WO2013134146A1 - Methods and compositions for treating viral infections by blocking type i interferon activity - Google Patents

Abstract

Disclosed herein are methods and compositions employing at least one inhibitor of Type I interferon receptor (IFNR) activation, e.g., an IFNR antagonist such as an IFNR1 antagonist, to block the activation of IFNRs in subjects. Such methods may be used to treat persistent or chronic viral infection, e.g., human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), and lymphocytic choriomeningitis virus (LCMV); to decrease type I interferon signaling, decrease one or more immunosuppressive factors (e.g., IL-10, PDL1, etc.); increase in the ratio of stimulatory to suppressive APCs; decrease in viral titers; and/or increase the cells associated with cell-mediated immunity (e.g., DC, macrophages, B cells, and NK cells) in subjects having a persistent or chronic viral infection.

Description

METHODS AND COMPOSITIONS FOR TREATING VIRAL INFECTIONS BY BLOCKING TYPE I

INTERFERON ACTIVITY

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Patent Application No. 61/607,735, filed 7 March 2012, and U.S. Patent Application No. 61/767,292, filed 21 February 2013, which are herein incorporated by reference in their entirety.

[0003] ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

[0004] This invention was made with Government support under Grant Nos. AI060567 and AI085043, awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0005] BACKGROUND OF THE INVENTION

[0006] 1. FIELD OF THE INVENTION

[0007] The present invention generally relates to methods and compositions for treating persistent or chronic viral infections.

[0008] 2. DESCRIPTION OF THE RELATED ART

[0009] At the onset of the viral infection, early Type I interferon (IFN-I) signals trigger antiviral immunity, which, in most cases, is able to control viral replication and reciprocally dampen IFN-I signals. Thus, many prior art treatments for viral infections comprise administering IFN-1 or an agonist of a Type I interferon receptor (IFNR), e.g., either the alpha subunit (IFNR1) or the beta subunit (IFNR2). In fact, administration of IFN-I early after lymphocytic choriomeningitis virus (LCMV) infection was recently demonstrated to enhance control of persistent LCMV infection (Wang, et al. (2012) Cell Host & Microbe 11, 631-642), and in many patients, IFN-I treatment in combination with the antiviral drug ribavirin is effective at eradicating hepatitis C virus (HCV) infection.

[0010] Unfortunately, despite initially robust antiviral immune activity, some viruses, including human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), and lymphocytic choriomeningitis virus (LCMV), outpace the immune response and establish persistent infections (Wilson & Brooks (2010) Immunol. Res. 48, 3-13; and Li, et al. (2009) Science 323, 1726-1729). In addition to virus-mediated evasion tactics, the host initiates an immunosuppressive response through various immunosuppressive factors, such as interleukin 10 (IL-10) and programmed death ligand 1 (PDL1), that actively suppress antiviral T cell responses and facilitate persistent infection (Barber, et al. (2006) Nature 439, 682-687; Brooks, et al. (2006) Nature Medicine 12, 1301-1309; Ejrnaes, et al. (2006) J Exp Med 203, 2461-2472; Day, et al. (2006) Nature 443, 350-354; Brockman, et al. (2009) Blood 1 14, 346-356; and Wilson, et al. (2012) Cell Host & Microbe 1 1 , 481-491). Unfortunately, many persistent and chronic viral infections are not responsive to interferon therapy (Chen, et al. (2005) Gastroenterology 128, 1437-1444; and Sarasin-Filipowicz, et al. (2008) PNAS USA 105, 7034-7039).

[001 1 ] SUMMARY OF THE INVENTION

[0012] In some embodiments, the present invention provides methods of treating a

persistent or chronic viral infection in a subject which comprise, consist essentially of, or consist of administering to the subject at least one inhibitor of Type I interferon receptor (IFNR) activation, preferably an IFNR antagonist, such as an IFNR1 antagonist. In some embodiments, the present invention provides methods of facilitating clearance of a virus which is the causative agent of a persistent or chronic infection in a subject which comprise, consist essentially of, or consist of administering to the subject at least one inhibitor of Type I interferon receptor (IFNR) activation, preferably an IFNR antagonist, such as an IFNR1 antagonist. In some embodiments, the present invention provides methods of decreasing one or more immunosuppressive factors in a subject having a persistent or chronic viral infection which comprise, consist essentially of, or consist of administering to the subject at least one inhibitor of Type I interferon receptor (IFNR) activation, preferably an IFNR antagonist, such as an IFNR1 antagonist. In some embodiments, the present invention provides methods of increasing one or more cells associated with cell-mediated immunity in a subject having a persistent or chronic viral infection which comprise, consist essentially of, or consist of administering to the subject at least one inhibitor of Type I interferon receptor (IFNR) activation, preferably an IFNR antagonist, such as an IFNR1 antagonist. In some embodiments, the present invention provides methods of increasing the ratio of stimulatory to suppressive antigen-presenting cells in a subject having a persistent or chronic viral infection which comprise, consist essentially of, or consist of administering to the subject at least one inhibitor of Type I interferon receptor (IFNR) activation, preferably an IFNR antagonist, such as an IFNR1 antagonist. In some embodiments, the present invention provides methods of reducing viral replication in a subject having a persistent or chronic viral infection which comprise, consists essentially of, or consists of administering to the subject at least one inhibitor of Type I interferon receptor (IFNR) activation, preferably an IFNR antagonist, such as an IFNR1 antagonist. In some embodiments, the methods of the present invention further comprise identifying the subject as having a high level of one or more immunosuppressive factors before administration of the at least one inhibitor of IFNR activation. In some embodiments, the methods of the present invention further comprise monitoring the efficacy of the treatment with the at least one inhibitor of IFNR activation by assaying the amount of one or more immunosuppressive factors in the subject after administration of the at least one inhibitor of IFNR activation. In some embodiments, the methods of the present invention further comprise potentiating the IFNy receptor and/or administering to the subject an agonist of the IFNy receptor. In some embodiments, the methods of the present invention further comprise administering to the subject a vaccine against the virus which is the causative agent of the persistent or chronic viral infection and/or an antagonist of an immunosuppressive cytokine. In some embodiments, the at least one inhibitor of IFNR activation is administered in an effective amount or a therapeutically effective amount. In some embodiments, the subject is human.

[0013] In some embodiments, the present invention provides uses of at least one IFNR antagonist, preferably an IFNR1 antagonist, for the manufacture of a medicament for treating a persistent or chronic viral infection in a subject. In some embodiments, the present invention provides an inhibitor of IFNR activation, e.g., an IFNR antagonist, preferably an IFNR1 antagonist, for use in treating a persistent or chronic viral infection. In some embodiments, the present invention provides an inhibitor of IFNR activation, e.g., an IFNR antagonist, preferably an IFNR1 antagonist, for treating a persistent or chronic viral infection in a subject, wherein an effective amount of the inhibitor of IFNR activation is administered to the subject after one or more immunosuppressive factors have been induced by the virus which is the causative agent of the persistent or chronic viral infection.

[0014] In some embodiments, the persistent or chronic viral infection is caused by human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), or lymphocytic choriomeningitis virus (LCMV).

[0015] In some embodiments, the inhibitor of IFNR activation is an antibody, preferably a monoclonal antibody. [0016] Both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed. The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute part of this specification, illustrate several embodiments of the invention, and together with the description serve to explain the principles of the invention.

