCL11, GADD45B, TNFSF10, CXCL2, VEGF, MAP3KB, DUSP5, APOBEC3G, and combinations thereof.

[0034] In other embodiments, said two or more interferon-responsive markers are two or more of IFI6, IFI27, and IFIT2.

[0035] In several embodiments, wherein said interferon is a type I interferon, a type II interferon, or a type III interferon. In some embodiments, combinations of interferons are used. In some embodiments, said interferon is an interferon alpha, interferon beta, interferon omega, interferon gamma, combinations thereof, or subtypes thereof. In some embodiments, said interferon is interferon alpha 2b. In several embodiments, said interferon alpha 2b is present in a concentration from about 1 to about 100,000 units per mL. In several embodiments, said interferon alpha 2b is present in a concentration of about 5,000 units per mL to about 15,000 units per mL.

[0036] In some embodiments, said solvent is phosphate buffered saline.

[0037] In some embodiments, said exposing is for a time between one hour and seven hours. In one embodiment, said exposing is for four hours. In one embodiment, said exposing occurs at about thirty-seven (37) degrees Celsius.

[0038] In several embodiments, the blood is whole blood and in some embodiments, said whole blood is heparinized.

[0039] In several embodiments, the subject has hepatitis. Thus, in several such embodiments, the interferon is administered as therapy to treat hepatitis. In several embodiments, said hepatitis is hepatitis C. In several embodiments, said hepatitis is hepatitis B.

[0040] There is also provided herein an ex vivo method for characterizing the potential responsiveness of a subject having hepatitis to interferon therapy comprising exposing a first sample of whole blood from the individual to interferon in a solvent for an amount of time sufficient for said interferon to alter the expression of an interferon-responsive marker; exposing a second sample of whole blood from the individual to the solvent without the interferon for said amount of time; quantifying the effect of said interferon as a change in expression of said interferon-responsive marker by measuring the amount of mRNA encoding said interferon-responsive marker in both said first whole blood sample and said second whole blood sample; comparing the quantified effect of interferon on expression of said interferon- responsive marker with a normal effect of interferon on expression of said interferon responsive marker, wherein said normal effect is calculated as an average of change in expression levels of a panel of control individuals; characterizing the potential responsiveness of said subject by identifying significant differences between the change in expression of said interferon-responsive marker in said subject and the average change in expression of said interferon-responsive marker in said panel of control individuals, wherein said subject is potentially responsive to said interferon therapy if the change in expression in said subject is substantially greater than the change in expression in said panel of control individuals, and wherein said subject is potentially non-responsive to said interferon therapy if the change in expression in said subject is substantially less than the change in expression of said panel of control individuals.

[0041] In several embodiments, said interferon-responsive marker is IFIT2. In several embodiments, said interferon-responsive marker is IFI27. In several embodiments, said interferon-responsive marker is IFI6.

[0042] Additionally, there is provided a method for characterizing the present immune status of a hepatitis patient, comprising exposing a first sample of whole blood from the patient to interferon in a solvent for an amount of time sufficient for said interferon to alter the expression one or more interferon-responsive markers; exposing a second sample of whole blood from the individual to the solvent without the interferon for said amount of time; quantifying the effect of said interferon as a change in expression of said one or more interferon-responsive markers by measuring the amount of mRNA encoding said one or more interferon- responsive markers in both said first whole blood sample and said second whole blood sample, wherein at least one of said one or more interferon-responsive markers are associated with reduced immune function; comparing the quantified effect of interferon on expression of said one or more interferon-responsive markers with a normal effect of interferon on expression of said one or more interferon-responsive markers, wherein said normal effect is calculated as an average of change in expression levels of a panel of control individuals; characterizing the present immune status of said hepatitis patient by identifying significant differences between the change in expression of said one or more interferon-responsive markers in said subject and the average change in expression of said one or more interferon- responsive markers in said panel of control individuals, wherein said subject is presently immuno-compromised if said change in expression in said interferon- responsive markers associated with reduced immune function is significantly different than the change in expression in said panel of control individuals.

[0043] In several embodiments, said one or more interferon-responsive markers are selected from the group consisting of TNFRSF5, SOCSl, CXCL10, AIM2, SOCSl, CXCL9, IL15RA, LGALS9, CXCLl l, GADD45B, TNFSFIO, CXCL2, VEGF, MAP3KB, DUSP5, and APOBEC3G.

BRIEF DESCRIPTION OF THE FIGURES

[0044] Figure 1 depicts the lack of induction of a housekeeping gene by interferon.

[0045] Figure 2 depicts the induction of TNFRSF in all disease categories.

[0046] Figure 3 depicts the induction of SOCSl in all disease categories

[0047] Figure 4 depicts the varied induction of CXCL10 across the disease categories.

[0048] Figure 5 depicts the varied induction of IFI6 across the disease categories and in particular between relapse and non-responders as compared to responders.

[0049] Figure 6 depicts the varied induction of IFI27 across the disease categories.

[0050] Figure 7 depicts preliminary data related to IFIT2 induction across disease categories.

[0051] Figures 8A-8V depicts compiled data summaries of mRNAs analyzed.

