CA2419894A1 - Process for high throughput screening of cpg-based immuno-agonist/antagonist - Google Patents

Abstract

The invention pertains to murine TLR9 and related TLR9s which include murine - specific amino acids, as well as nucleic acids which encode those polypeptides. The present invention also includes fragments and biologically functional variants of the murine TLR9. The invention further relates to methods of using such murine and non-murine TLR9 nucleic acids and polypeptides, especially in methods for screening for agonists and antagonis ts of immunostimulatory CpG nucleic acids. Also included are murine TLR9 inhibitors which inhibit murine TLR9 activity by inhibiting the expression o r function of murine TLR9. In a further aspect the present invention pertains to murine TLR7 and murine TLR8, as well as related TLR7 and TLR8 molecules whic h include murine-specific amino acids, as well as nucleic acids which encode those polypeptides. The present invention also includes fragments and biologically functional variants of the murine TLR7 and TLR8. Methods are included for screening for ligands of TLR7 and TLR8, as well as for inhibito rs and agonists and antagonists of signaling mediated by TLR7 and TLR8.

Description

PROCESS FOR HIGH THROUGHPUT SCREENING OF CpG-BASED
IMMUNO-AGONISTIANTAGONIST
Related Auplications This invention claims benefit of U.S. Provisional Application 60/233,035, filed September 15, 2000; U.S. Provisional Application 60/263,657, filed January 23, 2001; U.S.
Provisional Application 60/291,726, filed May 17, 2001; and U.S. Provisional Application 60/300,210, filed June 22, 2001.
1o Field of the Invention The invention pertains to signal transduction by immunostimulatory nucleic acids.
Background of the Invention Bacterial DNA is a potent immunomodulatory substance. Yamamoto S et al., Microbiol Immunol 36:983-997 (1992). It has been hypothesized to be a pathogen-derived ligand recognized by an unidentified pathogen recognition receptor that initiates a host of innate and adaptive immune responses. Wagner H, Adv Immunol 73:329-368 (1999).
CpG
motif containing oligodeoxynucleotides (CpG ODN) can mimic the biology of bacterial DNA. Krieg AM et al., Nature 374:546-549 (1995). CpG ODN and DNA vectors have 2o recently been shown to be of clinical value due to immunostimulatory, hematopoietic and adjuvant qualities.
The adaptive immune system appeared approximately 450 million years ago when a transposon that carried the forerunners of the recombinase activating genes, RAG-1 and RAG-2, was inserted into the germ line of early j awed vertebrates. Agarwal A.
et al., Nature 394:744 (1998). The ability to mount an adaptive immune response allowed organisms to remember the pathogens that they had already encountered, and natural selection made the adaptive immune response a virtually universal characteristic of vertebrates.
However, this did not lead to discarding the previous form of host defense, the innate immune system.
Indeed, this earlier form of host defense has been coopted to serve a second fiulction, 3o stimulating and orienting the primary adaptive immune response by controlling the expression of costimulatory molecules.

_2_ It had been surmised for a decade that cells of the innate immune system bear receptors for conserved molecular patterns associated with microbial pathogens. According to this model, when the protein antigens derived from pathogens are processed and presented as peptides that serve as the stimulus for specific T cell receptors, pattern recognition receptors (PRRs) on the antigen-presenting cells also induce the synthesis of costimulatory molecules, cytokines, and chemokines. These activated antigen-presenting cells serve to attract and activate the antigen-specific T cells that are essential to all adaptive immune responses. Janeway CAJ, Cold Spring Harbor Symp Quant Biol 54:1 (1989); Fearon DT et al., Science 272:50 (1996); and Medzhitov R et al., Cell 91:295 (1997). It was known that the l0 substances that can induce costimulation include bacterial lipopolysaccharide (LPS), synthetic double-stranded RNA, glycans, and mannans. Furthermore, experimental evidence indicated that the processed antigen ligand for the T cell had to be on the same cell as the costimulatory molecule. This is obviously of crucial importance for maintaining self tolerance; bystander presentation of costimulatory molecules would mean that tolerance would be lost whenever an infection occurred.
To validate this model, it was necessary to identify receptors for microbial patterns that, upon binding pathogen ligands, initiate signaling cascades leading to the production of costimulatory molecules and cytokines. Molecules such as mannose binding protein (MBP) do not qualify for this role, because they activate proteolytic cascades or promote 2o phagocytosis but are not known to induce costimulation. The break-through came with the identification of a human homologue of Drosophila Toll initially cloned as a cDNA and later named hTLR4 (for human Toll-like receptor). Medzhitov R et al., Nature 388:394 (1997);
Rock FL et al., Proc Natl Acad Sci USA 95:588 (1998); Chaudhary PM et al., Blood 91:4020-4027 (1998).
Toll-like receptors (TLRs) are a family of germline-encoded transmembrane proteins that facilitate pathogen recognition and activation of the innate immune system. Hoffinann JA et al., Science 284, 1313-1318 (1999); Rock FL et al., Proc Natl Acad Sci USA 95:588-593 (1998). TLRs engage conserved pathogen-derived ligands and subsequently activate the TLR/IL-1R signal transduction pathway to induce a variety of effector genes.
Medzhitov R et 3o al., Mol Cell 2:253-258 (1998); Muzio M et al., JExp Med 187:2097-2101 (1998).
So far, ten different mammalian TL,Rs have been described. Rock FL et al., Proc Natl Acad Sci USA 95:588-593 (1998); Chaudhary PM et al., Blood 91:4020-4027 (1998);
Takeuchi O et al., Gehe 231:59-65 (1999); Aderem A. et al., Nature 406:782-7 (2000). So far, genetic data suggest that the TLRs have unique functions and are not redundant. Ligands for and the function of most of these TLRs, aside from TLR2 and TLR4, remain to be elucidated.
It turns out that an LPS-binding and signaling receptor complex is assembled when hTLR4 interacts with LPS bound to CD14, a peripheral membrane protein held to the cell surface by a glycosyl-phosphoinositol tail. The presence of LPS binding protein (LBP) further increases signaling. The hTLR4 protein has a leucine-rich repeat sequence in its l0 extracellular domain that interacts with CD14 complexed with LPS. TLR4 then transduces the LPS signal across the membrane because destructive mutation of this gene lead to an LPS-unresponsive state in mice, which are also deficient in the clearance of Gram-negative bacteria. Poltorak A et al., Science 282:2085 (1998); Qureshi ST et al., JExp Med 189:615-625 (1999); Eden CS et al., Jlmmuraol 140:180 (1988). It has since become apparent that humans, like flies, have numerous Toll-like receptors (TLRs).
TLR4 and other TLRs have a cytoplasmic Toll/IL-1 receptor (TIR) homology domain.
This domain communicates with a similar domain on an adapter protein (MyD88) that interacts with TLR4 by means of a like:like interaction of TIR domains. The next interaction is between the adapter and a kinase, through their respective "death domains."
The kinase in 2o turn interacts with tumor necrosis factor (TNF) receptor-associated factor-6 (TRAF6).
Medzhitov R et al., Mol Cell 2:253 (1998); Kopp EB et al., Curr OpirZ Immunol 11:15 (1999).
After TRAF6, two sequential kinase activation steps lead to phosphorylation of the inhibitory protein hcB and its dissociation from NF-~cB. The first kinase is a mitogen-activated kinase kinase kinase (MAPI~K) known as NIK, for NF-~cB-inducing kinase. The target of this kinase is another kinase made up of two chains, called IxB kinase a (IKKa) and hcB kinase (3 (IKK(3), that together form a heterodimer of IKKa:IKI~(3, which phosphorylates IxB. NF-~cB
translocates to the nucleus to activate genes with ~cB binding sites in their promoters and enhancers such as the genes encoding interleukin-1 [3 (IL-1 (3), Ih-6, IL-8, the p40 protein of II,-12, and the costimulatory molecules CD80 and CD86.
The types of cells that respond to CpG DNA - B cells, dendritic cells (DCs) and macrophages - are also stimulated by other pathogen-derived pattern-recognition factors, such as LPS. In general, the PRRs of the innate immune system are situated on the cell surface, where they are probably best able to detect microbes. Although cell-surface proteins that bind DNA are well described, and have been proposed to mediate immune activation by CpG
motif (Liang H et al., JClin Invest 98:1119-1129 (1998)), this binding is sequence-s independent and does not bring about cell activation. Krieg AM et al., Nature 374:546-549 (1995); Yamamoto T et al., Microbiol Immunol 38:831-836 (1994); Hacker H et al., EMBO J
17:6230-6240 (1998). Because CpG ODNs that have been immobilized to prevent cell uptake are nonstimulatory (Krieg AM et al., Nature 374:546-549 (1995); Manzel L et al., Antisense Nucleic Acid Drug Dev 9:459-464 (1999)), it appears that CpG ODN
probably work by binding to an intracellular receptor. In support of this hypothesis, drugs such as chloroquine, which interfere with the endosomal acidification/processing of ODNs, specifically block the immune stimulatory effects of CpG DNA. Hacker H et al., EMBO J
17:6230-6240 (1998); Macfarlane DE et al., Jlmmunol 160:1122-1131 (1998); Yi AK et al., Jlmmunol 160:4755-4761 (1998). It has been proposed that an endosomal step is required for the CpG-induced signal transduction pathways. Hacker H et al., EMBO J
17:6230-6240 (1998); Yi AID et al., Jlmmunol 160:4755-4761 (1998). How the information contained in unmethylated CpG-motifs of bacterial DNA trigger changes in gene expression has not previously been discovered.
Since the receptor for bacterial DNA has been unknown, development of screening for optimal CpG motifs through direct binding analysis has been limited. An additional complication appears to be species-specific selectivity for CpG sequence, i.e., an optimal sequence for one species may not be optimal for another.
Summary of the Invention Nucleic acids encoding three Toll-like receptors, Toll-like receptor 7 (TLR7), TLR8, and TLR9 of the mouse have now been identified, isolated, cloned and sequenced by the inventors: The invention in general provides isolated nucleic acid molecules encoding TLRs and isolated fragments of those nucleic acid molecules; isolated TLR
polypeptides and isolated fragments of those polypeptides; expression vectors containing the foregoing nucleic acid molecules; host cells having the foregoing expression vectors; fusion proteins including the TLR polypeptides and fragments thereof; and screening methods useful for identifying, comparing, and optimizing agents which interact with these TLRs, particularly agents that alter the expression of and signaling associated with these TLR molecules. In preferred embodiments the screening methods are high throughput screening methods.
The invention in some aspects arises from the surprising discovery that TLR9 is involved in immunostimulatory nucleic acid (ISNA)-induced immunostimulation.
The invention also stems in part from the surprising discovery that TLR9 transducer immune activating signals in response to ISNA in a manner that is both sequence-specific and species-specific.
In a first aspect the invention provides isolated nucleic acid molecules which encode to full-length marine TLR9. According to this aspect of the invention, isolated nucleic acid molecules are provided which are selected from the group consisting of (a) nucleic acid molecules which hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence set forth as SEQ ID NO:1, and which code for a marine TLR9 having an amino acid sequence set forth as SEQ ID N0:3; (b) nucleic acid molecules that differ from the nucleic acid molecules of (a) in codon sequence due to degeneracy of the genetic code;
and (c) complements of (a) or (b). In a certain embodiment, the isolated nucleic acid molecule codes for SEQ ID N0:3, where SEQ ID N0:3 represents the deduced amino acid sequence of full-length marine TLR9. In some embodiments the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:1 or SEQ ID N0:2, where these 2o correspond to full-length cDNA and the open reading frame for marine TLR9, respectively.
The term "stringent conditions" as used herein refers to combined conditions based on parameters including salt, temperature, organic solvents, and optionally other factors with which the paractioner skilled in the art is familiar. Nucleic acid hybridization parameters may be found in references which compile such methods, e.g., Molecular Cloning: A
Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M.
Ausubel, et al., eds., John Wiley & Sons, Inc., New York. More specifically;
stringent conditions, as used herein, refers, for example, to hybridization at 65°C in hybridization buffer (3.5 x SSC, 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% bovine serum albumin, 2.SmM NaH2P04 (pH7), 0.5% SDS, 2mM EDTA). SSC is 0.15M sodium chloride/O.15M
sodium citrate, pH7; SDS is sodium dodecyl sulfate; and EDTA is ethylenediaminetetraacetic acid. After hybridization, the membrane upon which the DNA is transferred is washed with 2 x SSC at room temperature and then with 0.1 - 0.5 x SSC/0.1 x SDS at temperatures up to 68°C. There are other conditions, reagents, and so forth which can be used, which result in a similar degree of stringency. The skilled artisan will be familiar with such conditions, and thus they are not given here. It will be understood, however, that the skilled artisan will be able to manipulate the conditions in a manner to permit the clear identification of alleles of marine TLR nucleic acids of the invention. The skilled artisan also is familiar with the methodology for screening cells and libraries for expression of such molecules which then are routinely isolated, followed by isolation of the pertinent nucleic acid molecule and 1o sequencing.
The invention in a second aspect provides isolated TLR9 polypeptides or fragments thereof. The isolated TLR9 polypeptides or fragments thereof include at least one amino acid of a marine TLR9 selected from the group consisting of amino acids 2, 3, 4, 6, 7, 18, 19, 22, 38, 44, 55, 58, 61, 62, 63, 65, 67, 71, 80, 84, 87, 88, 91, 101, 106, 109, 117, 122, 123, 134, 136, 140, 143, 146, 147, 157, 160, 161, 167, 168, 171, 185, 186, 188, 189, 191, 199, 213, 217, 220, 227, 231, 236, 245, 266, 269, 270, 271, 272, 273, 274, 278, 281, 285, 297, 298, 301, 305, 308, 311, 322, 323, 325, 326, 328, 332, 335, 346, 348, 353, 355, 358, 361, 362, 365, 367, 370, 372, 380, 381, 382, 386, 389, 392, 394, 397, 409, 412, 413, 415, 416, 419, 430, 432, 434, 435, 438, 439, 443, 444, 446, 447, 448, 450, 451, 452, 454, 455, 459, 460, 463, 465, 466, 468, 469, 470, 472, 473, 474, 475, 478, 488, 489, 494, 495, 498, 503, 508, 510, 523, 531, 539, 540, 543, 547, 549, 561, 563, 565, 576, 577, 579, 580, 587, 590, 591, 594, 595, 597, 599, 601, 603, 610, 611, 613, 616, 619, 632, 633, 640, 643, 645, 648, 650, 657, 658, 660, 667, 670, 672, 675, 679, 689, 697, 700, 703, 705, 706, 711, 715, 716, 718, 720, 723, 724, 726, 729, 731, 735, 737, 743, 749, 750, 751, 752, 754, 755, 759, 760, 772, 774, 780, 781, 786, 787, 788, 800, 814, 821, 829, 831, 832, 835, 844, 857, 858, 859, 862, 864, 865, 866, 879, 893, 894, 898, 902, 910, 917, and 927 of SEQ ll~ N0:3, wherein the TLR9 polypeptide or fragment thereof has an amino acid sequence which is identical to a human TLR9 polypeptide or fragment thereof except for the at least one amino acid of marine TLR9. The TLR9 polypeptide or fragment thereof in certain embodiments according to this aspect of the invention further includes at least one amino acid of marine TLR9 selected from the group consisting of amino acids 949, 972, 975, 976, 994, 997, 1000, 1003, 1004, 1010, 7_ 1011, 101 ~, 1023, and 1027 of SEQ m N0:3. Thus specifically.excluded from this aspect of the invention are TLR9 fragments restricted to the C-terminal 95 amino acids and fragments thereof.
In certain embodiments the TLR9 polypeptide and fragments thereof according to this aspect of the invention exclude those TLR9 polypeptides and fragments thereof which differ from human TLR9 and fragments thereof only by one or more conservative amino acid substitutions at particular sites noted above. As is well known in the art, a "conservative amino acid substitution" refers to an amino acid substitution which generally does not alter the relative charge or size characteristics of the polypeptide in which the amino acid 1o substitution is made. Conservative substitutions of amino acids typically include substitutions made amongst amino acids within the following groups: methionine (M), isoleucine (n, leucine (L), valine (V); phenylalanine (F), tyrosine (Y), tryptophan (W); lysine (K), arginine (R), histidine (H); alanine (A), glycine (G); serine (S), threonine (T); glutamine (Q), asparagine (I~; and glutamic acid (E), aspartic acid (D).
According to this and other aspects of the invention, with reference to TLR
"polypeptides and fragments thereof," "fragments thereop' refers to polypeptide fragments having stretches of contiguous amino acid residues that are at least about ~
amino acids long.
Generally the fragments are at least about 10 amino acids long; more generally at least 12 amino acids long; often at least about 14 amino acids long; more often at least about 16 2o amino acids long; typically at least 1 ~ amino acids long; more typically at least 20 amino acids long; usually at least 22 amino acids long; and more usually at least 24 amino acids long. Certain preferred embodiments include larger fragments that are, for example, at least about 30 amino acids long, at least about 40 amino acids long, at Ieast about 50 amino acids long, at least about 100 amino acids long, at least about 200 amino acids long, and so on, up to and including fragments that are a single amino acid shorter than full-length TLR
polypeptide.
In certain embodiments, the human TLR9 has an amino acid sequence set forth as SEQ m N0:6.
In preferred embodiments, the isolated TLR9 polypeptides or fragments thereof 3o include an amino acid sequence selected from the group consisting of SEQ m N0:3 and fragments of SEQ m N0:3. In some embodiments according to this aspect of the invention, -g_ the isolated TLR9 polypeptides or fragments thereof include combinations of the foregoing human and marine TLR9 polypeptides.
In certain preferred embodiments the isolated TLR9 polypeptide or fragment thereof is an extracytoplasmic domain (also referred to herein as extracellular domain) of TLR9, or a portion thereof. As described in greater detail further herein, TLR7, TLRB, and TLR9 have certain structural and functional domains. Structural domains of these TLRs include but are not limited to an extracytoplasmic domain, a transmembrane domain, and a cytoplasmic domain. The eXtracytoplasmic domain extends into the lumen of endosomal/lysosomal vesicles. The cytoplasmic domain includes a Toll/interleukin-1 receptor-like domain (also to referred to as Toll/IL-1R domain, TIR homology domain, or TIR domain). In marine TLR9 the extracytoplasmic, transmembrane, and cytoplasmic domains correspond to amino acids 1 to about 819, about 820 to about 837, and about 838 to about 1032, respectively.
As mentioned above, it has been discovered according to the invention that TLR9 is involved in immune activation induced by certain nucleic acid molecules referred to in the art as immunostimulatory nucleic acids (ISNAs), including CpG nucleic acids. It is believed by the inventors that binding of ISNA to TLR9 leads to signal transduction involving the TIR
domain of TLR9. Thus in certain embodiments according to this aspect of the invention, the isolated TLR9 polypeptide or fragment thereof selectively binds to an ISNA, including an ISNA that is a CpG nucleic acid.
2o Also included according to this aspect of the invention are isolated TLR9 polypeptides or fragments thereof which axe portions of the extracytoplasmic domain believed by the inventors to interact with imrnunostimulatory nucleic acids such as CpG
nucleic acids. In certain embodiments such portions include an MBD motif set forth as any one of SEQ ID
NOs: 126, 127, 210, and 211. In certain embodiments portions of the extracytoplasmic domain believed by the inventors to interact with immunostimulatory nucleic acids include a CXXC motif set forth as any one of SEQ ID NOs: 196, 197, and 198.
According to a third aspect of the invention, isolated nucleic acid molecules are provided which encode the foregoing isolated TLR9 polypeptides or fragments thereof. The isolated nucleic acid molecules according to this aspect of the invention specifically exclude 3o certain expressed sequence tags (ESTs) identified by the following GenBank accession numbers: AA162495, AA197442, AA273731, AA794083, AA915125, AA968074, .