[0017] DESCRIPTION OF THE DRAWINGS

[0018] This invention is further understood by reference to the drawings wherein:

[0019] Figures 1A-1C show prolonged IFN-I signaling during persistent LCMV

infection.

[0020] Figure 1 A provides graphs indicating splenic virus titers +/- standard deviation

(SD) following acute LCMV-Arm (black) and persistent LCMV-C113 (gray) infection (left). Gene expression kinetics by microarray analysis of the indicated IFN-I receptor inducible genes, STAT genes and IFN-I responsive genes in whole spleen tissue following LCMV-Arm (black) or LCMV-C113 (gray) relative to na^'ive (uninfected) mice. Each value indicates the average of 3-4 mice per group per time point. Values are shown without error bars for clarity and the p-values for each gene are indicated in the

Supplementary Table. STAT: Signal Transducer and Activator of Transcription.

[0021] Figure IB provides bar graphs showing the IFNa (left) and IFNP (right) mRNA expression relative to HPRT in dendritic cells from na^'ive, LCMV-Arm or LCMV-C113 infected mice (day 9). N.D. = "not detected" and indicates that IFNa or IFNP transcripts were not detected after 40 cycles of amplification. HPRT mRNA expression was measurable in all samples. Each group is a pool of cells from 6-8 mice and is

representative of 2 independent experiments.

[0022] Figure 1C provides bar graphs showing the Mxl, OAS, and IRF3 mRNA

expression relative to HPRT in the indicated IL-10+ (GFP+, grey) or IL-10- (GFP-, black) dendritic cells (DC, top) or macrophages (bottom) 9 days after LCMV-C113 infection of Vert-X IL-10 reporter mice. Each group is a pool of cells from 6-8 mice and is representative of 2 independent experiments.

[0023] Figures 2A-2E evidence that immunosuppression during persistent LCMV

infection is dependent on type I interferon signaling. Wild-type C57BL/6 mice were treated with isotype or anti-IFNRl antibody beginning 1 day prior to LCMV-C113 infection. Each symbol in the scatter plots represents an individual mouse with bars indicating the mean of the group. In bar graphs, the data represent the average +/- SD of 3-6 mice per group. All data are representative of 2 or more independent experiments. *, p <0.05.

[0024] Figure 2A1 provides histograms illustrating PDLl expression (wild-type mice = right peaks and IFNR1-/- mice = left peaks) and bar graphs indicating the geometric mean fluorescence intensity (GMFI) +/- SD of PDLl on DC and macrophages on day 9 after LCMV-C113 infection of wild-type (left bars, B6) and IFNR1-/- (right bars) mice.

[0025] Figure 2A2 provides scatter plots indicating plasma IL-10 levels on day 9 after

LCMV-C113 infection and bar graphs indicating IL-10 production by cultured

splenocytes (18 hour culture in the absence of exogenous stimulation) isolated 9 days after LCMV-C113 infection from wild type (B6) or IFNR1-/- mice.

[0026] Figure 2A3 provides bar graphs providing the plasma viral titers in wild-type

C57BL/6 (B6) or IFNR1-/- mice on day 9 after LCMV-C113 infection.

[0027] Figure 2B1 provides bar graphs showing OAS (top), Mxl (middle) and IRF7

(bottom) mRNA expression relative to HPRT in spleen (left), liver (middle) and kidney (right) from naive, isotype (iso) or anti-IFNRl (alFNRl) antibody treated mice on day 9 after LCMV-C113 infection.

[0028] Figure 2B2 provides bar graphs showing OAS and Mxl expression in the

indicated FACSorted cell population from isotype (left bars) or anti-IFNRl (right bars) antibody treated mice. Each cell population is a pool from 6 mice and is representative of 2 independent experiments.

[0029] Figure 2C1 provides graphs indicating PDLl levels (left), plasma levels of IL-10

(scatter plots, middle), and plasma viral titers (scatter plots, right) on day 9 following infection of untreated mice (B6), isotype treated mice (iso), anti-IFNRl antibody treated mice (alFNRl) and untreated IFNR1-/- mice (IFNR1-/-).

[0030] Figure 2C2 is a graph showing plasma IL-10 levels at day 15 after LCMV-C113 infection of isotype treated mice (iso) and anti-IFNRl antibody treated mice (alFNRl).

[0031] Figure 2D1 provides FACS plots illustrating IL-10 reporter expression (GFP) by

Vert-X mice treated with isotype or anti-IFNRl antibody.

[0032] Figure 2D2 provides bar graphs illustrating the frequency of IL-10 expressing DC and the GMFI of PDLl expression by DC and the ratio of IL-10 non-producing

(stimulatory) to IL-10 producing (suppressive) DC in isotype treated mice (iso) and anti- IFNRl antibody treated mice (IFNR1). [0033] Figure 2E are H and E staining of spleens from naive mice (top) or on day 9 after

LCMV-C113 infection of mice treated with isotype (middle) or anti-IFNRl (bottom) antibody.

[0034] Figures 3A-3F evidence that treatment with an IFNR1 antagonist enhances

control of persistent infection and significantly alters the immune environment. Wild- type C57BL/6 mice were treated with isotype or anti-IFNRl antibody beginning 1 day prior to LCMV-C113 infection. Each symbol in the scatter plot represents an individual mouse with bars indicating the mean of the group. Dashed lines in the graphs indicate the level of detection of the plaque assay (200 PFU Data are representative of 2 or more independent experiments). *, p <0.05.

[0035] Figure 3 A provides graphs showing longitudinal plasma virus titers at the

indicated time points after infection.

[0036] Figure 3B provides graphs showing viral titers in plasma (left), liver (middle) and kidney (kidney) 30 days after infection.

[0037] Figure 3C provides graphs showing the quantity of total splenocytes and the indicated immune subsets in the spleen on day 9 following LCMV-C113 infection.

[0038] Figure 3D provides graphs indicating the total number, number of IFNy

expressing and of multi-cytokine producing / polyfunctional LCMV-GP61-80-specific CD4⁺ T cells and LCMV-GP33-41 -specific CD8⁺ T cells.

[0039] Figure 3E is a graph providing the plasma viral titers at day 30 after LCMV-C113 infection in mice that were either undepleted of cells and treated with isotype (iso/iso) or anti-IFNRl (alFNRl/iso) antibody, or depleted of CD4⁺ T cells or NK cells prior to infection and treated with anti-IFNRl antibody. X axis labels indicate antibody (Ab) treatments.

[0040] Figure 3F provides graphs showing the plasma virus titers at the indicated time point after LCMV-C113 infection in mice that were either treated with isotype or anti- IFNRl antibody with or without anti-IFNy antibody. X axis labels indicate antibody (Ab) treatments.

[0041] Figures 4A-4F evidence that treatment with an IFNR1 antagonist dampens the immunosuppressive program and facilitates clearance of persistent infection. C57BL/6 mice were infected with LCMV-C113 and treated with isotype or anti-IFNRl antibody beginning on day 25 after LCMV-C113 infection. Each symbol in the scatter plot represents an individual mouse and bar graphs indicate the average value +/- SD. All data are representative of 2-5 independent experiments. *, p <0.05.

