WO2011020094A1 - Use of synthetic single-stranded rna (ssrna) as a therapeutic agent to activate nod2-dependent immune response against viral infection - Google Patents

Abstract

Disclosed are methods, compositions, and kits for modifying an innate immune response in a subject, such as an innate immune response to a viral infection, employing single stranded RNA that can bind to a NOD2 polypeptide.

Description

DESCRIPTION

USE OF SYNTHETIC SINGLE-STRANDED RNA (SSRNA) AS A THERAPEUTIC AGENT TO ACTIVATE NOD2-DEPENDENT IMMUNE RESPONSE AGAINST

VIRAL INFECTION BACKGROUND OF THE INVENTION

The present application claims benefit of priority to U.S. Provisional Application Serial No. 61/234,105 filed August 14, 2009, the entire contents of which are hereby incorporated by reference.

This invention was made with government support under Grant No. AI069062 and Grant No. CAl 29246 awarded by the National Institutes of Health. The government has certain rights in the invention.

1. Field of the Invention

The present invention relates generally to the fields of immunology and infectious diseases. More particularly, it concerns methods and compositions for modulating an innate immune response in a subject involving single-stranded RNA and small molecules that bind to a NOD2 polypeptide.

2. Description of the Related Art

Innate immune antiviral responses are the first line of defense against virus infection (Kawai and Akira, 2006; Bose andBanerjee, 2003). Interferon-α/β (IFN) plays an important role during innate antiviral responses by activating the JAK-STAT signaling pathway (Stark et al., 1998). Virus-infected cells utilize pattern recognition receptors (PRRs) to recognize pathogen (virus) associated molecular patterns (PAMPs) and to trigger phosphorylation of the transcription factor interferon regulatory factor 3 (IRF3), which then translocates to the nucleus to transactivate IFN genes (Van Boxel-Dezaire et al, 2006). So far two classes of viral PRRs have been identified: the Toll-like receptors (TLRs) (Uematsu and Akira, 2007) and the RLH (RIG like helicases) receptors such as RIGI and Mda5 (O'Neill, 2006).

A third class of PRR includes members of the nucleotide binding domain (NBD) and leucine-rich-region (LRR) containing family (known as NLRs) of cytoplasmic proteins; these proteins respond to bacterial PAMPs to activate NF-κB and MAPK pathways (Basler and Garcia-Sastre, 2007; Martinon and Tschopp, 2005; Fritz et al., 2006). For example, the NLR N0D2 detects bacterial PAMPs including muramyl dipeptide (Kanneganti et al., 2007). However, to date, no NLRs were reported to respond to virus-specific PAMPs and activate an antiviral response.

Recently it was demonstrated that the NLR family member NLRXl interacts with IPS-I to negatively regulate the IFN pathway (Chen et al., 2009), and induces reactive oxygen species (ROS) formation (Franchi et al., 2009). Thus, members of NLR family of proteins may modulate (either positively or negatively) the host antiviral apparatus. In addition, N0D2 facilitates production of human β-defensin-2 (HBD2) after MDP stimulation, and HBD2 is also upregulated in human respiratory syncytial virus (RSV) infected cells (Moore et al., 2008; Tattoli et al., 2008).

Thus, there is the need for improved methods to modulate an innate immune response in a subject, such as for the purpose of treating and preventing viral infection. SUMMARY OF THE INVENTION

The present invention is based in part on the finding that nucleotide-binding oligomerization domain-2 protein (NOD2) could be activated to induce an innate immune response by single-stranded RNA (ssRNA). NOD2 has been found to act as a "susceptibility gene" during host defense against infections, such as infections by ssRNA viruses. Furthermore, lack of NOD2 gene confers an exaggerated and deadly lung disease following respiratory virus infection.

The present invention in part concerns methods of inhibiting, modulating, treating, or preventing a viral infection or the symptoms thereof in a subject, involving administering to a subject with a viral infection an effective amount of a pharmaceutical composition that includes: (a) a recombinant ssRNA or a small molecule, wherein the ssRNA or the small molecule binds to a NOD2 protein; and (b) a pharmaceutically acceptable carrier, wherein the viral infection is inhibited, modulated, treated, or prevented. The invention also concerns methods of inducing an innate immune response in a subject, involving administering to the subject a pharmaceutical composition that includes (a) a recombinant ssRNA or a small molecule, wherein said ssRNA or small molecule binds to a NOD2 protein; and (b) a pharmaceutically acceptable carrier, wherein the innate immune response is induced.

In some embodiments, the pharmaceutical composition consists essentially of (a) a recombinant ssRNA or a small molecule; and (b) one or more pharmaceutically acceptable carriers. In still further embodiments, the pharmaceutical composition consists of (a) a recombinant ssRNA or a small molecule; and (b) one or more pharmaceutically acceptable carriers.

The subject may be any subject, such as a mammal. For example, the mammal may be a human, a primate, a horse, a cow, a sheep, a goat, a rabbit, a dog, a cat, or a mouse. In particular embodiments, the mammal is a human. In more particular embodiments, the human is a patient with a congenital heart disease, a patient over age 60, a patient that has received an organ transplant, or a patient in need of interferon β or interferon α.

In particular embodiments, the subject is a subject with a viral infection. In specific embodiments, the viral infection is an infection due to a single-stranded RNA virus. For instance, the viral infection may be a respiratory virus infection. Non-limiting examples of viral infections due to single-stranded RNA viruses include infections due to a paramyxovirus, a rhabdovirus, a filovirus and an orthomyxovirus. Some examples of paramyxovirus include respiratory syncytial virus (RSV), Sendai virus, parainfluenza viruses (HPIV-I, HPIV -2, HPIV-3, or HPIV-4), measles virus, mumps virus, metapneumovirus, Hendra virus, Nipah virus. Examples of filovirus include Ebola virus, or Marburg virus. Examples of orthomyxovirus include influenza A virus, an influenza B virus, or an influenza C virus. Examples of rhabdovirus include rabies virus or vesicular stomatitis virus.

The ssRNA may be of any source, including natural sources and synthetic sources. It may be an RNA of any length, but in particular embodiments it is 10 to 30,000 nucleobases in length. In more limiting aspects of the invention, the ssRNA is 10 to 3,000 nucleobases in length, 10 to 300 nucleobases in length, or 10 to 100 nucleobases in length. The recombinant single-stranded RNA may be a ssRNA molecule derived from the genome of a ssRNA virus, such as any of the aforementioned viruses. The ssRNA may bind to any region of the NOD2 polypeptide but in particular embodiments it binds to the NBD domain of NOD2.

Composition that is administered to the subject may include any pharmaceutically acceptable carrier. Many such carriers are known in the art. Non-limiting examples include water, an alcohol, dimethylsulfoxide, or a lipid.

The composition is administered to the subject using any method known to those of ordinary skill in the art. For example, the composition may be administered by aerosol, by spray, intravenously, intradermally, intraarterially, intramuscularly, intrathecally, intratracheally, subcutaneously, orally, topically, intraperitoneally, via a drug-delivery device, or via a nanoparticle. In some embodiments, the nanoparticle is coated with the composition, or the composition components (ssRNA) are incorporated within the substance of the nanoparticle.

The ssRNA or small molecule may be comprised in a vector. For example, the vector may be a virus, a liposome, a dendritic cell, or other cell.

The composition may optionally include any additional agent. For example, the additional agent may be a secondary antiviral agent or an adjuvant. Non-limiting examples of secondary antiviral agents include oseltamivir and zanamivir. Non-limiting examples of adjuvants include aluminum phosphate or aluminum hydroxide. Other examples are discussed in the specification below.

In some aspects, the method further involves identifying a subject in need of inhibition, modulation, treatment, or prevention of a viral infection or the symptoms thereof. Examples of such subjects include neonates, patients with congenital heart disease, transplant patients, and any patient in need of an interferon.

Other aspects of the invention concern pharmaceutical compositions that include (a) a ssRNA or small molecule, wherein said RNA or small molecule binds to a nucleotide-binding oligomerization domain-2 (NOD2) protein; and (b) a pharmaceutically acceptable carrier. The ssRNA may be recombinant. It may be of any source or length, such as discussed above. In particular embodiments, the ssRNA is 10 to 30,000 nucleobases in length. In more particular embodiments, it is 10 to 3,000 nucleobases in length. The ssRNA may be derived from the genome of a single-stranded RNA virus. The composition may include any pharmaceutically acceptable carrier known to those of ordinary skill in the art. For example, the carrier may include water, an alcohol, dimethylsulfoxide, or a lipid. The composition may include any other component of a vaccine. For example, the composition may include one or more antigens or other agents capable of inducing an immune response in a subject. The composition may optionally include one or more secondary antiviral agents. Non- limiting examples include oseltamivir or zanamivir. The composition may furthe include nanoparticles. For example, the nanoparticular may be coated with a composition that includes the ssRNA as set forth herein.

Kits are also contemplated as part of the present invention. The kits may include one or more sealed vials, wherein (i) at least one vial includes a single-stranded RNA, wherein the ssRNA binds to a nucleotide-binding oligomerization domain-2 (NOD2) protein. The kit may optionally include other components, such as a syringe, a vial, or instructions for use. The kit may include one or more vials that includes a pharmaceutically acceptable carrier. The kit may include one or more vials that include a pharmaceutical composition of the present invention.

It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention.

The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternative are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or."

Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device and/or method being employed to determine the value.

As used herein the specification, "a" or "an" may mean one or more, unless clearly indicated otherwise. As used herein in the claim(s), when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one. As used herein "another" may mean at least a second or more.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES The following figures form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. IA, IB, 1C, ID, IE. ssRNA activates NOD2. (a) Activation of an IRF3 luciferase reporter in untreated (UT), ssRNA, and CpG DNA treated (6h) 293 cells transfected with pcDNA, human HA-NODl or human HA-N0D2. (b) RT-PCR analysis of N0D2 expression in A549 cells left untreated or stimulated for the indicated time periods with ssRNA. (c) Activation of IFN-β luciferase reporter in A549 cells transfected with either control siRNA or N0D2 siRNA and left untreated or stimulated (6h) with ssRNA. The luciferase assay results are presented as mean ± s.d. from three independent experiments. (d,e) Bone marrow-derived macrophages (BMM) or mouse embryonic fibroblasts (MEFs) were isolated from wild-type and N0D2-K0 mice and left untreated or stimulated with ssRNA for the indicated time periods. IFN-β production was measured by ELISA. Values represent the mean ± s.d. of three independent experiments.

