WO2010091265A1 - Compounds and methods for treating viral encephalitis - Google Patents

Abstract

The invention includes compositions, methods, and techniques for influencing or otherwise altering or regulating signaling that emanates from ligands, receptors, or pathways of TLR7, and/or IL- 12, and/or IL-23, and/or IL- 17, singly or in any combination. The invention includes inhibiting ligands, receptors, or pathways of TLR7, and/or IL- 12, and/or IL-23, and/or IL- 17 singly or in any combination using pharmacologic means, which includes agonist binding, antagonist binding and subsequent effects, and binding and subsequent effects of drugs or molecules that have variable or mixed effects on the TLR7 signaling pathway, and/or the IL- 12, and/or IL-23, and/or IL-17 pathways, whether singly or in any combination.

Description

Compounds and Methods for Treating Viral Encephalitis

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit pursuant to 35 U.S. C. § 119(e) of U.S.

Provisional Application No. 61/150,223, filed February 5, 2009 which is hereby incorporated by reference herein, as if set forth herein in its entirety.

GOVERNMENT CONTRACT RIGHTS This invention was made with government support under NIH A S 055749 and NIH

A150031 awarded by National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION Pioneering work by Charles Janeway, Jr. in the 1990s laid the bulk of the foundation for our current understanding of how humans and other complex organisms detect the presence of foreign invading organisms, and generate molecular signals that significantly impact the nature, magnitude, and duration of the response mounted by the organism to defend itself. Thanks to this work and intensive research over the ensuing years, it is now established that detection of foreign pathogens is accomplished by molecular receptors termed "pattern-recognition receptors" (abbreviated PRRs), because they directly bind molecular patterns (termed "pathogen-associated molecular patterns" [abbreviated PAMPs]) that are produced by pathogens but not by the host (Qureshi and Medzhitov, 2003; Yamamoto et al., 2004). Among these pattern recognition receptor systems that are ubiquitously found throughout the body of the organism are the Toll-like receptors (TLRs). Depending on the species, there are as many as twelve known TLRs. These are numbered, for example TLR-2, TLR-3, TLR-4, etc. Each specific type of TLR directly recognizes molecular patterns that are of a particular type, for example proteoglycans, or lipopolysaccharides, or various kinds of nucleic acids. Overlap in recognition of molecular patterns by distinct types of TLRs is relatively minimal. Hence, the body relies upon the integrity of the structure and function of each specific type of TLR in order to mount a proper defense against, in some cases, entire classes of pathogens. However, in many cases, pathogens (particularly bacteria) produce a number of different kinds of PAMPs that may be recognized by several distinct TLRs. Hence, TLRs function to drive innate immune responses that, in turn, fine-tune adaptive immunity (Qureshi and Medzhitov, 2003; Yamamoto et al., 2004).

It is now widely accepted that TLRs recognize a diverse set of pathogens, including bacteria (by TLR2, TLR4, TLR6, and TLR9), flagellated protozoans (by TLR5), and pathogenic fungi (by TLR2, TLR4, and TLR6) (Roeder et al., 2004; Yamamoto et al., 2004). TLR2 and TLR4 recognize viral proteins (Bieback et al., 2002; Kurt- Jones et al., 2000; Rassa et al., 2002), and our data demonstrate that TLR3 mediates host recognition of viral components (including double-stranded RNA [dsRNA] and the dsRNA analog poly(LC)) and intact virus including Lang reo virus and West Nile virus (WNV) (Alexopoulou et al., 2001; Edelmann et al., 2004; Town et al., 2006; Wang et al., 2004). Further, TLR7 and TLR8 are implicated in MyD88-dependent recognition of single-stranded (ss) RNA and ssRNA-producing viruses including vesicular stomatitis virus, influenza virus, humanparechoviris 1, andhuman immunodeficiency virus (Diebold et al., 2004; Heil et al., 2004; Lund et al., 2004; Schlaepfer et al., 2006; Triantafϊlou et al., 2005).

Of particular relevance for the claims detailed herein, TLR7 is thought to recognize single-stranded RNA ("ssRNA"), which is generally derived from certain types of viruses. West Nile virus ("WNV") is an example of a virus that produces ssRNA. WNV is classified as a ssRNA flavivirus, and is transmitted via mosquitoes, which can harbor the virus and infect humans and other target mammals including livestock (e.g., pigs, cows, and horses) with this virus after inflicting a bite. Infection of humans with WNV is now the most common cause of viral encephalitis, and is an increasingly alarming public health concern throughout the world (Campbell et al. 2002; Debiasi and Tyler, 2006; Gould and Fikrig, 2004; Gubler, 2007). In many cases, infection of humans is asymptomatic, however WNV infection can and does cause neurological symptoms and associated pathologies that can be lethal. These include meningitis and encephalitis, which are intense, acute inflammations of specific parts of the central nervous system (CNS), including the brain (Campbell et al., 2002; Davis et al., 2006). The work that led to this patent application established that WNV is indeed detected by TLR7. This work has also established that signaling initiated by WNV proceeds via the common TLR intracellular adaptor, MyD88. Furthermore, the work established that signaling by TLR7 that was initiated by WNV infection caused or otherwise led to recruitment of CD45+ leukocytes and CDl Ib+ macrophages to sites, target organs, and cells where WNV infection was present, including the brain and liver. In addition, the work shows that in the absence of TLR7, there are reductions in IL- 12 and IL-23 responses, and that genetic deficiency in interleukin (IL)- 12 p40 and IL-23 p40 (ILl 2b-/-) or IL-23 pl9 (IL23a-/-) caused or led to responses that were similar to those seen in TLR7-/- mice. In summary then, the work that led to this patent application demonstrated that TLR7 and responses that are dependent upon IL- 12 and/or IL-23 to WNV are vital host defense mechanisms, and that they act at least in part by enabling homing of immune cells to cells infected with WNV.

SUMMARY OF THE INVENTION

The present invention provides a method of manipulating TLR7-dependent, IL- 12-dependent, IL-23 -dependent, and/or IL-17-dependent effects. A variety of components of TLR7 and its downstream signaling system can serve as targets where one skilled in the art could intervene for a therapeutic or diagnostic purpose.

In one embodiment, the invention encompasses inhibiting TLR7 signaling by various methods, either by directly targeting the TLR7 receptor itself, or by affecting synthesis of the receptor, or by affecting degradation of the receptor, or by otherwise affecting the availability of the receptor.

In another embodiment, the invention encompasses inhibiting TLR7 signaling by various ways, methods, and techniques that are intended to directly target ligands and/or downstream adaptors and other signaling apparatus necessary to transmit the signals conveyed through TLR7, either by directly targeting such adaptors and/or other signaling apparatus, and/or by affecting the synthesis, degradation, or availability of such adaptors and/or signaling apparatus. In another embodiment, the invention encompasses stimulating TLR7 signaling by various methods, either by directly targeting the TLR7 receptor itself, or by affecting synthesis of the receptor, or by affecting degradation of the receptor, or by otherwise affecting the availability of the receptor.

In yet another embodiment, the invention encompasses stimulating TLR7 signaling by various ways, methods, and techniques that are intended to directly target ligands and/or downstream adaptors and other signaling apparatus necessary to transmit the signals conveyed through TLR7, either by directly targeting such adaptors and/or other signaling apparatus, and/or by affecting the synthesis, degradation, or availability of such adaptors and/or signaling apparatus.

In one embodiment, the invention encompasses inhibiting IL-23 signaling by various methods, either by directly targeting IL-23 and/or IL-23 receptor(s), or by affecting synthesis of IL-23 and/or its receptor(s), or by affecting degradation of IL-23 and/or its receptor(s), or by otherwise affecting the availability of IL-23 and/or its receptor(s).

In still another embodiment, the invention encompasses promoting or otherwise facilitating IL-23 signaling by various ways, methods, and techniques that are intended to directly target IL-23 and/or its receptor(s) and/or downstream adaptors and other signaling apparatus necessary to transmit the signals conveyed by IL-23, either by directly targeting such adaptors and/or other signaling apparatus, and/or by affecting the synthesis, degradation, or availability of such adaptors and/or signaling apparatus.

