WO2010091265A1 - Composés et procédés destinés au traitement d'une encéphalite virale - Google Patents

Composés et procédés destinés au traitement d'une encéphalite virale Download PDF

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WO2010091265A1
WO2010091265A1 PCT/US2010/023340 US2010023340W WO2010091265A1 WO 2010091265 A1 WO2010091265 A1 WO 2010091265A1 US 2010023340 W US2010023340 W US 2010023340W WO 2010091265 A1 WO2010091265 A1 WO 2010091265A1
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tlr7
virus
encephalitis
signaling pathway
molecule
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PCT/US2010/023340
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English (en)
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Terrence Town
Erol Fikrig
Richard Flavell
Fengwei Bai
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Yale University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV

Definitions

  • TLRs Toll-like receptors
  • TLR 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).
  • 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).
  • dsRNA double-stranded RNA
  • WNV West Nile virus
  • 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; Triantaf ⁇ lou et al., 2005).
  • ssRNA 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; Triantaf ⁇ lou et al., 2005).
  • TLR7 is thought to recognize single-stranded RNA (“ssRNA”), which is generally derived from certain types of viruses.
  • ssRNA single-stranded RNA
  • WNV 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.
  • livestock e.g., pigs, cows, and horses
  • 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.
  • IL interleukin
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • 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.
  • 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).
  • 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.
  • Figure 1 depicts the increased susceptibility of Tlr7—/— and Myd88 ⁇ / ⁇ mice, but not Tlr9—/— mice, after West Nile virus challenge.
  • 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 7 7 ZV 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).
  • Q-PCR Quantitative real-time PCR
  • D 3 postinfection peripheral blood, D6 perfused brain, or D6 perfused spleen samples from wild-type versus Tlr7—/— mice.
  • (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.
  • 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.
  • DAPI blue signal
  • Figure 4 comprised of Figures 4A through 4B inclusive, demonstrates 77r7-dependent macrophage homing in vitro.
  • loxO peritoneal thioglycollate-elicited macrophages
  • MOI 0.5, diluted from 1 :1 to 1 :50
  • MCP-I macrophage chemoattractant protein- 1
  • Figure 5 comprised of Figures 5 A through 5 C inclusive, demonstrates TLR7 and IL-23 signaling-dependent macrophage responses.
  • (B) Macrophages were stimulated with the TLR7 ligand loxoribine (loxO, from 0 to 250 ⁇ M as indicated) for 24 hr, and cell lysates were immunoblotted for IL-12R ⁇ l, IL-12R ⁇ 2, IL-23R, or actin (left). Densitometry (ratio of IL-12R ⁇ l to actin signal) is shown in right panel (n 3 for each condition).
  • FIG. 6 demonstrates that macrophage homing to West Nile virus is IL-23 signaling dependent.
  • 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.
  • SSC side scatter
  • 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.
  • 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 ⁇ M as indicated on the x-axis) for 24 h.
  • loxO the TLR7 agonist loxoribine
  • Macrophage supernatants were collected for IL- 12 p40 ELISA.
  • TOPRO3 was used as a nuclear counterstain (blue signal), and merged images are shown to the right.
  • 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.
  • 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.
  • WNV can interact with TLR7
  • 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.
  • 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.
  • a vaccine against infectious challenge or threat could, in whole or part, involve stimulation, inhibition, or both, of TLR7 signaling.
  • a cancer or cancers could be eradicated by a process that could involved stimulation, inhibition, or both, of TLR7 signaling.
  • 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.
  • 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.
  • inhibition i.e., inhibition
  • facilitation i.e., facilitation
  • 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.
  • an element means one element or more than one element.
  • antibody 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).
  • 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.
  • antigens can also be derived from other types of molecules, including lipids, recombinant or genomic DNA, RNA, and carbohydrates.
  • 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.
  • 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.
  • 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.
  • an antigen need not be encoded by a "gene" at all.
  • an antigen can be generated synthetically by various methods or can be derived from a biological sample.
  • 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.
  • 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.
  • 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.
  • 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.
  • a polynucleotide such as a gene, a cDNA, or an mRNA
  • 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.
  • RNA refers to ribonucleic acid.
  • donor antigen refers to an antigen expressed by the donor tissue to be transplanted into the recipient.
  • dsRNA refers to double-stranded ribonucleic acid.
  • ssRNA refers to single-stranded ribonucleic acid.
  • shRNA refers to short hairpin ribonucleic acid.
  • engine 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.
  • endogenous refers to any material from or produced inside an organism, cell, tissue, or system.
  • exogenous refers to any material introduced from or produced outside an organism, cell, tissue, or system.
  • expression is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.
  • fragment 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.