FIG. 2A, 2B, 2C, 2D. Activation of antiviral response by N0D2 in virus infected cells. (a,b) Activation of IRF3 and IFN-β luciferase reporter genes in mock infected and RSV infected 293 cells expressing pcDNA, HA-NODl, or HA-N0D2. In (a), luciferase was measured 6 h post-infection (pi.) and cells were infected with ultraviolet radiation (UV) treated or UV untreated RSV as indicated, (c) Plaque assay of VSV infectivity in 293 cells expressing pcDNA, HA-NODl or HA-N0D2. Crystal violet staining and VSV titer expressed as pfu per ml are shown, (d) RSV infectivity in 293 cells expressing pcDNA, HA-NODl or HA-N0D2. 100% infectivity represents the viral titer from cells expressing pcDNA. The plaque assay values represent the mean ± s.d. from three independent experiments. The luciferase assay results are presented as mean ± s.d. from three independent experiments.

FIG. 3A, 3B, 3C, 3D, 3E, 3F. N0D2 is required for IFN production, (a) RT-PCR analysis of N0D2 expression in mock and RSV infected A549 cells, (b) Activation of IRF3 luciferase reporter in mock and RSV infected (hours post-infection, p.i) A549 cells transfected with either control siRNA or N0D2 siRNA. The luciferase assay results are presented as mean ± s.d. from three independent experiments, (c) IFN-β production from mock and RSV infected primary normal human bronchial epithelial (NHBE) cells transfected with either control siRNA or N0D2 siRNA. (d-f) IFN-β production from mock and RSV infected alveolar macrophages (d), BMM (e) and MEFs (f) isolated from wild-type (WT) or N0D2-K0 mice. IFN-β was measured by ELISA and each value represents the mean ± s.d. from three independent experiments.

FIG. 4A, 4B, 4C, 4D, 4E. Activation of N0D2 by viral ssRNA genome, (a) Activation of IRF3 luciferase reporter in 293 cells expressing pcDNA, HA-NODl, HA- N0D2 that were left untreated (UT) or stimulated with RSV ssRNA genome (viral-ssRNA). Where indicated viral-ssRNA was treated with RNAse. The luciferase assay results are presented as mean ± s.d. from three independent experiments. (b,c) IFN-β production from BMM (b) and MEFs (c) isolated from wild-type (WT) or N0D2-K0 mice that were left untreated or stimulated with viral-ssRNA. IFN-β was measured by ELISA and each value represents the mean ± s.d. from three independent experiments, (d) 293 cells were transfected with pcDNA or HA-NOD2 and were mock infected or infected with RSV. At 4h or 8h postinfection, NOD2 was immunoprecipitated with HA-agarose and bound RNA was amplified using primers specific for GAPDH or RSV nucleocapsid (N) protein. The amplified products were analyzed on the agarose gel. (e) RSV ssRNA genome and total cellular mRNA was incubated with HA-N0D2 bound to HA-agarose beads. Bound RNA was amplified using the primers in (d). The amplified products were analyzed on the agarose gel. Total cellular mRNA amplified with GAPDH specific primers served as a positive control.

FIG. 5A, 5B, 5C. Role of MAVS during NOD2-mediated activation of the antiviral pathway, (a) IFN-β production from mock and RSV infected primary normal human bronchial epithelial (NHBE) cells transfected with either control siRNA, NOD2 siRNA or MAVS siRNA. (b) IFN-β production from mock and RSV infected MEFs isolated from wild- type (WT), N0D2-K0 or MAVS-KO mice, (c) IFN-β production from MEFs left untreated (UT) or stimulated with synthetic or viral (RSV) ssRNA. IFN-β was measured by ELISA and each value represents the mean ± s.d. from three independent experiments.

FIG. 6A, 6B, 6C, 6D, 6E. Interaction of MAVS with N0D2. (a) 293 cells were transfected with pcDNA, HA-N0D2 and-or GFP-MAVS and mock infected or infected with RSV. Lysates were immunoprecipitated with anti-HA agarose beads and bound proteins were immunoblotted with anti-GFP. (b) RSV-infected (4h) 293 cells co-expressing GFP-MAVS (green) and HA-N0D2 (red) were imaged using confocal microscopy. (c,d) Mock or RSV- infected (6h) A549 cells (c) or RSV-infected (4h) NHBE cells (d) were stained with anti- NOD2 and anti-MAVS and imaged by confocal microscopy to detect endogenous NOD2 and MAVS. (e) Double-labeled confocal immuno-fluorescent analysis (to detect endogenous NOD2 and MAVSof infected (4h) primary normal human bronchial epithelial (NHBE) cells with NOD2 (red) AND MAVS (green) antibodies. The merged yellow image shows co- localization of MAVS with NOD2 in infected NHBE cells.

FIG. 7A, 7B, 7C, 7E. NBD and LRR domains of NOD2 are essential for interaction with MAVS. (a) Schematic showing the various NOD2 constructs with deletion in specific domains. WT, wild-type; ΔCARD (NOD2 mutant lacking both CARD domains), ΔNBD (NOD2 mutant lacking the NBD domain) and ΔLRR (NOD2 mutant lacking the LRR domain), (b) 293 cells expressing various His-Myc tagged NOD2 constructs or pcDNA along with GFP- MAVS were lysed and lysates were incubated with Nickel-agarose (Ni-agarose). Following washing of the beads, the bound proteins were subjected to immunoblot analysis with anti-GFP. P; precipitation, (c) Lysates from cells in (b) were immunoblotted with anti- GFP to examine expression of GFP- MAVS. The first lane shows cells trans fected with pcDNA only, (d) Cell lysates obtain from 293 cells co-expressing WT and NOD2 deletion mutants (his-mys-NOD2-constructs) along with GFP-MAVS was incubated with Ni-agarose, followed by Western blotting with myc antibody (to detect his-myc tagged N0D2 protein constructs bound to the Ni-agarose beads). Please note that the WT and deleted version of N0D2 proteins are indicated with arrowheads.

FIG. 8 A, 8B, 8C, 8D, 8E, 8F. N0D2 is essential for host defense against virus infection, (a) IFN-β concentrations in the bronchoalveolar lavage (BAL) of RSV infected wild-type (WT) and N0D2-K0 mice. Values (n=four mice per group) are mean ± s.e.m. P < .05, by t-test when data were normally distributed, or by Mann- Whitney Rank sum test when data were not normally distributed, (b) RSV titer in the BAL (3d post-infection) of WT and N0D2-K0 mice. Values are mean ± s.e.m. P < .05, by t-test when data normally distributed, or by Mann- Whitney Rank sum test when data were not normally distributed, (c) H&E staining of lung sections obtained from RSV -infected WT and N0D2-K0 mice, (d) Neutrophil sequestration in lungs of RSV infected (2d post-infection) WT and N0D2-K0 mice was assessed by myeloperoxidase (MPO) activity assay with total lung homogenate. MPO activity is shown as percentage increase over enzyme activity in mock infected mice. Results are mean ± s.e., n=5, P < 0.05. (e) TUNEL staining of lung sections obtained from RSV infected WT and N0D2-K0 mice, (f) Survival of WT and N0D2-K0 mice infected with RSV (5x108 pfu per animal). P > 0.02 between N0D2-K0 and WT mice as deduced by Wilcoxon test.

FIG. 9A, 9B, 9C, 9D. Induction of IFN-β expression by ssRNA. (a) RT-PCR analysis of IFN- β expression in pcDNA, human HA-NODl and human HA-N0D2 transfected 293 cells treated with ssRNA. UT; untreated, (b) RT-PCR analysis of interferon- β (IFN- β) expression in UT, ssRNA and CpG treated 293 cells transfected with either pcDNA or HA- N0D2. (c) RT-PCR analysis of NODl and N0D2 expression in 293 cells transfected with either HA-NODl or HA-N0D2. (d) Activation of NF-κB-luciferase in non-transfected (NT), pcDNA, HA-NODl and HA-N0D2 transfected 293 cells that were either untreated (UT) or treated with MDP (10 μg/ml for 12h) or iE-DAP (10 μg/ml for 24h). The luciferase assay results are presented as mean ± s.d. from three independent experiments.

FIG. 1OA, 1OB, 1OC, 1OD, 1OE. N0D2 is required for antiviral response, (a) Efficiency of N0D2 specific siRNA was examined by analyzing N0D2 expression (by RT- PCR) in control or N0D2 siRNA transfected A549 cells +/- ssRNA treatment (6h). (b) Activation of IRF3-luciferase in untreated (UT) and ssRNA treated (6h) A549 cells transfected with either control siRNA or N0D2 siRNA. (c) IFN- β production from UT and po Iy-IC treated (10 μg/ml for 18h) bone marrow derived macrophages (BMM) and mouse embryo fibroblasts (MEFs) isolated from wild type (WT) or NOD2 knock-out (KO) mice. IFN- β was measured by ELISA assay and each value represents the mean ± s.d. from three independent experiments, (d) IFN-β concentrations in the serum and lung homogenate of WT and N0D2-K0 mice following poly-IC administration by intravenous or intranasal route, respectively. IFN-β values (n=8 mice/group) are presented as mean ± s.e.m. p < .05, by t-test when data were normally distributed, or by Mann- Whitney Rank sum test when data were not normally distributed, (e) Activation of IRF3-luciferase in 293 cells (expressing either pcDNA or HA-NOD2) infected (6h) with RSV in the presence of either control antibody (Con Ab) or RSV fusion (F protein) protein specific neutralizing antibody (F Ab). The luciferase assay results are presented as mean ± s.d. from three independent experiments.

FIG. HA, HB. NOD2 mediated antiviral response following infection with ssRNA viruses, (a) Activation of IRF3 -luciferase in human parainfluenza virus type-3 (HPIV-3) (0.5 MOI) or vesicular stomatitis virus (VSV) (0.5 MOI) infected (at indicated post-infection or p.i time-periods) 293 cells transfected with either pcDNA or HA-N0D2. (b) Activation of IRF3 -luciferase in vaccinia virus or RSV infected 293 cells transfected with either pcDNA or HA-N0D2. The luciferase assay results are presented as mean ± s.d. from three independent experiments.

FIG. 12A, 12B, 12C, 12D, 12E, 12F, 12G. N0D2 but not RIGI is required for antiviral response during early RSV infection, (a) Efficiency of N0D2 specific siRNA was examined by analyzing N0D2 expression (by RT-PCR) in control or N0D2 siRNA transfected A549 cells infected with RSV for 4h. (b) Activation of IFN-β-luciferase reporter gene in control siRNA or N0D2 siRNA trasfected A549 cells infected with RSV for 6h. The luciferase assay results are presented as mean ± s.d. from three independent experiments, (c) RT-PCR analysis of RIGI expression in RSV infected (4h - 16h post-infection) A549 cells, (d) Efficiency of RIGI specific siRNA was examined by analyzing RIGI expression in control siRNA or RIGI siRNA transfected A549 cells infected with RSV for 16h or 2Oh. (e) RT-PCR analysis of IFN-β expression in control siRNA or RIGI specific siRNA trasfected A549 cells infected with RSV for 2h-6h. (f) RT-PCR analysis of N0D2 expression in MEFs infected with RSV for Oh- 12h. (g) RT-PCR analysis of RIGI expression in MEFs infected with RSV for 0h-12h.