In one embodiment, the invention encompasses inhibiting IL- 12 signaling by various methods, either by directly targeting IL- 12 and/or IL- 12 receptor(s), or by affecting synthesis of IL- 12 and/or its receptor(s), or by affecting degradation of IL- 12 and/or its receptor(s), or by otherwise affecting the availability of IL- 12 and/or its receptor(s).

In still another embodiment, the invention encompasses promoting or otherwise stimulating IL- 12 signaling by various ways, methods, and techniques that are intended to directly target IL- 12 and/or its receptor(s) and/or downstream adaptors and other signaling apparatus necessary to transmit the signals conveyed by IL- 12, either by directly targeting such adaptors and/or other signaling apparatus, and/or by affecting the synthesis, degradation, or availability of such adaptors and/or signaling apparatus.

In one embodiment, the invention encompasses inhibiting IL- 17 signaling by various methods, either by directly targeting IL- 17 and/or IL- 17 receptor(s), or by affecting synthesis of the IL- 17 and/or its receptor(s), or by affecting degradation of IL- 17 and/or its receptor(s), or by otherwise affecting the availability of IL- 17 and/or its receptor(s).

In still another embodiment, the invention encompasses promoting or otherwise facilitating IL- 17 signaling by various ways, methods, and techniques that are intended to directly target IL- 17 and/or its receptor(s) and/or downstream adaptors and other signaling apparatus necessary to transmit the signals conveyed by IL- 17, either by directly targeting such adaptors and/or other signaling apparatus, and/or by affecting the synthesis, degradation, or availability of such adaptors and/or signaling apparatus.

BRIEF DESCRIPTION OF THE DRAWINGS For the purpose of illustrating the invention, there are depicted in the drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings.

Figure 1 depicts the increased susceptibility of Tlr7—/— and Myd88^~/^~ mice, but not Tlr9—/— mice, after West Nile virus challenge. Wild-type mice (n = 24), Tlr7—/— mice (n = 22), Myd88-/^~ mice (n = 13), and 7⁷ZVP-/- mice (n = 14) were i.p. infected with WNV (LD50) and monitored twice daily for 21 days for mortality. Data shown are represented as time after infection (days) on the x axis and percent survival (%) on the y axis. Kaplan- Meier survival analysis revealed significant differences between wild-type and Tlr7—/— or Myd88-/^~ mice, but not between wild-type and Tlr9—/— mice. Data shown are pooled from 2-4 independent experiments.

Figure 2, comprised of Figures 2A through 2E inclusive, depicts 7⁷ZV 7- and Myd88- dependent viral load and innate immune cytokine responses after West Nile virus challenge. Wild-type, Tlr7—/—, or Myd88^~/^~ mice were i.p. challenged with West Nile virus (LD50). (A) Quantitative real-time PCR (Q-PCR; mean unit-less ratio + 1 SEM) was performed for WNVE on day (D) 3 postinfection peripheral blood, D6 perfused brain, or D6 perfused spleen samples from wild-type versus Tlr7—/— mice. (B) Q-PCR results (mean unitless ratio + 1 SEM) for WNVE on D3 peripheral blood, D3 spleen, or D6 perfused brain samples from wild-type versus Myd88^~/- mice. (C) Q-PCR results (mean unitless ratio + 1 SEM) for blood innate immune cytokines in wild-type and Tlr7—/— mice on D3 after infection. (D) ELISA results (pg/mL; mean + 1 SEM) for IL-23 (left bars) or IL-12 p40 (right bars) in blood samples from wild-type, Myd88—/—, or Tlr7—/— mice on D2 Myd88^~/^~ mouse experiment) or D3 (Tlr7—/— mouse experiment) after infection. (E) Q-PCR results (mean unitless ratio (+ 1 SEM) for brain innate immune cytokines in wild-type and Tlr7—/— mice on D6 after infection. Data are pooled results from 2-4 similar independent experiments, with at least n = 3 (and up to n = 22) per group for each experiment. ***p < 0.001, **p < 0.01, and *p < 0.05 compared to wild- type mice.

Figure 3, comprised of Figures 3A through 3D inclusive, demonstrates that in vivo leukocyte homing to West Nile-infected cells is Tlr7 dependent. Wild-type (WT) or Tlr7-/^~ mice were i.p. challenged with WNV (LD50). Uninfected WT and Tlr7^~/^~ mice were euthanized and processed side-by-side as negative controls. (A) Perfused brains were isolated on day 6 postinfection, and WNV antigen (green signal) and CD45 (leukocyte common antigen, red signal) or CDl Ib (macrophage and microglia marker, red signal) were detected by immunofluorescence with a Zeiss ApoTome-equipped epifluorescence microscope (original magnification 63 x). (B) Perfused livers were isolated on day 3 postinfection, and WNV antigen (green signal) and CD45 (red signal) were imaged with a Zeiss Apo-Tome-equipped epifluorescence microscope (original magnification 20^χ). DAPI (blue signal) was used as a nuclear counterstain, and representative images are shown. Numbers of CD45+ or CDl Ib+ cells per image colocalized with WNV antigen+ areas (first number) and total CD45+ or CDl Ib+ cells per image (second number) are shown in the bottom right in (A) and (B). (C) Quantitative PCR for WNVE in wild-type or Tlr7—/— livers at day 3 p.i. Graph shows means + 1 SEM. (D) Quantitative PCR for 1123a in wild-type or Tlr7—/— livers at day 3 p.i. Graph shows means + 1 SEM. Similar results were observed in 2-4 independent experiments with at least n = 4 per group for each experiment. **p < 0.01 and *p < 0.05 compared to wild-type mice.

Figure 4, comprised of Figures 4A through 4B inclusive, demonstrates 77r7-dependent macrophage homing in vitro. (A) Peritoneal thioglycollate-elicited macrophages were prepared from wild-type or Tlr7—/— mice and placed in the upper chamber of transwell plates. In the lower chamber, the TLR7 agonist loxoribine (loxO, from 0 to 100 μM), supernatants from WNV-infected N2a cell lysates (MOI = 0.5, diluted from 1 :1 to 1 :50), or macrophage chemoattractant protein- 1 (MCP-I, 1000 μg/mL) was added for 6 hr. Glass coverslips placed in the lower chamber were recovered for confocal microscopy for CDl Ib (green signal). DAPI (blue signal) was used as a nuclear counterstain (original magnification 63 ^χ), and representative images are shown. (B) CDl Ib+ cells in high- power fields (original magnification 63 ^χ) were counted (n = 3 per condition), and data are presented as means + 1 SD. Similar results were obtained in 3-4 independent experiments. **p < 0.01 and *p < 0.05 compared to wild-type macrophages.

Figure 5, comprised of Figures 5 A through 5 C inclusive, demonstrates TLR7 and IL-23 signaling-dependent macrophage responses. (A) Peritoneal thioglycollate-elicited macrophages were prepared from wild-type (WT) or Tlr7-/- mice (key applies to [A] and [C]) and infected with WNV at MOI = 1. 24 hr thereafter, cell lysates were prepared for III 2b Q-PCR analysis (left, unitless ratio + 1 SEM) or immunoblot for IL-23 pl9 and actin as a loading control (middle). Densitometry (ratio of IL-23 pl9 to actin protein signal) is shown in right panel. (B) Macrophages were stimulated with the TLR7 ligand loxoribine (loxO, from 0 to 250 μM as indicated) for 24 hr, and cell lysates were immunoblotted for IL-12Rβl, IL-12Rβ2, IL-23R, or actin (left). Densitometry (ratio of IL-12Rβl to actin signal) is shown in right panel (n = 3 for each condition). (C) Macrophages (left) were infected with WNV (from 1.0 to 2.0 MOI), and cell lysates were harvested 24 hr later for immunoblot analysis of IL-12Rβl, IL-12Rβ2, IL-23R, or actin. Densitometry (ratio of target signal to actin) is shown in graphs below each immunoblot. Macrophages (right) were placed in the upper chamber of transwell plates, and IL-23 (from 0 to 10 ng/mL) was placed in the lower chamber. 6 hr later, CDl lb+ cells in high- power fields were counted on glass coverslips in the lower chamber by confocal microscopy (n = 3 per condition). **p < 0.01 and *p < 0.05 compared to wild-type macrophages at the same dose. Means ± ISD are shown in (B) and (C). Similar results were observed in 2-4 independent experiments.