AI428529, AI451215, AI463056, AI893951, AV142833, AV326033, AV353853, AW048117, AW048548, AW215685, AW549817, BB179985, BB215203, BB283380, BB285606, BB312895, BB497196, BB622397, BF016670, BF150116, BF161011, BF300296, BF385702, BF539367, BF784415, BG863184, BG922959, BG967012, BG974917, BI105291, BI153921, BI651868, BI653892, and W76964.
In a fouth aspect the invention provides isolated nucleic acid molecules which encode full-length marine TLR7. According to this aspect of the invention, isolated nucleic acid molecules are provided which are selected from the group consisting of (a) nucleic acid molecules which hybridize under stringent conditions to a nucleic acid molecule having a to nucleotide sequence set forth as SEQ ID N0:173, and which code for a marine TLR7 having an amino acid sequence set forth.as SEQ ID N0:175; (b) nucleic acid molecules that differ from the nucleic acid molecules of (a) in codon sequence due to degeneracy of the genetic code; and (c) complements of (a) or (b). In a certain embodiment, the isolated nucleic acid molecule codes for SEQ ID NO:175, where SEQ ID N0:175 represents the deduced amino acid sequence of full-length marine TLR7. In some embodiments the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID N0:173 or SEQ ID N0:174, where these correspond to full-length cDNA and the open reading frame for marine TLR7, respectively.
The invention in a fifth aspect provides isolated TLR7 polypeptides or fragments thereof. The isolated TLR7 polypeptides or fragments thereof include at least one amino acid of a marine TLR7 selected from the group consisting of amino acids 4, 8, 15, 16, 18, 21, 23, 24, 25, 27, 37, 39, 40, 41, 42, 44, 45, 61, 79, 83, 86, 89, 92, 96, 103, 109, 111, 113, 119, 121, 127, 128, 131, 145, 148, 151, 164, 172, 176, 190, 202, 203, 204, 205, 222, 225, 226, 228, 236, 238, 243, 250, 253, 266, 268, 271, 274, 282, 283, 287, 288, 308, 313, 314, 315, 325, 328, 331, 332, 341, 343, 344, 347, 351, 357, 360, 361, 362, 363, 364, 365, 366, 370, 371, 377, 378, 387, 388, 389, 392, 397, 398, 413, 415, 416, 419, 421, 422, 425, 437, 438, 440, 446, 449, 453, 454, 455, 456, 462, 470, 482, 486, 487, 488; 490, 491, 493, 494, 503, 505, 509, 511, 529, 531, 539, 540, 543, 559, 567, 568, 574, 583, 595, 597, 598, 600, 611, 613, 620, 624, 638, 645, 646, 651, 652, 655, 660, 664, 665, 668, 669, 672, 692, 694, 695, 698, 701, 704, 714, 720, 724, 727, 728, 733, 738, 745, 748, 755, 762, 777, 780, 789, 803, 846, 850, 851, 860, 864, 868, 873, 875, 884, 886, 888, 889, 890, 902, 903, 911, 960, 967, 970, 980, 996, 1010, 1018, 1035, and 1045 of SEQ 1D N0:175, wherein the TLR7 polypeptide or fragment thereof has an amino acid sequence which is identical to a human TLR7 polypeptide or fragment thereof except for the at least one amino acid of marine TLR7.
In certain embodiments the TLR7 polypeptide and fragments thereof according to this aspect of the invention exclude those TLR7 polypeptides and fragments thereof which differ from human TLR7 and fragments thereof only by one or more conservative amino acid substitutions at particular sites noted above.
In certain embodiments, the human TLR7 has an amino acid sequence set forth as SEQ ID N0:170.
l0 In preferred embodiments, the isolated TLR7 polypeptides or fragments thereof include an amino acid sequence selected from the group consisting of SEQ ID
N0:175 and fragments of SEQ ID N0:175. In some embodiments according to this aspect of the invention, the isolated TLR7 polypeptides or fragments thereof include combinations of the foregoing human and marine TLR7 polypeptides.
In certain preferred embodiments the isolated TLR7 polypeptide or fragment thereof is an extracytoplasmic domain of TLR7, or a portion thereof. In certain embodiments according to this aspect of the invention, the isolated TLR7 polypeptide or fragment thereof selectively binds to an ISNA, including an ISNA that is a CpG nucleic acid. Also included according to this aspect of the invention axe isolated TLR7 polypeptides or fragments thereof which are 2o portions of the extracytoplasmic domain believed by the inventors to interact with immunostimulatory nucleic acids such as CpG nucleic acids. In certain embodiments such portions include an MBD motif set forth as any one of SEQ ID NOs: 203, 204, 212, and 213.
In certain embodiments portions of the extracytoplasmic domain believed by the inventors to interact-with immunostimulatory nucleic acids include a CXXC motif set forth as any one,of SEQ ID NOs: 196, 199, and 200.
According to a sixth aspect of the invention, isolated nucleic acid molecules are provided which encode the foregoing isolated TLR7 polypeptides or fragments thereof. The isolated nucleic acid molecules according to this aspect of the invention specifically exclude certain ESTs identified by the following GenBank accession numbers: AA176010, 3o AA210352, AA241310, AA266000, AA266744, AA276879, AA288480, AA871870, AI119722, AI449297, AI466859, AI604175, AV322307, BB033376, BB116163, BB210788, BB464985, BB466708, BB636153, BF101884, BF124798, BF143871, BG067922, BG080980, BG082140, BG871070, BG964747, BG976560, BI150306, BI411471, and C87987.
In a seventh aspect the invention provides isolated nucleic acid molecules which encode full-length marine TLRB. According to this aspect of the invention, isolated nucleic acid molecules are provided which are selected from the group consisting of (a) nucleic acid molecules which hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence set forth as SEQ ID N0:190, and which code for a marine TLR8 having an amino acid sequence set forth as SEQ ID N0:192; (b) nucleic acid molecules that differ l0 from the nucleic acid molecules of (a) in codon sequence due to degeneracy of the genetic code; and (c) complements of (a) or (b). In a certain embodiment, the isolated nucleic acid molecule codes for SEQ ID N0:192, where SEQ ID N0:192 represents the deduced amino acid sequence of full-length marine TLRB. In some embodiments the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID N0:190 or SEQ ID N0:191 ~
where these correspond to full-length cDNA and the open reading frame for marine TLRB, respectively.
The invention in an eighth aspect provides isolated TLR8 polypeptides or fragments thereof. The isolated TLRB polypeptides or fragments thereof include at least one amino acid of a marine TLR8 selected from the group consisting of amino acids 5, 6, 9, 10, 14, 15, 18, 21, 22, 23, 24, 25, 26, 27, 28, 30, 39, 40, 41, 43, 44, 50, 51, 53, 55, 61, 67, 68, 74, 80, 85, 93, 98, 99, 100, 104, 105, 106, 107, 110, 114, 117, 119, 121, 124, 125, 134, 135, 138, 145, 155, 156, 157, 160, 161, 162, 163, 164, 166, 169, 170, 174, 180, 182, 183, 186, 187, 191, 193, 194, 196, 197, 199, 200, 207, 209, 210, 227, 228, 230, 231, 233, 234, 241, 256, 263, 266, 267, 268, 269, 272, 274, 275, 276, 280, 285, 296, 298, 299, 300, 303, 305, 306, 307, 310, 312, 320, 330, 333, 335, 343, 344, 345, 346, 347, 349, 351, 356, 362 365, 366, 375, 378, 379, 380, 381, 383, 384, 386, 387, 392, 402, 403, 408, 414, 416, 417, 422, 426, 427, 428, , 429, 430, 431, 433; 437, 438, 439; 440, 441, 444, 445, 449; 456, 461, 463, 471, 483, 486, 489, 490, 494, 495, 496, 505, 507, 509, 512, 513, 519, 520, 523, 537, 538, 539, 541, 542, 543, 545, 554, 556, 560, 567, 569, 574, 575, 578, 586, 592, 593, 594, 595, 597, 599, 602, 613, 617, 618, 620, 621, 623, 628, 630, 633, 639, 641, 643, 644, 648, 655, 658, 661, 663, 664, 666, 668, 677, 680, 682, 687, 688, 690, 692, 695, 696, 697, 700, 702, 703, 706, 714, 715, 726, 727, 728, 730, 736, 738, 739, 741, 746, 748, 751, 752, 754, 757, 764, 766, 772, 776, 778, 781, 784, 785, 788, 791, 795, 796, 801, 802, 806, 809, 817, 820, 821, 825, 828, 829, 831, 839, 852, 853, 855, 858, 863, 864, 900, 903, 911, 918, 934, 977, 997, 1003, 1008, 1010, 1022, 1023, 1024, 1026, and 1030 of SEQ ID N0:192, wherein the TLR8 polypeptide or fragment thereof has an amino acid sequence which is identical to a human TLRB
polypeptide or fragment thereof except for the at least one amino acid of marine TLRB.
In certain embodiments the TLRB polypeptide and fragments thereof according to this aspect of the invention exclude those TLR8 polypeptides and fragments thereof which differ from human TLR8 and fragments thereof only by one or more conservative amino acid to substitutions at particular sites noted above.
In certain embodiments, the human TLRB has an amino acid sequence set forth as SEQ ID N0:184.
In preferred embodiments, the isolated TLR8 polypeptides or fragments thereof include an amino acid sequence selected from the group consisting of SEQ ID
N0:192 and fragments of SEQ ID N0:192. In some embodiments according to this aspect of the invention, the isolated TLR8 polypeptides or fragments thereof include combinations of the foregoing human and marine TLR8 polypeptides.
In certain preferred embodiments the isolated TLR8 polypeptide or fragment thereof is an extracytoplasmic domain of TLRB, or a portion thereof. In certain embodiments according to this aspect of the invention, the isolated TLR8 polypeptide or fragment thereof selectively binds to an ISNA, including an ISNA that is a CpG nucleic acid. Also included according to this aspect of the invention are isolated TLR8 polypeptides or fragments thereof which are portions of the extracytoplasmic domain believed by the inventors to interact with immunostimulatory nucleic acids such as CpG nucleic acids. In certain embodiments such portions include an MBD motif set forth as any one of SEQ ID NOs: 205, 206, 214, and 215.
In certain embodiments portions of the extracytoplasmic domain believed by the inventors to interact with immunostimulatory nucleic acids include a CXXC motif set forth as any one of SEQ ID NOs: 196, 201, and 202.
According to a ninth aspect of the invention, isolated nucleic acid molecules are provided which encode the foregoing isolated TLR8 polypeptides or fragments thereof. The isolated nucleic acid molecules according to this aspect of the invention specifically exclude certain ESTs identified by the following GenBank accession numbers: AAl 16795, AA268605, AA920337, AI529457., AI849892, AV097766, AV117427, AV164719, AV169968, AW551677, BB143750, BB2I4171, BB243478, BB244318, BB254686, BB256660, BB258368, BB278984, BB291470, BB292008, BB364655, BB373674, BB428800, BB439876, BB444812, BB445724, BB465766, BB470182, BB535086, BB573907, BB573981, BB607650, BF135656, BF722808, BG299237, BG918020, BG919592, and W39977.
In a further aspect, the invention provides TLR expression vectors comprising the foregoing isolated nucleic acid molecules operably linked to a promoter. Thus in certain embodiments pertaining to TLR9, the expression vector includes an isolated nucleic acid molecule according to the first aspect or the third aspect of the invention, operably linked to a promoter. In other embodiments, relating to TLR7, the expression vector includes an isolated nucleic acid molecule according to the fourth aspect or the sixth aspect of the invention, operably linked to a promoter. In yet other embodiments, relating to TLRB, the expression vector includes an isolated nucleic acid molecule according to the seventh aspect or the ninth aspect of the invention, operably linked to a promoter.
The expression vectors according to this aspect of the invention are designed and constructed so that when they are introduced into a cell, under proper conditions they direct expression of the gene product encoded by the incorporated isolated nucleic acid molecule.
For example, the promoter can be constitutively active or it can be inducible or repressible upon interaction with a suitable inducer or repressor compound.
According to another aspect, host cells are provided that include a TLR
expression vector of the invention. While any suitable method can be used, an expression vector typically is introduced into a cell by transfection or transformation. The host cells transformed or transfected with the TLR expression vectors are in some embodiments co-transformed or co-transfected with another expression vector useful for the expression of another polypeptide. Alternatively, a host cell can be tranformed or transfected with an expression vector capable of directing expression of a TLR polypeptide or fragment thereof of the invention and (i) at least one additional TLR polypeptide or fragment thereof, or (ii) at least one non-TLR polypeptide or fragment thereof. In certain preferred embodiments, the host cell includes separate expression vectors for any combination of TLR7, TLRB, and TLR9. In some embodiments, a co-transformed or co-transfected expression vector may be useful for detection or regulation of TLR expression or TLR-related signaling.
Specifically, in certain preferred embodiments the host cell includes an expression vector providing a reporter construct capable of interacting with a TIR domain.
In another aspect, the invention provides agents which selectively bind the isolated TLR polypeptides and fragments thereof of the invention. In certain embodiments the agent does not bind a human TLR polypeptide or fragment thereof, wherein the human TLR is selected from human TLR7, TLRB, and TLR9. In certain embodiments the agent is a polypeptide, preferably one selected from the group consisting of monoclonal antibodies, l0 polyclonal antibodies, Fab antibody fragments, F(ab')2 antibody fragments;
Fv antibody fragments, antibody fragments including a CDR3 region, and fusion proteins and other polypeptides including any such antibodies or antibody fragments.
Also provided are agents which selectively bind the foregoing isolated nucleic acid molecules, preferably antisense nucleic acid molecules which selectively bind to any of the 15 foregoing isolated nucleic acid molecules encoding a TLR polypeptide or fragment thereof.
In some embodiments the agent is an isolated nucleic acid molecule which hybridizes under stringent conditions to an isolated nucleic acid moleucle provided according to any of the first, third, fourth, fifth, sixth, and eighth aspects of the invention. In certain preferred embodiments the agent is an isolated nucleic acid molecule having a nucleotide sequence 20 which is complementary to an isolated nucleic acid moleucle provided according to any of the first, third, fourth, fifth, sixth, and eighth aspects of the invention.
In still other aspects of the invention, methods for inhibiting TLR expression and TLR
signaling in a cell are provided. The methods include contacting the cell with an amount of an agent effective to inhibit TLR expression and TLR signaling in the cell, wherein the TLR
25 is selected from the group consisting of TLR7, TLR8, and TLR9. In some embodiments the agent brought into contact with the cell is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, Fab antibody fragments, F(ab')2 antibody fragments, Fv antibody fragments, antibody fragments including a CDR3 region, and fusion proteins and other polypeptides that include any such antibodies or antibody fragments. In some 3o embodiments the cell is contacted with an antisense nucleic acid specific for the TLR, in an amount effective to inhibit TLR expression in the cell. In some embodiments the cell is contacted with an agent such as a cytokine or small molecule, in an amount effective to inhibit TLR expression in the cell.
In yet another aspect the invention provides a method for identifying nucleic acid molecules which interact with a TLR polypeptide or a fragment thereof. The method involves contacting a TLR polypeptide selected from the group consisting of TLR7, TLRB, TLR9, and nucleic acid-binding fragments thereof with a test nucleic acid molecule; and measuring an interaction of the test nucleic acid molecule with the TLR
polypeptide or fragment thereof. Nucleic acid-binding fragments of TLRs preferably include the extracytoplasmic domain or subportions thereof, such as those which include at least an MBD
1o motif, a CXXC motif, or both an MBD motif and a CXXC motif.
In this and other aspects of the invention involving methods'of use of TLR
polypeptides and fragments thereof, in some embodiments the TLR polypeptide or fragment thereof is TLR7. Likewise in this and other aspects of the invention involving methods of use of TLR polypeptides and fragments thereof, in some embodiments the TLR
polypeptide or 1s fragment thereof is TLRB. Also in this and other aspects of the invention involving methods of use of TLR polypeptides and fragments thereof, in some embodiments the TLR
polypeptide or fragment thereof is TLR9.
In this and other aspects of the invention involving methods of use of TLR
polypeptides and fragments thereof, in some embodiments the TLR polypeptide or fragment 20 thereof is expressed in a cell. The cell expressing the TLR polypeptide or fragment thereof may naturally express the TLR polypeptide or fragment thereof, or it may be a host cell as provided by other aspects of the instant invention.
In this and other aspects of the invention involving methods of use of TLR
polypeptides and fragments thereof, in some embodiments the TLR polypeptide or fragment 25 thereof is an isolated TLR polypeptide or fragment thereof. In certain preferred embodiments the isolated TLR polypeptide or fragment thereof is immobilized on a solid support, for example a multiwell plate, a slide, a BIAcore chip, a bead, a column, and the like. The immobilization can be accomplished by any chemical or physical method suitable for the purpose of the assay to be performed according to the method of the invention.
3o In certain embodiments the TLR polypeptide or fragment thereof is fused with an Fc fragment of an antibody. The Fc fragment portion of such a fusion molecule may be useful, for example, for attaching the TLR polypeptide or fragment thereof to a substrate, or for providing a target for detecting the presence of the TLR polypeptide or fragment thereof. The Fc fragment can be selected from any suitable vertebrate species and will typically, but not necessarily, be derived from an antibody belonging to the IgG class of antibodies. For example, the Fc can be a human or a murine Fcy. In certain embodiments the TLR
polypeptide or fragment thereof is fused with an Fc fragment of an antibody with a specific cleavage site at or near the junction between the TLR polypeptide or fragment thereof and the Fc fragment. In one preferred embodiment the cleavage site is a thrombin protease recognition site. In a preferred embodiment the TLR polypeptide or fragment thereof fused to with the Fc fragment includes a TLR extracytoplasmic domain.
In certain embodiments the interaction involving the TLR polyeptide or fragment thereof and the test nucleic acid molecule is binding between the TLR
polypeptide or fragment thereof and the test nucleic acid molecule.
In certain embodiments according to this aspect of the invention, the measuring is accomplished by a method selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), biomolecular interaction assay (BIA), electromobility shift assay (EMSA), radioimmunoassay (RIA), polyacrylamide gel electrophoresis (PAGE), and Western blotting.
In certain embodiments the measuring is accomplished by a method comprising 2o measuring a response mediated by a TLR signal transduction pathway. For example, the response mediated by a TLR signal transduction pathway can be selected from the group consisting of induction of a gene under control of NF-xB promoter and secretion of a cytokine. In certain preferred embodiments the gene under control of NF-KB
promoter is selected from the group consisting of IL-8, IL-12 p40, NF-xB-luc, IL-12 p40-luc, and TNF-luc. In certain preferred embodiments the secreted cytokine is selected from the group consisting of IL-8, TNF-oc, and IL-12 p40.
In another embodiment the method according to this aspect of the invention can be used to determine if the test nucleic acid molecule is an immunostimulatory nucleic acid. The method involves the additional steps of comparing (a) the response mediated by a TLR signal transduction pathway as measured in the presence of the test nucleic acid molecule with (b) a response mediated by a TLR signal transduction pathway as measured in the absence of the test nucleic acid molecule; and determining the test nucleic acid molecule is an immunostimulatory nucleic acid when (a) exceeds (b).
In yet another embodiment the method according to this aspect of the invention can be used to determine if the response to the test nucleic acid molecule is stronger or weaker than a response to a reference nucleic acid molecule. The method involves the additional steps of comparing the response to a reference response when the TLR polypeptide is independently contacted with a reference nucleic acid molecule; and determining if the response is stronger or weaker than the reference response. In this embodiment the test nucleic acid molecule and the reference nucleic acid molecule are not able to compete or interact. For example, the to reference response can be a parallel control or a historical control.
In another embodiment the method involves the additional steps of comparing the response to a reference response when the TLR polypeptide is concurrently contacted with a reference nucleic acid molecule; and determining if the response is stronger or weaker than the reference response. In this embodiment the test nucleic acid molecule and the reference nucleic acid molecule are potentially able to compete or interact since they are both present, for example, in a single reaction. .
In another aspect the invention provides a screening method for identifying an immunostimulatory nucleic acid. The method according to this aspect involves contacting a functional TLR selected from the group consisting of TLR7, TLRB, and TLR9 with a test 2o nucleic acid molecule; detecting presence or absence of a response mediated by a TLR signal transduction pathway in the presence of the test nucleic acid molecule arising as a result of an interaction between the functional TLR and the test nucleic acid molecule; and determining the test nucleic acid molecule is an ISNA when the presence of a response mediated by the TLR signal transduction pathway is detected. A functional TLR refers to a TLR
polypeptide or fragment thereof that can bind with a ligand and as a consequence of the binding engage at least one step or additional polypeptide in a TLR signal transduction pathway.
In one embodiment the method according to this aspect of the invention includes the further step of comparing (a) the response mediated by the TLR signal transduction pathway arising as a result of an interaction between the functional TLR and the test nucleic acid molecule with (b) a response arising as a result of an interaction between the functional TLR
and a reference ISNA. In this and other screening assays of the instant invention, in preferred embodiments the screening method is performed on a plurality of test nucleic acids. In certain preferred embodiments the response mediated by the TLR signal transduction pathway is measured quantitatively, and the response mediated by the TLR signal transduction pathway associated with each of the plurality of test nucleic acid molecules is compared with a response arising as a result of an interaction between the functional TLR
and a reference ISNA.
In certain preferred embodiments a subset of the plurality of test nucleic acid molecules is selected based on the ability of the subset to produce a specific response mediated by the TLR signal transduction pathway. For example, the specific response can be to induction of a specific cytokine or panel of cytokines, e.g., Th1 cytokines, or, alternatively, inhibition of a specific cytokine or panel of cytokines, e.g., Th2 cytokines.
The specific response can be induction, or, alternatively, inhibition of a specific class or subclass of antibody or panel of classes or subclasses of antibodies, e.g., Thl-associated antibodies or Th2-associated antibodies. The specific response in some embodiments can be activation or inhibition of certain types of immune cells, e.g., B cells, dendritic cells (DCs), and natural killer (NK) cells. In some embodiments the specific response can be induction or inhibition of proliferation of certain types of immune cells, e.g., B cells, T cells, NK
cells, dendritic cells, monocytes/macrophages. The subset of the plurality of test nucleic acids is therefore selected on the basis of the common association between the test nucleic acids of the subset 2o and the particular type of response mediated by the TLR signal transduction pathway. The particular type of response mediated by the TLR signal transduction pathway is typically, but not necessarily, an immune cell response.
W certain embodiments the response mediated by a TLR signal transduction pathway is selected from the group consisting of induction of a gene under control of NF-~cB promoter and secretion of a cytokine. In certain preferred embodiments the gene under control of NF-xB promoter is selected from the group consisting of IL-8, IL-12 p40, NF-oB-luc, IL-12 p40-luc, and TNF-luc. In certain preferred embodiments the cytokine is selected from the group consisting of IL-8, TNF-a,, and IL-12 p40.
In certain preferred embodiments the reference ISNA is a CpG nucleic acid.
3o In certain preferred embodiments the test nucleic acid molecule is a CpG
nucleic acid.
According to this and other aspects of the invention involving functional TLR
in a screening assay, in some embodiments the functional TLR is expressed in a cell. In some embodiments the functional TLR is naturally expressed by the cell. In certain preferred embodiments the cell is an isolated mammalian cell that naturally expresses the functional TLR. Whether the cell expresses the TLR naturally or the cell expresses the TLR because an expression vector having an isolated nucleic acid molecule encoding the TLR
operatively linked to a promoter has been introduced into the cell, in some embodiments the cell further includes an expression vector comprising an isolated nucleic acid which encodes a reporter construct selected from the group consisting of IL-8, IL-12 p40, NF-~cB-luc, IL-12 p40-luc, and TNF-luc, operatively linked to a promoter.
1o Also according to this and other aspects of the invention involving functional TLR in a screening assay, in certain embodiments the functional TLR is part of a cell-free system.
Also according to this and other aspects of the invention involving functional TLR in a screening assay, in certain embodiments the functional TLR is part of a complex with another TLR. In certain preferred embodiments the complex is a complex of TLR9 and TLR7. In certain preferred embodiments the complex is a complex of TLR9 and TLRB.
Also according to this and other aspects of the invention involving functional TLR in a screening assay, in certain embodiments the functional TLR is part of a complex with a non-TLR protein selected from the group consisting of MyD88, IRAK, TRAF6, hcB, NF-~cB, and functional homologues and derivatives thereof.
2o Further according to this and and other aspects of the invention involving functional TLR in a screening assay, in certain embodiments the response mediated by a TLR signal transduction pathway is selected from the group consisting of induction of a gene under control of NF-xB promoter and secretion of a cytokine.
Also according to this and and other aspects of the invention involving functional TLR in a screening assay, in certain embodiments the gene under control of NF-xB promoter is selected from the group consisting of 1L-8, IL-12 p40, NF-~cB-luc, IL-12 p40-luc, and TNF-luc.
Also according to this and and other aspects of the invention involving functional TLR in a screening assay, in certain embodiments wherein the cytokine is selected from the 3o group consisting of IL-8, TNF-a, and IL-12 p40.
In a further aspect, the invention provides a screening method for comparing TLR