  • 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.
  • 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.
  • exogenous genetic material e.g., transgene, stable plasmid, integrating plasmid, naked genetic material, among other things
  • 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.
  • modulate is meant to refer to any change in biological state, i.e., increasing, decreasing, and the like.
  • 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.
  • 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.
  • pharmacologic refers to treatment, attempted treatment, or intended treatment by any drug, small molecule, or other type of molecule.
  • polynucleotide as used herein is defined as a chain of nucleotides.
  • nucleic acids are polymers of nucleotides, which can be hydrolyzed into monomeric "nucleotides.”
  • the monomeric nucleotides can be hydrolyzed into nucleosides.
  • 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 PCRTM, and the like, and by synthetic means.
  • 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.”
  • recognition 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.
  • 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.
  • recombinant DNA is defined as DNA produced by joining pieces of DNA from different sources.
  • recombinant polypeptide as used herein is defined as a polypeptide produced by using recombinant methods.
  • 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 includes numerous elements such as MyD88, IL-12, IL-23, and IL- 17; but is not limited to these elements.
  • 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.
  • 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.
  • the cells are cultured in vitro. It other embodiments, the cells are not cultured in vitro.
  • T-cell as used herein is defined as a thymus-derived cell that participates in a variety of cell-mediated immune reactions.
  • 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.
  • 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.
  • composition comprising the inhibitor of TLR7 or a component of the TLR7 signaling pathway can be any type of inhibitor.
  • 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.
  • facilitation of TLR7 or a component of the TLR7 signaling pathway is another intervention that arises directly from the disclosures herein.
  • facilitation of TLR7 or a component of the TLR7 signaling pathway can be by any means.
  • 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.
  • 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.
  • MMRRC Mutant Mouse Regional Resource Center
  • 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.
  • LD50 plaque-forming units
  • Ribonucleic acid was extracted from blood, spleen, liver, and brain tissue with the
  • 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.
  • mice were transcardially perfused with PBS.
  • IL- 12/-23 p40, TNF- ⁇ , 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
  • 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 ⁇ l Q-PCR master mix per well.
  • 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.
  • Western immunoblot was carried out as described (Wang et al., 2004).
  • cells were scraped into ice-cold lysis buffer (containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na 2 EDTA, 1 mM EGTA, 1% v/v Triton X- 100, 2.5 mM sodium pyrophosphate, 1 mM ⁇ -glycerophosphate, 1 mM Na 3 VO 4 , 1 ⁇ g/ml leupeptin, and 1 mM PMSF) and protein was quantified by the Bradford method. A 50 ⁇ g aliquot was then subjected to Nu-PAGE separation on gradient (4-12%) gels using MES buffer (Invitrogen).
  • MES buffer Invitrogen
  • membranes were incubated overnight at 4°C with primary antibodies against IL-12R ⁇ l (SantaCruz; 1 :100), IL-12R ⁇ 2 (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.
  • Tlr7—/— and Myd88-/- Mice are More Susceptible to Lethal WNV Infection.
  • 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-def ⁇ cient (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]).
  • TLR9 might cooperate with TLR7 in recognizing viral nucleic acid associated with murine cytomegalovirus (Zucchini et al., 2008).
  • 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.
  • Quantitative real-time polymerase chain reaction 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).
  • 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).
  • RNA expression of interferon- ⁇ (IFN- ⁇ ), IFN- ⁇ , interleukin (IL)-I ⁇ , IL-6, and tumor necrosis factor- ⁇ (TNF- ⁇ ) 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).
  • IL- 12 p40 the shared cytokine chain with IL-283 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).
  • livers another target organ of WNV infection
  • confocal microscopy revealed numerous CD45+ leukocytes in close vicinity of infected hepatocytes.
  • Tlr7—/— mice Similar to observations in brain, CD45+ cells were present, but were often found at a distance from WNV infected hepatocytes ( Figure 3B).
  • 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.
  • 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 ⁇ 10 5 macrophages were placed in the upper chamber.
  • loxO small molecule loxoribine
  • N2a WNV-infected neuroblastoma-2a
  • MCP-I macrophage chemoattractant protein- 1
  • 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- ⁇ , which further suggested that the above effect was TLR7 dependent ( Figure S2).
  • 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-12R ⁇ l subunit (van de Vosse et al, 2003).
  • IL-12R ⁇ 2 and IL-23R were not further inducible in Tlr7—/— and wild-type macrophages after loxO challenge, IL-12R ⁇ l 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.
  • IL-23 was playing the major role in TLR7-dependent macrophage chemotaxis in vitro.
  • 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).
  • 1123 a-/- mice had fewer infiltrating CDl Ib+ macrophages and microglia ( Figure 6) that were not clearly associated with WNV-infected brain cells.
  • proper infiltration and homing of immune cells to target WNV-infected cells required IL-23.
  • mice were infected with WNV (LD50) and brains were isolated on day 6 p.i..
  • 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.