FIG. 13. N0D2 expression in NHBE cells and silencing of N0D2 in NHBE cells. Efficiency of N0D2 specific siRNA was examined by analyzing N0D2 expression in control siRNA or N0D2 siRNA transfected NHBE cells infected with RSV.

FIG. 14. N0D2 is required for IFN production from influenza A [mouse-adapted influenza A/PR/8/34 (HlNl) virus] virus infected cells, (a) IFN-β production from mock and influenza A virus (Flu) infected (infected at 1 MOI for 4h and 8h) mouse embryo fibroblasts (MEFs) isolated from wild type (WT) and N0D2 knock-out (KO) mice, (b) IFN-β production from mock and Flu infected (infected at 1 MOI for 4h) bone marrow derived macrophages (BMM) isolated from WT and N0D2-K0 mice. Amount of IFN-β was measured by ELISA assay and each value represents the mean ± s.d. from three independent experiments.

FIG. 15. MAVS silencing efficiency in NHBE cells. Efficiency of MAVS specific siRNA was examined by analyzing MAVS expression (by RT-PCR) in control siRNA or MAVS siRNA transfected NHBE cells infected with RSV for 4h.

FIG. 16. MAVS is required for N0D2 mediated IFN production from influenza A [mouse-adapted influenza A/Pi?/8/34 (HlNl) virus] virus infected cells. IFN- production from mock and influenza A virus (Flu) infected (infected at 1 MOI for 4h) mouse embryo fibroblasts (MEFs) isolated from WT, N0D2-K0 and MAVS-KO mice. Amount of IFN- was measured by ELISA assay and each value represents the mean ± s.d. from three independent experiments.

FIG. 17A, 17B, 17C, 17D, 17E, 17F. Interaction of N0D2 with MAVS. (a) Mitochondrial and total cellular extract prepared from RSV infected (4h post-infection) 293 cells expressing HA-N0D2 was subjected to immunob lotting with anti-HA (to detect N0D2) and β-actin (to ascertain the purity of mitochondrial extract) antibody, (b) The band corresponding to the HA-N0D2 protein [in mitochondrial (Mito) and total cell (Total) extracts] from the immunob lot analysis in FIG. 17A was quantified by GeneTools program (Syngene) and the values (corresponding to the band intensities) are presented as a bar graph. The values are also presented to demonstrate the percentage of total N0D2 protein that is localized in mitochondria in mock (uninfected) cells vs. infected cells, (c) Confocal immuno- fluorescent analysis of RSV infected (at 3h post-infection) A549 cells following labeling with N0D2 (green) antibody (N0D2 antibody was purchased from Cayman Chemical company) and Mito-tracker (red, to detect mitochondria) (Mito-tracker Red was purchased from Invitrogen). The merged yellow image shows co-localization of NOD2 with mitochondria in infected cells, (d) Expression of GFP-MAVS in the total lysate of the samples used in FIG. 17A was monitored by immunob lotting with GFP antibody, (e) For the experiment shown in Fig. 6A, expression of GFP- MAVS and HA-N0D2 in the cell lysate (left panel) and amount of HA-N0D2 bound to anti-HA-agarose beads (right panel) was monitored by immunoblotting total lysates (25 μg) and anti-HA-agarose bound proteins with GFP and HA antibodies, (f) Left panel: Co-immunoprecipitation analysis was performed by immuno- precipitating (IP) RSV infected 293 cell (co-expressing HA-NODl and GFP-MAVS) lysate with anti-HA-agarose and immunoblotting with anti-GFP antibody. Expression of GFP- MAVS in the cell lysate (by immunoblotting 25 μg of total cellular lysate with anti-GFP antibody) is also shown in the lower panel. Right panel: Expression of HA-NODl in the cell lysate (25 μg total lysate) and amount of HA-NODl bound to anti-HA-agarose beads was examined by immunoblotting with anti-HA antibody.

FIG. 18A, 18B, 18C. IFN induction by MDP. (a) Activation of IRF3-luciferase reporter gene in ssRNA (1 μg/ml, 1Oh), MDP (10 μg/ml, 24h) treated and RSV infected (6h) 293 cells expressing either pcDNA, HA-N0D2 or HA-NODl. Control; untreated or mock infected cells. The luciferase assay results are presented as mean ± s.d. from three independent experiments, (b) IFN-β production from control, ssRNA (1 μg/ml, 24h), MDP (15 μg/ml, 24h) treated and RSV infected (6h) bone marrow derived macrophages (BMM) isolated from wild type (WT) and NOD2-knock-out (KO) mice, (c) IFN-β production from control, MDP treated (15 μg/ml, 24h) and RSV infected (6h) NHBE cells. Amount of IFN-β was measured by ELISA and each value represents the mean ± s.d. from three independent experiments.

FIG. 19A, 19B, 19C, 19D. Role of RICK during activation of antiviral response by

N0D2. (a) Endogenous RICK and HA-N0D2 expression in RSV infected (4h post-infection) HA-N0D2 expressing 293 cells transfected with either control shRNA or RICK shRNA. NT; non-transfected cells that is not expressing HA-N0D2. Please note that shRNA was utilized to silence expression of endogenous RICK in 293 cells, (b) RT-PCR analysis of IFN-β expression in mock and RSV infected 293 cells (expressing HA-N0D2) transfected with control shRNA or RICK shRNA. The first lane of the gel shows that 293 cells transfected with RICK shRNA in the absence of HA-N0D2 expression failed to induce IFN-β following RSV infection, (c) Efficiency of RICK specific shRNA was examined by analyzing endogenous RICK expression (by RT-PCR) in control or RICK shRNA transfected NHBE cells infected with RSV. (d) IFN-β production from mock and RSV infected NHBE cells transfected with either control shRNA, NOD2 shRNA or RICK shRNA. Amount of IFN-β was measured by ELISA and each value represents the mean ± s.d. from three independent experiments.

FIG. 2OA, 2OB. Interactions of RICK and RIGI with MAVS and NOD2. (a) Left panel: Co-immunoprecipitation analysis was performed by immuno-precipitating (IP) 293 cell [co-expressing HA-NOD2/GFP-MAVS, HA-RIGI/GFP-MAVS, HA-NOD2/GFP (empty GFP expressing vector), HA-RIGI/GFP)] lysate with anti-HA antibody (covalently conjugated to agarose beads) and immunob lotting with anti-GFP antibody. In the lower panel, amount of HA-NOD2 and HA-RIGI bound to anti-HA-agarose beads is also shown by immunob lotting the HA-agarose bound proteins with anti-HA antibody. Right panel: Expression of GFP- MAVS and GFP in the cell lysate was analyzed by immunob lotting 12 μg of total cellular lysate with anti-GFP antibody, (b) Left panel: Co-immunoprecipitation analysis was performed by immuno-precipitating (IP) 293 cell [co-expressing HA- NOD2/GFP-MAVS, HA-NOD2/GFP-RICK, HA-NOD2/GFP (empty GFP expressing vector) +/- RSV infection or MDP treatment (20 μg/ml, 3h)] lysate with anti-HA antibody (covalently conjugated to agarose beads) and immunoblotting with anti-GFP antibody. In the lower panel, amount of HA-NOD2 bound to anti-HA-agarose beads is also shown by immunoblotting the HA-agarose bound proteins with anti-HA antibody. Right panel: Expression of GFP-MAVS, GFP-RICK and GFP in the cell lysate was analyzed by immunoblotting 12 μg of total cellular lysate with anti-GFP antibody.

FIG. 21A, 21B, 21C, 21D. Role of NF-κB in NOD2 mediated IFN gene expression, (a) NHBE cells transfected with NF-κB luciferase plasmid were infected with non-infectious recombinant adenoviruses expressing GFP (Ad-GFP) or IκB-super-repressor (Ad-SR). The GFP and IKB-SR expressing cells were infected with RSV and luciferase activity was measured at 16h post-infection. The luciferase assay results are presented as mean ± s.d. from three independent experiments. Please note that while RSV activated NF -KB in control GFP expressing cells, expression of IKB-SR led to drastic reduction in NF -KB activity, (b) RT-PCR analysis of IFN-β expression in GFP or IKB-SR expressing NHBE cells infected with RSV for 4h. (c) RT-PCR analysis of TNF-α expression in GFP or IKB-SR expressing NHBE cells infected with RSV for 16h. (d) The band corresponding to IFN and TNF from the RT-PCR analysis in Supplemental This figure was quantified by GeneTools program (Syngene) and the values (corresponding to the band intensities) are presented as a bar graph. 100% represents band intensity from RSV infected cells expressing control GFP.

FIG. 22A, 22B. LRR and NBD domains of NOD2 are required for induction of IFN response, (a) Cell lysates obtained from 293 cells co-expressing wild-type (WT) and N0D2 deletion mutants (his-myc-NOD2 constructs) along with GFP-MAVS was incubated with Ni- agarose, followed by immunoblotting with myc antibody (to detect his-myc tagged NOD2 protein constructs bound to the Ni-agarose beads). Please note that the WT and deleted version of N0D2 proteins are indicated with arrowheads. P; precipitated, (b) Activation of IRF3-luciferase reporter gene in mock infected and RSV infected (6h post-infection) 293 cells expressing pcDNA, WT N0D2 and various NOD2 deletion mutants [ΔCARD (NOD2 mutant lacking both CARD domains), ΔNBD (N0D2 mutant lacking the NBD domain) and ΔLRR (NOD2 mutant lacking the LRR domain)] . The luciferase assay results are presented as mean ± s.d. from three independent experiments.

FIG. 23A, 23B, 23C, 23D. NOD2 expression in RSV infected mice lungs and pro- inflammatory cytokine and chemokine production from RSV infected wild-type (WT) and

N0D2-K0 mice, (a) RT-PCR analysis of murine NOD2 expression in lungs following infection of wild type mice with RSV (5 X 106 pfu/animal) for Id and 4d. Concentrations of

TNF-α (b), IL-10 (c) and RANTES (d) were measured in bronchoalveolar (BAL) specimens at Id and 6d post-infection by FACS analysis using the BD Cytometric mouse cytokine/chemokine Bead Array kit (BD BioSciences). The cytokine values (n=4 mice/group) are presented as mean ± s.e.m. p < .05, by t-test when data were normally distributed, or by

Mann- Whitney Rank sum test when data were not normally distributed.