signaling activity of a test compound with an ISNA. The method entails contacting a functional TLR selected from the group consisting of TLR7, TLRB, and TLR9 with a reference ISNA and detecting a reference response mediated by a TLR signal transduction pathway; contacting a functional TLR selected from the group consisting of TLR7, TLRB, and TLR9 with a test compound and detecting a test response mediated by a TLR
signal transduction pathway; and comparing the test response with the reference response to compare the TLR signaling activity of the test compound with the ISNA.
In certain embodiments according to this aspect of the invention, the reference ISNA
is a CpG nucleic acid.
l0 In certain embodiments according to this aspect of the invention, the test compound is a polypeptide. In certain embodiments the test compound is part of a combinatorial library of compounds.
In certain embodiments the functional TLR is contacted with the reference ISNA
and the test compound independently. Accordingly, in certain embodiments the screening method is a method for identifying an ISNA mimic, and the test compound is determined to be an ISNA mimic when the test response is similar to the reference response obtained with the reference ISNA. A test response is similar to the reference response when the test and reference responses are qualitatively alike, even if not quantitatively alike.
Thus, for example, the test and reference responses are considered alike when both responses include 2o induction of a Thl-like immune response. The test response can be quantitatively less than, about the same as, or greater than the reference response.
In certain other embodiments the functional TLR is contacted with the reference ISNA
and the test compound concurrently to produce a test-reference response mediated by a TLR
signal transduction pathway, wherein the test-reference response may be compared to the reference response. In certain preferred embodiments the screening method is a method for identifying an ISNA agonist, wherein the test compound is an ISNA agonist when the test-reference response is greater than the reference response. In certain preferred embodiments the screening method is a method for identifying an ISNA antagonist, wherein the test compound is an ISNA antagonist when the test-reference response is less than the reference response.
In a further aspect the invention provides a screening method for identifying species specificity of an ISNA. The method according to this aspect of the invention involves contacting a functional TLR selected from the group consisting of TLR7, TLRB, and TLR9 of a first species with a test ISNA; contacting a functional TLR selected from the group consisting of TLR7, TLRB, and TLR9 of a second species with the test ISNA;
measuring a response mediated by a TLR signal transduction pathway associated with the contacting the functional TLR of the first species with the test ISNA; measuring a response mediated by the TLR signal transduction pathway associated with the contacting the functional TLR of the second species with the test ISNA; and comparing (a) the response mediated by a TLR signal transduction pathway associated with the contacting the functional TLR of the first species 1o with the test ISNA with (b) the response mediated by the TLR signal transduction pathway associated with the contacting the functional TLR of the second species with the test ISNA.
In preferred embodiments the TLR of the first species corresponds to the TLR
of the second species, e.g., the TLR of the first species is human TLR9 and the TLR of the second species is murine TLR9. In certain embodiments the functional TLR may be expressed in a cell, part of cell-free system, or part of a complex with another TLR or with a non-TLR
protein, as previously described.
In yet another aspect the invention provides a method for identifying lead compounds for a pharmacological agent useful in the treatment of disease associated with TLR9 signaling activity. The method according to this aspect of the invention involves providing a cell 2o comprising a TLR9 polypeptide or fragment thereof as provided in the second aspect of the invention; contacting the cell with a candidate pharmacological agent under conditions which, in the absence of the candidate pharmacological agent, cause a first amount of signaling activity; and determining a second amount of TLR9 signaling activity as a measure of the effect of the pharmacological agent on the TLR9 signaling activity, wherein a second amount of TLR9 signaling activity which is less than the first amount indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which reduces TLR9 signaling activity and wherein a second amount of TLR9 signaling activity which is greater than the first amount indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which increases TLR9 signaling activity.
3o These and other aspects of the invention are described in greater detail below.