[0042] Figure 4A1 provides graphs showing the longitudinal plasma viral titers in mice at the indicated time points. The percent of mice at each time point exhibiting detectible virus titers is summarized in the kinetic graph (Figure 4A2). The shaded area in Figure 4A2 represents the time of treatment. The data are combined from 2 experiments.

[0043] Figure 4B provides graphs showing the viral titers in the serum (left), liver

(middle), and kidney (right) at day 46 after LCMV-C113 infection.

[0044] Figure 4C provides graphs showing the OAS (left), Mxl (middle) and IRF7

(right) mR A expression relative to HPRT in splenocytes from naive mice (right bars) or on day 30 after LCMV-C113 infection in mice treated with isotype (iso) or anti-IFNRl antibody (alFNRl).

[0045] Figure 4D provides graphs indicating GMFI of PDL1 on dendritic cells (left) and plasma IL-10 levels (right) on day 30 after LCMV-C113 infection in mice treated with isotype (iso) or anti-IFNRl antibody (alFNRl).

[0046] Figures 5A and 5B show cytokine and type I interferon expression in the spleen during acute and persistent infection.

[0047] Figure 5A are graphs showing the cytokine gene expression in whole spleen

tissue on the indicated day following persistent LCMV-C113 compared to acute LCMV- Arm infection as determined by microarray analysis. Increase on the y-axis indicates elevated expression in persistent infection compared to acute infection.

[0048] Figure 5B are graphs showing IFNa and IFNP gene expression in LCMV-Arm

(left bars of each set) or LCMV-C113 (right bars of each set) normalized to na^'ive spleen levels in whole spleen samples as determined by microarray analysis. An increase on the y-axis indicates elevated expression in the specified infection compared to na^'ive mice. Each bar indicates the average expression of the indicated cytokine for 3-4 mice per group per time point. Error bars are omitted for clarity.

[0049] Figures 6A-6E evidence that IFN-I signaling supports immunosuppression.

[0050] Figure 6A1 are graphs providing the plasma IL-10 levels (left) and viral titers

(right) at the indicated day following LCMV-C113 infection in wild-type C57BL/6 (squares) and IFNR1-/- (circles) mice. Figure 6A2 are graphs providing the viral titers at day 9 following LCMV-Arm infection of C57BL/6 vs. IFNR1-/- mice and isotype vs. anti-IFNRl antibody treated mice. Figure 6A3 shows LCMV nucleoprotein (NP) staining in dendritic cells on day 9 after LCMV CI 13 infection in isotype and anti-IFNRl antibody treated mice. Figure 6A4 shows flow plots gated on LCMV NP+ dendritic cells. Note, despite the increased amount of LCMV-NP+ dendritic cells in anti-IFNRl antibody treated mice, no alteration in the DC subsets staining positive or LCMV-NP is observed. 6A5 shows LCMV-NP+ macrophages in isotype (left plot) and anti-IFNRl (right plot) antibody treated mice. Data are representative of the average ± SD of 4-6 mice per group and 2 or more independent experiments. *, p <0.05.

[0051] Figure 6B are bar graphs showing PDLl mRNA expression (left) in FACSorted

DC and macrophages following in vitro treatment with media (left bars of each set) or IFNP (right bars of each set). PDLl mRNA was normalized to expression of HPRT and each group is a pool of cells from 6-8 mice. The results are representative of 2 independent experiments. IL-10 production (right) quantified in the culture supernatant of splenocytes from LCMV-C113 infected wild-type mice stimulated with media alone (left bar) or with IFNP (right bar) for 18 hours. Data are representative of the average ± SD of 4-5 mice per group and 2 independent experiments. *, p <0.05.

[0052] Figure 6C provides graphs showing the amounts of the indicated cytokines in the plasma on day 9 in mice treated with isotype (iso) or anti-IFNRl antibody (alFNRl) beginning day -1 prior to LCMV-C113 infection. Inflammasome dependent cytokines were quantified in the plasma 9 days after infection. The bar graphs indicate the average +/- SD of DC (left) and macrophages (right) exhibiting enzymatically active caspase 1.

[0053] Figure 6D are whole spleen images. H and E staining of spleen from naive mice or on day 9 after LCMV-C113 infection of mice treated with isotype (iso) or anti-IFNRl antibody beginning prior to infection.

[0054] Figure 7 provides graphs showing the LCMV-specific and total IgG production following treatment with an IFNR1 antagonist. Wild-type mice were treated with isotype (iso) or anti-IFNRl antibody (alFNRl) beginning 1 day prior to LCMV-C113 infection. The graphs represent LCMV-specific IgG and total IgG levels in the plasma (ng/ml) on day 9 (left plots) and day 30 (right plots) after infection. Data represent two independent experiments comprised of 3-5 mice per group. *, p <0.05.

[0055] Figure 8 are tables evidencing that IFN signaling is significantly enhanced in persistent compared to acute LCMV infection. P-values are shown for microarray analysis for the interferon receptor inducible genes, STAT genes and interferon responsive genes on the indicated day following persistent LCMV-C113 compared to acute LCMV-Arm infection. Shaded cells indicate significantly elevated expression in persistent compared to acute infection.

[0056] DETAILED DESCRIPTION OF THE INVENTION

[0057] As set forth in the experiments below, the fundamental role of chronic IFN-I signaling in driving the suppressive state and preventing elimination of persistent virus infection is demonstrated. The experiments herein demonstrate that when the viral burden exceeds the ability of the acute immune response, IFN-I signals are maintained and potentiate multiple parameters of immune dysfunction associated with persistent infection including the expression of multiple immunosuppressive factors, splenic disorganization and the development of immunoregulatory APC populations. However, as disclosed herein, contrary to teachings in the prior art, blocking the activation of the Type I interferon receptors (IFNRs), e.g., by treatment with an antagonist of IFNR (IFNR antagonist), reverses many of the immune dysfunctions associated with persistent viral infection and facilitates decreased viral replication and increased viral clearance. In fact, as exemplified herein, blocking (i.e., preventing or inhibiting) the activation of IFNR with an antagonist of IFNRl (IFNRl antagonist) diminished chronic immune activation and immunosuppression, restored lymphoid tissue architecture, increased multiple immune parameters associated with control of virus replication, and facilitated viral clearance in subjects having persistent viral infections. It should be noted that prior to the present invention, because of the dual nature of IFNRs, i.e., in acute viral infections, activation of IFNRs aids in the control and clearance of the infection, and in persistent and chronic viral infections, the continued activation of IFNRs prevent or inhibit the ability of one's immune system to control and clear the infection, it was unknown whether blocking the activation of IFNRs would actually result in the control and clearance of persistent and chronic viral infections.

[0058] Therefore, in some embodiments, the present invention is directed to methods of treating and/or inhibiting a chronic or persistent viral infection in a subject which comprise blocking the activation of IFNRs in the subject. In some embodiments, the IFNR is an IFNRl . In some embodiments, the IFNR is an IFNR2. In some

embodiments, the subject is an animal. In some embodiments, the subject is a human. In some embodiments, the subject is in need of treatment. Subjects in need of treatment include those who have a chronic or persistent viral infection and those who are or will be exposed to a virus which causes a chronic or persistent viral infection in subjects. In some embodiments, the chronic or persistent viral infection is caused by a Human Immunodeficiency Virus (HIV), a Hepatitis B Virus (HBV), a Hepatitis C Virus (HCV), or a Lymphocytic Choriomeningitis Virus (LCMV).