FIG. 24. Loss of body weight of RSV infected mice. Body weight of RSV infected

(5X106 pfu/animal) wild-type (WT) (n=6 mice/day) and NOD2-KO (n=6 mice/day) mice during the course of infection. Results are expressed as the % change in body weight from the baseline (uninfected animals) and the results represent mean ± s.e.m. (by t-test p < 0.05, WT vs. NOD2-KO mice).

FIG. 25. NOD2 is required for IFN production during early influenza A virus infection of mice. IFN-β concentrations in the bronchoalveolar lavage (BAL) of influenza A [mouse-adapted influenza A/PR/8/34 (HlNl) virus] infected (1 X 105 pfu/mouse) WT and

NOD2 KO mice were measured at 2d post-infection. IFN-β was measured by ELISA and each value (n=5 mice/group) represents mean ± s.e.m. p < .05, by t-test when data were normally distributed, or by Mann- Whitney Rank sum test when data were not normally distributed.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS The present invention is in part based on the finding that NOD2 plays an important role in host antiviral defense mechanisms. The present inventors have found that NOD2 can function as a cytopasmic viral pattern recognition receptor by triggering an antiviral response via activation of interferon regulatory factor-3 9IRF3 and interferon-β. N0D2 -deficient mice fail to produce IFN efficiently and are highly susceptible to respiratory RNA virus such as human respiratory syncytial virus and influenza A virus, leading to enhanced viral pathogenesis and disease progression.

A. Nucleic Acids and Single-Stranded RNA

Various aspects of the present invention require polynucleotides encoding a N0D2 polypeptide or N0D2 polypeptide equivalent. The polynucleotide may be a nucleic acid segment encoding a N0D2 polypeptide as set forth above. Other aspects of the present invention include methods and compositions employing a single-stranded RNA that binds to a N0D2 polypeptide or polypeptide equivalent.

The polynucleotides may be derived from any source known to those of ordinary skill in the art. For example, the polynucleotide may be synthesized using any method known to those of ordinary skill in the art or obtained from natural sources. Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods.

"Nucleic acid" or "polynucleotide" used herein may mean at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof. A single strand provides a probe that may hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions. Nucleic acids may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. A single-stranded sequence may include a portion of sequence that is double stranded, such as a hairpin sequence.

A nucleic acid will generally contain phosphodiester bonds, although nucleic acid analogs may be included that may have at least one different linkage, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite linkages and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, which are incorporated by reference.

Nucleic acids containing one or more non-naturally occurring or modified nucleotides are also included within one definition of nucleic acids. The modified nucleotide analog may be located for example at the 5'-end and/or the 3'-end of the nucleic acid molecule. Representative examples of nucleotide analogs may be selected from sugar- or backbone- modified ribonucleotides. It should be noted, however, that also nucleobase-modified ribonucleotides, i.e. ribonucleotides, containing a non-naturally occurring nucleobase instead of a naturally occurring nucleobase such as uridines or cytidines modified at the 5 -position, e.g. 5-(2-amino)propyl uridine, 5-bromo uridine; adenosines and guanosines modified at the 8-position, e.g. 8-bromo guanosine; deaza nucleotides, e.g. 7-deaza-adenosine; O- and N- alkylated nucleotides, e.g. N6-methyl adenosine are suitable. The 2'-OH-group may be replaced by a group selected from H, OR, R, halo, SH, SR, NH.sub.2, NHR, NR.sub.2 or CN, wherein R is Csub.l-C.sub.6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I. Modified nucleotides also include nucleotides conjugated with cholesterol through, e.g., a hydroxyprolinol linkage as described in Krutzfeldt et al. (2005); Soutschek et al. (2004); and U.S. Patent Publication No. 20050107325, which are incorporated herein by reference. Modified nucleotides and nucleic acids may also include locked nucleic acids (LNA), as described in U.S. Patent No. 20020115080, which is incorporated herein by reference. Additional modified nucleotides and nucleic acids are described in U.S. Patent Publication Nos. 20050182005, which is incorporated herein by reference. Modifications of the ribose- phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments, to enhance diffusion across cell membranes, or as probes on a biochip.

Mixtures of naturally occurring nucleic acids and analogs may be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made. It may be advantageous to combine portions of the genomic DNA with cDNA or synthetic sequences to generate specific constructs. For example, where an intron is desired in the ultimate construct, a genomic clone will need to be used. Introns may be derived from other genes. The cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.

In some embodiments, the polynucleotide encoding a N0D2 polypeptide or polypeptide equivalent as set forth above is operatively coupled to a promoter. "Promoter" as used herein may mean a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. A promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial expression and/or temporal expression of same. A promoter may also comprise distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A promoter may be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter may regulate the expression of a gene component constitutively, or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter and the CMV IE promoter.

Within certain embodiments expression vectors are employed to express a nucleic acid of interest, such as a miRNA that encodes a NOD2 polypeptide as set forth herein. Expression requires that appropriate signals be provided in the vectors, and which include various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells. Elements designed to optimize messenger RNA stability and translatability in host cells also are defined. The conditions for the use of a number of dominant drug selection markers for establishing permanent, stable cell clones expressing the products are also provided, as is an element that links expression of the drug selection markers to expression of the polypeptide.

There are a number of ways in which expression vectors may introduced into cells. In certain embodiments of the invention, the expression construct comprises a virus or engineered construct derived from a viral genome. One of the preferred methods for in vivo delivery involves the use of an adenovirus expression vector. "Adenovirus expression vector" is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express an antisense polynucleotide that has been cloned therein. In this context, expression does not require that the gene product be synthesized. Other examples of vectors include retroviruses, lentiviruses, and so forth.

Several non-viral methods for the transfer of expression constructs into cultured mammalian cells also are contemplated by the present invention. These include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al, 1990) DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al, 1986; Potter et al, 1984), direct microinjection (Harland and Weintraub, 1985), DNA- loaded liposomes (Nicolau and Sene, 1982; Fraley et al., 1979) and lipofectamine-DNA complexes, cell sonication (Fechheimer et al., 1987), gene bombardment using high velocity microprojectiles (Yang et al., 1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use.

In a further embodiment of the invention, the expression construct may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.

Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful. Wong et al., (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells. Nicolau et al., (1987) accomplished successful liposome-mediated gene transfer in rats after intravenous injection.

Other expression constructs which can be employed to deliver a nucleic acid encoding a particular gene into cells are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993).

In other embodiments, the delivery vehicle may comprise a ligand and a liposome. For example, Nicolau et al. (1987) employed lactosyl-ceramide, a galactose-terminal asialganglioside, incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes. Thus, it is feasible that a nucleic acid encoding a particular gene also may be specifically delivered into a cell type by any number of receptor-ligand systems with or without liposomes. For example, epidermal growth factor (EGF) may be used as the receptor for mediated delivery of a nucleic acid into cells that exhibit upregulation of EGF receptor. Mannose can be used to target the mannose receptor on liver cells. Also, antibodies to CD5 (CLL), CD22 (lymphoma), CD25 (T-cell leukemia) and MAA (melanoma) can similarly be used as targeting moieties.

In a particular example, the oligonucleotide may be administered in combination with a cationic lipid. Examples of cationic lipids include, but are not limited to, lipofectin,

DOTMA, DOPE, and DOTAP. The publication of WO/0071096, which is specifically incorporated by reference, describes different formulations, such as a DOTAP: cholesterol or cholesterol derivative formulation that can effectively be used for gene therapy.

B. Antigens and Vaccines

Certain embodiments of the present invention involves the use of polypeptides and/or nucleic acid sequences disclosed herein to "immunize" subjects or as "vaccines." As used herein, "immunization" or "vaccination" means increasing or activating an immune response against an antigen. It does not require elimination or eradication of a condition but rather contemplates the clinically favorable enhancement of an immune response toward an antigen. The vaccine may be a prophylactic vaccine or a therapeutic vaccine. A prophylactic vaccine comprises one or more epitopes associated with a disorder for which the individual may be at risk. Therapeutic vaccines comprise one or more epitopes associated with a particular disorder affecting the individual, such as tumor associated antigens in cancer patients.

As used herein, "vaccine" means an organism or material that contains an agent that is capable of inducing an immunoprotective response in a subject in an innocuous form. The vaccine is designed to trigger an immunoprotective response. The vaccine may be recombinant or non-recombinant. When inoculated into a non-immune host, the vaccine will provoke an immune response against an organism or material, but will not cause disease.

The immune response may also be a T cell response, such as increased antigen presentation to T cells, or increased proliferation of T cells.

The single stranded RNA and/or N0D2 polypeptides as set forth herein are administered with the intent of inducing an immune response. Depending on the intended mode of administration, the compounds of the present invention can be in various pharmaceutical compositions. The compositions will include a unit dose of the selected polypeptide in combination with a pharmaceutically acceptable carrier and, in addition, can include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, and excipients. "Pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable.

Preparation of vaccines and immunizing agents is generally well understood in the art, as exemplified by U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, all incorporated herein by reference. Typically, such vaccines are prepared as injectables either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccines.

Examples of physiologically acceptable carriers include saline solutions such as normal saline, Ringer's solution, PBS (phosphate-buffered saline), and generally mixtures of various salts including potassium and phosphate salts with or without sugar additives such as glucose. The active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. Nontoxic auxiliary substances, such as wetting agents, buffers, or emulsifiers may also be added to the composition. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. In one embodiment of the invention, adjuvants are not required for immunization. Parenteral administration, if used, is generally characterized by injection. Sterile injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.

The vaccine compositions set forth herein may comprise an adjuvant and/or a carrier. Adjuvants are any substance whose admixture into the vaccine composition increases or otherwise modifies the immune response to an antigen. Adjuvants could for example be selected from the group consisting of: A1K(SO4)2, AlNa(SO4)2, A1NH(SO4)4, silica, alum, AI(OH)3, Ca3(PO4)2, kaolin, carbon, aluminum hydroxide, muramyl dipeptides, N-acetyl- muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-nornuramyl-L-alanyl-D- isoglutamine (CGP 11687, also referred to as nor-MDP), N-acetylmuramyul-L-alanyl-D- isoglutaminyl-L-alanine-2-(l "2" -dipalmitoyl-s- n-glycero-3-hydroxphosphoryloxy)- ethylamine (CGP 19835 A, also referred to as MTP-PE), RIBI (MPL+TDM+CWS) in a 2% squalene/Tween-80.RTM. emulsion, lipopolysaccharides and its various derivatives, including lipid A, Freund's Complete Adjuvant (FCA), Freund's Incomplete Adjuvants, Merck Adjuvant 65, polynucleotides (for example, poly IC and poly AU acids), wax D from Mycobacterium, tuberculosis, substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella, liposomes or other lipid emulsions, Titermax, ISCOMS, Quil A, ALUN (see U.S. Pat. No 5,554,372), Lipid A derivatives, choleratoxin derivatives, HSP derivatives, LPS derivatives, synthetic peptide matrixes or GMDP, Interleukin 1 , Interleukin 2, Montanide IS A-51 and QS-21.