Brief Description of the Figures FIG. 1 is two paired bar graphs showing (A) the induction of NF-~cB and (B) the amount of IL-8 produced by 293 fibroblast cells transfected with human TLR9 in response to exposure to various stimuli, including CpG-ODN, GpC-ODN, LPS, and medium.
FIG. 2 is a bar graph showing the induction of NF-KB produced by 293 fibroblast cells transfected with marine TLR9 in response to exposure to various stimuli, including CpG-ODN, methylated CpG-ODN (Me-CpG-ODN), GpC-ODN, LPS, and medium.
FIG.3 is a series of gel images depicting the results of reverse transcriptase-polymerase chain reaction (RT-PCR) assays for marine TLR9 (mTLR9), human TLR9 l0 (hTLR9), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in untransfected control 293 cells, 293 cells transfected with mTLR9 (293-mTLR9), and 293 cells transfected with hTLR9 (293-hTLR9).
FIG. 4 is a graph showing the degree of induction of NF-xB-luc by various stimuli in stably transfected 293-hTLR9 cells.
15 FIG. 5 is a graph showing the degree of induction of NF-xB-luc by various stimuli in stably transfected 293-mTLR9 cells.
FIG. 6 is an image of a Coomassie-stained polyacrylamide gel depicting the presence of soluble hTLR9 in the supernatants of yeast cells transfected with hTLR9, either induced (lane 1) or not induced (lane 2).
2o FIG. 7 is a graph showing proliferation of human B cells in response to various stimuli, including Escherichia coli (E. coli) DNA, DNase-digested E. coli DNA, CpG-ODN, GpC-ODN, and LPS.
FIG. 8 is two paired bar graphs showing induction of (top) IL-8 and (bottom) TNF in plasmacytoid dendritic cells (CD123+ DC) and monocyte-derived dendritic cells (MDDC) in 25 response to various stimuli, including E. coli DNA, DNase-digested E. coli DNA, CpG-ODN, GpC-ODN, and LPS.
FIG. 9 is a series of images of stained gels showing results of semi-quantitative RT-PCR comparing relative levels of human TLR9, TLR2, and TLR4 mRNA expression in human peripheral blood cells: MDDC (lane 1), purified CD14+ monocytes (lane 2), B cells 30 (lane 3), CD123+ DC (lane 4), CD4+ T cells (lane 5), and CD8+ T cells (lane 6). GAPDH is a control for equalizing amounts of cDNA.

FIG. 10 is a pair of graphs showing amounts of IL-12 induced in (A) human peripheral blood mononuclear cells (PBMC) and (B) marine splenocytes in response to shown concentrations of various ODN, including ODN 2006 (filled circles), 2006-GC (open circles), 1668 (filled triangles), and 1668-GC (open triangles).
FIG. 11 is a quartet of graphs depicting responsiveness of 293 cells transfected with hTLR9 (left panels) or mTLR9 (right panels) upon stimulation with shown concentrations of various ODN, including ODN 2006 (filled circles), 2006-GC (open circles), 1668 (filled triangles), and 1668-GC (open triangles). Responses are shown in terms of induction of NF-~cB-luc (upper panels) and IL-8 (lower panels).
l0 FIG. 12 is a bar graph depicting the dose-response of 293-hTLR9 cells to E.
coli DNA
(black bars) and to DNase-digested E. coli DNA (gray bars).
FIG. 13 is a pair of graphs showing the responsiveness of (A) 293-hTLR9 and (B) 293-mTLR9 cells to shown concentrations of phosphodiester versions of ODN 2006 (filled circles), 2006-GC (open circles), 1668 (filled triangles), and 1668-GC (open triangles).
15 Fig. 14 is a pair of graphs showing the responsiveness of 293-hTLR9 and 293-mTLR9 cells to shown concentrations of ODN 5002 (filled circles) and ODN 5007 (open circles).
FIG. 15 is a bar graph showing the response of 293 cells transfected with mTLR9 to CpG-ODN 1668 is inhibited in a dose-dependent manner by co-exposure to non-CpG-ODN
PZ2.
2o FIG. 16 is a bar graph showing the response of 293-hTLR9 cells to CpG-ODN
(black bars) or to TNF (gray bars) in the presence of shown amounts of blocking non-CpG-ODN.
FIG. 17 is a bar graph showing blockade of response of 293-hTLR9 cells to CpG-ODN, but not to IL-1 or TNF, in the presence of Bafilomycin A (gray bars).
Control treatment with dimethyl sulfoxide (DMSO) is shown in black bars.
25 FIG. 18 is a graph showing the effect of varying concentrations of dominant negative human MyD88 on the induction of NF-~B in 293-hTLR9 cells stimulated with CpG-ODN
(open circles), TNF-a (filled circles), or control (filled triangles).
FIG. 19 is a series of three Western blot images showing the response of various polyclonal antibodies to purified hTLR9-FLAG and mTLR9-FLAG: upper panel, anti-human 3o and anti-mouse intracellular; middle, anti-mouse extracellular; and lower, anti-human extracellular. Arrows indicate position of TLR9 in each blot.

FIG. 20 is a bar graph depicting the responsiveness of native form hTLR9 and hTLR9 variant form hTLR9-CXXCm to various stimuli at different concentrations.
FIG. 21 is a bar graph depicting the responsiveness of native form mTLR9 and mTLR9 variant form mTLR9-CXXCm to various stimuli at different concentrations.
FIG. 22 is a bar graph showing the responsiveness of native form mTLR9, mTLR9 variant form mTLR9-Phmut, and mTLR9 variant form mTLR9-MBDmut to various stimuli at different concentrations.
FIG. 23 is a bar graph showing the responsiveness of native form hTLR9, hTLR9 variant form hTLR9-PHmut, and hTLR9 variant form hTLR9-MBDmut to various stimuli at to different concentrations.
FIG. 24 is a bar graph showing the responsiveness of native form mTLR9 and mTLR9 variant form mTLR9-TIRh to various stimuli at different concentrations.
FIG. 25 is a bar graph showing the responsiveness of native form hTLR9 and hTLR9 variant form hTLR9-TIRm to various stimuli at different concentrations.
15 FIG. 26 is a series of linear maps representing various features of human TLR7, TLR~, and TLR9 polypeptides.
FIG. 27 is an image of a silver stained polyacrylamide gel and schematic representation of a fusion protein in which the extracellular domain of human TLR9 (hTLR9) is fused to a human IgGl Fc domain (hIgG-Fc) with a thrombin protease recognition site 2o interposed. From left to right, the gel was loaded with (1) supernatant of transfectants; (2) lysates of transfectants, treated with thrombin; (3) untreated lysates of transfectants; (4) molecular weight markers; (5) supernatant of mock transfectants; (6) lysates of mock transfectants, treated with thrombin; and (7) untreated lysates of mock transfectants. Soluble hTLR9 and Fc are the products released from intact hTLR9-IgG-Fc following thrombin 25 treatment. Molecular weights are indicated along the right side of the silver stain gel image.
Brief Description of Selected Seguences SEQ ID NO:1 is the nucleotide sequence encoding a cDNA for marine TLR9.
SEQ ID N0:2 is the nucleotide sequence encoding the coding region of marine TLR9.
3o SEQ ID N0:3 is the amino acid sequence of a marine TLR9 encoded by SEQ ID
NO:1.