[0059] In some embodiments, the activation of IFNRs is blocked (i.e., prevented or

inhibited) by way of antagonizing the IFNRs in the subject with one or more IFNR antagonists. In some embodiments, the activation of the IFNRs is prevented or inhibited by way of blocking the ability of the ligands, i.e., interferon alphas and interferon beta, to activate the IFNRs. In particular, as the experiments herein evidence that blocking the activation of IFNRs (by way of antagonizing the IFNRs) is beneficial in controlling persistent viral infection, blocking the ligands, i.e., interferon alphas and interferon beta, that bind to and activate IFNRs will also prevent or inhibit the IFNRs from being activated and will result in the same beneficial result in controlling persistent viral infection.

[0060] In some embodiments, at least one IFNR antagonist is administered to the subject.

As used herein, an "IFNR antagonist" refers to an agent that antagonizes a mammalian IFNR, preferably a human IFNRl (Chill, et al. (2003) Structure 11(7), 791-802; and Uze, et al. (2007) Curr. Top. Microbiol. Immunol. 316, 71-95). As used herein, an "IFNRl antagonist" refers to an agent that antagonizes a mammalian IFNRl . Similarly, as used herein, an "IFNR2 antagonist" refers to an agent that antagonizes a mammalian IFNR2. Examples of IFNR antagonists include compounds such as antibodies, chemicals, IFNR binding proteins, IFNR receptor mimics, proteins such as those disclosed in US

7,285,526, US 20110027282, and EP 1250358, and antibodies such as those disclosed in US 7,662,381, and WO 200686586, and the like. In some embodiments, the IFNR antagonist is an antibody, e.g., MAR1-5A3, (Satie, et al. (2011) J. Biol. Chem. 286, 23280-23295) or the human equivalent (i.e., an antibody that specifically binds and antagonizes a human IFNR, e.g., MEDI546 (a human antibody that blocks IFNRl (Medlmmune LLC)), 64G12 (a monoclonal antibody that binds to IFNRl and neutralizes the activity of all type I interferons (Bristol-Myers Squibb Company)), MDX-1185 (a fully human antibody that is an IFNRl antagonist (Bristol-Myers Squibb Company)), and the like. The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they retain, or are modified to comprise, a ligand-specific binding domain. [0061] In some embodiments, at least one interferon inhibitor is administered to the subject. As used herein, an "interferon inhibitor" refers to an agent that inhibits the ability of an interferon, i.e., an interferon alpha or interferon beta, to activate a mammalian IFNR, preferably a human IFNR1. Although there are many different interferons (13 main interferon alphas (1, 2, 4, 5, 6, 7, 8, 10, 13, 14, 16, 17 and 21) and one interferon beta) and variants, a variety of interferon inhibitors are known in the art and/or are commercially available. For example, sifalimumab which is a fully human monoclonal antibody against interferon alpha, mAb Al which is a neutralizing antibody against interferon beta is commercially available from Biolegend or eBioscience and clone MMHB-3 is commercially available from PBL Interferon Source. Additionally, various antibodies against one or more interferons can readily be made using methods known in the art.

[0062] As used herein, "inhibitors of IFNR activation" refers to IFNR antagonists and interferon inhibitors. It is important to note that immunosuppressive cytokines as disclosed in US 20110008332 are the downstream result of IFNR activation, i.e., activation of an IFNR causes expression and synthesis of immunosuppressive cytokines. The activity and function of such cytokines are broad and diverse. Thus, even in view of US 20110008332, prior to the present invention, it was unknown whether the inhibition of IFNR activation upstream would actually result in the ability to control persistent and chronic viral infections in light of the dual nature of IFNRs - in acute viral infections, activation of IFNRs aids in the control and clearance of the infection; but, as evidenced herein, in persistent and chronic viral infections, the continued activation of IFNRs prevent or inhibit the ability of one's immune system to control and clear the infection.

[0063] Thus, in some embodiments, the present invention is directed to treating a subject having a persistent or chronic viral infection which comprises administering to the subject at least one inhibitor of IFNR activation, preferably an IFNR antagonist such as an IFNR1 antagonist. The at least one inhibitor of IFNR activation, may be administered to the subject before, during, and/or after infection by the virus which is the causative agent of the persistent or chronic viral infection. In some embodiments, the at least one inhibitor of IFNR activation is administered to a subject who has been determined to be suffering with a persistent or chronic viral infection.

[0064] In some embodiments, the at least one inhibitor of IFNR activation is

administered in an effective amount or a therapeutically effective amount. As used herein, an "effective amount" is the amount of the at least one inhibitor of IFNR activation which is capable of preventing or inhibiting the activation of the IFNRs in a subject. In some embodiments, the at least one IFNR antagonist is administered in an effective amount or a therapeutically effective amount. An "effective amount" of an IFNR antagonist is an amount that antagonizes some or all of the IFNRs in a subject. As used herein, a "therapeutically effective amount" is the amount of the at least one inhibitor of IFNR activation which results in the desired therapeutic effect (e.g., a decreased level of one or more immunosuppressive factors (e.g., IL-10, PDLI, etc.) associated with a persistent or chronic viral infection, a decreased viral titer, etc., a decrease in expression of one or more IFN-I genes, and/or an increase in IFNy) as compared to controls (e.g., untreated subjects). In some embodiments, the

therapeutically effective amounts of the at least one inhibitor of IFNR activation range from about 1-50 mg per kg body weight, preferably about 8-40 mg per kg body weight. One skilled in the art may readily determine the effective amounts and therapeutically effective amounts for human subjects using the methods described herein and/or drawing correlations from animal models and the dose administered may be more or less based on the given subject, the particular inhibitor of IFNR activation, severity of disease, age, tolerance, etc.

[0065] Treatment of a subject with the at least one inhibitor of IFNR activation according to the present invention can include a single treatment or a series of treatments. It will be appreciated that the actual dosages will vary according to the particular composition, the particular formulation, the mode of administration, and the particular subject and condition being treated. It will also be appreciated that the effective dosage used for treatment may increase or decrease over the course of a particular treatment. Optimal dosages for a given set of conditions may be ascertained by those skilled in the art using conventional dosage-determination tests in view of the experiments herein. Changes in dosage may result and become apparent by standard diagnostic assays known in the art.

[0066] In some embodiments, the present invention is directed to pharmaceutical

compositions for use in the treatment of chronic or persistent viral infections which comprise at least one inhibitor of IFNR activation and a pharmaceutically acceptable carrier or a diluent. As used herein, a "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration and known in the art. Except insofar as any conventional media or agent is incompatible with the active pharmaceutical ingredient, use thereof in the compositions is contemplated. In some embodiments, the amount of the at least one inhibitor of IFNR activation in the pharmaceutical composition is an effective amount. The pharmaceutical compositions of the invention may be prepared in a unit-dosage form appropriate for the desired mode of administration. It will be appreciated that the preferred route will vary with the condition and age of the subject, the nature of the condition to be treated, and the given composition.