DETAILED DESCRIPTION

[0052] In several embodiments described herein, ex vivo methods are provided for predicting the responsiveness of a subject to interferon (IFN) therapy. IFN is used clinically for cancer, hepatitis, multiple sclerosis, etc. However, it is not effective for every patient, and responsive subjects are not readily identifiable by present methods. Currently, genotyping of IL28 is known to be associated with the clinical outcome of IFN therapy for hepatitis. However, this association is not 100% accurate. Moreover, once IFN therapy is started, it must continue for several months until responsiveness to the therapy can be characterized. In some patient populations, even a short delay in receiving a successful therapy can be the difference between life and death. Thus, methods are provided herein by which the efficacy of IFN therapy can be predicted for each patient. Thus, IFN therapy can effectively be screened and administered only to those who are likely to respond. Not only does this improve the likelihood of an improved patient outcome (reduced "false effective" therapies), but it also decreases the healthcare cost by eliminating the misuse of expensive IFN therapy to non-responder patients as well as the troubleshooting to the adverse reactions associated with IFN.

[0053] In several embodiments, the methods involve collection of peripheral whole blood from an individual that is potentially responsive to IFN (e.g., for hepatitis therapy) and stimulation of the whole blood with IFN to screen for changes in expression of one or more IFN-responsive markers. In several embodiments, detection of changes in the mRNA encoding one or more of the IFN- responsive markers is related to the individual's potential responsiveness to IFN therapy for hepatitis. In other embodiments, methods are provided for the monitoring of the ongoing responsiveness of an individual IFN therapy for hepatitis. In some embodiments, peripheral whole blood is collected from an individual before and after administration of IFN, and evaluated for changes in expression of a panel of IFN- responsive markers. The changes in expression are then used to evaluate the individual's ongoing responsiveness to, and the efficacy of, IFN therapy for hepatitis. In several embodiments, the methods described herein are used to predict the responsiveness to, or monitor the efficacy of, IFN administration in order to treat hepatitis infection (or a related or unrelated malignancy).

[0054] HBV is known to have infected approximately one-third of the world's population, more than 2 billion people. Of these infected individuals, over 350 million are chronic carriers of the virus. Acute hepatitis B can cause various symptoms, including liver inflammation, vomiting, jaundice, but rarely death. Chronic hepatitis B, however, may eventually lead to liver scarring (fibrosis and/or cirrhosis) and liver cancer, a cancer known to have poor responsiveness to chemotherapy regimes.

[0055] HBV can be classified into one of four major serotypes (adr, adw, ayr, ayw) based on the antigenic epitopes present on the viral proteins present on the exterior surface of the viral particle. Additionally, eight genotypes of HBV exist, according to overall nucleotide sequence variation of the HBV genome. Each genotype has a distinct geographical distribution and is often used for researching and cataloging the evolution and transmission of the virus. Clinically, however, variations between genotypes may greatly affect the severity of the infection, the overall response to treatment, recurrence of infection and risk of long term complications.

[0056] While acute hepatitis C infections are often asymptomatic, HCV has a high propensity to establish chronic infection and result in liver disease, which may include advanced scarring (cirrhosis). In some cases, those with cirrhosis will go on to develop liver failure or other complications of cirrhosis, including liver cancer. HCV has a rapid turnover rate within a host, and is also known to have error prone replication, which may play a role in allowing the virus to evade the host immune response for long periods of time.

[0057] Similarly to HBV, HCV exists in numerous different genotypes, with a total number ranging from six to eleven, depending on the classification methods used. Responsiveness of treatment varies across the genotypes, and can also affect the duration of treatment necessary to elicit a positive response against the infection.

[0058] IFN therapy, in various forms or combinations (including IFNa- 2b), plays a critical role in the treatment of patients with hepatitis infections. It is often used in combination with various other therapeutic agents, but in patients who are unable to receive IFN, successful treatment is far more difficult. In some embodiments, the IFN administered to an individual is classified as a type I interferon, a type II interferon, or a type III interferon. In some embodiments, the interferon administered is chosen from the group consisting of IFN-alpha, IFN-beta, IFN-omega, IFN-gamma, combinations thereof, and subtypes thereof. In certain embodiments, the IFN administered is either IFN-alpha or IFN-gamma. In some such embodiments, the IFN-alpha is IFN-alpha-2b.

[0059] Despite the beneficial therapeutic effects, side effects of IFN treatment may be severe. Symptoms can include decreased platelet count (which may lead to bleeding/bruising problems), depression, fatigue, increased risk of infection, post-administration flu-like symptoms (fever, chills, headache, muscle aches and pains, diarrhea), and possible tissue damage at sites of administration. Other documented side-effects include anorexia, congestion, increased heart rate, confusion, low white blood cell count, low red blood cell count, an increase in liver enzymes and/or triglycerides, skin rashes, mild hair loss, local and/or systemic swelling (edema), cough or difficulty breathing. Such side effects would be potentially harmful to a healthy individual, and could be devastating to an individual already suffering from an infection, a malignancy, or from side effects brought on by other therapeutic agents.

[0060] Thus, there exists a need for predicting if an individual having hepatitis will benefit from IFN therapy, as administration of IFN without positive effect may simply elicit unwanted and unnecessary side effects in non-responsive patients. Additionally, given the large number of people who have hepatitis or a form of cancer, a means to identify IFN responsive individuals would assist in providing tailored care to those who would benefit, and allowing non-responsive individuals to seek alternative therapeutic regimes that would be beneficial. Moreover, identification of markers that allow for categorization of responsive and non- responsive individuals would enable care-givers to monitor the efficacy of therapy in an on-going fashion, and may assist in the identification of individuals who develop a refractory response to IFN therapy.