SEQ m N0:173 is the nucleotide sequence encoding a cDNA for marine TLR7.
SEQ ID N0:174 is the nucleotide sequence encoding the coding region of marine TLR7.
SEQ ID N0:175 is the amino acid sequence of a marine TLR7 encoded by SEQ ID
N0:173.
SEQ ID N0:190 is the nucleotide sequence encoding a cDNA for marine TLRB.
SEQ ID N0:191 is the nucleotide sequence encoding the coding region of marine TLRB.
SEQ ID N0:192 is the amino acid sequence of a marine TLR8 encoded by SEQ ID
1o N0:190.
Detailed Description of the Invention The present invention in one aspect involves the identification of cDNAs encoding mouse TLR9, referred to herein as marine TLR9 and, equivalently, mTLR9. The nucleotide sequence of the cDNA for marine TLR9 is presented as SEQ ID NO:1, the coding region of the cDNA for marine TLR9 is presented as SEQ ID N0:2, and the amino acid sequence of the marine TLR9 is presented as SEQ ID N0:3. The closely related human TLR9 (equivalently, hTLR9) was deposited in GenBank under accession numbers AF245704 and NM 017742.
2o The nucleotide sequence of the cDNA for marine TLR9 presented as SEQ ID
NO:1 is 3200 nucleotides long and includes the open reading frame (ORF, bases 40-3135) presented as SEQ m N0:2 which spans 3096 nucleotides (excluding the stop codon). The amino acid sequence of the marine TLR9 presented as SEQ m N0:3 is 1032 amino acids (aa) long, and it is believed to include an extracellular domain (aa 1-819), a transmembrane domain (aa 820-837), and an intracellular domain (aa 838-1032).
The amino acid sequence of human TLR9 (SEQ ID NO:6) and the amino acid sequence of the marine TLR9 (SEQ ID N0:3) are thus both 1032 amino acids long:
Comparison of the aligned amino acid sequences for the marine and the human molecules reveals a single base insertion at as 435 of the marine TLR9 and a single base deletion at as 860 of the human TLR9. (See Table 4 below.) Whereas much of the polypeptide presented herein is identical to human TLR9, marine TLR9 has several single amino acid differences. These differences in amino acids are specifically amino acids 2, 3, 4, 6, 7, 18, 19, 22, 38, 44, 55, 58, 61, 62, 63, 65, 67, 71, 80, 84, 87, 88, 91, 101, 106, 109, 117, 122, 123, 134, 136, 140, 143, 146, 147, 157, 160, 161, 167, 168, 171, 185, 186, 188, 189, 191, 199, 213, 217, 220, 227, 231, 236, 245, 266, 269, 270, 271, 272, 273, 274, 278, 281, 285, 297, 298, 301, 305, 308, 311, 322, 323, 325, 326, 328, 332, 335, 346, 348, 353, 355, 358, 361, 362, 365, 367, 370, 372, 380, 381, 382, 386, 389, 392, 394, 397, 409, 412, 413, 415, 416, 419, 430, 432, 434, 435, 438, 439, 443, 444, 446, 447, 448, 450, 451, 452, 454, 455, 459, 460, 463, 465, 466, 468, 469, 470, 472, 473, 474, 475, 478, 488, 489, 494, 495, 498, 503, 508, 510, 523, 531, 539, 540, 543, 547, 549, 561, l0 563, 565, 576, 577, 579, 580, 587, 590, 591, 594, 595, 597, 599, 601, 603, 610, 611, 613, 616, 619, 632, 633, 640, 643, 645, 648, 650, 657, 658, 660, 667, 670, 672, 675, 679, 689, 697, 700, 703, 705, 706, 711, 715, 716, 718, 720, 723, 724, 726, 729, 731, 735, 737, 743, 749, 750, 751, 752, 754, 755, 759, 760, 772, 774, 780, 781, 786, 787, 788, 800, 814, 821, 829, 831, 832, 835, 844, 857, 858, 859, 862, 864, 865, 866, 879, 893, 894, 898, 902, 910, 917, 927, 949, 972, 975, 976, 994, 997, 1000, 1003, 1004, 1010, 1011, 1018, 1023, and 1027 of SEQ ID N0:3 In some forms the mouse protein mTLR9 contains a signal sequence at the N-terminus (amino acids 1-26) which allows transport to the endoplasmic reticulum and subsequently to the cell surface or intracellular compartments. A transmembrane region (amino acids 820-837) anchors the protein to the cell membrane. The cytoplasmic tail contains a Toll/IL-1 receptor (T1R) homology domain which is believed to function in signaling upon ligand binding. Leucine-rich-repeats (LRR) can be found in the extracellular region (a common feature of TLRs) and may be involved in ligand binding or dimerization of the molecule.
Both mouse and human TLR9 have an N-terminal extension of approximately 180 amino acids compared to other TLRs. An insertion also occurs at amino acids 253-268, which is not found in TLRs 1-6 but is present in human TLR7 and human TLRB.
(See Figure 26.) This insert has two CXXC motifs which participate in forming a CXXC domain.
The CXXC domain resembles a zinc forger motif and is found in DNA-binding proteins and in certain specific CpG binding proteins, e.g., methyl-CpG binding protein-1 (MBD-1).
3o Fujita N et al., Mol Cell Biol 20:5107-5118 (2000). Both human and mouse domains occur at as 253-268:

CXXC motif GNCXxCxXXXXXCXXC SEQ ID N0:196 Human TLR9: GNCRRCDHAPNPCMEC SEQ ID N0:197 Murine TLR9: GNCRRCDHAPNPCMIC SEQ ID N0:198 An additional motif involved in CpG binding is the MBD motif, also found in MBD-1, listed below as SEQ ID N0:125. Fujita, N et al., Mol Cell Biol 20:5107-18 (2000); Ohki I
et al., EMBO J 18:6653-6661 (1999). Amino acids 524-554 of hTLR9 and as 525-555 of mTLR9 correspond to the MBD motif of MBD-1 as shown:
to MBD motif MBD-1 R-XXXXXXX-R-X-D-X-Y-XXXXXXXXX-R-S-XXXXXX-Y SEQ ID N0:125 hTLR9 Q-XXXXXXX-K-X-D-X-Y-XXXXXXXXX-R-L-XXXXXX-Y SEQID N0:126 mTLR9 Q-XXXXXXX-K-X-D-X-Y-XXXXXXXXX-Q-L-XXXXXX-Y SEQID N0:127 hTLR9 Q-VLDLSRN-K-L-D-L-Y-HEHSFTELP-R-L-EALDLS-Y SEQID N0:210 mTLR9 Q-VLDLSHN-K-L-D-L-Y-HWKSFSELP-Q-L-QALDLS-Y SEQID N0:211 Although the signaling functions of MBD-1 and TLR9 are quite different, the core D-X-Y is involved in CpG binding and is conserved. The C-terminal octamer S-XXXXXX-Y of the MBD motif may not be involved in binding and the S is not conserved by TLR9.
The other mismatches are highly conserved or moderately conserved; example R to K or R
to Q. These changes could explain MBD-1 as a methyl-CpG binder and TLR9 as a non-methyl-CpG
binder. The differences between mouse and human TLR9 may explain inter-species differences in CpG-motif sequence selectivity. See Figure 14 for inter-species sequence selectivity.
As discussed in Example 11 below and shown in Figures 22 and 23, the D-X-Y
core of this MBD motif occurs as D-L-Y in both mTLR9 (aa 535-537) and hTLR9 (aa 534-536).
Substitution of A for D and A for Y in the D-X-Y core, resulting in A-L-A in place of D-L-Y, 3o destroys receptor activity for mTLR9 and hTLR9 alike.
The invention involves in one aspect murine TLR9 nucleic acids and polypeptides, as _~8_ well as therapeutics relating thereto. The invention also embraces isolated functionally equivalent variants, useful analogs and fragments of the foregoing nucleic acids and polypeptides; complements of the foregoing nucleic acids; and molecules which selectively bind the foregoing nucleic acids and polypeptides.
The murine TLR9 nucleic acids and polypeptides of the invention are isolated.
As used herein with respect to nucleic acids, the term "isolated" means: (i) amplified ira vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, as by cleavage and gel separation; or (iv) synthesized by, for example, chemical synthesis. An isolated nucleic acid is one which is readily manipulable by recombinant DNA
to techniques well known in the art. Thus, a nucleotide sequence contained in a vector in which 5' and 3' restriction sites are known or for which PCR primer sequences have been disclosed is considered isolated, but a nucleic acid sequence existing in its native state in its natural host is not. An isolated nucleic acid may be substantially purified, but need not be. For example, a nucleic acid that is isolated within a cloning or expression vector is not pure in that it may 15 comprise only a tiny percentage of the material in the cell in which it resides. Such a nucleic acid is isolated, however, as the term is used herein because it is readily manipulable by standard techniques known to those of ordinary skill in the art. An isolated nucleic acid as used herein is not a naturally occurring chromosome.
As used herein with respect to polypeptides, "isolated" means separated from its 2o native environment and present in sufficient quantity to permit its identification or use.
Isolated, when referring to a protein or polypeptide, means, for example: (i) selectively produced by expression cloning or (ii) purified as by chromatography or electrophoresis.
Isolated proteins or polypeptides may be, but need not be, substantially pure.
The term "substantially pure" means that the proteins or polypeptides are essentially free of other 25 substances with which they may be found in nature or ih vivo systems to an extent practical and appropriate for their intended use. Substantially pure polypeptides may be produced by techniques well known in the art. Because an isolated protein may be admixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the protein may comprise only a small percentage by weight of the preparation. The protein is nonetheless 3o isolated in that it has been separated from the substances with which it may be associated in living systems, i.e., isolated from other proteins.

As used herein a marine TLR9 nucleic acid refers to an isolated nucleic acid molecule which codes for a marine TLR9 polypeptide. Such nucleic acid molecules code for marine TLR9 polypeptides which include the sequence of SEQ ID N0:3 and fragments thereof. The nucleic acid molecules include the nucleotide sequences of SEQ ID NO:1, SEQ ID
N0:2, and nucleotide sequences which differ from the sequences of SEQ ID NO:1 and SEQ ID
N0:2 in codon sequence due to the degeneracy of the genetic code. The marine TLR9 nucleic acids of the invention also include alleles of the foregoing nucleic acids, as well as fragments of the foregoing nucleic acids. Such fragments can be used, for example, as probes in hybridization assays and as primers in a polymerase chain reaction. Preferred marine TLR9 nucleic acids to include the nucleic acid sequence of SEQ ID NO:1 and SEQ ID N0:2.
Complements of the foregoing nucleic acids also are embraced by the invention.
As used herein a marine TLR9 nucleic acid or marine TLR9 polypeptide also embraces homologues and alleles of marine TLR9. In general homologues and alleles typically will share at least 40% nucleotide identity and/or at least 50%
amino acid identity to the sequences of specified nucleic acids and polypeptides, respectively. Thus homologues and alleles of marine TLR9 typically will share at least 40% nucleotide identity and/or at least 50% amino acid identity to the sequences of marine TLR9 nucleic acids and TLR9 polypeptides, respectively. In some instances homologues and alleles will share at least 50%
nucleotide identity and/or at least 65% amino acid identity and in still other instances will 2o share at least 60% nucleotide identity and/or at least 75% amino acid identity. Preferably the homologues and alleles will share at least 80% nucleotide identity and/or at least 90% amino acid identity, and more preferably will share at least 90% nucleotide identity and/or at least 95% amino acid identity. Most preferably the homologues and alleles will share at least 95%
nucleotide identity and/or at least 99% amino acid identity. The homology can be calculated using various publicly available software tools developed by the National Center for Biotechnology Information (NCBI, Bethesda, Maryland) that can be obtained through the Internet (ftp:/ncbi.nlin.nih.gov/pub/). Exemplary tools include the BLAST
system available from the NCBI at http://www.ncbi.nlm.nih.gov, used with default settings.
Pairwise and ClustalW alignments (BLOSUM30 matrix setting) as well as I~yte-Doolittle hydropathic analysis can be obtained, for example, using' the MacVector sequence analysis software (Oxford Molecular Group). Watson-Crick complements of the foregoing nucleic acids also axe embraced by the invention.
Alleles of the marine TLR9 nucleic acids of the invention can be identified by conventional techniques. For example, alleles of marine TLR9 can be isolated by hybridizing a probe which includes at least a fragment of SEQ ID NO:1 or SEQ ID N0:2 under stringent conditions with a cDNA library and selecting positive clones. Thus, an aspect of the invention is those nucleic acid sequences which code for marine TLR9 polypeptides and which hybridize to a nucleic acid molecule consisting of SEQ m NO:1 or SEQ m N0:2 under stringent conditions.
In screening for marine TLR nucleic acids, a Southern blot may be performed using the foregoing stringent conditions, together with a radioactive probe. After washing the membrane to which the DNA is finally transferred, the membrane can be placed against x-ray film to detect the radioactive signal. Corresponding non-radioactive methods are also well known in the art and can be used to similar effect.
The marine TLR nucleic acids of the invention also include degenerate nucleic acids which include alternative codons to those present in the native materials. For example, serine residues are encoded by the codons AGC, AGT, and TCA, TCC, TCG and TCT. Each of the six codons is equivalent fox the purposes of encoding a serine residue. Thus, it will be apparent to one of ordinary skill in the art that any of the serine-encoding nucleotide triplets may be employed to direct the protein synthesis apparatus, ih vitro or in vivo, to incorporate a 2o serine residue into an elongating marine TLR polypeptide. Similarly, nucleotide sequence triplets which encode other amino acid residues include, but are not limited to: CCA, CCC, CCG and CCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG (arginine codons);
ACA, ACC, ACG and ACT (threonine codons); AAC and AAT (asparagine codons); and ATA, ATC and ATT (isoleucine codons). As is well known by those of ordinary skill in the art, other specific amino acid residues may be encoded similarly by multiple nucleotide sequences. Thus, the invention embraces degenerate nucleic acids that differ from the biologically isolated nucleic acids in codon sequence due to the degeneracy of the genetic code. The above-noted codon degeneracy notwithstanding, it is well appreciated by those skilled in the art that there are certain codon usage preferences in specific host organisms, 3o such that in practice it may be preferred to select or to avoid certain degenerate codons in a particular host.