Figure 6 demonstrates that macrophage homing to West Nile virus is IL-23 signaling dependent. Wild-type (n = 6), 1112a-/- (n = 4), 1112b-/- (n = 4), and 1123a-/- (n = 3) mice were infected with West Nile virus (LD50). Brains were isolated on day 6 after infection and immunostained for confocal microscopy with antibodies against CDl Ib (green signal) and WNV antigen (red signal) to reveal microglia and infiltrating macrophages in WNV-infected brain regions. TOPRO3 was used as a nuclear counterstain (blue signal) and merged images are shown to the right. Numbers of CDl lb+ cells per image co localized with WNV antigen+ areas (first number) and total CDl Ib+ cells per image (second number) are shown in the bottom right. Similar results were obtained in 2-4 independent experiments.

Figure 7, comprised of Figures 7 A through 7C inclusive, depicts characterization of brain immune cells in wild-type, Tlr7—/—, Myd88—/—, 1112-/-, and 1123-/- mice after West Nile virus infection. Brain flow cytometry results are shown from 2-3 independent experiments on day 6 post-WNV infection (LD50; n = 1-5 mice per genotype and numbers represent percentages ± SD for the indicated gates). The 37:70% Percoll interface was collected and stained for CD45, CD4, CD8, CDl Ib, and F4/80 antigen as indicated in (A) (right, applies for all panels). A minimum of 100,000 events were collected for flow cytometry analysis, and dot plots show side scatter (SSC) on the x axis and CD45 log fluorescence intensity on the y axis. Numbers represent percentages of positive cells within gated regions. (A) Brain flow cytometry results are shown from wild-type vs. Tlr7—/— mice. (B) Brain flow cytometry data are shown for wild-type compared to Myd88^~/- or 1112a—/— mice. (C) Brain flow cytometry data are shown for wild-type compared to 1123a-/- or 1112b-/- mice.

Figure 8, comprised of Figures 8A and 8B, demonstrates reduced infiltrating leukocytes and increased viral load in Myd88-/- mouse brains after West Nile virus challenge. Panels are representative bright-field photomicrographs (original magnification = x 40) from wild-type or Myd88-/- mouse brain sections at day 6 post-infection. (A) CD45 immunohistochemistry is shown in olfactory bulb (B) WNV antigen immunohistochemistry is shown in olfactory bulb (upper panels) or brainstem (lower panels) from wild-type or Myd88-/- mice. Brain sections were nuclear counter-stained with hematoxylin (blue signal). Similar results were observed in 2-3 independent experiments.

Figure 9, comprised of Figures 9A and 9B, demonstrates macrophage 77r7-dependent cytokine responses. Peritoneal thioglycollate-elicited macrophages were prepared from wild-type or Tlr7-/- mice and treated with the TLR7 agonist loxoribine (loxO, from 0 to 200 μM as indicated on the x-axis) for 24 h. (A) Macrophage supernatants were collected for IL- 12 p40 ELISA. (B) Macrophage supernatants were collected for TNF-α ELISA. Data are presented as mean values + 1 SEM with n = 3 for each condition presented. Similar results were observed in 2 independent experiments.

Figure 10, comprised of Figures 1OA and 1OB, demonstrates that brain leukocyte homing and mouse survival after lethal West Nile infection is IL-23 -dependent (A) Wild-type (n = 6), III 2a-/- (n = 4), Il 12b-/- (n = 4), and 1123a-/- (n = 3) mice were infected with WNV (LD50), brains were isolated on day 6 after infection, and immunostained with antibodies against CD45 (green signal) and West Nile virus (WNV) antigen (red signal) to reveal infiltrating leukocytes in WNV-infected brain regions. TOPRO3 was used as a nuclear counterstain (blue signal), and merged images are shown to the right. Numbers of CD45+ cells per image co-localized with WNV antigen+ areas (first number) and total CD45+ cells per image (second number) are shown in the bottom right of merged images. Similar results were obtained in 2 independent experiments. (B) (left) Wild-type (n = 43) and 1112a-/- mice (n = 38), (middle) wild-type (n = 30) and III 2b-/- mice (n = 22), or (right) wild-type (n = 20) and Il 23 a-/- mice (n = 9) were i.p. infected with West Nile virus (LD50) and monitored twice daily for 21 days for mortality. Data shown are represented as time after infection (days) on the x-axis and percent survival (%) on the y- axis. Kaplan-Meier survival analysis revealed significant differences between wild-type and III 2b-/- or 1123a-/- mice (*P < 0.05, **P < 0.01), but not between wild-type and III 2a-/- mice. Data shown are pooled from 2-4 independent experiments. DETAILED DESCRIPTION OF THE INVENTION

The present invention encompasses composition, methods, approaches, and techniques to treat WNV infections and the encephalitis and menigitis and other brain and central nervous system inflammatory conditions that the viral infection causes. However, the present invention is not limited to treating these conditions instigated by or exacerbated by WNV, and the present invention is likely to apply to other, non-WNV forms of viral encephalitis.

A skilled artisan in the field will readily understand that, just as WNV can interact with TLR7, in the same or a similar way, other pathogens or PAMPs or DAMPs may also interact with TLR7 and lead to similar inflammations, whether in the brain and central nervous system, or in some other cell, tissue, organ, or topological region of the body. Examples include other forms of encephalitis including but not limited to Japanese encephalitis, Eastern equine encephalitis, or other flavivirus-induced or bacterial encephalitidies. Based on the disclosures herein, the intervention described could be used to treat or cure any number of problems, diseases, pathologies, or abnormal conditions that are caused by or contributed to by TLR7 signaling. Therefore, the interventions described herein are not limited to WNV or conditions, diseases, and pathologies caused by or contributed to by WNV.

The interventions described here could also be used to affect TLR7 signaling in situations where no disease is currently present, for example, but not limited to, situations where protection from infections or other disease challenges is sought, for example, but not limited to, vaccines, or various strategies to protect from cancer or cancers, or eradicate existing cancer or cancers.

Hence, for example, a skilled artisan would readily appreciate that, based on the disclosure presented herein, a vaccine against infectious challenge or threat could, in whole or part, involve stimulation, inhibition, or both, of TLR7 signaling.

A skilled artisan would also readily appreciate that, based on the disclosure presented herein, a cancer or cancers could be eradicated by a process that could involved stimulation, inhibition, or both, of TLR7 signaling. Likewise, a skilled artisan would understand from the disclosures presented herein, that protection from a cancer or cancers could similarly involve stimulation, inhibition, or both, of TLR7 signaling, particularly since cancers often involve modified or mutant nucleic acids, which could thereby interact with TLR7.

A skilled artisan would also readily understand that, based on the disclosures herein, everything in the preceding paragraph could also apply to IL- 12, IL-23 or IL- 17 signaling, hence this invention disclosure embodies the effects of IL- 12, IL-23 and IL- 17, whether those effects are transmitted or delivered through the known receptors for IL-23 and IL- 17, or whether those effects are transmitted or delivered in ways that are independent of the known receptors for IL-23 and IL- 17.

Furthermore, a skilled artisan will readily understand that, based on the disclosures herein, signaling could be affected in either a negative (i.e., inhibition) or positive (i.e., facilitation) manner, depending upon what type of effect is desired, and what situation, condition, disease, pathology, tissue, cell, body region, or normal or abnormal physiologic condition the intervention is applied in, and also depending upon how the duration of the effect should be affected, and whether or not it is desirable to turn the effect on or off.

Definitions

As used herein, each of the following terms has the meaning associated with it in this section. The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object(s) of the article. By way of example, "an element" means one element or more than one element.

The term "about" will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. The term "affect" as used herein refers to any method, technique, approach, intervention, or treatment that causes or leads to a detectable biological change.

The term "antibody" as used herein, refers to an immunoglobulin molecule, which is able to specifically bind to a specific epitope on an antigen molecule or group of antigen molecules. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoactive portions of intact immunoglobulins. Most antibodies are tetramers of immunoglobulin molecules. The antibodies in the present invention may exist in a variety of forms, for example including (but not limited to) polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, single chain antibodies, humanized antibodies, and other types of chemically or otherwise artificially modified or altered antibodies or fragments derived therefrom (Harlow et al., 1988; Houson et al., 1988; Bird et al., 1988).