[0067] In some embodiments, the at least one inhibitor of IFNR activation is

administered in conjunction with a vaccine against the virus which is the causative agent for the persistent or chronic viral infection and/or an antagonist of an immunosuppressive cytokine. See e.g., US 20110008332. Thus, in some embodiments, the pharmaceutical compositions further comprise (a) a vaccine against the virus which is the causative agent of the persistent or chronic viral infection and/or (b) an antagonist of an

immunosuppressive cytokine. Nevertheless, in some embodiments, the at least one inhibitor of IFNR activation and the vaccine against the virus which is the causative agent for the persistent or chronic viral infection and/or an antagonist of an

immunosuppressive cytokine are administered as separate compositions. Thus, according to some of these embodiments, the at least one inhibitor of IFNR activation is

administered before, at the same time, and/or after administration of the vaccine and/or the antagonist of an immunosuppressive cytokine.

[0068] In some embodiments, the present invention is directed to kits which comprise the at least one inhibitor of IFNR activation packaged together with: one or more reagents for assaying the virus which is the causative agent for the persistent or chronic viral infection; one or more reagents for assaying an immunosuppressive factor; a device for administering the at least one inhibitor of IFNR activation; a vaccine against the virus which is a causative agent for the persistent or chronic viral infection; an antagonist of an immunosuppressive cytokine; or a combination thereof.

[0069] As a high IFN-I signature (e.g., high levels of one or more immunosuppressive factors, high levels of interferon proteins, and/or high levels of IFN-I gene expression) in a subject having a viral infection may indicate that treatment with an interferon or an agonist of IFNR may be ineffective, in some embodiments, the methods of the present invention further comprise identifying the subject as having a high IFN-1 signature before administration of the at least one inhibitor of IFNR activation. In some embodiments, the methods further comprise monitoring the efficacy of the treatment with the at least one inhibitor of IFNR activation by assaying the levels of one or more immunosuppressive factors in the subject and/or determining whether the subject exhibits a high IFN-I signature after administration of the at least one inhibitor of IFNR activation.

[0070] While expression of the interferon proteins themselves is often hard to detect in mammalian plasma, the activity of IFNRs can be measured by expression of genes downstream of the IFNRs (i.e., the IFN-I gene expression signature). For example, the expression level of one or more of the following genes downstream of IFNR (IFN-I genes) may be used to characterize a subject as having a high IFN-I signature: Mxl, Oasl, Oas3, Oasll, Oasl2, Mx2, Oasla, Oaslg, IRF1, IRF4, IRF7, IFR9, Ifi44, Ifitl, Ifit2, Ifit3, CXCL10 (IP-10). Expression of all these genes are elevated 1.5-4 fold in the spleen of subjects during persistent infection compared to no infection. Thus, in some embodiments, when expression of one or more of these genes are up-regulated by about 1.5-4 fold or more in the spleen of the subject, the subject is characterized as having a high IFN-I signature in need of treatment with an inhibitor of IFNR according to the present invention. The IFN-I signature in a subject may be determined by assaying the level of IFN activity in peripheral blood mononuclear cells (PBMC) using methods known in the art, e.g., ELISA and/or reverse-transcription polymerase chain reaction (RT-PCR).

[0071] The experiments herein demonstrate that chronic IFN-I signaling during persistent infection derails multiple critical parameters of productive immunity associated with virus clearance and implicates a multifaceted restoration of the immune environment as the mechanism underlying the enhanced ability to control infection when IFN-I signaling is inhibited. Specifically, the accelerated clearance of persistent infection following treatment with an IFNR1 antagonist requires CD4⁺ T cells and enhanced IFNy. Thus, in some embodiments, the methods of the present invention exclude antagonizing the IFNy receptor. In some embodiments, the methods of the present invention further include potentiating the IFNy receptor and/or administering to the subject an agonist of the IFNy receptor. In some embodiments, the methods of the present invention are performed in conjunction with other therapies, e.g., vaccination, and methods and treatments for enhancing immune function. [0072] As disclosed herein, in subjects having persistent or chronic viral infections, treatment with an IFNR1 antagonist results in about a 2-500 fold decrease one or more immunosuppressive factors (e.g., IL-10, PDLl, etc.); about a 20% increase in the ratio of stimulatory to suppressive APCs; about a 1-4 fold decrease in viral titers; and/or about a 2-4 fold increase in cells associated with cell-mediated immunity (e.g., DC,

macrophages, B cells, and NK cells), when compared to a negative control. As set forth in the experiments below, these results are from the administration of an IFNR1 antagonist by itself. Thus, in some embodiments, the methods and compositions according to the present invention include the use of another active ingredient such as a vaccine against the virus which is a causative agent for the persistent or chronic viral infection and/or an antagonist of an immunosuppressive cytokine. However, in some of these embodiments, the methods and compositions may include the use of an agonist of the IFNy receptor.

[0073] The following examples are intended to illustrate but not to limit the invention.

[0074] MATERIALS AND METHODS

[0075] Mice and Virus

[0076] C57BL/6 (wild type) were purchased from The Jackson Laboratory (Bar Harbor,

ME).

[0077] IFNR1-/- mice were provided by Dr. Genhong Cheng and Dr. Dorian McGavern

(NINDS/NIH) (Muller et al. (1994) Science 264: 1918-1921).

[0078] Vert-X IL- 10/GFP reporter mice (Madan, et al. (2009) Journal of Immunology

183(4): 2312-2320) were generously provided by Christopher Karp at the Cincinnati Children's Hospital Research Foundation, and the University of Cincinnati College of Medicine, Cincinnati, Ohio (current address is The Bill & Melinda Gates Foundation).

[0079] LCMV-GP61 -80-specific CD4 TCR transgenic (SMART A) mice. (Oxenius, et al.

(1998) European Journal of Immunology 28(1): 390-400).

[0080] Six-ten week old mice were used for all experiments. All mice were housed

under specific pathogen- free conditions and mouse handling conformed to the

requirements of the University of California, Los Angeles Animal Research Committee guidelines. Mice were infected intravenously (i.v.) via the retro-orbital sinus with 2 x 10⁶ plaque forming units (PFU) of LCMV-Arm or LCMV-Cl 13. Virus stocks were prepared and viral titers were quantified as described previously (Brooks, et al. (2005) Journal of Virology 79(16): 10514-10527). [0081] In vivo Anti-IFNRl Antibody Treatment

[0082] C57BL/6 mice were treated intraperitoneally (i.p.) with anti-IFNRl antibody

(clone MAR1-5A3; Leinco Technologies, St. Louis MO) either starting prior to infection: day -1 (500 day 0 (500 day 2 (250 μg), day 4 (250 μg) and day 6 (250 μg) or therapeutically in the midst of the established persistent infection: day 25 (500 μg), day 27 (500 μg), and day 29 (250 μg).

[0083] Cytokine Quantification

[0084] IL-10, IFNy and IL-18 levels were determined using cytokine specific Quantikine

ELISA kits (R & D Systems, Minneapolis, MN). Optical density values were read using a Synergy 2 plate reader (BioTek, Winooski, VT) at 450 nm. Luminex was performed using the MILLIPLEX MAP Mouse Cytokine/Chemokine 22-plex kit (Millipore, Billerica, MA). Samples were analyzed on a Bio-Plex 200 System with HTF and Bio- Plex Manager 6.1 Software (Bio-Rad, Hercules, CA).