The invention also provides modified nucleic acid molecules which include additions, substitutions and deletions of one or more nucleotides. The modified nucleic acid molecules according to this aspect of the invention exclude fully native human TLR9 nucleic acid molecules (GenBank Accession No. AF245704 (SEQ m N0:4) or GenBank Accession No.
NM 017442 (SEQ m N0:5)). In preferred embodiments, these modified nucleic acid molecules and/or the polypeptides they encode retain at least one activity or function of the unmodified nucleic acid molecule and/or the polypeptides, such as signaling activity, etc. In certain embodiments, the modified nucleic acid molecules encode modified polypeptides, preferably polypeptides having conservative amino acid substitutions as are described elsewhere herein. The modified nucleic acid molecules are structurally related to the unmodified nucleic acid molecules and in preferred embodiments are sufficiently structurally related to the unmodified nucleic acid molecules so that the modified and unmodified nucleic acid molecules hybridize under stringent conditions known to one of skill in the art.
For example, modified nucleic acid molecules which encode polypeptides having single amino acid changes can be prepared. Each of these nucleic acid molecules can have one, two or three nucleotide substitutions exclusive of nucleotide changes corresponding to the degeneracy of the genetic code as described herein. Likewise, modified nucleic acid molecules which encode polypeptides having.two amino acid changes can be prepared which have, e.g., 2-6 nucleotide changes. Numerous modified nucleic acid molecules like these will be readily envisioned by one of skill in the art, including for example, substitutions of nucleotides in codons encoding amino acids 2 and 3, 2 and 4, 2 and 5, 2 and 6, and so on. In the foregoing example, each combination of two amino acids is included in the set of modified nucleic acid molecules, as well as all nucleotide substitutions which code for the amino acid substitutions. Additional nucleic acid molecules that encode polypeptides having additional substitutions (i.e., 3 or more), additions or deletions (e.g., by introduction of a stop codon or a splice site(s)) also can be prepared and are embraced by the invention as readily envisioned by one of ordinary skill in the art. Any of the foregoing nucleic acids or polypeptides can be tested by routine experimentation for retention of structural relation or activity to the nucleic acids and/or polypeptides disclosed herein.
The invention also provides isolated fragments of SEQ ID NO:1 and SEQ m N0:2.
The fragments can be used as probes in Southern blot assays to identify such nucleic acids, or they can be used in amplification assays such as those employing PCR. Smaller fragments are those comprising 12, 13, 14, 15, 16, 17, 18, 20, 22, 25, 30, 40, 50, or 75 nucleotides, and every integer therebetween, and are useful, e.g., as primers for nucleic acid amplification procedures. As known to those skilled in the art, larger probes such as 200, 250, 300, 400 or more nucleotides are preferred for certain uses such as Southern blots, while smaller fragments will be preferred for uses such as PCR. Fragments also can be used to produce fusion proteins for generating antibodies or determining binding of the polypeptide fragments.
Likewise, fragments can be employed to produce non-fused fragments of the marine TLR9 polypeptides, useful, for example, in the preparation of antibodies, in immunoassays, and the like. The foregoing nucleic acid fragments further can be used as antisense molecules to inhibit the expression of marine TLR9 nucleic acids and polypeptides, particularly for therapeutic purposes as described in greater detail below.
The invention also includes functionally equivalent variants of the marine TLR9, which include variant nucleic acids and polypeptides which retain one or more of the functional properties of the marine TLR9. Preferably such variants include the marine-specific N-terminal domain (e.g., amino acids 1-819 or amino acids 1-837 of SEQ m N0:3).
For example, variants include a fusion protein which includes the extracellular and transmembrane domains of the marine TLR9 (i.e., amino acids 1-837) which retains the ability to interact with extracellular molecules in a manner similar to intact marine TLR9.
Alternative variants include, for example, a fusion protein which includes the cytoplasmic domain of marine TLR9 (i.e., amino acids 838-1032) which retains the ability to interact with intracellular molecules in a manner similar to intact marine TLR9. Still other functionally equivalent variants include truncations, deletions, point mutations, or additions of amino acids to the sequence of SEQ m N0:3 which retain functions of SEQ m N0:3. For example, the FLAG peptide sequence (DYKDDDDK) can be added at the N-terminal end, or green fluorescent protein (GFP) can be added at the C-terminal end. All such addition variant polypeptides are preferably made by translation of modified nucleic acids based on SEQ m NO:1 or SEQ m N0:2 with corresponding appropriate coding nucleic acid sequence appended thereto with maintenance of the proper reading frame.
Functionally equivalent variants also include a marine TLR9 which has had a portion (e.g., of the N-terminus) removed or replaced by a similar domain from another TLR (e.g., a "domain-swapping" variant). Examples of such domain-swapping variants include at least two types: those involving swapping a TLR9 domain from one species with a TLR9 domain from another species, and those involving swapping a TLR domain from TLR9 with a TLR
domain from another TLR. In certain preferred embodiments the swapping involves corresponding domains between the different TLR molecules. It is believed that certain such domain-swapping variants are not functionally equivalent in a literal sense, insofar as they can function in a manner superior to either or both intact parent TLR
molecules from which a particular domain-swapping variant derives. For example, the TLR/11,-1R
signaling mediated by human TLR9 could be limited, not by the capacity of its extracellular domain to interact with CpG ODN, but rather by the capacity of its cytoplasmic domain to engage the TLR/IZ,-1R signaling pathway. In such a circumstance, it could be advantageous to substitute a more potent cytoplasmic domain from a TLR9 from another species. Such a domain-swapping variant, e.g., chimeric hTLR9/mTLR9, could be used in screening assays for CpG
immuno-agonist/antagonists to increase the sensitivity of the assay, without changing the species specificity.
Other functionally equivalent variants will be known to one of ordinary skill in the art, as will be methods for preparing such variants. The activity of a functionally equivalent variant can be determined using the methods provided herein, and in references that have described assays using other TLRs and TLRs of other species. Such variants are useful, hater alia, for evaluating bioavailability of drugs, in assays for identification of compounds which bind to and/or regulate the signaling function of the marine TLR9, and for determining the portions of the marine TLR9 which are required for signaling activity.
Variants which are non-functional also can be prepared as described above.
Such variants are useful, for example, as negative controls in experiments testing TLR9 signaling activity. Examples of non-functional variants include those incorporating a mutation of proline at as 915 to histidine (P915H) which renders both mTLR9 and hTLR9 nonfunctional with respect to signaling. Father examples of non-functional variants include those incorporating a mutation of the D-X-Y core of the MBD motif to A-L-A, as discussed above, to render both mTLR9 and hTLR9 nonfunctional with respect to CpG DNA binding.
A marine TLR9 nucleic acid, in one embodiment, is operably linked to a gene expression sequence which can direct the expression of the marine TLR9 nucleic acid within a eukaryotic or prokaryotic cell. A "gene expression sequence" is any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, which facilitates the efficient transcription and translation of the nucleic acid to which it is operably linked.
With respect to marine TLR9 nucleic acid, the "gene expression sequence" is any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, which facilitates the efficient transcription and translation of the marine TLR9 nucleic acid to which it is operably linked. The gene expression sequence may, for example, be a mammalian or viral promoter, such as a constitutive or inducible promoter. Constitutive mammalian promoters include, but are not limited to, the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, [3-actin promoter, and other constitutive promoters. Exemplary viral promoters which function constitutively in eukaryotic cells include, for example, promoters from the simian virus (e.g., SV40), papilloma virus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus (RSV), cytomegalovirus (CMV), the long terminal repeats (LTR) of Moloney marine leukemia virus and other retroviruses, and the thymidine kinase (TK) promoter of herpes simplex virus. Other constitutive promoters are known to those of ordinary skill in the art.
The promoters useful as gene expression sequences of the invention also include inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, the metallothionein (MT) promoter is induced to promote transcription and 2o translation in the presence of certain metal ions. Other inducible promoters are known to those of ordinary skill in the art.
In general, the gene expression sequence shall include, as necessary, 5' non-transcribing and 5' non-translating sequences involved with the initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT
sequence, and the like. Especially, such 5' non-transcribing sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably,joined marine TLR9 nucleic acid. The gene expression sequences optionally include enhancer sequences or upstream activator sequences as desired.
Generally a nucleic acid coding sequence and a gene expression sequence are said to be "operably linked" when they axe covalently linked in such a way as to place the transcription and/or translation of the nucleic acid coding sequence under the influence or control of the gene expression sequence. Thus the marine TLR9 nucleic acid sequence and the gene expression sequence are said to be "operably linked" when they are covalently linked in such a way as to place the transcription and/or translation of the marine TLR9 coding sequence under the influence or control of the gene expression sequence. If it is desired that the marine TLR9 sequence be translated into a functional protein, two DNA
sequences are said to be operably linked if induction of a promoter in the 5' gene expression sequence results in the transcription of the marine TLR9 sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the marine TLR9 sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a gene expression sequence would be operably linked to a marine TLR9 nucleic acid sequence if the gene expression sequence were capable of effecting transcription of that marine TLR9 nucleic acid sequence such that the resulting transcript might be translated into the desired protein or polypeptide.
The marine TLR9 nucleic acid molecules and the marine TLR9 polypeptides (including the marine TLR9 inhibitors described below) of the invention can be delivered to the eukaryotic or prokaryotic cell alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating: (1) delivery of a nucleic acid or polypeptide to a target cell, (2) uptake of a nucleic acid or polypeptide by a target cell, or (3) expression of a nucleic acid molecule or polypeptide in a target cell. In this particular setting, a "vector" is any vehicle capable of facilitating: (1) delivery of a marine TLR9 nucleic acid or polypeptide to a target cell, (2) uptake of a marine TLR9 nucleic acid or polypeptide by a target cell, or (3) expression of a marine TLR9 nucleic acid molecule or polypeptide in a target cell.
Preferably, the vectors transport the marine TLR9 nucleic acid or polypeptide into the target cell with reduced degradation relative to the extent of degradation that would result in the absence of the vector. Optionally, a "targeting ligand" can be attached to the vector to selectively deliver the vector to a cell which expresses on its surface the cognate receptor (e.g., a receptor, an antigen recognized by an antibody) for the targeting ligand. In this manner, the vector (containing a marine TLR9 nucleic acid or a marine TLR9 polypeptide) can be selectively delivered to a specific cell. In general, the vectors useful in the invention are divided into two classes: biological vectors and chemical/physical vectors. Biological vectors are more useful for delivery/uptake of marine TLR9 nucleic acids to/by a target cell.
Chemical/physical vectors are more useful for delivery/uptake of marine TLR9 nucleic acids or marine TLR9 proteins to/by a target cell.
Biological vectors include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the nucleic acid sequences of the invention, and free nucleic acid fragments which can be linked to the nucleic acid sequences of the invention. Viral vectors are a preferred type of biological vector and include, but are not limited to, nucleic acid sequences from the following viruses: retroviruses, such as Moloney marine leukemia virus; Harvey marine sarcoma virus; marine mammary tumor virus; Rous sarcoma virus;
adenovirus;
adeno-associated virus; SV40-type viruses; polyoma viruses; poxviruses;
Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; and polio virus. One can readily employ other vectors not named but known in the art.
Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses, the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. In general, the retroviruses are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered 2o retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell line with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in I~riegler, M., "Gerae Transfer and Expression, A
Laboratory Manual, " W.H. Freeman Co., New York (1990) and Murray, E.J., ed., "Methods irz Molecular Biology " vol. 7, Humana Press, Inc., Clifton, New Jersey (1991):
Another preferred virus for certain applications is the adeno-associated virus (AAV), a double-stranded DNA virus. The AAV can be engineered to be replication-deficient and is capable of infecting a wide range of cell types and species. It further has advantages, such as heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the AAV can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression. In addition, wild-type AAV infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the AAV
genomic integration is a relatively stable event. The AAV can also function in an extrachromosomal fashion.
Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. See, e.g., Molecular Cloning: A
1o Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Cells are genetically engineered by the introduction into the cells of heterologous DNA (RNA) encoding a marine polypeptide or fragment or variant thereof. That heterologous DNA (RNA) is placed under operable control of transcriptional elements to permit the expression of the heterologous DNA in the host cell.
Preferred systems for mRNA expression in mammalian cells are those such as pRc/CMV (available from Invitrogen, Carlsbad, CA) that contain a selectable marker such as a gene that confers 6418 resistance (which facilitates the selection of stably transfected cell lines) and the human CMV enhancer-promoter sequences. Additionally, suitable for expression in primate or canine cell lines is the pCEP4 vector (Invitrogen), which contains an Epstein Barr virus (EBV) origin of replication, facilitating the maintenance of plasmid as a multicopy extrachromosomal element. Another expression vector is the pEF-BOS
plasmid containing the promoter of polypeptide Elongation Factor 1 a, which stimulates efficiently transcription in vitro. The plasmid is described by Mishizuma and Nagata (Nucleic Acids Res 18:5322 (1990)), and its use in transfection experiments is disclosed by, for example, Demoulin (Mol Cell Biol 16:4710-4716 (1996)). Still another preferred expression vector is an adenovirus, described by Stratford-Perncaudet, which is defective for E1 and E3 proteins (J Clin Invest 90:626-630 (1992)).
In addition to the biological vectors, chemical/physical vectors may be used to deliver a nucleic acid or polypeptide to a target cell and facilitate uptake thereby.
As used herein, a "chemical/physical vector" refers to a natural or synthetic molecule, other than those derived from bacteriological or viral sources, capable of delivering an isolated nucleic acid or polypeptide to a cell. As used herein with respect to a marine TLR9 nucleic acid or polypeptide, a "chemical/physical vector" refers to a natural or synthetic molecule, other than those derived from bacteriological or viral sources, capable of delivering the isolated marine TLR9 nucleic acid or polypeptide to a cell.
' A preferred chemical/physical vector of the invention is a colloidal dispersion system.
Colloidal dispersion systems include lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system of the invention is a liposome. Liposomes are artificial membrane vesicles which are useful as a delivery vector in to vivo or ih vitro. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2 - 4.0 ~m can encapsulate large macromolecules. RNA, DNA, and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley et al., Tends Biochem Sci 6:77 (1981)). In order for a liposome to be an efficient nucleic acid transfer vector, one or more of the following characteristics should be present: (1) encapsulation of the nucleic acid of interest at high efficiency with retention of biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information.
Liposomes may be targeted to a particular tissue by coupling the liposome to a 2o specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein. Ligands which may be useful for targeting a liposome to a particular cell will depend on the particular cell or tissue type. Additionally when the vector encapsulates a nucleic acid, the vector may be coupled to a nuclear targeting peptide, which will direct the marine TLR9 nucleic acid to the nucleus of the host cell.
Liposomes are commercially available from Gibco BRL, for example, as LIPOFECT1NTM and LIPOFECTACETM, which are formed of cationic lipids such as N-[1-(2, 3 dioleyloxy)-propyl]-N, N, N-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDAB). Methods for making liposomes are well known in the art and have been described in many publications.
3o Other exemplary compositions that can be used to facilitate uptake by a target cell of nucleic acids in general, and nucleic acids encoding the marine TLR9 in particular, include calcium phosphate and other chemical mediators of intracellular transport, microinjection compositions, electroporation and homologous recombination compositions (e.g., for integrating a marine TLR9 nucleic acid into a preselected location within a target cell chromosome).
~ The invention also embraces so-called expression kits, which allow the artisan to prepare a desired expression vector or vectors. Such expression kits include at least separate portions of the previously discussed coding sequences. Other components may be added, as desired, as long as the previously mentioned sequences, which are required, are included.
It will also be recognized that the invention embraces the use of the marine l0 cDNA sequences in expression vectors to transfect host cells and cell lines, be these prokaryotic (e.g., E. coli), or eukaryotic (e.g., 293 fibroblast cells (ATCC, CRL-1573), MonoMac-6, THP-1, U927, CHO cells, COS cells, yeast expression systems and recombinant baculovirus expression in insect cells). Especially useful are mammalian cells such as human, pig, goat, primate, rodent, guinea pig, etc. They may be of a wide variety of tissue types, and include primary cells and cell lines. The expression vectors require that the pertinent sequence, i.e., those nucleic acids described supra, be operably linked to a promoter.
The invention also provides isolated marine TLR9 polypeptides which include the amino acid sequences of SEQ ID N0:3 and fragments thereof, encoded by the marine TLR9 nucleic acids described above. Marine TLR9 polypeptides also embrace alleles, functionally 2o equivalent variants and analogs (those non-allelic polypeptides which vary in amino acid sequence from the disclosed marine TLR9 polypeptides by 1, 2, 3, 4, 5, or more amino acids) provided that such polypeptides retain TLR9 activity. Non-functional variants also are embraced by the invention; these are useful as antagonists of TLR9 signaling function, as negative controls in assays, and the like. Such alleles, variants, analogs and fragments are useful, for example, alone or as fusion proteins for a variety of purposes including as a component of assays.