[0061] In several embodiments, IFN stimulation of whole blood obtained from an individual (or IFN administration to the individual) alters the expression of one or more markers that are associated with one or more disease conditions. In certain embodiments, the expression of one or more of the markers is induced, while in other embodiments, the expression of one or more of the markers is down- regulated. In certain embodiments, the induction or down-regulation of the expression of one or more of the markers is statistically significant, as measured with standard statistical analyses with p < 0.05 representing a statistically significant change. In several embodiments, a significant increase in the expression of one or more IFN-responsive markers is an indication that an individual will be responsive to IFN administration. In several embodiments, a significant decrease in the expression of one or more IFN-responsive markers is an indication that an individual may fail to respond to IFN administration. [0062] In some embodiments, the makers responsive to IFN therapy include one or more of interferon alpha- inducible protein 6 (IFI6), IFI27, and interferon-induced protein with tetratricopeptide repeats 2 (IFIT2). In some embodiments, the makers responsive to IFN therapy include one or more of LGALS9, IFI6, IFIT2, CXCL2, IL15A, MAP3KB, IFI27, TLR3, CXCL9, APEBEC3G, GADD45B, OAS2, VEGF, CXCL11, DUSP5, and AIM2.

[0063] In several embodiments, blood is collected from mammals, preferably humans. In some embodiments, the blood collected is whole blood. Multiple experimental protocols directed to determining expression screening of markers responsive to a drug have been done in isolated leukocyte preparations. Such isolated populations are often preferred because the variety of lymphocytes in whole blood may preclude detection of induction of a specific mRNA in a small subset of lymphocytes. Moreover, with numerous complex biochemical interactions between the multiple types of lymphocytes, there is the possibility that use of a whole blood preparation inhibits or modifies the induction and measurement reactions. Furthermore, certain stimulatory agents (e.g., IFN) which are used in several embodiments, may interact with plasma proteins or plasma factors, and thereby exhibit decreased or reduced activity. However, when used as presented in several embodiments as described herein, whole blood unexpectedly produces reproducible, accurate, and physiologically relevant results that allow the characterization of the future or ongoing responsiveness of an individual to IFN. However, while preferred embodiments employ whole blood, in other embodiments, blood cells separated from plasma may also be used, as well as isolated leukocyte preparations.

[0064] In several preferred embodiments, the collected whole blood is heparinized upon collection. In several embodiments, the collected whole blood is stored at 4° C until the stimulation protocol. In several embodiments, the whole blood is collected into a tube containing IFN (or the solvent therefore) and thus, is stimulated upon collection. Such samples are thereafter stored until analysis.

[0065] In brief, certain embodiments of the device comprise a multi-well plate that contains a plurality of sample-delivery wells, a leukocyte-capturing filter underneath the wells, and an mRNA capture zone underneath the filter which contains immobilized oligo(dT). In certain embodiments, the device also contains a vacuum box adapted to receive the filter plate to create a seal between the plate and the box, such that when vacuum pressure is applied, the blood is drawn from the sample- delivery wells across the leukocyte-capturing filter, thereby capturing the leukocytes and allowing non-leukocyte blood components to be removed by washing the filters. In other embodiments, other means of drawing the blood samples through out of the sample wells and through the across the leukocyte-capturing filter, such as centrifugation or positive pressure, are used. In preferred embodiments of the device, leukocytes are captured on a plurality of filter membranes that are layered together. In several embodiments, the captured leukocytes are then lysed with a lysis buffer, thereby releasing mRNA from the captured leukocytes. The mRNA is then hybridized to the oligo(dT)-immobilized in the mRNA capture zone. Further detail regarding the composition of lysis buffers that may be used in several embodiments can be found in United States Patent Application No.:l l/376,018, filed March 15, 2006, which is currently pending and which is incorporated in its entirety by reference herein. In several embodiments, cDNA is synthesized from oligo(dT)-immobilized mRNA. In preferred embodiments, the cDNA is then amplified using real time PCR with primers specifically designed for amplification of infection-associated markers. Primers that are used in such embodiments are shown in Table 1. Further details about the PCR reactions used in some embodiments are also found in United States Patent Application No.: 11/376,018.

[0066] In several embodiments, the blood sample is combined with IFN and/or a control agent. In several embodiments, the control agent induces little or no response in the blood samples. In certain embodiments, the control agent is the same solvent used to carry the IFN. As discussed above, in some embodiments, various IFN are used, IFN-alpha, IFN-beta, IFN-omega, IFN-gamma, combinations thereof, and subtypes thereof. In certain embodiments, the control agent is phosphate- buffered saline (PBS). In other embodiments, other inert control agents may be used such as control IgG (serving as a control for those stimulating agents that are antibodies) or DMSO. In still additional embodiments, no particular control agent is used (e.g., IFN-stimulated samples are compared to those having received no stimulation).

[0067] In several embodiments, a small volume of the previously stimulated blood from each sample is processed to allow determination of the levels of mRNA encoding one or more IFN markers in the blood. In some embodiments, the levels of mRNA encoding one or more IFNoc responsive markers will change significantly in response to the IFN incubation. To determine these mRNA levels, the erythrocytes and blood components other than leukocytes are removed from the whole blood sample. In preferred embodiments, the leukocytes are isolated using a device for isolating and amplifying mRNA. Embodiments of this device are described in more detail in United States Patent Application Nos.: 10/796,298, 11/525,515, 11/376,018, 11/803,593, 11/803,594, and 11/803,663, each of which is incorporated in its entirety by reference herein.

[0068] In several embodiments, on the same day of blood draw, a small volume of whole blood is combined with IFN (or control agent) and incubated at 37°C for a period of time. In some embodiments, the incubation period is about 4 hours. In some embodiments, incubation is for greater than 4 hours, while in other embodiments, incubation is for less than 4 hours. After incubation, all blood samples are stored frozen at -80°C until analysis.