[0085] RNA Microarray

[0086] C57BL6 mice were either left uninfected (naive) or infected with LCMV-Arm or

CI 13 (n = 3-4 mice per group). Spleens were isolated on day 5, 9 and 30 after infection and immediately frozen in RNA Later (Qiagen) at 1 mg/ml tissue. Whole spleens were subsequently homogenized, and RNA from splenic homogenates were isolated with the RNeasy extraction kit (Qiagen). RNAs were evaluated using an Agilent Bioanalyzer, labeled using the Ambion WT labeling kit, and hybridized to the Affymetrix Mouse Genes ST 1.0 microarray which were scanned and summarized using Affymetrix

Expression Console and RMA16. RMA normalized data was referenced to uninfected spleen samples and genie probe sets with RMA > 6.0 that differed between the different sample treatments were identified using ANOVA with Benjamini Hochberg FDR - corrected p < 0.05 and further ranked by relative fold-differences between LCMV-Arm and C113 infected sample groups.

[0087] Isolation and Adoptive Transfer of Virus Specific T Cells

[0088] To specifically identify and isolate virus-specific T cells, CD4⁺ T cells and CD8⁺

T cells were negatively selected (StemCell Technologies) from the spleens of na^'ive Ly5.1+ SMARTA mice, respectively. Following purification 5000 SMARTA cells were co-transferred i.v. into 6-8 week old Ly5.2+ C57BL/6 mice. We avoided the problems associated with using large, non-physiologic numbers of transferred transgenic T cells by only transferring low numbers of transgenic T cells (Marzo et al., Nat Immunol 6, 793 (Aug, 2005); and Brooks et al. Journal of Virology 79, 10514 (2005)). Mice were infected with LCMV one day after cell transfer.

[0089] Flow Cytometry

[0090] Analysis of immune cell subsets was performed by staining directly ex vivo for surface expression of CD45-Pacific Orange or Pacific Blue, CD1 lc-Pacific Blue or PE, Thyl . l-FITC or PE, Thyl .2-PerCP, NKl . l-PerCPCy5, B220-APCCy7, CD1 lbPeCy7, F4/80-PE or APC, MHC Class II-PE, CD4-Pacific Blue, CD8-Pacific Blue all obtained from BioLegend or BD Pharmingen. MHC tetramers were obtained from the NIH. IL- 10 expression was determined by GFP expression in the Vert-X IL-10-GFP reporter mouse. Active caspase 1 was quantified by flow cytometry utilizing the FAM-FLICA Caspase-1 activity kit from (Immunochemistry, Bloomington, MN). LCMV viral antigen was quantified by flow cytometric analysis using the anti-LCMV nucleoprotein-specific antibody mAbl 13. Flow cytometric analysis was performed using the Digital LSR II and FACSVerse (Becton Dickinson).

[0091] Purification of Dendritic Cells and Macrophages

[0092] IL-10 producing and non-producing DC and macrophages were sorted from

spleen following B cell depletion (CD19 MACS beads, Miltenyi) as follows: IL-10 expressing DC (GFP+, CD45+, Thy 1.2-, NK1.1-, CD1 lc+ bright), non-IL-10-producing DC (GFP-, CD45+, Thyl .2-, NK1.1-, CD1 lc+ bright), IL-10 expressing macrophage (GFP+, CD45+, Thyl .2-, NK1.1-, F4/80+), and non-IL-10 producing macrophages (GFP-, CD45+, Thyl .2-, NK1.1-, F4/80+). Cells were sorted using a FACSVantage fluorescence-activated cell sorter (Becton Dickinson). Post sort purity was > 98%.

[0093] ΙΓΝβ Treatment In vitro

[0094] Bulk splenocytes were cultured in complete media supplemented with

recombinant IFNP (PBL Interferon, Piscataway, NJ) at a concentration of 250 units/mL for 24 hours.

[0095] Histology

[0096] Na^'ive and day 9 LCMV-C113 infected anti-IFNRl or isotype antibody-treated spleens were excised and fixed for 24 hours in 4% paraformaldehyde, then paraffin embedded, sectioned and stained with Hematoxylin and Eosin (H & E). Embedding, H & E staining and tissue scanning for image analysis were performed by the Translational Pathology Core Laboratory at UCLA.

[0097] Quantitative RT-PCR

[0098] RNA purified from sorted dendritic cells or macrophages, whole splenocytes, or tissue homogenates was isolated with the RNeasy extraction kit (Qiagen). RNA was normalized for input and amplified directly using the One-Step RT-PCR kit (Qiagen). Mxl, OAS, IRF3, IRF7, PDL1, and HPRT were amplified using Applied Biosystems Assays-on-Demand TaqMan pre-made expression assays. IFNa and ΙΚΝβ primer sequences were previously described (Hahm, et al. Immunity 22, 247 (Feb, 2005)). RNA expression was normalized to HPRT.

[0099] LCMV-Specific Antibody ELISA

[0100] To quantify LCMV-specific IgG, LCMV-C1 13 was used to coat 96-well

Maxisorp ELISA plates (Nunc) overnight. Plates were blocked with 3% BSA/PBS/0.05% tween-20. Subsequently, plasma isolated from the indicated mice was incubated on the LCMV coated plates. Plates were washed and incubated with an HRP-labeled goat anti- mouse IgG antibody (Invitrogen), followed by the addition of o-phenylenediamine substrate in 0.05 M phosphate citrate buffer. The reaction was stopped with 2N H₂S0₄ and the optical density (O.D.) values were read using an ELISA plate reader (Synergy 2, BioTek) at 490 nm. The concentration of LCMV-specific IgG was interpolated from a standard curve generated from a serial dilution of purified mouse IgG (Invitrogen; 500 ng/ml -0.49 ng/ml) incubated on plates coated with goat anti-mouse IgG (Invitrogen).

[0101] Statistical Analysis

[0102] Student's t-tests (two-tailed, unpaired) and log-rank Mantel-Cox and Gehan-

Breslow tests (for clearance curve; Figure 4C) were performed using the GraphPad Prism 5 software (GraphPad Software Inc.).

[0103] IFN - 1 Signaling Modulates Immunosuppression in Persistent Viral Infections

[0104] In order to identify the fundamental mechanisms that orchestrate

immunosuppression during virus infection, mice were infected with LCMV-Armstrong (Arm) which induces a robust T cell response that resolves infection within 8-10 days; or LCMV-Clone 13 (CI 13) which generates a persistent infection due to the up-regulation of an immunosuppressive factor (e.g., IL-10, PDLl) that suppress antiviral immune responses. Enigmatically, expression of immunosuppressive factors is similar in the early stages of both acute and persistent LCMV infection, but then wanes in acute infection, allowing for productive immunity; or is maintained/ increased in persistent infection.

[0105] To reveal factors exhibiting similar kinetics that might be utilized to sense virus replication dynamics and control immune responses, R A microarray based splenic network analysis was performed using methods known in the art. Tissue-wide cytokine expression patterns were found to be strikingly similar in acute and persistent infections with only very few differences, including no cytokines previously associated with regulation of PDLl or IL-10 expression during persistent infection (Fig. 5 A). Analogous to virus clearance kinetics, IFN-I receptor stimulated genes, STAT genes, and IFN-I responsive genes were initially similarly expressed in LCMV- Arm and CI 13 infections, but then rapidly dissipated as acute infection resolved, whereas they remained elevated in the presence of persisting virus replication (Fig. 1 A and Fig. 8).