Fragments of a polypeptide preferably are those fragments which retain a distinct functional capability of the intact polypeptide, in particular as a receptor of various molecules.
Accordingly, fragments of a TLR9 polypeptide preferably are those fragments which retain a 3o distinct functional capability of the TLR9 polypeptide, in particular as a receptor of various molecules. Of particular interest are fragments that bind to ISNAs, including, for example, fragments that bind CpG nucleic acids. Other functional capabilities which can be retained in a fragment of a polypeptide include signal transduction (e.g., TLR/IL-1R
signaling by marine TLR9), interaction with antibodies and interaction with other polypeptides (such as would be found in a protein complex). Those skilled in the art are well versed in methods that can be applied for selecting fragments which retain a functional capability of the marine TLR9.
Confirmation of the functional capability of the fragment can be carried out by synthesis of the fragment and testing of the capability according to standard methods. For example, to test the signaling activity of a marine TLR9 fragment, one inserts or expresses the fragment in a cell in which signaling can be measured. Such methods, which are standard in the art, are to described further herein.
The invention embraces variants of the marine TLR9 polypeptides described above.
As used herein, a "variant" of a polypeptide is a polypeptide which contains one or more modifications to the primary amino acid sequence of a polypeptide.
Accordingly, a "variant"
of a marine TLR9 polypeptide is a polypeptide which contains one or more modifications to the primary amino acid sequence of a marine TLR9 polypeptide. Modifications which create a marine TLR9 variant can be made to a marine TLR9 polypeptide for a variety of reasons, including 1) to reduce or eliminate an activity of a marine TLR9 polypeptide, such as signaling; 2) to enhance a property of a marine TLR9 polypeptide, such as signaling, binding affinity for nucleic acid ligand or other ligand molecule, protein stability in an expression 2o system, or the stability of protein-protein binding; 3) to provide a novel activity or property to a marine TLR9 polypeptide, such as addition of an antigenic epitope or addition of a detectable moiety, e.g., luciferase, FLAG peptide, GFP; 4) to establish that an amino acid substitution does or does not affect molecular signaling activity; or 5) reduce immunogenicity of a marine TLR9 polypeptide. Modifications to a marine TLR9 polypeptide are typically made to the nucleic acid which encodes the marine TLR9 polypeptide, and can include deletions, point mutations, truncations, amino acid substitutions and additions of amino acids or non-amino acid moieties. Alternatively, modifications can be made directly to the polypeptide, such as by cleavage, addition of a linker molecule, addition of a detectable moiety (for example, biotin, fluorophore, radioisotope, enzyme, or peptide), addition of a 3o fatty acid, and the like.
Modifications also embrace fusion proteins comprising all or part of the marine TLR9 amino acid sequence. One of skill in the art will be familiar with methods fox predicting the effect on protein conformation of a change in protein sequence, and can thus "design" a variant marine TLR9 according to known methods. One example of such a method is described by Dahiyat and Mayo in Science 278:82-87 (1997), whereby proteins can be designed de novo. The method can be applied to a known protein to vary a only a portion of the polypeptide sequence. By applying the computational methods of Dahiyat and Mayo, specific variants of a marine TLR9 polypeptide can be proposed and tested to determine whether the variant retains a desired conformation.
Variants include marine TLR9 polypeptides which are modified specifically to alter a to feature of the polypeptide unrelated to its physiological activity. For example, cysteine residues can be substituted or deleted to prevent unwanted disulfide linkages.
Similarly, certain amino acids can be changed to enhance expression of a marine TLR9 polypeptide by eliminating proteolysis by proteases in an expression system (e.g., dibasic amino acid residues in yeast expression systems in which KEX2 protease activity is present).
Mutations of a nucleic acid which encode a marine TLR9 polypeptide preferably preserve the amino acid reading frame of the coding sequence, and preferably do not create regions in the nucleic acid which are likely to hybridize to form secondary structures, such as hairpins or loops, which can be deleterious to expression of the variant polypeptide.
Mutations can be made by selecting an amino acid substitution, or by random 2o mutagenesis of a selected site in a nucleic acid which encodes the polypeptide. Variant polypeptides are then expressed and tested for one or more activities to determine which mutation provides a variant polypeptide with a desired property. Further mutations can be made to variants (or to non-variant marine TLR9 polypeptides) which are silent as to the amino acid sequence of the polypeptide, but which provide preferred codons for translation in a particular host. The preferred codons for translation of a nucleic acid in, e.g., E. coli, axe well known to those of ordinary skill in the art. Still other mutations can be made to the noncoding sequences of a muririe TLR9 gene or cDNA clone to enhance.
expression of the polypeptide.
The activity of variants of marine TLR9 polypeptides can be tested by cloning the 3o gene encoding the variant marine TLR9 polypeptide into a prokaryotic or eukaryotic (e.g., mammalian) expression vector, introducing the vector into an appropriate host cell, expressing the variant marine TLR9 polypeptide, and testing for a functional capability of the marine TLR9 polypeptides as disclosed herein. For example, the variant marine polypeptide can be tested for ability to provide signaling, as set forth below in the examples.
Preparation of other variant polypeptides may favor testing of other activities, as will be known to one of ordinary skill in the art.
The skilled artisan will also realize that conservative amino acid substitutions may be made in marine TLR9 polypeptides to provide functionally equivalent variants of the foregoing polypeptides, i.e., variants which retain the functional capabilities of the marine TLR9 polypeptides. As used herein, a "conservative amino acid substitution"
refers to an 1o amino acid substitution which does not alter the relative charge or size characteristics of the polypeptide in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g., Molecular Cloning: A
Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley ~ Sons, Inc., New York.
Exemplary functionally equivalent variants of the marine TLR9 polypeptides include conservative amino acid substitutions of SEQ ID N0:3. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, 2o W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.
Conservative amino acid substitutions in the amino acid sequence of marine polypeptide to produce functionally equivalent variants of marine TLR9 typically are made by alteration of the nucleic acid sequence encoding marine TLR9 polypeptides (e.g., SEQ ID
NO:1 and SEQ ID N0:2). Such substitutions can be made by a variety of methods known to one of ordinary skill in the art. For example, amino acid substitutions may be made by PCR-directed mutation, site-directed mutagenesis according to the method of Kunkel (Kunkel, Proc Natl Acad Sci USA 82:488-492 (1985)), or-by chemical synthesis of a gene encoding a marine TLR9 polypeptide. The activity of functionally equivalent fragments of marine TLR9 polypeptides can be tested by cloning the gene encoding the altered marine TLR9 polypeptide 3o into a bacterial or mammalian expression vector, introducing the vector into an appropriate host cell, expressing the altered marine TLR9 polypeptide, and testing for the ability of the marine TLR9 polypeptide to mediate a signaling event. Peptides which are chemically synthesized can be tested directly for function.
A variety of methodologies well known to the skilled practitioner can be utilized to obtain isolated marine TLR9 polypeptide molecules. The polypeptide may be purified from cells which naturally produce the polypeptide by chromatographic means or immunological recognition. Alternatively; an expression vector may be introduced into cells to cause production of the polypeptide. In another method, mRNA transcripts may be microinjected or otherwise introduced into cells to cause production of the encoded polypeptide. Translation of mRNA in cell-free extracts such as the reticulocyte lysate system also may be used to to produce polypeptide. Those skilled in the art also can readily follow known methods for isolating marine TLR9 polypeptides. These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography and immune-affinity chromatography.
The invention as described herein has a number of uses, some of which are described elsewhere herein. For example, the invention permits isolation of the marine polypeptide molecules by, e.g., expression of a recombinant nucleic acid to produce large quantities of polypeptide which may be isolated using standard protocols. As another example; the isolation of the marine TLR9 gene makes it possible for marine TLR9 to be used in methods for assaying molecular interactions involving TLR9.
2o As discussed further in the Examples below, it has been discovered according to one aspect of the invention that responsiveness to ISNA can be reconstituted in ISNA-unresponsive cells by introducing into ISNA-unresponsive cells an expression vector that directs the expression of marine TLR9 (and certain homologues and variants thereof). Cells so reconstituted also exhibit responses to substances other than phosphorothioate ISNA, e.g., E. coli DNA, phosphodiester CpG-ODN, and even methylated CpG-ODN.
Also as discussed further in the Examples below, it has been discovered according to certain aspects of the instant invention that TLR9 not only confers upon cells the ability to signal in response to binding ISNA, but also confers both sequence specificity and species specificity to such signaling responses. Thus marine TLR9 signaling in response to CpG-3o ODN 166, reportedly an optimal marine ISNA, was found to be significantly stronger than the corresponding marine TLR9 signaling response to CpG-ODN 2006, reportedly an optimal human ISNA. The converse was also found to be true, i.e., human TLR9 signaling in response to CpG-ODN 2006 was found to be significantly stronger than the corresponding human TLR9 signaling response to CpG-ODN 1668. Furthermore, it has been discovered according to the instant invention that certain types of cells preferentially express TLR9. For r5 example, TLR9 is strongly expressed in B cells and plasmacytoid dendritic cells (CD123+
DC), but only weakly by T cells, monocyte-derived dendritic cells (MDDC), and CD 14+
monocytes. In contrast, TLR2 and TLR4 are strongly expressed by MDDC and CD
14+
monocytes, but relatively weakly by B cells, CD123+ DC, and T cells.
The invention also embraces agents which bind selectively to the marine TLR9 l0 nucleic acid molecules or polypeptides as well as agents which bind to variants and fragments of the polypeptides and nucleic acids as described herein. The agents include polypeptides which bind to marine TLR9, and antisense nucleic acids, both of which are described in greater detail below. The agents can inhibit or increase marine TLR9-mediated signaling activity (antagonists and agonists, respectively).
15 Some of the agents are inhibitors. A marine TLR9 inhibitor is an agent that inhibits marine TLR9-mediated signaling across a cell membrane.
As used herein "TLR9 signaling" refers to an ability of a TLR9 polypeptide to activate the TLR/IL-1R (TIR) signaling pathway, also referred to herein as the TLR
signal transduction pathway. Without meaning to be held to any particular theory, it is believed that 20 the TLR/IL-1R signaling pathway involves signaling via the molecules myeloid differentiation marker 88 (MyD88) and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), leading to activation of kinases of the IxB kinase complex and the c-jun NH2-terminal kinases (e.g., JNK 1l2). Hacker H et al., JExp Mecl 192:595-600 (2000). A
molecule which inhibits TLR9 activity (an antagonist) is one which inhibits TLR9-mediated 25 activation of the TLR/IL-1R signaling pathway, and a molecule which increases TLR9 signaling (an agonist) is one which increases TLR9-mediated activation of the TLR/IL,-1R
signaling pathway. Changes in TLR9 activity can be measured by assays such as those disclosed herein, including expression of genes under control of xB-sensitive promoters and enhancers. Such naturally occurring genes include the genes encoding IL-1 (3, IL-6, IL-8, the 30 p40 subunit of interleukin 12 (IL-12p40), and the costimulatory molecules CD80 and CD86.
Other genes can be placed under the control of such regulatory elements (see below) and thus serve to report the level of TLR9 signaling. Additional nucleotide sequence can be added to SEQ ID N0:1 or SEQ ID N0:2, preferably to the 5' or the 3' end of SEQ ID N0:2, to yield a nucleotide sequence encoding a chimeric polypeptide that includes a detectable or reporter moiety, e.g., FLAG, luciferase (luc), green fluorescent protein (GFP) and others known by those skilled in the art. These are discussed in greater detail in the Examples below.
In one embodiment the marine TLR9 inhibitor is an antisense oligonucleotide that selectively binds to a marine TLR9 nucleic acid molecule, to reduce the expression of marine TLR9 (or TLR9 of another species) in a cell. This is desirable in virtually any medical condition wherein a reduction of TLR9 signaling activity is desirable.
to As used herein, the term "antisense oligonucleotide" or "antisense"
describes an oligonucleotide that is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide, or riiodified oligodeoxyribonucleotide which hybridizes under physiological conditions to DNA comprising a particular gene or to an mRNA
transcript of that gene and, thereby, inhibits the transcription of that gene and/or the translation of that 15- mRNA. The antisense molecules are designed so as to interfere with transcription or translation of a target gene upon hybridization with the target gene or transcript. Those skilled in the art will recognize that the exact length of the antisense oligonucleotide and its .
degree of complementarity with its target will depend upon the specific target selected, including the sequence of the target and the particular bases which comprise that sequence. It 2o is preferred that the antisense oligonucleotide be constructed and arranged so as to bind selectively with the target under physiological conditions, i.e., to hybridize substantially more to the target sequence than to any other sequence in the target cell under physiological conditions.
Based upon SEQ ID NO:1 and SEQ ID N0:2, or upon allelic or homologous genomic 25 and/or cDNA sequences, one of skill in the art can easily choose and synthesize any of a number of appropriate antisense molecules for use in accordance with the present invention.
In order to be sufficiently selective and potent for inhibition, such antisense oligonucleotides should comprise at least 10 and, more preferably, at least 15 consecutive bases which are complementary to the target, although in certain cases modified oligonucleotides as short as 7 3o bases in length have been used successfully as antisense oligonucleotides.
Wagner RW et al., Nat Biotechrlol 14:840-844 (1996). Most preferably, the antisense oligonucleotides comprise a complementary sequence of 20-30 bases. Although oligonucleotides may be chosen which are antisense to any region of the gene or mRNA transcripts, in preferred embodiments the antisense oligonucleotides correspond to N-terminal or 5' upstream sites such as translation initiation, transcription initiation or promoter sites. In addition, 3'-untranslated regions may be targeted. Targeting to mRNA splicing sites has also been used in the art but may be less preferred if alternative mRNA splicing occurs. In addition, the antisense is targeted, preferably, to sites in which mRNA secondary structure is not expected (see, e.g., Sainio et al., Cell Mol Neurobiol 14(5):439-457 (1994)) and at which polypeptides are not expected to bind. Thus, the present invention also provides for antisense oligonucleotides which are to complementary to allelic or homologous cDNAs and genomic DNAs corresponding to marine TLR9 nucleic acid containing SEQ m NO:l or SEQ m N0:2.
In one set of embodiments, the antisense oligonucleotides of the invention may be composed of "natural" deoxyribonucleotides, ribonucleotides, or any combination thereof.
That is, the 5' end of one native nucleotide and the 3' end of another native nucleotide may be i5 covalently linked, as in natural systems, via a phosphodiester internucleoside linkage. These oligonucleotides may be prepared by art-recognized methods which may be carried out manually or by an automated synthesizer. They also may be produced recombinantly by vectors.
In preferred embodiments, however, the antisense oligonucleotides of the invention 2o also may include "modified" oligonucleotides. That is, the oligonucleotides may be modified in a number of ways which do not prevent them from hybridizing to their target but which enhance their stability or targeting or which otherwise enhance their therapeutic effectiveness.
The term "modified oligonucleotide" as used herein describes an oligonucleotide in which (1) at least two of its nucleotides are covalently linked via a synthetic internucleoside 25 linkage (i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide) and/or (2) a chemical group not normally associated with nucleic acids has been covalently attached to the oligonucleotide. Preferred synthetic internucleoside linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, 30 carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters and peptides.
The term "modified oligonucleotide" also encompasses oligonucleotides with a covalently modified base and/or sugar. For example, modified oligonucleotides include oligonucleotides having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position. Thus modified oligonucleotides may include a 2'-O-alkylated ribose group. In addition, modified oligonucleotides may include sugars such as arabinose instead of ribose. The present invention, thus, contemplates pharmaceutical preparations containing modified antisense molecules that are complementary to and hybridizable with, under physiological conditions, nucleic acids encoding marine TLR9 polypeptides, together with pharmaceutically acceptable carriers.
1o Agents which bind marine TLR9 also include binding peptides and other molecules which bind to the marine TLR9 polypeptide and complexes containing the marine polypeptide. When the binding molecules are inhibitors, the molecules bind to and inhibit the activity of marine TLR9. When the binding molecules are activators, the molecules bind to and increase the activity of marine TLR9. To determine whether a marine TLR9 binding agent binds to marine TLR9 any known binding assay may be employed. For example, the binding agent may be immobilized on a surface and then contacted with a labeled marine TLR9 polypeptide. The amount of marine TLR9 which interacts with the marine binding agent or the amount which does not bind to the marine TLR9 binding agent may then be quantitated to determine whether the marine TLR9 binding agent binds to marine TLR9.
The marine TLR9 binding agents include molecules of numerous size and type that bind selectively or preferentially to marine TLR9 polypeptides, and complexes of both marine TLR9 polypeptides and their binding partners. These molecules may be derived from a variety of sources. For example, marine TLR9 binding agents can be provided by screening degenerate peptide libraries which can be readily prepared in solution, in immobilized form or as phage display libraries. Combinatorial libraries also can be synthesized of peptides containing one or more amino acids. Libraries further can be synthesized of peptoids and non-peptide synthetic moieties.
Phage display can be particularly effective in identifying binding peptides useful according to the invention. Briefly, one prepares a phage library (using, e.g., m13, fd, or lambda phage), displaying inserts from 4 to about 80 amino acid residues using conventional procedures. The inserts may represent, for example, a completely degenerate or biased array.