Table 1 : Primer Sequences for RT-PCR Amplification

CD25 CAGAAGTCATGAAGCCCAAGTG 71 GGCAAGCACAACGGATGTCT 72

CTLA4 CATGCCTCCTCTTCTTCCTTGA 73 GGAGGGTGCCACCATGACTA 74

Granzyme B GCGGTGGCTTCCTGATACAA 75 CCAAGGTGACATTTATGGAGCTT 76

CD8A CCGAGAGAACGAGGGCTACTATT 77 GCACGAAGTGGCTGAAGTACAT 78

CD16 GTTTGGCAGTGTCAACCATCTC 79 AAAAGGAGTACCATCACCAAGCA 80

CD32A GCTGACGGCGGCTACATG 81 GAGGAAGAGTCAGGTAGATGTTTTTATCA 82

CD64 CTGGCAGTGGGAATAATGTTTTT 83 CACTTTTTCTTTCTTTTCAGTTCTTTGCG 84

IgG Fc CAGCCGGAGAACAACTACAAGAC 85 GCTGCCACCTGCTCTTGTC 86

ARG AGACACCAGAAGAAGTAACTCGAACA 87 TCCCGAGCAAGTCCGAAAC 88

MPO ACTGCCTGGGTTCCAATCC 89 TGTTTAAGGAGGGTAATTTGCTCAA 90

TLR2 GAAGAGTGAGTGGTGCAAGTATGAA 91 ATGGCAGCATCATTGTTCTCATC 92

TLR4 GATTGCTCAGACCTGGCAGTT 93 TGTCCTCCCACTCCAGGTAAGT 94

GM-CSF GGCCCCTTGACCATGATG 95 TCTGGGTTGCACAGGAAGTTT 96

IFNy GGAGACCATCAAGGAAGACATGA 97 GCTTTGCGTTGGACATTCAA 98

TGF CTGCTGAGGCTCAAGTTAAAAGTG 99 TGAGGTATCGCCAGGAATTG T 100

CD4 AAATGCCACACGGCTCTCA 101 GGGTGCTGTGCTTCTGTGAAC 102

STAT 1 GTGGAAAGACAGCCCTGCAT 103 ACTGGACCCCTGTCTTCAAGAC 104

STAT 3 GCCAGAGAGCCAGGAGCAT 105 GGTGTCACACAGATAAACTTGGTCTT 106

STAT 4 CATTTGGTACAACGTGTCAACCA 107 TGTGGCAGGTGGAGGATTATTA 108

VEGF CGCAGCTACTGCCATCCAAT 109 TGGCTTGAAGATGTACTCGATCTC 110

POMC ACGAGGGCCCCTACAGGAT i l l TGATGATGGCGTTTTTGAACA 112

GBP AGAAGTGAAGGCGGGAATTTATT 113 ATCCCCTTCCTCGGTTCCT 114

XAF1 CCTAGAGGAGATAAAGCAGCCTATGA 115 AAGCTAACCACCGGCATTTCT 116

AIM2 GGTGAAACCCCGAAGATCAA 117 CTGGACTACAAACAAACCATTCACA 118

SHP2 TCCAGATGGTGCGGTCTCA 119 CCTGCGCTGTAGTGTTTCAATATAA 120

SOCS 1 GGAACTGCTTTTTCGCCCTTAGC 121 CTGAAAGTGCACGCGGATGCT 122

SLP76 CCGTTATCAGAAGGAAAGTCAAGTT 123 ATATCTGACACAGACAGAAAGTCCTCTT 124

G1P2 CAAATGCGACGAACCTCTGA 125 CCGCTCACTTGCTGCTTCA 126

BST2 GAGATCACTACATTAAACCATAAGCTTCAG 127 TCTCACGCTTAAGACCTGGTTTT 128

IRF7 TCCCCACGCTATACCATCTACCT 129 ACAGCCAGGGTTCCAGCTT 130

LCK TTAAGTGGACAGCGCCAGAA 131 CCCAAAAGACCACACATCTGACT 132

IFIT2 TAAGAAAAAGTGCTCGGAGAGCTC 133 CCGACGGCCATGAAGGT 134

IFI6 TTCAGTTGGAACTGGAAGGG 135 ACCTGCACCCTTACTTGCAT 136

IFI27 ACATGAAAGAGATGCCAGGG 137 CAGATGTTGCCCAGGAGATT 138

Alternative primers derived from publicly available sequences for the genes above may also be used

[0069] In several embodiments, the methods described herein are used to monitor an individual's responsiveness to ongoing IFN administration, e.g., for the treatment of hepatitis. In some such embodiments, a first blood sample is obtained from the individual. In some embodiments, the first blood sample is obtained prior to the administration of any IFN to the individual. In other embodiments, the individual has received IFN in the past, and will again in the future. In some embodiments, the first blood sample is obtained at a time between two administrations of IFNoc-2b, preferably just prior to an administration. A second blood sample is obtained from the individual at a time after the administration of IFN. In certain embodiments, this time is several hours, though in other embodiments, the time is several weeks, and in some embodiments up to several months. In other embodiments, additional samples are taken serially over the course of several months. The blood samples obtained from the individual are then frozen until expression analysis, which is performed as described above. Evaluation of expression levels of IFN responsive markers can thus be used to monitor the progress (i.e., efficacy) of IFN administration. In some embodiments, a significant difference in expression of one or more IFN responsive markers between the post-IFN administration blood sample and the pre-IFN administration blood sample indicates that therapy is effective. In other embodiments, a lack of any significant difference in expression of one or more IFN responsive markers between the post-IFN and pre-IFN administration blood samples indicates that therapy is not effective. In still other embodiments, this protocol is adapted to monitor the efficacy of other putative therapeutic agents.