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Abstract

La présente invention concerne des compositions, des procédés, et des techniques destinés à influencer ou autrement modifier ou réguler la signalisation émanant de ligands, de récepteurs, ou de voies de TLR7, et/ou de l'IL- 12, et/ou de l'IL-23, et/ou de l'IL- 17, seuls ou en combinaison. L'invention comprend l'inhibition de ligands, de récepteurs, ou de voies de TLR7, et/ou de l'IL- 12, et/ou de l'IL-23, et/ou de l'IL-17 seuls ou dans une combinaison quelconque en utilisant un moyen pharmacologique, qui comprend la liaison à un agoniste, la liaison à un antagoniste et les effets subséquents, et la liaison et les effets subséquents de médicaments ou de molécules qui ont des effets variables ou mixtes sur la voie de signalisation de TLR7, et/ou sur les voies de l'IL-12, et/ou de l'IL-23, et/ou de l'IL-17, seuls ou dans une combinaison quelconque.
PCT/US2010/023340 2009-02-05 2010-02-05 Composés et procédés destinés au traitement d'une encéphalite virale WO2010091265A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050054590A1 (en) * 2003-09-05 2005-03-10 Averett Devron R. Administration of TLR7 ligands and prodrugs thereof for treatment of infection by hepatitis C virus
US20060269936A1 (en) * 2005-03-24 2006-11-30 Jaromir Vlach Screening assay for TLR7, TLR8 and TLR9 agonists and antagonists
US20080131466A1 (en) * 2006-09-26 2008-06-05 Infectious Disease Research Institute Vaccine composition containing synthetic adjuvant

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050054590A1 (en) * 2003-09-05 2005-03-10 Averett Devron R. Administration of TLR7 ligands and prodrugs thereof for treatment of infection by hepatitis C virus
US20060269936A1 (en) * 2005-03-24 2006-11-30 Jaromir Vlach Screening assay for TLR7, TLR8 and TLR9 agonists and antagonists
US20080131466A1 (en) * 2006-09-26 2008-06-05 Infectious Disease Research Institute Vaccine composition containing synthetic adjuvant

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
AKIRA: "Toll-like Recptor Signalling", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 278, no. 40, 3 October 2003 (2003-10-03), pages 38105 - 38108 *
JIANG ET AL.: "CD14 is required for MyDD88-independent signaling", NATURE IMMUNOLOGY, vol. 6, no. 6, June 2005 (2005-06-01), pages 565 - 570 *

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