Other agents which stimulate the immune response can also be administered to the subject. For example, other cytokines are also useful in vaccination protocols as a result of their lymphocyte regulatory properties. Many other cytokines useful for such purposes will be known to one of ordinary skill in the art, including interleukin- 12 (IL- 12) which has been shown to enhance the protective effects of vaccines, GM-CSF and IL- 18. Thus cytokines can be administered in conjunction with antigens and adjuvants to increase the immune response to the antigens.

A vaccine composition according to the present invention may comprise more than one different adjuvant. Furthermore, the invention encompasses a therapeutic composition further comprising any adjuvant substance including any of the above or combinations thereof.

In certain embodiments, the vaccine composition includes a carrier. The carrier may be any suitable carrier known to the person skilled in the art, for example a protein or an antigen presenting cell. Examples include serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones, such as insulin or palmitic acid. For immunization of humans, the carrier must be a physiologically acceptable carrier acceptable to humans and safe. However, tetanus toxoid and/or diptheria toxoid are suitable carriers in one embodiment of the invention. Alternatively, the carrier may be dextrans for example sepharose.

The timing of administration of the vaccine and the number of doses required for immunization can be determined from standard vaccine administration protocols. Typically a vaccine composition will be administered in two doses. The first dose will be administered at the elected date and a second dose will follow at one month from the first dose. A third dose may be administered if necessary, and desired time intervals for delivery of multiple doses of a particular agent can be determined by one of ordinary skill in the art employing no more than routine experimentation.

For each recipient, the total vaccine amount necessary can be deduced from protocols for immunization with other vaccines. The exact amount of antigen-HCH2 polymer required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the particular fusion protein used, its mode of administration, and the like. Generally, dosage will approximate that which is typical for the administration of other vaccines, and will preferably be in the range of about 10 ng/kg to 1 mg/kg.

Methods for the preparation of mixtures or emulsions of polypeptides disclosed herein and adjuvant are well known to those of skill in the art of vaccination (see, e.g. Plotkin and Orenstein, 2004).

In some embodiments, the vaccine composition includes antigen presenting cells. The antigen presenting cell can be a dendritic cells (DC). DC may be cultivated ex vivo or derived in culture from peripheral blood progenitor cells (PBPC) and peripheral blood stem cells (PBSC). The dendritic cells may be prepared and used in therapeutic procedures according to any suitable protocol known to those of ordinary skill in the art. It will be appreciated by the person skilled in the art that the protocol may be adopted to use with patients with different HLA types and different diseases.

C. Pharmaceutical Preparations

Pharmaceutical preparations of NOD2 polypeptides, or antibodies and/or antibody fragments for administration to a subject are contemplated by the present invention.

1. Formulations

Any type of pharmaceutical preparation is contemplated by the current invention.

One of skill in art would be familiar with the wide range of types of pharmaceutical preparations that are available, and would be familiar with skills needed to generate these pharmaceutical preparations.

In certain embodiments of the present invention, the pharmaceutical preparation will be an aqueous composition. Aqueous compositions of the present invention comprise an effective amount an NOD2 polypeptide or single stranded RNA and the like, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Aqueous compositions of gene therapy vectors expressing any of the foregoing are also contemplated.

The phrases "pharmaceutical preparation suitable for delivery" or "pharmacologically effective" of "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate.

As used herein, "pharmaceutical preparation" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

The biological material should be extensively dialyzed to remove undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle, where appropriate. The active compounds will then generally be formulated for administration by any known route, such as parenteral administration. The preparation of an aqueous composition containing an active agent of the invention disclosed herein as a component or active ingredient will be known to those of skill in the art in light of the present disclosure.

An agent or substance of the present invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. A person of ordinary skill in the art would be familiar with techniques for generation of salt forms. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.

The present invention contemplates NOD2 polypeptides that will be in pharmaceutical preparations that are sterile solutions for intravascular injection or for application by any other route. A person of ordinary skill in the art would be familiar with techniques for generating sterile solutions for injection or application by any other route. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients familiar to a person of skill in the art. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.

The active agents disclosed herein may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so. Multiple doses can also be administered.

In addition to the compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g., tablets or other solids for oral administration; liposomal formulations; time release capsules; and any other form currently used, including cremes. One may also use nasal solutions or sprays, aerosols or inhalants in the present invention.

Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders. A person of ordinary skill in the art would be familiar with well-known techniques for preparation of oral formulations. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 75% of the weight of the unit, or preferably between 25-60%. The amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained.

The use of liposomes and/or nanoparticles is also contemplated for the introduction of the modulator of cell death or gene therapy vectors into host cells. The formation and use of liposomes is generally known to those of skill in the art.

Administration of the pharmaceutical compositions of the present invention may be by any method known to those of ordinary skill in the art. For example, administration may be topical, local, regional, systemic, by aerosol, by spray, intravenous, intradermal, intraarterial, intramuscular, intrathecal, intratracheal, subcutaneous, or intraperitoneal. Oral compositions are also contemplated by the present invention.

2. Dosage

An effective amount of the therapeutic or preventive agent is determined based on the intended goal, for example treatment or prevention of a bacterial infection in a subject. The quantity to be administered, both according to number of treatments and dose, depends on the subject to be treated, the state of the subject and the protection desired. Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual.

For example, a dose of the therapeutic agent may be about 0.0001 milligrams to about

1.0 milligrams, or about 0.001 milligrams to about 0.1 milligrams, or about 0.1 milligrams to about 1.0 milligrams, or even about 10 milligrams per dose or so. Multiple doses can also be administered. In some embodiments, a dose is at least about 0.0001 milligrams. In further embodiments, a dose is at least about 0.001 milligrams. In still further embodiments, a dose is at least 0.01 milligrams. In still further embodiments, a dose is at least about 0.1 milligrams. In more particular embodiments, a dose may be at least 1.0 milligrams. In even more particular embodiments, a dose may be at least 10 milligrams. In further embodiments, a dose is at least 100 milligrams or higher.

In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.

The dose can be repeated as needed as determined by those of ordinary skill in the art. Thus, in some embodiments of the methods set forth herein, a single dose is contemplated. In other embodiments, two or more doses are contemplated. Where more than one dose is administered to a subject, the time interval between doses can be any time interval as determined by those of ordinary skill in the art. For example, the time interval between doses may be about 1 hour to about 2 hours, about 2 hours to about 6 hours, about 6 hours to about 10 hours, about 10 hours to about 24 hours, about 1 day to about 2 days, about 1 week to about 2 weeks, or longer, or any time interval derivable within any of these recited ranges.

3. Secondary Treatment

Certain embodiments of the claimed invention provide for a method of treating or preventing a disease in a subject. Some of the methods set forth herein involve administering to the subject one or more secondary forms of therapy directed to the treatment or prevention of the disease. In particular embodiments, the disease is a viral infection. Any such therapy known to those of ordinary skill in the art is contemplated as a secondary form of therapy.

D. Treatment and Prevention of Disease

"Treatment" and "treating" as used herein refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition. For example, treatment of pneumonia may involve administration of a therapeutic agent for the reduction in symptoms of pneumonia, such as reduction in cough or improvement in respiratory function.

The term "therapeutic benefit" or "therapeutically effective" as used throughout this application refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of this condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease.

"Prevention" and "preventing" are used according to their ordinary and plain meaning to mean "acting before" or such an act. In the context of a particular disease or health-related condition, those terms refer to administration or application of an agent, drug, or remedy to a subject or performance of a procedure or modality on a subject for the purpose of blocking the onset of a disease or health-related condition.

The disease to be treated or prevented may be any viral infection. For example, the viral infection may be an infection or the respiratory tract. E. Kits

The technology herein includes kits. For example, a kit may include, for example, one or more components such as a sealed containing including a NOD2 polypeptide or a nucleic acid encoding a N0D2 polypeptide or a single-stranded RNA. The kits may optionally include a reagent, an instruction sheet, and other elements useful to practice the technology described herein. These physical elements can be arranged in any way suitable for carrying out the invention.

Kits can include further buffers, enzymes, labeling compounds, and the like. Any of the compositions described herein may be comprised in a kit.

The kit may include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a single vial. The kits of the present invention also will typically include a means for containing the nucleic acids or polypeptides set forth herein, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, such as a sterile aqueous solution.

However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.

The container means will generally include at least one vial, test tube, flask, bottle, syringe and/or other container means, into which the nucleic acid formulations are placed, preferably, suitably allocated. The kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent.

The kits of the present invention will also typically include a means for containing the vials in close confinement for commercial sale.

F. Examples

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLES

Example 1

Activation of Innate Immune Antiviral Response by NOD2 Methods

Virus and cell culture. RSV (A2 strain) and VSV were propagated in HeIa and BHK cells, respectively (Kota et al, 2008; Bose et al, 2003). Influenza A [AAPi?/8/34 (HlNl)] virus was grown in the allantoic cavities of 10-day-old embryonated eggs. All viruses were purified by centrifugation (two times) on discontinuous sucrose gradients. Human lung epithelial A549 cells and 293 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin, streptomycin, and glutamine. Primary normal human bronchial epithelial (NHBE) cells (from Lonza) were maintained in bronchial epithelial growth medium (BEGM) according to the supplier's instruction.

Luciferase assay. 293 cells were transfected (Lipofectamine 2000 from Invitrogen) with lμg of various plasmids (HA-NOD2, HA-NODl, pcDNAβ.l, IRF3 -luciferase, IFN-β- luciferase) and 100 ng of pRL-null-renilla luciferase. 293 cells were then infected or treated with RSV, ssRNA, or CpG DNA. A549 cells were transfected (Lipofectamine 2000 from Invitrogen) with 8OnM of NOD2 siRNA or control siRNA. 24 h post-siRNA transfection, cells were co-transfected with pRL-null-renilla luciferase (100 ng), IRF3 -luciferase (lμg) or IFN-β-luciferase (lμg). After 24h, cells were either infected with 0.5 MOI of RSV or treated with lμg per ml ssRNA40-LyoVec (Invivogen) for different time periods. Luciferase activity was measured using Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol. Transfection efficiency was normalized by measuring expression of renilla luciferase. Luciferase units were measured by standard methodology.