[0070] After the completion of PCR reaction, the mRNA (as represented by the amount of PCR-amplified cDNA detected) for one or more IFN markers is quantified. In certain embodiments, quantification is calculated by comparing the amount of mRNA encoding one or more IFN markers to a reference value. In other embodiments, the reference value is expression level of a gene that is not induced by IFN, e.g., a house-keeping gene, such as beta-2 microglobulin. In other embodiments, a house keeping gene is used as a correction factor, such that the ultimate comparison is the induced expression level of one or more IFN markers as compared to the same marker from a non-induced (control) sample. In still other embodiments, the reference value is zero, such that the quantification of one or more IFN markers is represented by an absolute number. In several embodiments a ratio comparing the expression of one or more IFN-responsive to one or more IFN-non-responsive markers is made. In still other embodiments, no normalization is made.

[0071] In several embodiments, a multivariate analysis is used to characterize (e.g., predict) an individual's potential responsiveness to IFN therapy (e.g., for hepatitis). Thus, in several embodiments disease categorizations are used in the multivariate analysis to allow grouping of an individual into a certain predictive group. In some embodiments, the groups may be effectively binary, e.g., responder versus non-responder. However, in several embodiments, the groups correlate with degrees of responsiveness to IFN therapy (e.g., partial responder, responder with relapse, etc.).

[0072] After stimulation of blood samples and quantification (e.g., by calculating fold change) of each gene of interest after exposure to each stimulus of interest, the results are categorized in order to characterize (e.g., predict) each individual's responsiveness to IFN therapy for hepatitis.

[0073] The quantified data (e.g., fold change of each individual gene due to each individual stimulus) may optionally be converted, for example by taking the log(10) of the fold change, in order to normalize the distribution of the data prior to further analysis.

[0074] The quantified data is then processed by discriminant analysis, which allows prediction of an individual's group membership (e.g., responsiveness) based on the combination of variables (change in the amount of certain IFN- associated mRNA and stimulants). As discussed in Example 1, because clinical progression was known for each individual, the methods disclosed herein allow for the prediction of group membership (e.g., responsiveness) when only the mRNA quantification data is known. Standard, art accepted methods for discriminant analysis were used to generate parameters for each group (e.g., responders without relapse (S), responders with relapse(R), and non-responders (NR)) and each combination of mRNA of interest and stimulant agent. A prediction of group membership can be thereafter be made utilizing these parameters and the fold change data for each gene of interest and each stimulant. A non-limiting example of this calculation is provided below:

Predictor Value = Parameter Constant for Group 1 + [Parameter value(G_ene x and stimulant A)] + [Parameter value(_Gene x and stimulant B)] + [Parameter value(Gene Y and stimulant A)]+ [Parameter value(Gene Y and stimulant B)] + [ . · . ]

[0075] The pattern in the example formula above is thus repeated until each combination of gene of interest with each stimulant agent is accounted for, and repeated for each of the groups. Thereafter, the calculated Predictor Value is then used to make the prediction of group membership for that individual. In this example (and Example 1) the largest Predictor Value of each of the three groups is used to predict membership of the individual within that group. For example if the Predictor Value is 13 for S, 10 for R, and 5 for N, that individual would be categorized as a member of the S group, and thereby be characterized as likely to be responsive to IFN therapy for hepatitis.

[0076] In several embodiments, the methods described herein are used to monitor an individual's responsiveness to ongoing hepatitis therapy using IFN. In some such embodiments, a first blood sample is obtained from the individual. In some embodiments, the first blood sample is obtained prior to the administration of any IFN treatment to the individual. In other embodiments, the individual has received a treatment in the past, and will again in the future. In some embodiments, the first blood sample is obtained at a time between two administrations of IFN therapy, preferably just prior to an administration. A second blood sample is obtained from the individual at a time after the administration of IFN therapy. In certain embodiments, this time is several hours, though in other embodiments, the time is several weeks, and in some embodiments up to several months. In other embodiments, additional samples are taken serially over the course of several months. The blood samples obtained from the individual are then frozen until expression analysis, which is performed as described above. Evaluation of expression levels of IFN-induced markers can thus be used to monitor the progress (i.e., efficacy) of IFN therapy, as discussed above. In some embodiments, maintenance of the disease status categorization of a patient over time (e.g., no relapse) indicates that the patient is responsive to therapy. Changes in category (e.g., no relapse to relapse) indicate that the patient's responsiveness to the therapy is diminished.

[0077] In several embodiments of the methods disclosed herein, artifacts introduced by the analysis itself are eliminated as much as possible, which aids in a more accurate characterization of the complex immune function in each individual. The ex vivo assay systems and methods disclosed herein minimize technical variation associated with blood manipulation, mRNA purification, cDNA synthesis, and PCR.

EXAMPLES

[0078] Specific embodiments will be described with reference to the following examples which should be regarded in an illustrative rather than a restrictive sense.

Example 1- Ex vivo screening of IFN responsive Markers Equipment Preparation and Experimental Subject

[0079] Ten of 5xl0⁷ units/mL interferon a2b (IFN) was added into 4 mL heparin blood collection tubes, and lyophilized. Each tube was then re-capped and made to have a negative vacuum pressure by aspirating air from the capped tube. Control tubes were also prepared in the same way, but without any IFN. Blood was collected from eight (8) healthy donors (HC) and thirty-six 36 Hepatitis C patients (HCV) who had received IFN therapy in the past. Among the 36 HCV patients, fourteen (14) patients were classified as IFN therapy responders without relapse (S), nine (9) patients were responders but had a relapse (R), and 13 patients were non- responders (N). These categorizations were based on 6 months follow-up of the titer of HCV using a standard viral load test.