The term "antigen" or "Ag" (the latter being the abbreviation for "antigen") as used herein is defines as a molecule that provokes an immune response. This is ofter referred to an an "antigenic" response or an "antigen-driven" immune response, since not all immune responses directly involve antigens. The immune response instigated by an "antigen" may involve either antibody production, or the activation of specific immunologically-competent cells, or the recruitment of immune effector cells that have some role in immune defenses, or the retention of such cells in a particular area or region of the body where infection or invasion of the body by foreign organisms may be occurring (or such a region where recruitment, retention, or activation of immune effector cells occurs in response to other molecules such as "danger signals" that are not necessarily produced by invading or infecting or colonizing foreign organisms, or one or more combinations of these. Those persons skilled and knowledgeable in the art and science will understand that any macromolecule, including virtually all proteins or peptides, but not necessarily limited to proteins or peptides, can serve as an antigen. Hence, antigens can also be derived from other types of molecules, including lipids, recombinant or genomic DNA, RNA, and carbohydrates. Furthermore, antigens may be molecules that are composed of one or more classes of such molecules, including, but not limited to, for example, protein antigens that have carbohydrate moieties associated, bound, or otherwise attached to them. A skilled artisan will understand that any DNA, which comprises a nucleotide sequence or sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes and "antigen" as that term is used herein. Furthermore, skilled artisans will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene or genes, but may be encoded by portions of distinct genes, or fused or otherwise modified or altered sequences of genes. It is furthermore obvious, to the skilled artisan, that the present invention includes, but is not limited to, the use of partial nucleotide sequences with or without modifications such as changes in the associated carbohydrates or phosphate or other chemical groups, and that these nucleotide sequences may be arranged in various combinations or spatial arrangements to elicit the desired immune response. One skilled in the art will further understand that an antigen need not be encoded by a "gene" at all. It is readily apparent, to a skilled artisan, that an antigen can be generated synthetically by various methods or can be derived from a biological sample. Such a biological sample can include, but is not limited to, a tissue sample, a tumor sample, a cell, a part of a cell, an organelle within a cell, or a biological fluid.

The term "antisense" refers particularly to the nucleic acid sequence of the non- coding strand of a double-stranded DNA molecule encoding a polypeptide, or to a sequence which is substantially homologous to the non-coding strand. As defined herein, an antisense sequence is complementary to the sequence of a double-stranded DNA molecule encoding a polypeptide. It is not necessary that the antisense sequence be complementary solely to the coding portion of the coding strand of the DNA molecule. The antisense sequence may be complementary to regulatory sequences specified on the coding strand of a DNA molecule encoding a polypeptide, which regulatory sequences control expression of the coding sequences.

As used herein, the term "DNA" refers to deoxyribonucleic acid.

"Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates or guides for synthesis of other polymers and macromolecules in biological processes having a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Therefore, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings that are publicly available and readily understood by skilled artisans, and the non- coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

As used herein, the term "RNA" refers to ribonucleic acid. The term "donor antigen" refers to an antigen expressed by the donor tissue to be transplanted into the recipient.

The term "dsRNA" refers to double-stranded ribonucleic acid.

The term "ssRNA" refers to single-stranded ribonucleic acid.

The term "shRNA" refers to short hairpin ribonucleic acid. As used herein, the term "engineer" refers to any manipulation of a cell that results in a detectable change in the cell, wherein the manipulation includes but is not limited to inserting a polynucleotide and/or polypeptide heterologous to the cell and mutating a polynucleotide and/or polypeptide native to the cell. A polynucleotide or polypeptide is "heterologous" to a cell if it is not part of the polynucleotides and polypeptides expressed in the cell as it exists in nature, i.e., it is not part of the wild-type of that cell. A polypeptide or polynucleotide is instead "native" to a cell if it is part of the polynucleotides and polypeptides expressed in the cell as it exists in nature, i.e., it is part of the wild-type of that cell. As used herein, "endogenous" refers to any material from or produced inside an organism, cell, tissue, or system.

As used herein, the term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue, or system.

As used herein, the term "expression" is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.

The term "fragment," as used herein, is a portion of an antibody or antibodies that differ in length from the length of a reference antibody, but retains some or all of the essential properties of the reference antibody. Similarly, a protein fragment can exist that is part of a larger parent protein. One example of a retained essential property would be the ability of the fragment antibody to bind to an antigen or part of an antigen, much like the reference antibody, and thereby alter the properties or function of a molecule, for example TLR7.

As used herein, the term "genetically engineered" refers to a modification of the inherent genetic material of a microorganism (e.g., one or more of the deletion such as a gene knockout, addition, or mutation of one or more nucleic acid residues within the genetic material), addition of exogenous genetic material to a microorganism (e.g., transgene, stable plasmid, integrating plasmid, naked genetic material, among other things), causing the microorganism to alter its genetic response due to external or internal signaling (e.g., environmental pressures, chemical pressures, among other things, or any combination of these or similar techniques for altering the overall genetic makeup of the organism.

As used herein, the term "modulate" is meant to refer to any change in biological state, i.e., increasing, decreasing, and the like. For example, the term "modulate" may refer to the ability to positively or negatively regulate the expression or activity of TLR7 and/or downstream adaptor molecules such as MyD88.

Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase "nucleotide sequence" that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain one or more introns.

The term "pharmacologic" as used herein refers to treatment, attempted treatment, or intended treatment by any drug, small molecule, or other type of molecule. The term "polynucleotide" as used herein is defined as a chain of nucleotides.

Furthermore, nucleic acids are polymers of nucleotides, which can be hydrolyzed into monomeric "nucleotides." The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

The term "polypeptide" as used herein is defined as a chain of amino acid residues, usually having a defined sequence. As used herein the term polypeptide is mutually inclusive of the terms "peptide" and "protein."

The term "recognition," as used herein, is defined as any interaction, regardless of the nature of the biological response or lack of biological response, that involves any molecule or collection of molecules or assembled molecules. As a non-limiting example, "recognition" of some molecule or molecules or assembly of molecules or organism or virus or portion of virus or cell by the TLR7 signaling pathway can occur by direct binding or by some other means, including but not limited to steric interaction, covalent binding, coordinate-covalent interaction, or indirect interaction or interactions, or other types of interactions, whether by the TLR7 receptor itself or by molecules directly or indirectly associated with the TLR7 signaling pathway. As used herein, the term "recombinant DNA" is defined as DNA produced by joining pieces of DNA from different sources.

The term "recombinant polypeptide" as used herein is defined as a polypeptide produced by using recombinant methods. The terms "signaling" or "signaling pathway" as used herein refer to any of the components within or outside of a cell that enable a stimulus to produce a biological effect. One embodiment would be the "TLR7 signaling pathway", which is stimulated by

WNV or ssRNA and includes numerous elements such as MyD88, IL-12, IL-23, and IL- 17; but is not limited to these elements.

As used herein, a "substantially purified" cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cells that have been separated from the cells with which they are naturally associated in the natural state. In some embodiments, the cells are cultured in vitro. It other embodiments, the cells are not cultured in vitro.

The term "T-cell" as used herein is defined as a thymus-derived cell that participates in a variety of cell-mediated immune reactions.

The term "B-cell" as used herein is defined as a cell derived from the bone marrow and/or spleen. B cells can develop into plasma cells, which can produce antibodies.

As used herein, the term "virus" is defined as a particle consisting of nucleic acid (RNA or DNA) enclosed in a protein coat, with or without an outer lipid envelope, which is capable of replicating within a whole cell.

Description The composition comprising the inhibitor of TLR7 or a component of the TLR7 signaling pathway can be any type of inhibitor. For example, and without limitation, the inhibitor can be selected from the group consisting of a small interfering RNA (siRNA), a microRNA (miRNA), short hairpin RNA (shRNA), other forms of RNA interference, an antisense nucleic acid, a ribozyme, an expression vector encoding a dominant negative mutant transgene, an intracellular antibody, a peptide, a tetramer, and a small molecule.

As disclosed herein, and by similar logic to the preceding strategy on inhibiting TLR7 or a component of the TLR7 signaling pathway, likewise facilitation of TLR7 or a component of the TLR7 signaling pathway is another intervention that arises directly from the disclosures herein. And, similarly, such facilitation of TLR7 or a component of the TLR7 signaling pathway can be by any means. For example, and without limitation, the facilitation can be accomplished by one or more of a group consisting of, but not limited to, siRNA, miRNA, shRNA, other forms of RNA interference, an antisense nucleic acid, a ribozyme, an expression vector encoding a transgene, an intracellular antibody, a peptide, a tetramer, and a small molecule.