RT-PCR. The primers used to detect the various genes by RT-PCR are provided in

Table 1. TABLE 1 - Primers and siRNAs

RT-PCR primers

siRNAs

siRNA. All the siRNAs were ordered from Qiagen. The sequences of siRNA oligonucleotide used in the current study are provided in a supplementary table. As a negative control, AllStars Negative Control siRNA from Qiagen (catalog number 1027281, proprietary sequence) was used. A549 or 293 cells were transfected with siRNAs using

Lipofectamine 2000 (Invitrogen) and NHBE cells were transfected with siRNAs with PrimeFect Primary Cell siRNA Transfection Reagent (Lonza) according to the manufacturer's protocol.

Viral infection. 293 or A549 cells were infected with purified RSV at 0.5 multiplicity of infection (MOI) in serum free antibiotic free OPTI-MEM medium (GIBCO). Following adsorption for 1.5h at 37°C, cells were washed twice with serum containing DMEM and the infection was continued in the presence of serum containing DMEM for the specified time points. MEFs were infected with purified RSV or influenza A (A/PR/S/34 virus) at 1 MOI in serum free antibiotic free OPTI-MEM medium.

Co-immunoprecipitation. 293 cells were transfected with indicated tagged constructs and were then infected with RSV. Cell pellets were lysed (in TBS containing 1% Triton- XlOO) and sonicated. All lysates were incubated for 12h (at 4°C) with anti-HA-agarose beads (Sigma- Aldrich). Proteins bound to washed anti HA-agarose were eluted at pH 2.8. Eluted proteins were subjected to immunoblot analysis with anti-GFP (Santa Cruz) or anti-HA (Sigma, clone HA-7)

Immunofluorescence analysis. Cells plated on four well glass chamber slides were transfected with indicated tagged constructs. Cells were then infected with RSV (1 MOI) for 4h or 6h. Following infection, cells were fixed with 3.7% formaldehyde and permeabilized and blocked in the permeabilizing buffer containing Triton X-IOO (0.2%) and BSA (3%), and then incubated with either anti-HA (Sigma), anti-NOD2 (Cayman Chemical company) or anti-MAVS (Cell Signaling Technology) antibodies for 1 hr at 37°C. The washed cells were then incubated with the secondary antibody (Vector laboratories). Finally, the washed cells were mounted and the imaging of the cells was carried out using Zeiss LSM510 META laser scanning confocal microscopy.

Interaction of NOD2 with viral ssRNA. 293 cells were transfected with HA-N0D2 and then infected with RSV. Lysates were immunoprecipitated with anti-HA agarose for 4h at 4°C. After washing the beads with TBS, Tri-reagent was added to isolate bound RNA. RT- PCR was performed using RSV nucleocapsid (N protein) protein and GAPDH specific primers. For the cell-free interaction assay, lysate obtained from 293 cells expressing HA- N0D2 was incubated with HA-agarose beads. The HA-N0D2 bound to the beads were incubated with RSV ssRNA genome or total cellular mRNA (cellular mRNA was isolated by using the RNeasy minikit) for 45min incubation at 4°C. Beads were washed and RNA isolated from the washed beads was amplified using primers described above.

Virus infection of mice. 6-8-week old pathogen-free C57BL/6 and N0D2-K0 (C57BL/6J background) mice were obtained from Jackson laboratory. We further back- crossed these N0D2-K0 mice to the C57BL/6 genetic background for a total of eight generations. Genome wide SNP analysis on these animals (Harlan Laboratories, Inc.), revealed that wild-type and N0D2-K0 mice are genetically identical, with the exception of the N0D2 deletion (data not shown). Mice were anesthetized using inhaled methoxyfluorane and intranasally inoculated with RSV (5 x 10⁶ pfu per animal) in 100 μl of low serum Opti- MEM medium (Invitrogen). Uninfected control animals were sham-inoculated with 100 μl of Opti-MEM. For another set of studies, mice were infected intranasally with RSV at 5^χ10 pfu per animal and the survival of infected mice was followed for 18 d.

TUNEL assay and MPO assay. Formalin fixed lungs were stained using the in situ TUNEL assay kit from Promega. Lung neutrophil content was assessed by measuring myeloperoxidase (MPO) activity (Bubeck et al, 2007; Wilmott et al, 1998).

Generation of NOD2 mutants. The NOD2 cDNA was cloned into pcDNA6-Myc- His vector (Invitrogen) and deletion mutants of NOD2 were constructed by PCR.

Treatment with synthetic and viral ssRNA. Cells were treated lμg per ml of synthetic ssRNA that is already conjugated with trasfection reagent (ssRNA40-LyoVec from Invivogen). For isolation of viral ssRNA, purified RSV virion particles were centrifuged for 4h at 28,000 rpm using SW32Ti rotor. The ssRNA genome was isolated from the viral pellet by using the RNeasy minikit. Cells were transfected with viral ssRNA using Lipofectamine 2000 from Invitrogen.

Isolation of MEFs and macrophages. Alveolar macrophages were collected by centrifuging bronchoalveolar lavage fluid at 2500 rpm for 10 min at 4°C. After washing the cell pellet was seeded in a 24-well plate. MEFs were prepared as described previously ⁵⁰. Bone marrow-derived macrophages were obtained from femurs and tibias of wild-type and NOD2-KO mice and were cultured for 6-8 days.

ELISA. ELISA was performed using human or mouse IFN-β specific ELISA kits

(PBL interferon source).

Results

ssRNA induces IFN production via NOD2. To study the involvement of NLR proteins in antiviral responses, various HA-tagged human NLR proteins (e.g. NODl, NOD2,

IPAF, NAIP, NOD3) were expressed in 293 cells, which do not endogenously express most

NLRs. These cells were treated with synthetic ssRNA and analyzed IFN production and

IRF3 activation. The studies involved utilization of ssRNA because several highly pathogenic viruses including paramyxoviruses (RSV, Sendai virus, human parainfluenza viruses, measles virus), rhabdoviruses (rabies virus, vesicular stomatitis virus) and orthomyxoviruses (influenza viruses) contain ssRNA genomes, and because ssRNA activates PRRs including TLR7, TLR8 and RIGI (O'Neill, 2006; basler and Garcia-Sastre, 2007). NOD2 but not NODl facilitated ssRNA-induced IRF3 activation (FIG. IA) and IFN-β production (FIG. 9A, 9B). Such activation was lacking in cells treated with CpG DNA (FIG. IA, 9A, 9B). HA-tagged NOD constructs were expressed in high amounts in transfected 293 cells (FIG. 9C); in addition, HA-NOD2 and HA-NODl proteins were functional as they facilitated activation of NF-κB in 293 cells treated with MDP or iE-DAP, respectively (FIG. 9D).

To establish the physiological relevance of NOD2-mediated ssRNA-induced activation of IRF3, the role of endogenous N0D2 in inducing IRF3-IFN following ssRNA treatment was next evaluated. For these studies, human lung epithelial A549 cells were utilized since these cells are permissive to the majority of viruses that contain ssRNA genomes and endogenously express various PRRs. Treatment of these cells with ssRNA resulted in increased N0D2 expression (FIG. IB); NODl expression remained unchanged. NOD2-specifϊc siRNA diminished N0D2 expression in ssRNA-treated A549 cells (FIG. 10A), and impaired ssRNA-induced activation of IRF3 and IFN-β production (FIG. 1C, FIG. 10B).

Similarly, ssRNA-induced IRF3 activation and IFN-β production was reduced in bone marrow derived macrophages (BMM) and mouse embryo fibroblast (MEF) isolated from N0D2-K0 compared to wild-type mice (FIG. ID, IE). In contrast, wild-type and N0D2-K0 BMM and MEFs produced similar amounts of IFN-β after treatment with poly-IC (dsRNA that activates TLR3) (FIG. 10C). The ability of poly-IC to induce IFN (via TLR3) in both wild-type and N0D2-K0 MEFs showed that IRF3-IFN pathway is intact in N0D2-K0 animals. Furthermore, poly-IC administration in vivo resulted in production of similar concentrations of IFN-β in wild-type and N0D2-K0 animals (FIG. 10D). Thus these results obtained with cell lines and primary cells demonstrated that activation of N0D2 by ssRNA results in IFN-β production.

NOD2 facilitates virus-induced IFN production. To further validate the ability of N0D2 to launch an antiviral response, we infected 293 cells with RSV. RSV induced activation of IRF3 and IFN-β in 293 cells expressing HA-N0D2 but not in 293 cells expressing HA-NODl (FIG. 2A, 2B). Inactivation of virion particles with ultra-violet (UV) light abolished the ability of RSV to activate IRF3 in N0D2 expressing cells (FIG. 2A). The inability of UV inactivated RSV to activate IRF3 indicated that an intact viral RNA genome is essential for N0D2 activation. The direct role of viral components in N0D2 activation was further confirmed by the loss of IRF3 activation following inhibition of RSV cellular entry with an RSV neutralizing antibody (specific for the RSV fusion or F protein (FIG. 10E).

The functional significance of N0D2 in antiviral responses was established using the

IFN -sensitive vesicular stomatitis virus (VSV). Plaque assay analysis of VSV titers obtained from 293 cells expressing HA-N0D2 or HA-NODl revealed a marked reduction in viral titer in cells expressing HA-N0D2 (FIG. 2C). Like VSV, RSV titers were lower in N0D2 expressing cells compared to NODl expressing cells (FIG. 2D). Human parainfluenza virus type 3 (Bose et al, 2001) and VSV (Bose et al, 2003) also activated IRF3 in N0D2 expressing 293 cells (FIG. HA). In contrast, vaccinia virus— a DNA virus— failed to activate IRF3 in N0D2 expressing cells (FIG. HB). These results demonstrated that, like the RLH receptors like RIGI and Mda5, N0D2 can function as a cytoplasmic PRR for viral ssRNA.

The role of endogenous N0D2 in inducing IRF3-IFN in response to RSV infection was next evaluated. Previous studies described activation of IRF3 and production of IFN-β from RSV infected A549 cells as early as 2h post-infection (Jamaluddin et al, 2001). Although uninfected A549 cells did not express detectable amounts of N0D2, RSV infection resulted in increased N0D2 expression within 2h post-infection (FIG. 3A). RSV infection failed to induce NODl expression (data not shown). N0D2 siRNA markedly diminished N0D2 expression after RSV infection (FIG. 12A) and resulted in reduced activation of IRF3 and production of IFN-β after RSV infection (FIG. 3B, FIG. 12B). The effect was more pronounced during early (4h and 6h) compared to late (1Oh) time points after infection (FIG. 3B). This result suggests that N0D2 is critical for the early antiviral response, whereas during late infection time periods other PRRs (e.g. RIGI) may activate the IRF3-IFN pathway.