Blood collection and Induction

[0080] Blood from the subjects was drawn directly into control or IFN tubes. Blood volume was approximately 1.5 mL/tube. After the blood draw, each tube was immediately mixed, and incubated at 37 °C for 4 hours. After incubation, blood collection tubes were stored frozen at -80°C freezer until analysis.

mRNA Analysis

[0081] For analysis, control and IFN treated samples were thawed on ice. 96-well filterplates were assembled with leukocyte reduction membranes (Leukosorb; Pall) and placed over oligo(dT)-immobilized collection plates. 150 μΐ_^ of 5 mmol/L Tris (pH 7.4) was applied to wet the filter membranes. After centrifugation at 120g for 1 min at 4 °C to remove the Tris solution from the membranes, 50 μΐ_^ of the stimulated whole blood samples was applied to each well and immediately centrifuged at 120g for 2 min at 4 °C. The wells were then washed once with 300 μΐ_^ of phosphate-buffered saline. After centrifugation at 2000g for 5 min at 4 °C to remove the saline solution, 60 μΐ_^ of stock lysis buffer [5 g/L N-lauroylsarcosine, 4x standard saline citrate, 10 mmol/L Tris-HCl (pH 7.4), 1 mmol/L EDTA, 1 mL/L IGEPAL CA- 630 (substitute of NP-40), 1.79 mol/L guanidine thiocyanate (all from Sigma)], supplemented with 10 mL/L 2-mercaptoethanol (Bio-Rad), 0.5 g/L proteinase K (Pierce), 0.1 g/L salmon sperm DNA (5 Prime Eppendorf/Brinkman), 0.1 g/L Escherichia coli tRNA (Sigma), 5 nmol/L each of the specific reverse primers, and 10¹⁰ molecules/L of synthetic RNA34 (as external control), was added to each well of the filterplates. The plates were then incubated at 37 °C for 10 min, placed over oligo(dT)-immobilized collection microplates (GenePlate; RNAture), and centrifuged at 2000g for 5 min at 4 °C. After overnight storage at 4 °C, the microplates were washed 3 times with 100 μΐ_^ of plain lysis buffer and then 3 times with 150 μΐ_^ of wash buffer [0.5 mol/L NaCl, 10 mmol/L Tris (pH 7.4) 1 mmol/L EDTA] at 4 °C.

[0082] cDNA was synthesized directly in each well by addition of 30 μΐ_^ of buffer containing lx reverse transcription buffer [50 mM KC1, 10 mM Tris-HCl (pH 8.3), 5.5 mM MgCl₂, 1 nL^L Tween 20], 1.25 mM each deoxynucleoside triphosphate, 4 units of rRNasin, and 80 U of MMLV reverse transcriptase (Promega; without primers) and incubation at 37 °C for 2 h. From each 30-μί reaction, 4 μL· of cDNA was transferred directly to 384- well PCR plates, and 5 μΐ_^ of TaqMan universal master mixture (Applied Biosystems) and 1 μΐ. of 5 μΜ each of the forward and reverse primers for IFN-induced markers or beta-actin (see Table 1) were added. PCR was carried out in a PRISM 7900HT (Applied Biosystems), with 1 cycle of 95 °C for 10 min followed by 45 cycles of 95 °C for 30 s, 55 °C for 30 s, and 60 °C for 1 min. Each gene was amplified in separate wells. The cycle threshold (Ct), i.e., the cycle at which certain amounts of PCR products (based on fluorescence) were generated, was determined with analytical software (SDS; Applied Biosystems). The ACt were determined by subtracting each Ct of IFNa-2b treated sample from each Ct of PBS-treated control sample, and the fold increase was calculated by 2^A-ACt.

[0083] As shown in Figure 1, beta-2-microglobulin (B2M) was not induced (e.g., fold change is approximately 1) in any of the disease categories after stimulation with IFN -2b. This demonstrated that B2M could be used as a negative control, in some embodiments. HCV represents the sum of all of the data for the S, R, and N groups (e.g., HCV is a cohort of data that is not divided based on the known clinical outcome).

[0084] Figure 2 depicts the expression of tumor necrosis factor receptor superfamily type 5 (TNFRSF5) in response to IFNa-2b stimulation of blood samples. In contrast to Figure 1, the HC group showed induction in response to IFNa-2b. This suggests that, in some embodiments, TNFRSF5 could be used as a positive control for IFNa-2b stimulation. While there was induction of TNFRSF5 in HCV, S, R, and N categories, there was no statistical significance among the five groups.

[0085] Figure 3 shows data related to the expression of suppressor of cytokine signaling 1 (SOCS1) in response to IFNa-2b treatment. Similar to TNFRSF5, SOCS1 induced in the healthy control subjects. A portion of the samples from the HCV, S, R, and N categories were also induced. However, the remaining samples were not induced and as a result, no statistical significance was found between the five disease categories.

[0086] Figure 4 depicts the results of expression of the chemokine CXCL10 in response to IFNa-2b treatment of the blood samples. All samples from healthy controls were induced. In contrast, only a portion of the samples from the HCV group were induced, and thus the induction of CXCL10 in HCV was significantly lower than that of HC (p=0.004). This reduced induction may represent the underlying immunocompromised status of HCV subjects. However, among the subcategories of HCV, there was no statistical significance detected between the S, R, and N groups. As such, CXCL10 can be used, in some embodiments, as a positive control for IFNa-2b stimulation. However, the lack of detectable differences between CXCL10 induction in the S, R, and N groups suggests that this marker may have limited use as a predictors of successful IFN therapy. In other words, markers that are likely to predict successful IFN therapy are differentially induced/expressed between the responsive, relapse, and non-responsive groups.