[0106] Although at the whole-organ level, expression of the IFNa and IFNP genes

themselves were not elevated above uninfected mice (Fig. 5B), IFNa and IFNP transcripts were present in dendritic cells (DC) during persistent infection, whereas their expression was undetectable in naive or at day 9 following LCMV- Arm infection (Fig. IB). Interestingly, utilizing the Vert-X IL-10-GFP reporter mouse, OAS and Mxl (genes directly stimulated by IFN-I signaling) expression was observed to be specifically enriched in the immunoregulatory antigen-presenting cell (APC) that co-express the highest levels of PDLl and IL-10 and can suppress antiviral T cell responses. Notably, expression of interferon regulatory factor 3 (IRF3, a gene involved in the IFN-I response, but not directly activated by IFN-I signaling), was not differentially increased in immunoregulatory APCs (Fig. 1C), thereby indicating that sustained IFN-I signaling modulates multiple parameters of immunosuppression during viral persistence.

[0107] Next, the impact of prolonged IFN-I signaling toward potentiating

immunosuppression in vivo was determined. Levels of virus replication correlate with expression of the immunosuppressive factors PDLl and IL-10 during virus infection. However, despite the elevated levels of viral replication and viral antigen in IFNR1-/- compared to wild type mice, PDLl and IL-10 were decreased in IFNR1-/- mice during LCMV-C113 infection (Fig. 2A, Fig. 6B). Further, cells from LCMV-C113 infected wild- type mice increased PDLl and IL-10 expression in response to IFNP (Fig. 6C). Of note, IFNRl-/- mice failed to clear LCMV-Arm (Fig. 6 A) which is consistent with the antiviral and immune stimulatory effect of IFN-I during viral infection. Thus, the disconnect between IL-10/PDL1 expression and virus titers in LCMV-C113 infected IFNRl-/- mice indicates that IFN-I signaling is a critical component in a sensing system translating virus clearance kinetics into expression of the immunosuppressive program in vivo.

[0108] To delineate the underlying role of IFN-I in inducing immunosuppression,

separate from potential abnormalities of life-long genetic deficiency in IFNRl-/- mice, wild-type mice were treated with an IFNRl antagonist, i.e., MAR1-5A3 (an anti-IFNRl antibody which blocks IFN-I signaling by binding to the IFNRl), beginning 1 day prior to LCMV-C113 infection. The IFNRl antagonist diminished Mxl, OAS, and IRF7 expression in multiple tissues and cell types (Fig. 2B), thereby indicating the ability to potently inhibit IFN-I signaling in vivo. Analogous to persistently infected IFNRl -/- mice, the anti-IFNRl antibody led to decreased PDLl and IL-10 expression and elevated virus titers compared to isotype antibody treated LCMV-C113 infected mice (Fig. 2C). Interestingly, IL-10 levels rapidly rebounded when treatment with the anti-IFNRl antibody was withdrawn (day 15; Fig. 2C), thereby indicating the sensitive surveillance and rapid modulation of the immunosuppressive state through IFN-I signaling.

[0109] Heightened IFN-I signaling can inhibit inflammasome activity in some situations.

However, despite higher levels of virus replication and antigen (Fig. 2C, Fig. 6B), reduced amounts of IL-1, IL-18 and inflammasome activation were observed following treatment with the anti-IFNRl antibody during persistent infection (Fig. 6D), thereby indicating IFN-I stimulates inflammasome activity during persistent infection and that blocking IFN-I signaling with an IFNRl antagonist decreases chronic inflammation. The reduced levels of inhibitory factors and chronic activation following treatment with the anti-IFNRl antibody were not indicative of a global down-regulation of proinflammatory cytokines, and in fact, expression of interferon-gamma (IFNy) was substantially elevated following treatment with the anti-IFNRl antibody (Fig. 6D). Thus, treatment with an IFNRl antagonist during persistent viral infection reduces

immunosuppression and chronic inflammation resulting from the persistent viral infection.

[0110] Treatment with an IFNRl antagonist, e.g., the anti-IFNRl antibody, prior to

infection also decreased the levels of IL-10- and PDLl - expressing immunoregulatory APCs that normally emerge in conjunction with persistent virus replication (Fig. 2D), thereby leading to a population shift toward stimulatory APCs possessing enhanced T cell stimulatory capacity. Interestingly, treatment with the anti-IFNRl antibody prevented the splenic disorganization that normally accompanies persistent infections (Fig. 2E, Fig. 6E) and is associated with faulty immune interactions and the inability to control infection. Specifically, animals treated with the anti-IFNRl antibody exhibited highly organized and demarcated splenic red pulp, marginal zone and white pulp regions, whereas isotype treated mice displayed the characteristic loss of structure during persistent infection (Fig. 2E, Fig. 6E). Thus, because of the preserved splenic

organization following treatment with an IFNR1 antagonist, immune cells are better positioned to interact and based on the population shift, the interactions will be with stimulatory instead of suppressive APCs.

[0111] Given the dampening of immunosuppression and enhanced pro-inflammatory environment observed following treatment with the anti-IFNRl antibody, how blocking IFN-I signaling prior to infection contributes to control of persistent infection was determined. Although virus titers were initially increased in mice treated with the anti- IFNRl antibody (day 8), by 30 days, viremia was dramatically reduced compared to isotype treatment and many of the mice had already controlled infection (Fig. 3A).

Further, virus titers were decreased in multiple compartments, including the kidney (a life-long persistent reservoir of LCMV-C113) (Fig. 3B), thereby demonstrating the enhanced control of a persistent viral infection by blocking early IFN-I signaling.

Mirroring findings in IFNR1-/- mice, treatment with the anti-IFNRl antibody led to persistent infection with LCMV-Arm and accompanying T cell exhaustion in wild type mice (Fig. 6A and data not shown), thereby indicating the initial antiviral and immune stimulatory requirements for IFN-I to control acute viral infection. Together, these results highlight the duality of IFN-I during viral infection - acute IFN-I signals possess antiviral and immune stimulatory potential to promote clearance of infection, whereas sustained IFN-I signaling induces many of the dysfunctions associated with persistent viral infections and ultimately impedes viral clearance.

[0112] Effect of IFNR1 Antagonist on Cell-Mediated Immunity and Viral Clearance

[0113] Treatment with an IFNR1 antagonist, i.e., anti-IFNRl antibody, prior to infection was found to induce a dramatic numerical increase in many immune subsets 9 days after infection (i.e., at a time when virus was elevated in both isotype and anti-IFNRl treated mice), including DCs, macrophages, B cells, CD4 T cells, CD8 T cells and NK cells (Fig. 3C). Treatment with the anti-IFNRl antibody increased the total number and amount of functional virus-specific CD4⁺ T cells (including total IFNy and

polyfunctional cytokine producing cells; Fig. 3D), thereby indicating enhanced antiviral CD4 T cell activity. However, despite an overall increase in B cells and CD4 T cells, LCMV-specific antibody titers were not elevated in anti-IFNRl treated mice at day 9 or 30 after infection, although total IgG levels were increased early (Fig. 7A). Unlike virus- specific CD4⁺ T cells, virus-specific CD8⁺ T cell numbers and cytokine production were similar or slightly reduced when IFN-I signaling was blocked (Fig. 3D), thereby suggesting that virus-specific CD8⁺ T cell responses are not major contributors to the enhanced control of virus replication following treatment with an IFNR1 antagonist.