Indeed, we (FIG. 12C) and others (Liu et al, 2007) have shown that RIGI expression in A549 cells is detectable only at late time points post-infection with RSV. In addition, the early antiviral response was independent of RIGI because silencing of RIGI had no effect in IFN-β expression during early RSV infection (FIG. 12D, 12E). Like A549 cells, MEFs did not express abundant N0D2 until early time points after RSV infection, and RIGI was undetectable until late time points after RSV infection (FIG. 12F, 12G). Thus temporal expression of N0D2 and RIGI during early and late infection, respectively, may facilitate optimal sustained IFN production from virus-infected cells. Next, the role of N0D2 in primary normal human bronchial epithelial cells (NHBE) was examined, as these cells constitute the major cell type infected by RSV in humans. RSV rapidly induced N0D2 expression in NHBE cells (FIG. 13). N0D2 was essential for IFN production, as N0D2 siRNA markedly reduced IFN- production from RSV infected NHBE cells (FIG. 3C). The critical role of N0D2 was further established by demonstrating that BMM, MEFs and alveolar macrophages derived from N0D2-K0 mice produced less IFN- than wild-type counterparts after RSV infection (FIG. 3D-F). N0D2-K0 MEFs and BMM also exhibited defective IFN- production following influenza A/Pi?/8/34 (HlNl) virus infection (FIG. 14). Collectively these data reveal an important role for endogenous N0D2 in the induction of antiviral immune responses.

NOD2 interacts with viral ssRNA. The role of the viral ssRNA genome (viral- ssRNA) in N0D2 activation was next investigated. Viral-ssRNA isolated from purified RSV virion particles activated IRF3 only in N0D2 expressing cells (FIG. 4A). An intact ssRNA genome was required for N0D2 activation since treatment of the viral-ssRNA with RNAse abolished IRF3 activation (FIG. 4A). Viral-ssRNA utilizes N0D2 for IFN production, as diminished IFN-β production following viral-ssRNA treatment was observed in MEFs and BMM isolated from N0D2-K0 compared to wild-type mice (FIG. 4B, 4C).

The role of viral-ssRNA as an activator of N0D2 was further demonstrated by observing interaction of viral ssRNA with N0D2 within a cellular milieu. HA-N0D2 from RSV-infected 293 cells that were transfected with HA-N0D2 were immunoprecipitated, and amplified bound RNA with either GAPDH (control) or RSV nucleocapsid (N) protein specific primers. These experiments revealed association of N0D2 with viral but not control RNA (FIG. 4D). In a cell-free assay, HA-N0D2 bound to HA-agarose beads was incubated with RSV ssRNA genome or mRNA isolated from cells. After incubation, bound RNA was amplified with either GAPDH (control) or RSV nucleocapsid (N) protein specific primers; we detected interaction of N0D2 with RSV ssRNA (FIG. 4E). In contrast, GAPDH mRNA (which is enriched in total cellular mRNA) did not associate with N0D2 (FIG. 4E). We also observed failure of NODl to interact with viral ssRNA genome (data not shown). These results demonstrate that interaction of viral ssRNA genome with N0D2 results in its activation and subsequent induction of IFN production.

MAVS is required for NOD2-mediated responses. We next focused on the mechanism utilized by N0D2 to activate IRF3-IFN. As both RIGI and N0D2 posses CARD (caspase recruitment domain) domains (Basler and Garcia- S astre, 207; Martinon and Tschopp, 2005; Fritz et al, 2006; Kanneganti et al, 2007), it was speculated that similar to RIGI, N0D2 may also interact with MAVS. In addition, a recent study showed that the NLR family member NLRXl interacts with mitochondrial localized MAVS via its nucleotide binding domain (NBD), a domain also found in NOD2 (Moore et al, 2008). Interaction of NOD2 with MAVS was essential for NOD2- mediated activation of antiviral responses, as MAVS siRNA (FIG. 15) diminished RSV-induced IFN-β production from infected NHBE cells to an extent similar as NOD2 siRNA (FIG. 5A). Similar observations were noted in MEFs derived from MAVS-KO mice that were infected with RSV or transfected with viral or synthetic ssRNA (FIG. 5B). Influenza A virus also required MAVS for IFN production, as IFN-β production from NOD2-KO and MAVS-KO MEFs was reduced to a similar extent following influenza A infection (FIG. 16). These results demonstrate that MAVS is critical for virus-induced NOD2-mediated IFN production.

The interaction of NOD2 with MAVS was next examined. Initially the ability of activated NOD2 to translocate to the mitochondria was investigated. Immunoblot analysis of mitochondrial extract from RSV-infected NOD2 expressing cells revealed that although approximately 6%-7% of NOD2 is localized in mitochondria in uninfected cells, RSV infection resulted in enrichment (40%-45% of total cellular NOD2) of NOD2 in mitochondria (FIG. 17A, 17B). Immunofluorescence analysis also revealed co-localization of endogenous NOD2 with mitochondria in RSV-infected A549 cells (FIG. 17C). To study the interaction of NOD2 with MAVS, 293 cells were transfected with HA-NOD2 and GFP tagged MAVS. Co- immunoprecipitation analysis revealed interaction of NOD2 with MAVS (FIG. 6A), and this interaction was enhanced following RSV infection. Additional controls associated with FIG. 6A are shown in FIG. 17D, 17E. In contrast to NOD2, NODl failed to interact with MAVS (FIG. 17F). Double labeled immunofluorescence studies with RSV-infected 293 cells expressing GFP-MAVS and HA-N0D2 confirmed co-localization of N0D2 and MAVS (FIG. 6B). Similarly, RSV infection of A549 (FIG. 6C) and NHBE (FIG. 6D) cells enhanced co localization of endogenous N0D2 with endogenous MAVS. These results demonstrated that N0D2 interacts with MAVS during virus infection.

N0D2 can activate NF-κB and MAPK pathways via the kinase RICK (also known as Rip2, CARDIAK, CCK and Ripk2) (Inohara et al, 2002; Franchi et al, 2008). Bacterial products like MDP specifically stimulate NOD2 and result in NF-κB activation via RICK. However, treatment of NOD2 expressing 293 cells with MDP did not activate IRF3 (FIG. 18A). Likewise, MDP treatment of NHBE and BMM did not result in IFN-β production (FIG. 18B, 18C). In addition, RICK may not play a major role in IFN induction by N0D2 since silencing endogenous RICK expression did not alter N0D2 mediated IFN-β production in RSV infected cells (FIG. 19). We also examined the efficiency of interaction between N0D2 and MAVS compared to RIGI and MAVS. Although both N0D2 and RIGI associated with MAVS, the RIGI-MAVS interaction was slightly more efficient than N0D2-MAVS interaction (FIG. 20A). In addition, we also observed that in RSV-infected cells, N0D2 more efficiently interacted with MAVS than with RICK (FIG. 20B). However, NOD2 efficiently interacted with RICK in cells stimulated with the well-established NOD2 stimulator MDP (FIG. 20B). Thus, NOD2 utilizes either RICK or MAVS depending on the stimulus (e.g. MDP vs. ssRNA) to activate either IRF3 or NF-κB.

In addition to IRF3, NF-κB activation is required for IFN gene expression. Although IRF3 alone is capable of inducing IFN gene transcription, the transactivating function of NF- KB synergistically acts with IRF3 to promote optimal IFN expression (Wathelet et al, 1998). This is also true for NOD2 mediated IFN expression, as suppressing NF-κB activity in RSV infected cells diminished IFN expression via activated NOD2 by 30%-35%; as expected, expression of the NF-κB-dependent TNF gene in RSV infected cells was reduced by 80% (FIG. 21). Based on these results, we speculate that NOD2 activated by stimulation by viral ssRNA interacts with MAVS to induce activation of both IRF3 and NF-κB in a manner similar to that of RLHs (Basler and Garcia-Sastre, 2007). In contrast, NOD2 activated by bacterial products (e.g., MDP) activates NF-κB via RICK. Further detailed studies are required to investigate the role of NOD2 in activating NF -KB pathway during virus infection and the role of MAVS and RICK during these events.

MAVS interacts with LRR-NBD domains of NOD2. The CARD domain of RIGI promotes its association with MAVS, whereas NLRXl utilizes its NBD domain to interact with MAVS. Thus, we next investigated the role of NBD, CARD and LRR domains of NOD2 in MAVS association. For these studies, we generated various His-Myc tagged versions of NOD2 deletion mutants - ΔCARD (NOD2 mutant lacking both CARD domains), ΔNBD (NOD2 mutant lacking the NBD domain) and ΔLRR (NOD2 mutant lacking the LRR domain) (FIG. 7A). These mutants were expressed in 293 cells along with GFP-MAVS. Lysates obtained from these cells were precipitated with nickel-agarose beads and, after washing, the proteins bound to the beads were subjected to immunob lotting with anti-GFP. While ΔCARD was capable of interacting with MAVS, both ΔNBD and ΔLRR failed to associate with MAVS (FIG. 7B). Comparable amounts of GFP- MAVS protein were expressed in the various NOD2 mutant expressing cells (FIG. 7C). In addition, similar amounts of His-Myc tagged N0D2 mutants were bound to the Nickel-agarose beads during the experimental condition used to study interaction of MAVS with N0D2 mutants (FIG. 22A). These results indicate that unlike RIGI, the CARD domains of N0D2 are not important for its interaction with MAVS. However, the NBD and LRR domains of N0D2 are required for MAVS association.

This conclusion was further validated by examining the functionality of the N0D2 mutants in activating IRF3. Infection of 293 cells transfected with either wild-type or mutant (ΔCARD, ΔNBD, ΔLRR) N0D2 constructs revealed that wild-type N0D2 and ΔCARD, but neither ΔNBD nor ΔLRR, induced IRF3 activation after infection with RSV (FIG. 22B).