The embodiments described immediately above relating to inhibiting or facilitating TLR7 signaling can and are similarly applied to IL- 12, IL-23 and IL- 17, by all means described above, but are not limited to such means.

Experimental Examples

The invention is further described in detail by reference to the following experimental examples. These examples are provided for illustrative purposes only. These examples are not intended to be limiting in any way, unless otherwise specified. Therefore, the invention should in no way be construed as being limited in any manner to the following specific examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching derived from the examples provided herein.

The materials and methods employed in the experiments disclosed herein are now described in the sections immediately below.

Mice

Tlr7-/- (Lund et al, 2004), Myd88-/- (Adachi et al, 1998), and Tlr9-/- (Hemmi et al, 2003) mice were bred to the C57BL/6 background by backcrossing for 10 successive generations. 1112a-/- (Mattner et al., 1996) and 1112b-/- (Magram et al., 1996) mice on a C57BL/6 background were obtained from Jackson Laboratories. 1123a-/- mice (Cua et al., 2003) on a mixed C57BL/6 3129 background were obtained from the Mutant Mouse Regional Resource Center (MMRRC). We performed all experiments on 8- to 12-week- old mice, and mouse groups were rigorously age- and sex-matched for each infection experiment. All experiments were performed with approval from the Yale Animal Resources Center Institutional Animal Care and Use Committee.

Virus Infection We inoculated mice intraperitoneally with 2000 plaque-forming units (p.f.u.) (LD50) of WNV isolate 2741 in 100 ml of PBS with 5% gelatin as previously described (Wang et al., 2004). Mice were observed for up to 21 days after infection and we checked them twice daily for morbidity (including lethargy, anorexia, and difficulty ambulating) and mortality.

Quantitative PCR

Ribonucleic acid was extracted from blood, spleen, liver, and brain tissue with the

RNeasy kit (QIAGEN). RNA was used to synthesize complementary (c) DNA with the ProSTAR First-strand RT-PCR kit (Stratagene). The flurogenic probes and primers that we used have been described elsewhere (Wang et al., 2004). Probes contained a 50 reporter, FAM, and a 30 quencher, TAMRA (Applied Biosystems). The assay was performed on an iCycler (Bio-Rad). The thermal cycling consisted of 95°C for 3.5 min and 48 cycles of 95°C for 30 s and 60⁰C for 1 min. To normalize the samples, the same amount of cDNA was used in the Actb Q-PCR. The ratio of the amount of amplified gene compared with the amount of Actb cDNA represented the relative amounts in each sample.

Immunofluorescence Imaging Brains and livers were rapidly isolated, fixed in 4% paraformaldehyde (PFA) overnight at 4°C, and cryoprotected in a graded series of sucrose (10%, 20%, and 30%, each overnight at 4°C). Spleens from infected mice sacrificed day 3 postinfection were used as a positive control to ensure specificity of WNV antibodies (data not shown). Para-median sagittal sections were cut at 25 mm with a cryostat. Tissue sections were PAP pen (Zymed Laboratories) applied and preblocked in serum-free protein block (Dakocytomation) for 30 min at ambient temperature. Sections were then reacted overnight at 4°C with various combinations of primary antibodies against CDl Ib (Serotec; 1 :200), CD45 (Serotec, 1 :200), or WNV antigen (from J. F. Anderson; 1 :250). After three rinses in PBS, sections were reacted with appropriate secondary antibodies conjugated with Alexa Fluor 488, 594, or 647 for 1 hr at ambient temperature. After three additional rinses in PBS, sections were then nuclear counterstained with DAPI or TOPRO3 (Invitrogen) and mounted in fluorescence mounting medium (ProLong Gold). Images were acquired in independent channels with a Zeiss ApoTome-equipped fluorescence microscope or a Zeiss LSM510 META confocal microscope. Immune cells in brain and liver and numbers of immune cells colocalized with WNV-infected target cells were counted in a blind fashion with Zeiss Axiovision software.

Histology and Immunohistochemistry Mice were transcardially perfused with PBS. We removed brains and spleens and placed them in 4% paraformaldehyde overnight at 4°C, and routinely embedded in paraffin. We processed antigen retrieval at 90⁰C for 30 min in 10% (v/v) target retrieval solution (Dakocytomation) or by treatment with 88% formic acid for 8 min. Endogenous peroxidase activity was quenched in 0.3% H₂O₂ for 30 min at room temperature. We performed the staining using the Vectastain Elite ABC kit coupled to the 3 '-3 diaminobenzadine substrate (Vector Laboratories). Rat antibody specific for mouse CD45 (clone YW 62.3, Serotec) or mouse ascitic fluid WNV antigen antibodies were used. Sections were digitized with Kodak scientific imaging software (Eastman Kodak).

Enzyme-Linked Immunosorbance Assay (ELISA)

ELISA was carried out based on previously published methods (Wang et al., 2004). IL- 12/-23 p40, TNF-α, and IL-23 pl9 ELISA kits were purchased from R&D Systems or eBioscience, and the assays were performed in accordance with the manufacturer's instruction. Results are expressed as pg of cytokine per mL of cell culture medium.

Chemokines and Receptors, Common Cytokines and Toll-Like Receptor Signaling Pathway Q-PCR Arrays

One million peritoneal thioglycollate-elicited macrophages were prepared from wild-type or 77rr^Amice and challenged with WNV isolate 2741 (MOI = 1.0) in 6-well culture plates in 2 ml medium and incubated in a 37°C, 5% CO₂ incubator. Macrophages were collected at 24 h after washes with PBS for total RNA extraction by Qiagen RNeasy Mini kit (Valencia, CA). The RT² Profiler™ PCR Array kits were purchased from SuperArray Bioscience Corporation (Frederick, MD). The assay procedures followed the kit user manual. Briefly, one microgram of total RNA from the TIrT ^'or wild-type macrophages was transcribed into the first strand cDNA and loaded into 96-well PCR array plates with 25 μl Q-PCR master mix per well. After performing Q-PCR, the resulting threshold cycle values (C_t) for all genes were exported into the company-provided Data Analysis Template Excel files for comparison of gene expression between TIrT^1' and wild-type macrophages. The Q-PCR array experiments for each analysis were repeated two to three times with similar results.

Cytokine Antibody Array One million peritoneal thioglycollate-elicited macrophages were prepared from wild-type or TlrT^μ mice and challenged with WNV isolate 2741 (MOI = 1.0) in 6-well culture plates in 2 ml medium and incubated in a 37°C, 5% CO₂ incubator. Supernatants were collected at 24 h for protein level cytokine expression analysis using Mouse Cytokine Antibody Array 3 which detects 62 cytokines (RayBiotech., Norcross, GA). Briefly, one ml of TIrT^' or wild-type macrophage supernatant was incubated with arrayed antibody support membrane overnight at 4°C. After several washes, the membranes were incubated with biotinylated detection antibody for 2 h at room temperature. Then, membranes were incubated with HRP-labeled-streptavidin for 2 h at room temperature after washes. Cytokine expression signals were detected by exposing membranes to Kodak X-omat AR film for analysis.

Western Immunoblot

Macrophages were isolated as described above and plated in 6-well culture plates (1 x 10⁶ cells/well) and infected with WNV (MOI = 1.0 to 2.0) or challenged with the Tlr7 agonist loxoribine (0 to 200 μM) for 24 h. Western immunoblot was carried out as described (Wang et al., 2004). Briefly, cells were scraped into ice-cold lysis buffer (containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% v/v Triton X- 100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 μg/ml leupeptin, and 1 mM PMSF) and protein was quantified by the Bradford method. A 50 μg aliquot was then subjected to Nu-PAGE separation on gradient (4-12%) gels using MES buffer (Invitrogen). After rinsing in TBS containing 0.05% Tween-20 (TBS-T) and blocking in block buffer (TBS-T plus 5% nonfat milk), membranes were incubated overnight at 4°C with primary antibodies against IL-12Rβl (SantaCruz; 1 :100), IL-12Rβ2 (SantaCruz; 1 :100), IL-23R (abeam; 1 :500), IL-23 pl9 (SantaCruz; 1 :100), or actin (SantaCruz; 1 :500). Membranes were rinsed three times in TBS-T, and incubated at for 1 h at ambient temperature with appropriate secondary HRP-conjugated antibodies (Amersham; 1 :4000). After extensive rinses in TBS-T, ECL substate was added (Pierce) and membranes were exposed to film. Band densities were quantified by first digitizing images into a Windows-based computer using an Alpha Innotech FluorChem 8800 Imager and then using Scion Image for Windows software, release beta 4.0.2, to calculate background-subtracted band density ratios.