[0114] Based on the increase in NK cells, virus-specific CD4⁺ T cells and systemic IFNy levels (Fig. 3C, Fig. 3D, Fig. 6D), the role of each of these factors in accelerating virus clearance following treatment with an IFNR1 antagonist, i.e., anti-IFNRl antibody, was examined. Despite increased NK cell numbers, NK cell depletion did not affect viral clearance mediated by the anti-IFNRl antibody (Fig. 3E). On the other hand, CD4 depletion prior to infection abrogated the accelerated virus control engendered by treatment with the anti-IFNRl antibody (Fig. 3E), thereby implicating CD4⁺ T cells as targets of IFN-I mediated immunosuppression and key effectors of viral clearance following treatment with an IFNR1 antagonist.

[0115] To assess the role of increased IFNy in viral clearance facilitated by blocking

IFN-I signaling with an IFNR1 antagonist, mice were treated with anti-IFNRl antibody and/or IFNy antibody at the time of LCMV-C113 infection (i.e., day -1 to 6).

Interestingly, the accelerated clearance of persistent infection following treatment with the anti-IFNRl antibody was abrogated in mice co-treated with the anti-IFNy antibody (Fig. 3F). Of note, mice treated with the anti-IFNy antibody alone died about 35 days after infection, whereas mice receiving both the anti-IFNRl antibody and the anti-IFNy antibody survived and cleared the viral infection similar to untreated mice. Together, these results indicate that IFNR1 antagonists, e.g., anti-IFNRl antibodies, stimulate accelerated clearance of persistent viral infections through CD4⁺ T cell and IFNy dependent mechanisms. [0117] Effect of IFNR1 Antagonist on Viral Replication

[0118] Based on the sustained IFN-I signature and immune dysfunction through

persistent infection, whether blocking IFN-I signaling with an IFNR1 antagonist during an established persistent infections impacts virus replication was examined. Treatment with an IFNR1 antagonist, i.e., an anti-IFNRl antibody, beginning 25 days after infection accelerated control of persistent viral infection compared to isotype treatment (Fig. 4A). The enhanced control of infection occurred despite the initial increase in virus titers immediately after treatment with the anti-IFNRl antibody (Fig. 4A), thereby indicating that treatment with an IFNR1 antagonist facilitates a reduction in viral replication.

Further, treatment with the anti-IFNRl antibody in the midst of persistent viral infection reduced virus titers in multiple compartments (Fig. 4B). The natural control of persistent LCMV beginning about 50 days after infection prevented longer-term plasma analyses as all mice controlled viremia by day 60 after infection (not shown). Treatment with the anti-IFNRl antibody beginning 25 days after infection also reduced the IFN-I gene expression signature and decreased IL-10 and PDL1 levels (Fig. 4C, Fig. 4D), thereby demonstrating that IFN-I continues to be a key component of an immunologic surveillance system and stimulator of immunosuppression throughout persistent viral infection. Thus, blocking chronic IFN-I signaling via an IFNR1 antagonist in vivo reduces viral replication in persistent viral infections.

[0119] To the extent necessary to understand or complete the disclosure of the present invention, all publications, patents, and patent applications mentioned herein are expressly incorporated by reference therein to the same extent as though each were individually so incorporated.

[0120] Having thus described exemplary embodiments of the present invention, it should be noted by those skilled in the art that the within disclosures are exemplary only and that various other alternatives, adaptations, and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments as illustrated herein, but is only limited by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a persistent or chronic viral infection in a subject which comprises, consists essentially of, or consists of administering to the subject at least one inhibitor of Type I interferon receptor (IFNR) activation, preferably an IFNR antagonist, such as an IFNR1 antagonist.

2. A method of facilitating clearance of a virus which is the causative agent of a persistent or chronic infection in a subject which comprises, consists essentially of, or consists of

administering to the subject at least one inhibitor of Type I interferon receptor (IFNR) activation, preferably an IFNR antagonist, such as an IFNR1 antagonist.

3. A method of decreasing one or more immunosuppressive factors in a subject having a persistent or chronic viral infection which comprises, consists essentially of, or consists of administering to the subject at least one inhibitor of Type I interferon receptor (IFNR) activation, preferably an IFNR antagonist, such as an IFNR1 antagonist.

4. A method of increasing one or more cells associated with cell-mediated immunity in a subject having a persistent or chronic viral infection which comprises, consists essentially of, or consists of administering to the subject at least one inhibitor of Type I interferon receptor (IFNR) activation, preferably an IFNR antagonist, such as an IFNR1 antagonist.

5. A method of increasing the ratio of stimulatory to suppressive antigen-presenting cells in a subject having a persistent or chronic viral infection which comprises, consists essentially of, or consists of administering to the subject at least one inhibitor of Type I interferon receptor (IFNR) activation, preferably an IFNR antagonist, such as an IFNR1 antagonist.

6. A method of reducing viral replication in a subject having a persistent or chronic viral infection which comprises, consists essentially of, or consists of administering to the subject at least one inhibitor of Type I interferon receptor (IFNR) activation, preferably an IFNR antagonist, such as an IFNR1 antagonist.

7. The method according to any one of the preceding claims, which further comprises identifying the subject as having a high level of one or more immunosuppressive factors before administration of the at least one inhibitor of IFNR activation.

8. The method according to any one of the preceding claims, which further comprises monitoring the efficacy of the treatment with the at least one inhibitor of IFNR activation by assaying the amount of one or more immunosuppressive factors in the subject after

administration of the at least one inhibitor of IFNR activation.

9. The method according to any one of the preceding claims, which further comprises potentiating the IFNy receptor and/or administering to the subject an agonist of the IFNy receptor.

10. The method according to any one of the preceding claims, which further comprises administering to the subject a vaccine against the virus which is the causative agent of the persistent or chronic viral infection and/or an antagonist of an immunosuppressive cytokine.

11. The method according to any one of the preceding claims, wherein the at least one inhibitor of IFNR activation is administered in an effective amount or a therapeutically effective amount.

12. The method according to any one of the preceding claims, wherein the subject is human.

13. Use of at least one inhibitor of IFNR activation, preferably an IFNR antagonist, such as an IFNR1 antagonist, for the manufacture of a medicament for treating a persistent or chronic viral infection in a subject.

14. An inhibitor of IFNR activation, preferably an IFNR1 antagonist, such as an IFNR1 antagonist, for use in treating a persistent or chronic viral infection.

15. An inhibitor of IFNR activation, preferably an IFNR1 antagonist, such as an IFNR1 antagonist, for treating a persistent or chronic viral infection in a subject, wherein an effective amount of the inhibitor of IFNR activation is administered to the subject after one or more immunosuppressive factors have been induced by the virus which is the causative agent of the persistent or chronic viral infection.

16. The method according to any one of the preceding claims, wherein the persistent or chronic viral infection is caused by human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), or lymphocytic choriomeningitis virus (LCMV).

17. The method according to any one of the preceding claims, wherein the inhibitor of IFNR activation is an antibody, preferably a monoclonal antibody.