[0087] For example, Figure 5 depicts the expression of interferon alpha- inducible protein 6 (IFI6). As shown, induction of IFI6 occurred in all of the HC samples, while induction in the HCV samples was significantly less (p=0.008). Again, this may be representative of the underlying immune impairment of HCV subjects. Within the various clinical categories, IFI6 expression was also different. Responders without relapse (S) showed significantly more induction of IFI6 as compared to both relapse subjects (p=0.04) and non-responders (p=0.05). This differential expression is used, in several embodiments, as a basis for use of IFI6 as a predictor of successful IFN therapy.

[0088] Table 2 shows the data presented in Figure 5 in a tabular format and separated with by > 25 fold or < 25 fold induction thresholds as the cutoff for predicting responders versus non-responders, respectively.

Table 2: Prediction rates based on expression of IFI6

[0089] Using a Chi-squared analysis and the known clinical outcome of the patients in the S, R, and N groups, these data indicate that of 10 patients with > 25 fold induction in IFI6, 9 of those were clinically responsive without relapse. As such, induction of IFI6 by > 25 fold is 90% accurate as a predictor of successful IFN therapy. Induction of IFI6 by less than < 25 fold was also 71% accurate as a predictor of un-successful clinical outcomes in response to IFN therapy (12 of 17 patients were non-responsive). Thus, in several embodiments, IFI6 is used as a predictor for successful IFN therapy in hepatitis subjects. In some embodiments, IFI6 induction results in greater than 70% accuracy in predicting successful therapy. In some embodiments, the accuracy is greater than 80%, greater than 90%, greater than 95%, or in some embodiments, 100% accurate.

[0090] Additionally, IFI27 has been identified as a marker that is useful, in some embodiments, as a predictor of successful IFN therapy. As shown in Figure 7, there was modest induction in several of the HC samples, but there was no significant difference identified between the HC and HCV groups. However, when the HCV group was categorized based on clinical outcome, several important findings were detected. Those patients that were clinically responsive did not show a significant difference between HC or HCV subjects. This suggests that, in some embodiments, an increase in IFI27 may result in successful therapy (e.g., similar to a normal control). Likewise, there was no difference detected between the responders with relapse and the HC group. This suggests, that, at least a portion of the subjects may at least initially have successful IFN therapy, despite the possibility of relapse. The non- responder group showed no induction of IFI27 (significantly less than HC, p=0.05). Thus, in several embodiments, induction of IFI27 that is not significantly different from normal controls indicates a likelihood of success of IFN therapy. Moreover, when a lack of IFI27 induction is detected, a subject is more likely to fail to respond to IFN therapy. This is essentially negative identification of successful therapy (e.g., rather than predicting successful therapy, unsuccessful therapy can also be predicted by a lack of induction).

[0091] Table 3 shows the data of Figure 6 in tabular format with > 3 fold or <3 fold induction thresholds as the cutoffs for responders and non-responders to IFN therapy.

Table 3: Prediction rates based on expression of IIFI27

[0092] As described above, a Chi squared analysis was used to correlate the known clinical outcome of the patients with the level of induction of IFI27. When induction of IFI27 was > 3 fold, the accuracy of predicting responsive patients without relapse was 73%. When induction was < 3 fold, IFI27 predicted non- responders with 75% accuracy. Thus, IFI27 is used, in several embodiments as a marker of predicting whether a subject with hepatitis is likely to be responsive or non- responsive to IFN therapy.

[0093] Figure 7 depicts another marker useful for predicting successful IFN therapy. IFIT2 induction was analyzed in the HCV, S, R, and N groups as described above and in 3 normal controls (HC). While no statistical differences were detected among the R, S, or N groups, a Chi-squared analysis revealed that IFIT2 induction is still quite accurate at predicting responders versus non-responders. Table 4 shows that with a threshold of > 100 fold change for responders and < 100 fold change for non-responders, prediction of successful IFN therapy is 80% accurate and prediction of unsuccessful therapy is 77% accurate.

Table 4: Prediction rates based on expression of IFIT2

[0094] While the markers above provide relatively high accuracy of predicting successful IFN therapy, in several embodiments information related to the induction of expression of multiple markers can be used to achieve even higher accuracy for each of the disease categories. Table 5 shows the results of multivariate discriminate analysis using IFN-induced expression data. As described above, the expression data are used to calculate a Predictor Value, which is used to place each subject into a predicted category, in this case, responder, responder with relapse, and non-responder. Table 5 shows predictions based on IFI6 and IFI27 expression data. As a result of the data described above for those markers, the prediction that a subject would be a non-responder was 86% accurate, the prediction that a subject would respond, but possibly relapse was 80%. However, the prediction that a subject would be responsive to IFN therapy was 100%. Table 5: Summary of overall prediction rate

Claims

What is claimed is:

1. A method for identifying a subject having hepatitis as likely to respond to interferon therapy, comprising:

obtaining at least a first and a second sample of whole blood from the subject;

exposing the first sample of whole blood from said subject having hepatitis to interferon in a solvent for an amount of time sufficient for said interferon to alter the expression of two or more interferon-responsive markers,

wherein said two or more interferon-responsive markers are selected from the group consisting of IFI6, IFI27, IFIT2, TNFRSF5, SOCS1, CXCL10, AIM2, SOCS1, CXCL9, IL15RA, LGALS9, CXCL11, GADD45B, TNFSF10, CXCL2, VEGF, MAP3KB, DUSP5, APOBEC3G,

exposing the second sample of whole blood from said subject having hepatitis to the solvent without the interferon for said amount of time,

quantifying the expression of said two or more interferon-responsive markers by measuring the amount of mRNA encoding said one or more interferon-responsive markers in both said first whole blood sample and said second whole blood sample by a method selected from the group consisting of reverse-transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, northern blotting, fluorescence activated cell sorting, ELISA, and mass spectrometry;

evaluating the potential responsiveness of said subject to said interferon therapy by:

a) predetermining a parameter by multivariate discriminant analysis for each combination of said two or more interferon-responsive markers and said interferon using a plurality of patients in one or more of a plurality of groups with known clinical responsiveness to interferon therapy for hepatitis,

b) calculating a predictor value for said subject for each of said plurality of groups; and c) comparing said predictor values with said predetermined parameters in order to categorize said subject within one of said plurality of groups of known clinical responsiveness; and

identifying the subject as likely to respond to interferon therapy when said subject is categorized in any of said plurality of groups of known clinical responsiveness.

2. The method of Claim 1, wherein said two or more interferon-responsive markers are selected from the group consisting of IFI6, IFI27, and IFIT2.

3. The method of Claim 1, wherein said interferon is chosen from the group consisting of a type I interferon, a type II interferon, and a type III interferon.

4. The method of Claim 1, wherein said interferon is chosen from the group consisting of interferon alpha, interferon beta, interferon omega, interferon gamma, combinations thereof, and subtypes thereof.

5. The method of Claim 1, wherein said interferon is interferon alpha 2b.

6. The method of Claim 5, wherein said interferon alpha 2b is present in a concentration from about 1 to about 1 x 10 units per mL.

7. The method of Claim 6, wherein said interferon alpha 2b is lyophilized in a blood collection tube prior to said exposing.

8. The method of Claim 1, wherein said solvent is phosphate buffered saline.

9. The method of Claim 1, wherein said exposing is for a time between about one hour and about seven hours.

10. The method of Claim 9, wherein said exposing is for about four hours.

11. The method of Claim 1, wherein said exposing occurs at about thirty-seven (37) degrees Celsius.

12. The method of Claim 1, wherein said whole blood is heparinized.

13. The method of Claim 1, wherein said hepatitis is hepatitis C.

14. The method of Claim 1, wherein said hepatitis is hepatitis B

15. A method for identifying a subject having hepatitis as likely to respond to interferon therapy, comprising:

obtaining at least a first and a second sample of whole blood from the subject;

exposing the first sample of whole blood from the individual to interferon in a solvent for an amount of time sufficient for said interferon to alter the expression of an interferon-responsive marker,

wherein said interferon-responsive marker is one or more of IFIT2, IFI27, and IFI6;

exposing the second sample of whole blood from the individual to the solvent without the interferon for said amount of time;

quantifying the effect of said interferon as a change in expression of said interferon-responsive marker by measuring the amount of mRNA encoding said interferon-responsive marker in both said first whole blood sample and said second whole blood sample by a method selected from the group consisting of reverse-transcription polymerase chain reaction (RT- PCR), real-time RT-PCR, northern blotting, fluorescence activated cell sorting, ELISA, and mass spectrometry;

comparing the quantified effect of interferon on expression of said interferon-responsive marker with a normal effect of interferon on expression of said interferon responsive marker, wherein said normal effect is calculated as an average of change in expression levels of a panel of control individuals; evaluating the potential responsiveness of said subject by identifying significant differences between the change in expression of said interferon- responsive marker in said subject and the average change in expression of said interferon-responsive marker in said panel of control individuals and

a) indentifying said subject as potentially responsive to said interferon therapy when said identified significant differences are substantially greater in the subject as compared to the panel of control individuals, or b) indentifying said subject as potentially non-responsive to said interferon therapy when said identified significant differences are substantially less in the subject as compared to the panel of control individuals.

16. A method for identifying a patient having hepatitis as likely to respond to an immune based therapy, comprising:

obtaining at least a first and a second sample of whole blood from the patient;

exposing the first sample of whole blood from the patient to interferon in a solvent for an amount of time sufficient for said interferon to alter the expression one or more interferon-responsive markers,

wherein said one or more interferon-responsive markers are selected from the group consisting of TNFRSF5, SOCS1, CXCL10, AIM2, SOCS1, CXCL9, IL15RA, LGALS9, CXCL11, GADD45B, TNFSF10, CXCL2, VEGF, MAP3KB, DUSP5, APOBEC3G;

quantifying the effect of said interferon as a change in expression of said one or more interferon-responsive markers by measuring the amount of mRNA encoding said one or more interferon-responsive markers in both said first whole blood sample and said second whole blood sample by a method selected from the group consisting of reverse-transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, northern blotting, fluorescence activated cell sorting, ELISA, and mass spectrometry,

wherein at least one of said one or more interferon-responsive markers are associated with reduced immune function;

comparing the quantified effect of interferon on expression of said one or more interferon-responsive markers with a normal effect of interferon on expression of said one or more interferon-responsive markers, wherein said normal effect is calculated as an average of change in expression levels of a panel of control individuals;

identifying significant differences between the change in expression of said one or more interferon-responsive markers in said subject and the average change in expression of said one or more interferon-responsive markers in said panel of control individuals;

characterizing said patient as immuno-compromised if said change in expression in said interferon-responsive markers associated with reduced immune function is significantly different than the change in expression in said panel of control individuals; and

identifying said patient as not-likely to respond to an immune-based therapy if said patient is characterized as immuno-compromised.