Statistical Analysis

We calculated standard errors of the means (SEM) and standard deviations (SD) and analyzed data by nonpaired Student's t test for single mean comparisons or ANOVA and post-hoc testing for multiple comparisons of the means. Survival curve comparisons were conducted with the log-rank test (equivalent to the Mantel-Haenszel test, Prism software).

Example 1:

Tlr7—/— and Myd88-/- Mice Are More Susceptible to Lethal WNV Infection. We used the mouse model of WNV encephalitis to examine the role of TLR7 and MyD88 in viral recognition in vivo. Mice were challenged with 2 x 10³ plaque-forming units (p.f.u.) of WNV corresponding to a dose at which approximately 50% of wild-type (C57BL/6J) animals survive (LD50) and were monitored twice daily for mortality. TLR7-deficient (Tlr7—/—) mice were significantly more susceptible (9% survival) to lethal WNV infection than were wild-type control mice (50% survival, p < 0.05; Figure 1). The adaptor molecule MyD88 is required for TLR7 signaling ([Diebold et al, 2004], [Hemmi et al., 2002] and [Lund et al., 2004]), and our data indicated a similar pattern of survival results after WNV infection of MyD88-defϊcient (Myd88^~/-) mice (15% survival) compared to wild-type controls (p < 0.05; Figure 1). TLR9 recognizes bacterial DNA containing unmethylated CpG motifs (Hemmi et al., 2000) and, like TLR7, requires MyD88 for signaling ([Bauer et al., 2001] and [Hemmi et al., 2003]). Furthermore, it has recently been suggested that TLR9 might cooperate with TLR7 in recognizing viral nucleic acid associated with murine cytomegalovirus (Zucchini et al., 2008). However, our results suggested that increased susceptibility of Tlr7—/— mice to WNV infection was specific, because TLR9-deficient (7W-/-) mice infected with WNV at LD50 (43% survival) were not significantly different from controls (50% survival, p > 0.10; Figure 1).

Example 2: Viral Load and Cytokines in Tlr7^~/- and Mvd88-/^~ Mice after WNV Infection. We next examined viral load in the brain as well as systemically. WNV was detected at high amounts in the spleen, liver, and blood at days 2-3 postinfection (pi.) and in the brain at day 6 p.i. (Wang et al., 2004). Quantitative real-time polymerase chain reaction (Q-PCR) measuring WNV envelope gene [WNVE) revealed approximate 3 -fold increased RNA abundance compared to control mice in Tlr7—/— mice (p < 0.05; Figure 2A) and Myd88^~/- mice (p < 0.05; Figure 2B) in blood at days 2-3 p.i. There was also a modest (2-fold) but significant (p < 0.05) increase in Myd88^~/- splenic WNVE RNA expression (Figure 2B) at day 3 p.i., which did not reach significance in Tlr7—/— mice at day 3 (data not shown) but was significantly higher at day 6 p.i. (p < 0.01; Figure 2A). Strikingly, WNVE RNA expression was markedly (8-fold) elevated in Tlr7—/— brains at day 6 p.i. (p < 0.05; Figure 2A) and was also significantly (3-fold) increased in Myd88^~/- brains at day 6 p.i. (p < 0.05; Figure 2B).

Furthermore, infectious viral plaque formation assay was carried out on Tlr7—/— and control brains at day 6 p.i. with WNV (LD50). Consistent with WNVE Q-PCR results, there was a striking increase in infectious virus recovered from Tlr7—/— brains (n = 4; 1.33 x 107 pfu/g of brain ± 0.9 x 107 SEM) versus wild-type brains (n = 5; 0.04 x 107 pfu/g of brain ± 0.03 x 107 SEM) that trended toward statistically significant (p = 0.07). Additionally, cytokine abundance was measured in blood and brain. Surprisingly, RNA expression of interferon-α (IFN-α), IFN-β, interleukin (IL)-I β, IL-6, and tumor necrosis factor-α (TNF-α) were all significantly (*p < 0.05, **p < 0.01, ***p < 0.001) increased in blood from WNV-infected Tlr7—/— mice versus controls at day 3 p.i. (Figure 2C).

Despite a generalized increase in systemic innate cytokine RNAs after WNV infection of Tlr7-/^~ mice, IL- 12 p40 (the shared cytokine chain with IL-23) RNA (III 2b) was significantly (p < 0.05) reduced in blood samples from Tlr7—/— mice versus controls (Figure 2C), and a similar pattern of results was noted for secreted heterodimeric IL-23 protein in Myd88^~/- mice compared with controls early after infection (p < 0.05; Figure 2D). Also, in concert with reduced IL-12 p40 RNA in blood from Tlr7—/— mice versus controls, there was significant (p < 0.05) reduction in IL-12 p40 protein concentrations in blood plasma (Figure 2D). Quantification of IL-12 p35, IL-12 p40, and IL-23 pl9 RNAs in brains of Tlr7-/- mice versus controls at day 6 p.i. disclosed significant (*p < 0.05, **p < 0.01) reductions in IL-12 p35 and IL-23 pl9 RNAs in Tlr7-/^~ mice (Figure 2E). Example 3:

Immune Cell Homing to WNV-Infected Cells In Vivo depends on TLR7 and MyD88. Next, we performed immunofluorescence analysis of WNV antigen and CDl Ib (microglial and macrophage marker) or CD45 (leukocyte marker) in brains of Tlr7—/— versus control mice at day 6 p.i. Our focus was on the olfactory bulb, because this brain region is most sensitive to WNV infection and brain inflammation (Wang et al., 2004). Consistent with WNVE Q-PCR, these analyses revealed increased WNV antigen immunoreactivity in brains of infected Tlr7—/— mice compared with infected wild-type animals (Figure 3A). Similar results were obtained in other brain regions, including cerebral cortex, brainstem, cerebellum, and striatum (data not shown). In wild-type mice, CDl Ib+ microglia and macrophages and CD45+ leukocytes were found in close apposition to (often in direct contact with) infected neurons. However, despite increased WNV burden, CDl lb+ and CD45+ immune cells failed to home to infected brain cells in Tlr7—/— mice, although they were detected in these mice at a distance from WNV- infected brain cells (Figure 3A). As expected, uninfected (control) brain sections from wild-type or Tlr7—/— mice did not display signal for WNV antigen, CD45, or CDl Ib (Figure 3A). A more severe phenotype was observed in infected Myd88^~/- brains, where CD45+ cells were nearly absent (Figure 8A), despite increased WNV burden (Figure 8B).

To determine whether impaired immune cell homing in infected TIr 7-/- mice was specific to the brain, we also analyzed livers (another target organ of WNV infection) (Venter et al., 2005) from wild-type versus Tlr7—/— mice at day 3 p.i.. In wild-type mice, confocal microscopy revealed numerous CD45+ leukocytes in close vicinity of infected hepatocytes. However, a different pattern of results was evident in Tlr7—/— mice. Similar to observations in brain, CD45+ cells were present, but were often found at a distance from WNV infected hepatocytes (Figure 3B). This phenotype was even more striking when considering that infected TIr 7-/- mouse livers had nearly 6-fold higher abundance of WNVE RNA copies compared with wild-type controls (p < 0.01; Figure 3C). Furthermore, this effect was associated with significantly (p < 0.05) reduced IL-23 pl9 RNA in Tlr7—/— versus wild-type mouse livers (Figure 3D). To determine whether this effect was owed to a generalized defect in TIr 7—/— immune cells, macrophages were recovered from the peritoneal cavity at 4 days after thioglycollate injection and enumerated. There was a modest nonsignificant trend for more cells in the Tlr7—/— mice (n = 4 mice per group, means ± SD for wild-type versus Tlr7^~/^~ mice: 7.16 x 10⁶ ± 1.94 x 10⁶ versus 9.14 x 10⁶ ± 1.61 x 10⁶; p = 0.17), indicating that macrophage recruitment, locomotion, and homing were intact in these animals. When taken together, these data suggested that TLR7 mediated immune cell homing to WNV-infected target cells in vivo and that this effect was associated with IL- 12 and IL-23 responses.