Role of NOD2 in host antiviral defense. The physiological role of N0D2 by infecting wild-type and N0D2-K0 mice with RSV was then assessed. The mouse model of RSV infection mimics virus infection in humans, as infected mice can develop disease states resembling pneumonia (Jafri et al, 2004; Bolger et al, 2005); in addition, mice induce a robust antiviral response characterized by production of IFN-β and expression of IFN- dependent genes like Mx during early RSV infection (within 12h post-infection) (Jafri et al, 2004; Bolger et al, 2005; Guerrero-Plata et al, 2005a; Guerrero-Plata et al, 2005b; Pletneva et al, 2008; Chavez-Bueno et al, 2005). Moreover, RSV is sensitive to IFN in infected mice because as low as 200 units per ml of IFN inhibits RSV infection in mice by 100 fold (Guerrero-Plata et al, 2005). It is important to mention that during RSV infection of mouse respiratory tract, IFN is induced early during infection (at 12h-2d post-infection) but its production is lost at 3d post-infection (Jafri et al, 2004; Bolger et al, 2005; Guerrero-Plata et al, 2005a; Guerrero-Plata et al, 2005b; Pletneva et al, 2008; Chavez-Bueno et al, 2005). This observation suggests that IFN is important to restrict RSV spread during early infection and the production of IFN dictates the clinical outcome of the disease (for e.g. lung inflammation and apoptosis of airway cells).

Wild-type and NOD2-KO mice were infected with sublethal dose of RSV (5x10⁶ pfu per animal delivered by intra-nasal inoculation) followed by collection of lungs and bronchoalveolar lavage (BAL) fluid at different time periods. We observed expression of murine NOD2 in RSV-infected lungs at Id post-infection, and such expression was lost at 4d post-infection (FIG. 23A). This result suggested an important role for NOD2 in IFN expression, as the IFN induction kinetics correlated with the NOD2 expression kinetics (Jafri et al, 2004; Bolger et al, 2005; Guerrero-Plata et al, 2005a; Guerrero-Plata et al, 2005b; Pletneva et al, 2008; Chavez-Bueno et al, 2005). Compared to wild-type mice, N0D2-K0 mice showed diminished IFN-β production in the respiratory tract and increased viral titer (FIG. 8A, 8B).

It is well known that RSV causes lung disease by inducing pneumonia, a massive inflammation of the lungs (Hippenstiel et al, 2006). Higher virus burden ultimately results in enhanced inflammation and exaggerated lung disease due to flooding of alveolar spaces with edema fluid. This occurs as a result of enhanced permeability of the epithelial barrier due to apoptosis of airway epithelial cells. RSV infection resulted in more severe lung pathology in N0D2-K0 mice (as deduced by H&E staining of lung sections 3 and 5 days post-infection) (FIG. 8C). Massive peribronchial lymphocytic inflammation and filling of the lumen with exudates of infiltrating neutrophils and mucus was noted. Neutrophils constitute the major immune cells infiltrating the lung of RSV-infected mice and humans and high number of these cells in the airway causes severe immunopathology associated with RSV clinical disease (Yasui et al, 2005; Wang and Forsyth, 2000; Wang et al, 1998). To examine neutrophil accumulation in the lungs, we performed a myeloperoxidase (MPO) activity assay (Bubeck et al, 2007; Wilmott et al, 1998) with lung homogenates of RSV infected wild-type and NOD2-KO mice. Higher RSV-induced enhancement of neutrophil activity was visible in the lung tissue of NOD2-KO (-35%) compared to wild-type (-4%) mice (FIG. 8D). The enhanced inflammation in the respiratory tract of NOD2-KO mice was also confirmed by higher concentrations of pro-inflammatory cytokines and chemokines (e.g. tumor necrosis factor, IL-10, RANTES) in the BAL of infected NOD2-KO compared to wild-type animals (FIG. 23B-23D). High RSV load has been associated with enhanced apoptosis of airway epithelial cells and infiltrating neutrophil granulocytes, which contributes to the development of lung lesions and injury (Welliver et al, 2007). In deed, in situ apoptosis analysis of lung sections by TUNEL assay revealed enhanced apoptosis in the lungs of N0D2-K0 compared to wild-type animals infected with RSV (FIG. 8E).

Notably, RSV-infected N0D2-K0 mice lost significantly more body weight and exhibited reduced survival than wild-type counterparts (FIG. 24, FIG. 8F). Diminished IFN-β production in the BAL of N0D2-K0 compared to wild-type mice infected with influenza A virus was also observed (FIG. 25). These results demonstrated that NOD2 is a critical component of host antiviral defense mechanisms. All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Claims

1. A method of inhibiting, modulating, treating, or preventing a viral infection or the symptoms thereof in a subject, comprising administering to a subject with a viral infection an effective amount of a pharmaceutical composition comprising:

a) a recombinant single-stranded RNA or a small molecule, wherein said RNA or said small molecule binds to a nucleotide-binding oligomerization domain-2 (NOD2) protein; and b) a pharmaceutically acceptable carrier,

wherein the viral infection is inhibited, modulated, treated, or prevented.

2. The method of claim 1 , wherein the subject is a mammal.

3. The method of claim 2, wherein the mammal is a human.

4. The method of claim 3, wherein the human is an infant, a patient with a congenital heart disease, a patient over age 60, a patient that has received an organ transplant, or a patient in need of interferon β or interferon α.

5. The method of claim 1, wherein the viral infection is an infection due to a single- stranded RNA virus.

6. The method of claim 1 , wherein the viral infection is a respiratory virus infection.

7. The method of claim 1 , wherein the viral infection is an infection due to a a paramyxovirus, a rhabdovirus, a filovirus, or an orthomyxovirus.

8. The method of claim 7, wherein the viral infection is due to a paramyxovirus that is respiratory syncytial virus (RSV), Sendai virus, parainfluenza virus, measles virus, mumps virus, metapneumonia virus, Hendra virus, or Nipah virus.

9. The method of claim 7, wherein the viral infection is due to an orthomyxovirus that is an influenza A virus, an influenza B virus, or an influenza C virus.

10. The method of claim 8, wherein the parainfluenza virus is HPIV-I , HPIV-2, HPIV-3, or HPIV-4.

11. The method of claim 5, wherein the viral infection is a fϊlo virus that is Ebola virus or Marburg virus.

12. The method of claim 6, wherein the rhabdovirus is rabies virus or vesicular stomatitis virus.

13. The method of claim 1, wherein the recombinant single-stranded RNA is 10 to 30,000 nucleobases in length.

14. The method of claim 13, wherein the recombinant single-stranded RNA is 10 to 3,000 nucleobases in length.

15. The method of claim 14, wherein the recombinant single-stranded RNA is 10 to 300 nucleobasis in length.

16. The method of claim 15, wherein the recombinant single-stranded RNA is 10 to 100 nucleobases in length.

17. The method of claim 1, wherein the recombinant single-stranded RNA is a single- stranded RNA molecule derived from the genome of a single-stranded RNA virus.

18. The method of claim 1, wherein the pharmaceutically acceptable carrier comprises water, an alcohol, dimethylsulfoxide, or a lipid.

19. The method of claim 1, wherein the RNA or small molecule binds to the NBD domain ofNOD2.

20. The method of claim 1, wherein the composition is administered by aerosol, by spray, intravenously, intradermally, intraarterially, intramuscularly, intrathecally, intratracheally, subcutaneously, orally, topically, intraperitoneally, via a drug-delivery device, or via a nanoparticle.

21. The method of claim 20, wherein the nanoparticle is coated with the composition.

22. The method of claim 1, wherein the single-stranded RNA or small molecule is comprised in a vector.

23. The method of claim 22, wherein the vector is a cell or a virus.

24. The method of claim 22, wherein the vector is a liposome.

25. The method of claim 23, wherein the cell is a dendritic cell.

26. The method of claim 1, further comprising administering to the subject a secondary antiviral therapy.

27. The method of claim 26, wherein the secondary antiviral therapy is oseltamivir or zanamivir.

28. The method of claim 1, wherein the composition further comprises an adjuvant.

29. The method of claim 28, wherein the adjuvant is aluminum phosphate or aluminum hydroxide.

30. The method of claim 1, further comprising identifying a subject in need of inhibition, modulation, treatment, or prevention of a viral infection or the symptoms thereof.

31. A method of inducing an innate immune response in a subject, comprising

administering to the subject a pharmaceutical composition comprising:

a) a recombinant single-stranded RNA or a small molecule, wherein said RNA or small molecule binds to a nucleotide-binding oligomerization domain-2 (NOD2) protein; and b) a pharmaceutically acceptable carrier,

wherein the innate immune response is induced.

32. The method of claim 31 , wherein the subject is a human.

33. The method of claim 31, wherein the recombinant single-stranded RNA is 10 to 30,000 nucleobases in length.

34. The method of claim 33, wherein the recombinant single-stranded RNA is 10 to 3,000 nucleobases in length.

35. The method of claim 31, wherein the recombinant single-stranded RNA is a single- stranded RNA molecule derived from the genome of a single-stranded RNA virus.

36. The method of claim 31, wherein the pharmaceutically acceptable carrier comprises water, an alcohol, dimethylsulfoxide, or a lipid.

37. The method of claim 31, wherein the RNA or small molecule binds to the NBD domain of NOD2.

38. The method of claim 31, wherein the composition is administered by aerosol, by spray, intravenously, intradermally, intraarterially, intramuscularly, intrathecally, intratracheally, subcutaneously, orally, topically, intraperitoneally, or via a drug-delivery device.

39. A pharmaceutical composition comprising:

a) a recombinant single-stranded RNA or small molecule, wherein said RNA or small molecule binds to a nucleotide-binding oligomerization domain-2 (NOD2) protein; and

b) a pharmaceutically acceptable carrier.

40. The composition of claim 39, wherein the recombinant single-stranded RNA is 10 to 30,000 nucleobases in length.

41. The composition of claim 40, wherein the recombinant single-stranded RNA is 10 to 3,000 nucleobases in length.

42. The composition of claim 39, wherein the recombinant single-stranded RNA is a single-stranded RNA molecule derived from the genome of a single-stranded RNA virus.

43. The composition of claim 39, wherein the pharmaceutically acceptable carrier comprises water, an alcohol, dimethylsulfoxide, or a lipid.

44. The composition of claim 39, wherein the RNA or small molecule binds to the NBD domain of NOD2.

45. The composition of claim 39, wherein the composition comprises a secondary antiviral agent.

46. The composition of claim 45, wherein the secondary antiviral agent is oseltamivir or zanamivir.

47. The composition of claim 39, wherein the composition further comprises a nanoparticle.

48. The composition of claim 47, wherein the nanoparticle is coated with the composition.

49. The composition of claim 39, wherein the composition further comprises one or more antigens.

50. A kit comprising one or more sealed vials, wherein (i) at least one vial comprises a recombinant single-stranded RNA, wherein said RNA binds to a nucleotide-binding oligomerization domain-2 (NOD2) protein.

51. The kit of claim 50, further comprising a syringe.

52. The kit of claim 50, comprising one or more vials comprising a pharmaceutically acceptable carrier.

53. The kit of claim 50, wherein at least one vial comprises a composition as set forth in any of claims 39-49.