Example 4:

Recognition of WNV Triggers TLR7 and IL-23 -Dependent Macrophage Chemotaxis In Vitro.

To determine whether reduced immune cell homing to WNV-infected cells in TIr 7-/- mice could be recapitulated in vitro, we established a transwell chemotaxis assay. Because CDl Ib+ macrophage cell homing to infected target cells was inhibited in Tlr7—/— mice, peripheral macrophages were elicited from Tlr7—/— and wild-type mice for in vitro chemotaxis analyses. Lower chambers of transwell plates (containing a glass coverslip) were loaded with a dose range of the TLR7 small molecule agonist loxoribine (loxO), supernatants from WNV-infected neuroblastoma-2a (N2a) lysates, or macrophage chemoattractant protein- 1 (MCP-I, as a positive control), and 1 ^χ 10⁵ macrophages were placed in the upper chamber. After 6 hr, lower-chamber glass coverslips were recovered and immunolabeled with CDl Ib antibody for confocal microscopy. Wild-type macrophage migration was increased in response to loxO, infected N2a lysate supernatants, and MCP-I. Yet, although Tlr7—/— macrophages increased migration toward MCP-I, indicating an intact chemotactic response, they were refractive to loxO or infected N2a lysate supernatants (Figure 4A). Quantitation revealed significantly (*p < 0.05, **p < 0.01, ***p < 0.001) reduced chemotaxis of Tlr7—/— macrophages toward loxO (at 50 or 100 μM) or infected N2a lysate supernatants (multiplicity of infection [MOI] = 0.5, from undiluted to 1 :50), but not toward MCP-I (Figure 4B). We also infected N2a cells with MOI = 1 of WNV and prepared cell lysate supernatants and noted similar effects with this material on Tlr7—/— versus wild-type macrophages transwell migration (data not shown).

Next, we determined whether the inhibition of migration of Tlr7—/— macrophages in response to WNV infection was associated with reduced IL- 12 and IL-23 amounts. Tlr7—/— and wild-type macrophages were infected with WNV (MOI = 1) and assayed for IL- 12 p40 RNA and IL-23 pl9 protein by Q-PCR and immunoblot, respectively. Infected Tlr7-/^~ macrophages produced significantly (p < 0.01) less IL-12 p40 RNA compared with wild-type cells, and WNV-induced IL-23 pl9 protein was also clearly reduced in infected TIr 7-/- macrophages (Figure 5A). Additionally, TIr 7-/- macrophages were completely nonresponsive to a dose-range of loxO when measuring IL-12 p40 or TNF-α, which further suggested that the above effect was TLR7 dependent (Figure S2).

In our model, we proposed that brain-resident macrophages (microglia) initially produced IL-12 and IL-23 upon TLR7 recognition of brain-penetrating WNV and that infiltrating macrophages and other leukocytes would then migrate in response to this signal. If this were the case, then one might expect reduced IL-12 receptor (R) and IL-23R expression to underlie hyporesponsiveness of TIr 7-/- macrophages to WNV-induced chemotaxis. In an effort to address this possibility, Tlr7—/— or wild-type macrophages were challenged with a dose-range of loxO, and protein expression of IL-12R and IL-23R was determined. Much as IL-12 and IL-23 share the IL-12 p40 subunit (Cooper and Khader, 2007), their receptors also form heterodimers sharing the common chain IL-12Rβl subunit (van de Vosse et al, 2003). Although IL-12Rβ2 and IL-23R were not further inducible in Tlr7—/— and wild-type macrophages after loxO challenge, IL-12Rβl was induced in wild-type macrophages, but Tlr7^~/^~ macrophages were nonresponsive (Figure 5B). It was next determined whether WNV infection of wild-type versus Tlr7—/— macrophages could produce a similar effect. Similar to loxO challenge, WNV at MOI = 1 or 2 induced IL-12Rβl expression in wild-type macrophages, but this response was inhibited in Tlr7—/— macrophages (Figure 5C). Finally, we examined whether IL-23 or IL-12 could directly affect TLR7-dependent macrophage chemotaxis in vitro by using our transwell assay. Results showed that IL-23 dose dependently (from 2.5 to 10 ng/mL) augmented chemotaxis of wild-type but not Tlr7—/— macrophages (Figure 5C). However, both wild- type and Tlr7—/— macrophages were refractive to IL-12 p70 chemotaxis (n = 3 transwells per group, means ± SD for wild-type versus Tlr7—/— macrophages for cells per high power field, 0 ng: 4 cells ± 1 cell versus 4.33 cells ± .58 cells; 2.5 ng: 4.67 cells ± .58 cells versus 5.33 cells ± .58 cells; 5.0 ng: 4.67 cells ± .58 cells versus 5.33 cells ± .58 cells; 10 ng: 4.33 cells ± .58 cells versus 4.67 cells ± .58 cells). Thus, IL-23 was playing the major role in TLR7-dependent macrophage chemotaxis in vitro. To determine whether the effects we observed in TIr 7—/— macrophages might be due to a compensatory TIr response, TIr Q-PCR array was performed on WNV-infected macrophages, and only Tlr7 RNA was substantially altered after infection in Tlr7—/— macrophages (data not shown). Further, Q-PCR arrays were carried out for chemokines and chemokine receptors, and cytokines and cytokine receptors, to identify any additional targets of TLR7 after WNV infection, but there were no obvious alterations (data not shown). Finally, supernatants from infected macrophages were assayed by protein cytokine array and we did not observe obvious differences between wild-type and Tlr7—/— macrophages (data not shown). Thus, according to unbiased approaches, additional TLR7-dependent targets of WNV were not identified in macrophages.

Example 5:

Immune Cell Homing to WNV-infected Cells Is TLR7-IL-23 Dependent In Vivo. Our data thus far suggested that recognition of WNV by TLR7 caused IL-23-dependent immune cell homing. To directly test this hypothesis in vivo, wild-type, IL-12 p35- defϊcient (III 2a-/-), IL-12 p40-deficient (1112b-/-), or IL-23 pl9-defϊcient (1123a-/-) mice were infected with WNV and we assessed infected brains for presence of WNV and infiltrating leukocytes by confocal microscopy on day 6 p.i.. Wild-type and 1112a—/— mice demonstrated CDl Ib+ macrophages and microglia (Figure 6) and CD45+ leukocytes (Figure 10A) in close apposition to infected brain cells. However, in both III 2b-/- and 1123a—/— mice, CDl lb+ macrophages and microglia (Figure 6) and CD45+ leukocytes (Figure 10A) were not clearly co localized to foci of WNV-infected cells. Further, 1123 a-/- mice had fewer infiltrating CDl Ib+ macrophages and microglia (Figure 6) that were not clearly associated with WNV-infected brain cells. Thus, proper infiltration and homing of immune cells to target WNV-infected cells required IL-23.

To better define and enumerate brain immune cells, wild-type, Tlr7—/—, Myd88—/—, III 2a-/-, 1123a—/—, or 1112b—/— mice were infected with WNV (LD50) and brains were isolated on day 6 p.i.. We applied flow cytometry methodology to single-cell suspensions of harvested brains to characterize numbers of brain-infiltrating macrophages (based on CD45^luCDl lb+ status; [Juedes and Ruddle, 2001] and [Town et al, 2008]), brain-resident microglia (by CD45 CDl lb+F4/80 Ag+; [Juedes and Ruddle, 2001] and [Town et al., 2008]), and brain-infiltrating CD45^luCD4+ and CD45^luCD8+ T cells in these mice. In concert with our confocal microscopic analyses, brain-infiltrating macrophages were reduced by 59% to as much as 98% when comparing wild-type to Tlr7^~/^~, Myd88^~/-, 1112b—/—, or 1123a-/- mice, whereas 1112a-/- mice appeared similar to wild-type WNV-infected brains (Figure 7). A generally similar pattern of results was evident when considering CD4+ and CD8+ T cells, which were attenuated between 55% and 89% when comparing wild-type to Tlr7—/—, Myd88—/—, or Il 12b—/— mice, whereas Il 12a—/— or 1123 a-/- mice appeared similar to wild-type WNV-infected brains (Figure 7). However, a different pattern of results emerged when considering brain-resident microglia, which did not obviously differ from wild-type WNV-infected brains except for an apparent 75% reduction in III 2b—/— mice (Figure 7). We also analyzed B cells by CD 19 expression, but did not detect consistent differences between these mouse genotypes (data not shown).

To determine whether impaired leukocyte homing to WNV-infected brains was due to a general defect in leukocyte expansion or differentiation, or cell death in the periphery, we analyzed spleens from the same animals as above and did not detect any differences between genotypes (data not shown). Interestingly, similar effects are reported in other neuroinflammatory conditions where peripheral leukocytes infiltrate into the central nervous system (CNS) of (1) experimental autoimmune encephalomyelitis-induced mice or (2) Alzheimer's mouse models in the absence of innate immune TGF-β signaling, but populations of these cells in the periphery are essentially unaltered ([Laouar et al., 2008] and [Town et al., 2008]). Taken together, these brain flow cytometry results corroborated our confocal microscopic analyses and strengthened our conclusion that brain infiltration or homing of leukocytes (specifically, macrophages and T cells) to WNV-infected CNS was dependent on TLR7-MyD88-IL-23 signaling.

Finally, we determined whether survival after lethal WNV encephalitis was dependent on IL-12, IL-23, or both. Thus, wild-type, 1112a—/—, 1112b—/—, or 1123a—/— mice were infected with WNV (LD50) and monitored twice daily for survival. Based on our findings of IL-23 -dependent immune cell homing to WNV-infected brain cells, we hypothesized reduced survival in 1112b—/— and 1123a.—/— but not 1112a—/— mice after lethal WNV challenge. Results were consistent with this hypothesis. Specifically, although 1112a—/— mice did not differ from wild-type controls (42% survival for both groups, p > 0.10; Figure 10B), both Il 12b—/— mice (27% survival versus 53% for wild-type controls, p < 0.05; Figure 10B) and 1123 a—/— mice (0% survival versus 25% for wild-type controls, p < 0.01; Figure 10B) were more susceptible to lethal WNV infection. Collectively, these results showed that survival after lethal WNV challenge required intact IL-23 as opposed to IL- 12 responses.

Claims

1. A method of affecting TLR7-dependent signaling for the purpose of treating encephalitis, the method comprising stimulating the TLR7 signaling pathway in a cell in an organism in which TLR7 is naturally expressed, or is induced to be expressed by a natural or artificial means.

2. The method of Claim 1, wherein encephalitis is caused by West Nile virus or at least a portion of a West Nile virus.

3. The method of Claim 1, wherein encephalitis is caused by Eastern Equine

Encephalitis virus or at least a portion of a Eastern Equine Encephalitis virus.

4. The method of Claim 1, wherein encephalitis is caused by Japanese Encephalitis virus or at least a portion of a Japanese Encephalitis virus.

5. The method of Claim 1, wherein encephalitis is caused by a flavivirus or at least a portion of a flavivirus.

6. The method of Claim 1, wherein encephalitis is caused by a virus or at least a portion of a virus other than a flavivirus.

7. The method of Claim 1, wherein encephalitis is caused by a bacteria or at least a portion of a bacteria.

8. The method of Claim 1, wherein encephalitis is caused by a foreign pathogen or at least a portion of a foreign pathogen other than a virus or bacteria.

10. The method of Claim 1, wherein said TLR7 signaling pathway is the TLR7-MyD88 signaling pathway.

11. The method of Claim 1, wherein said TLR7 signaling pathway is the TLR7 signaling pathway that does not require signaling through the MyD88 adaptor.

12. The method of Claim 1, wherein a molecule or molecules used to influence TLR7 signaling is a drug or small molecule.

13. The method of Claim 1, wherein a molecule or molecules used to influence TLR7 signaling is an antibody or antibodies, and wherein the molecule or molecules are modified, unmodified, humanized, or consist of a fragment, or are chimeric hybrid forms of an antibody or antibodies or fragments thereof.

14. The method of Claim 1, wherein a molecule or molecules used to influence TLR7 signaling are lipids, carbohydrates, amino acids, or single-stranded nucleic acids, and wherein the molecule or molecules are naturally occurring, endogenously synthesized, exogenously synthesized, or artificially created, or double- stranded nucleic acids whether naturally occurring, endogenously synthesized, exogenously synthesized, or artificially created, with or without additional chemical or other types of modifications.

15. The method of Claim 1 , wherein the TLR7 signaling pathway is the TLR7- IL- 12 signaling pathway.

16. The method of Claim 1 , wherein the TLR7 signaling pathway is the TLR7-

IL-23 signaling pathway.

17. A method of affecting TLR7-dependent signaling for the purpose of treating encephalitis, the method comprising inhibiting the TLR7 pathway in a cell in an organism in which TLR7 is naturally expressed, or is induced to be expressed by a natural or artificial method.

18. The method of Claim 17, wherein encephalitis is caused by West Nile virus or at least a portion of a West Nile virus.

19. The method of Claim 17, wherein encephalitis is caused by Eastern Equine Encephalitis virus or at least a portion of a Eastern Equine Encephalitis virus.

20. The method of Claim 17, wherein encephalitis is caused by Japanese Encephalitis virus or at least a portion of a Japanese Encephalitis virus.

21. The method of Claim 17, wherein encephalitis is caused by a flavivirus or at least a portion of a flavivirus.

22. The method of Claim 17, wherein encephalitis is caused by a virus or at least a portion of a virus other than a flavivirus.

23. The method of Claim 17, wherein encephalitis is caused by a bacteria or at least a portion of a bacteria.

24. The method of Claim 17, wherein encephalitis is caused by a foreign pathogen or at least a portion of a foreign pathogen other than a virus or bacteria.

25. The method of Claim 17, wherein said TLR7 signaling pathway is the TLR7-MyD88 signaling pathway.

26. The method of Claim 17, wherein said TLR7 signaling pathway is the TLR7 signaling pathway that does not require signaling through the MyD88 adaptor.

27. The method of Claim 17, wherein the molecule or molecules used to influence TLR7 signaling is a drug or small molecule.

28. The method of Claim 17, wherein the molecule or molecules used to influence TLR7 signaling is an antibody or antibodies, and wherein the molecule or molecules are modified, unmodified, humanized, or consist of a fragment, or are chimeric hybrid forms of an antibody or antibodies or fragments thereof.

29. The method of Claim 17, wherein the molecule or molecules used to influence TLR7 signaling are lipids, carbohydrates, amino acids, or single-stranded nucleic acids whether naturally occurring, endogenously synthesized, exogenously synthesized, or artificially created, or double-stranded nucleic acids, and wherein the molecule or molecules are naturally occurring, endogenously synthesized, exogenously synthesized, or artificially created, with or without additional chemical or other types of modifications.

30. The method of Claim 17, wherein the TLR7-dependent signaling pathway is the TLR7-IL-12 signaling pathway.

31. The method of Claim 17, wherein the TLR7-dependent signaling pathway is the TLR7-IL-23 signaling pathway.

32. A method of enhancing recognition of a virus by the TLR7 signaling pathway, comprising manipulating the TLR7 signaling pathway in a cell in an organism in which TLR7 is naturally expressed, or is induced to be expressed by a natural or artificial means, wherein the virus is West Nile virus, flavivirus, or at least a portion of West Nile virus or flavivirus, or a molecule derived from West Nile virus or flavivirus, wherein the molecule is unmodified or modified endogenously, exogenously, or artificially.

33. A method of inhibiting recognition of a virus by the TLR7 signaling pathway, comprising manipulating the TLR7 signaling pathway in a cell in an organism in which TLR7 is naturally expressed, or is induced to be expressed by a natural or artificial means, wherein the virus is West Nile virus, flavivirus, or at least a portion of West Nile virus or flavivirus, or a molecule derived from West Nile virus or flavivirus, wherein the molecule is unmodified or modified endogenously, exogenously, or artificially.

34. A method of treating an inflammatory central nervous system disease, comprising manipulating the TLR7 signaling pathway via a pharmarcologic approach.