One then can select phage-bearing inserts which bind to the marine TLR9 polypeptide. This process can be repeated through several cycles of reselection of phage that bind to the marine TLR9 polypeptide. Repeated rounds lead to enrichment of phage bearing particular sequences. DNA sequence analysis can be conducted to identify the sequences of the expressed polypeptides. The minimal linear portion of the sequence that binds to the marine TLR9 polypeptide can be determined. One can repeat the procedure using a biased library containing inserts containing part or all of the minimal linear portion plus one or more additional degenerate residues upstream or downstream thereof. Yeast two-hybrid screening methods also may be used to identify polypeptides that bind to the marine TLR9 1o polypeptides. Thus, the marine TLR9 polypeptides of the invention, or a fragment thereof, can be used to screen peptide libraries, including phage display libraries, to identify and select peptide binding partners of the marine TLR9 polypeptides of the invention.
Such molecules can be used, as described, for screening assays, for purification protocols, for interfering directly with the functioning of marine TLR9 and for other purposes that will be apparent to those of ordinary skill in the art.
The invention also embraces agents which bind selectively to certain regulatory sequences associated with the marine TLR9 nucleic acid molecules described herein. The agents include polypeptides which bind to transcription and translation regulatory sequences of marine TLR9, and antisense nucleic acids, both of which are described in greater detail below. The agents can inhibit or increase marine TLR9 expression, as well as signaling activity (antagonists and agonists, respectively). Agents which bind selectively to regulatory sequences associated with the marine TLR9 nucleic acid molecules can be identified using methods familiar to those of skill in the art. For example, a promoter region including at least 100, 200, 300, 400, 500, or more nucleotides upstream (5') of the coding region of marine TLR9 can be identified by isolating, from appropriate genomic DNA, such nucleotide sequences using the sequences of SEQ m NO:1 or SEQ m NO:2 as primers or as probes, and then inserting the promoter region DNA into an appropriate expression vector so as to control the expression of TLR9 or some other reporter gene, introducing the TLR9 promoter vector into an appropriate host cell, and screening for TLR9 or reporter expression by those cells following their incubation in the presence and absence of various test agents.
A reporter gene other than TLR9 can include, for example, an enzyme, a cytokine, a cell surface antigen, luciferase, chloramphenicol acetyl transferase (CAT), etc. An agent that inhibits expression of TLR9 or the reporter under the control of the TLR9 promoter is classified as a TLR9 expression inhibitor. Conversely, an agent that augments expression of TLR9 or reporter under the control of the TLR9 promoter is classified as a TLR9 expression enhancer. It was discovered according to the instant invention, for example, that the cytokine IL-4 inhibits the expression of TLR9. In this manner it is possible to identify agents that can be administered in conjunction with ISNA, for example by local administration, to enhance response to the ISNA. Such an enhancing effect might be desirable, for example, in the setting of immunization or vaccination. Conversely, it it is possible to identify agents that can be-1o administered in conjunction with a ISNA, for example by local administration, to inhibit response to the ISNA. Such an inhibiting response might be desirable, for example, in the setting of gene replacement therapy.
Therefore the invention generally provides efficient methods of identifying pharmacological agents or lead compounds for agents useful in the treatment of conditions associated with TLR9 activity and the compounds and agents so identified.
Generally, the screening methods involve assaying for compounds which inhibit or enhance signaling through marine TLR9. Such methods are adaptable to automated, high throughput screening of compounds. Examples of such high throughput screening methods are described in U.S.
patents 6,103,479; 6,051,380; 6,051,373; 5,998,152; 5,876,946; 5,708,158;
5,443,791;
5,429,921; and 5,143,854.
A variety of assays for pharmacological agents are provided, including labeled ira vitro protein binding assays, signaling assays using detectable molecules, etc. For example, protein binding screens are used to rapidly examine the binding of candidate pharmacological agents to a marine TLR9. The candidate pharmacological agents can be derived from, for example, combinatorial peptide or nucleic acid libraries. Convenient reagents for such assays are known in the art. An exemplary cell-based assay of signaling involves contacting a cell having a marine TLR9 with a candidate pharmacological agent under conditions whereby the induction of a detectable molecule can occur. Specific conditions are well known in the art and are described, for example, in Hacker H et al., JExp Med 192:595-600 (2000), and 3o references cited therein. A reduced degree of induction of the detectable molecule in the presence of the candidate pharmacological agent indicates that the candidate pharmacological agent reduces the signaling activity of marine TLR9. An increased degree of induction of the detectable molecule in the presence of the candidate pharmacological agent indicates that the candidate pharmacological agent increases the signaling activity of marine TLR9.
Marine TLR9 used in the methods of the invention can be added to an assay mixture as an isolated polypeptide (where binding of a candidate pharmaceutical agent is to be measured) or as a cell or other membrane-encapsulated space which includes a marine TLR9 polypeptide. In the latter assay configuration, the cell or other membrane-encapsulated space can contain the marine TLR9 as a polypeptide or as a nucleic acid (e.g., a cell transfected with an expression vector containing a marine TLR9). In the assays described herein, the 1o marine TLR9 polypeptide can be produced recombinantly, isolated from biological extracts, or synthesized in vitro. Marine TLR9 polypeptides encompass chimeric proteins comprising a fusion of a marine TLR9 polypeptide with another polypeptide, e.g., a polypeptide capable of providing or enhancing protein-protein binding, enhancing signaling capability, facilitating detection, or enhancing stability of the marine TLR9 polypeptide under assay conditions. A
polypeptide fused to a marine TLR9 polypeptide or fragment thereof may also provide means of readily detecting the fusion protein, e.g., by immunological recognition or by fluorescent labeling.
The assay mixture also comprises a candidate pharmacological agent. Typically, a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a 2o different response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration of agent or at a concentration of agent below the limits of assay detection. Candidate pharmaceutical agents encompass numerous chemical classes, although typically they are organic compounds. Preferably, the candidate pharmacological agents are small organic compounds, i.e., those having a molecular weight of more than 50 yet less than about 2500. Polymeric candidate agents can have higher molecular weights, e.g., oligonucleotides in the range of about 2500 to about 12,500.
Candidate agents comprise functional chemical groups necessary for structural interactions with polypeptides, and may include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups and more preferably at least three of 3o the functional chemical groups. The candidate agents can comprise cyclic carbon or heterocyclic structure andlor axomatic or polyaromatic structures substituted with one or more of the above-identified functional groups. Candidate agents also can be biomolecules such as nucleic acids, peptides, saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs of the above, or combinations thereof and the like. Where the agent is a nucleic acid, the agent typically is a DNA or RNA molecule, although modified nucleic acids having non-natural bonds or subunits are also contemplated.
Candidate agents are obtained from a wide variety of sources, including libraries of natural, synthetic, or semisynthetic compounds, or any combination thereof.
For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, l0 synthetic organic combinatorial libraries, phage display libraries of random peptides, and the like. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural and synthetically produced libraries and compounds can be readily modified through conventional chemical, physical, and biochemical means. Further, known pharmacological agents may be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amidification, etc., to produce~structural analogs of the agents.
Therefore, a source of candidate agents are libraries of molecules based on known TLR9 ligands, e.g., CpG oligonucleotides shown herein to interact with TLR9, in which the structure of the ligand is changed at one or more positions of the molecule to contain more or 2o fewer chemical moieties or different chemical moieties. The structural changes made to the molecules in creating the libraries of analog inhibitors can be directed, random, or a combination of both directed and random substitutions and/or additions. One of ordinary skill in the art in the preparation of combinatorial libraries can readily prepare such libraries based on existing TLR9 ligands.
A variety of other reagents also can be included in the mixture. These include reagents such as salts, buffers, neutral proteins (e.g., albumin), detergents, etc. which may be used to facilitate optimal protein-protein and/or protein-nucleic acid binding. Such a reagent may also reduce non-specific or background interactions of the reaction components. Other reagents that improve the efficiency of the assay such as protease inhibitors, nuclease 3o inhibitors, antimicrobial agents, and the like may also be used.
The mixture of the foregoing assay materials is incubated under conditions whereby, but for the presence of the candidate pharmacological agent, the marine TLR9 mediates TLR/IL-1R signaling. For determining the binding of a candidate pharmaceutical agent to a marine TLR9, the mixture is incubated under conditions which permit binding.
The order of addition of components, incubation temperature, time of incubation, and other parameters of the assay may be readily determined. Such experimentation merely involves optimization of the assay parameters, not the fundamental composition of the assay. Incubation temperatures typically are between 4°C and 40°C. Incubation times preferably are minimized to facilitate rapid, high throughput screening, and typically are between 1 minute and 10 hours.
After incubation, the level of signaling or the level of specific binding between the marine TLR9 polypeptide and the candidate pharmaceutical agent is detected by any convenient method available to the user. For cell-free binding type assays, a separation step is often used to separate bound from unbound components. The separation step may be accomplished in a variety of ways. For example, separation can be accomplished in solution, or, conveniently, at least one of the components is immobilized on a solid substrate, from which the unbound components may be easily separated. The solid substrate can be made of a wide variety of materials and in a wide variety of shapes, e.g., microtiter plate, microbead, dipstick, resin particle, etc. The substrate preferably is chosen to maximize signal-to-noise ratios, primarily to minimize background binding, as well as for ease of separation and cost.
Separation may be effected for example, by removing a bead or dipstick from a reservoir, emptying or diluting a reservoir such as a microtiter plate well, rinsing a bead, particle, chromatographic column or filter with a wash solution or solvent.
The separation step preferably includes multiple rinses or washes. For example, when the solid substrate is a microtiter plate, the wells may be washed several times with a washing solution, which typically includes those components of the incubation mixture that do not participate in specific bindings such as salts, buffer, detergent, non-specific protein, etc.
Where the solid substrate is a magnetic bead, the beads may be washed one or more times with a washing solution and isolated using a magnet.
Detection may be effected in any convenient way for cell-based assays such as measurement of an induced polypeptide within, on the surface of, or secreted by the cell.
Examples of detection methods useful in such cell-based assays include fluorescence-activated cell sorting (FAGS) analysis, bioluminescence, fluorescence, enzyme-linked immunosorbent assay (ELISA), reverse transcriptase-polymerase chain reaction (RT-PCR), and the like.
A variety of methods may be used to detect the label, depending on the nature of the label and other assay components. For example, the label may be detected while bound to the solid substrate or subsequent to separation from the solid substrate. Labels may be directly detected through optical or electron density, radioactive emissions, nonradiative energy transfers, etc., or indirectly detected with antibody conjugates, streptavidin-biotin conjugates, etc. Methods for detecting the labels are well known in the art.
The marine TLR9 binding agent may also be an antibody or a functionally active l0 antibody fragment. Antibodies are well known to those of ordinary skill in the science of immunology. As used herein, the term "antibody" means not only intact antibody molecules but also fragments of antibody molecules retaining specific target binding ability. Such fragments are also well known in the art and are regularly employed both in vitro and in vivo.
In particular, as used herein, the term "antibody" means not only intact immunoglobulin molecules but also the well-known active fragments F(ab')Z and Fab. F(ab')2 and Fab fragments which lack the Fc fragment of intact antibody clear more rapidly from the circulation and may have less non-specific tissue binding than an intact antibody (Wahl RL et al., JNucl Med 24:316-325 (1983)).
Monoclonal antibodies may be made by any of the methods known in the art utilizing 2o marine TLR9, or a fragment thereof, as an immunogen. Alternatively the antibody may be a polyclonal antibody specific for marine TLR9 which inhibits marine TLR9 activity. The preparation and use of polyclonal antibodies are also known to one of ordinary skill in the art.
Significantly, as is well known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W.R. (1986) Tlae Experimental Foundations of Modern Immunology, Wiley &
Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). The pFc' and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region, designated 3o an F(ab')2 fragment, retains both of the antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd.
The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.
Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the 1o paratope (see, in general, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FRl through FR4) separated respectively by three complementarity determining regions (CDRl through CDR3).
The CDRs, and in particular the CDR3 regions, and more particularly the heavy chain CDR3, are largely responsible for antibody specificity.
The sequences of the antigen-binding Fab' portion of the anti-marine TLR9 monoclonal antibodies identified as being useful according to the invention in the assays provided above, as well as the relevant FR and CDR regions, can be determined using amino acid sequencing methods that are routine in the art. It is well established that non-CDR
regions of a mammalian antibody may be replaced with corresponding regions of non-specific or hetero-specific antibodies while retaining the epitope specificity of the original antibody.
This technique is useful for the development and use of "humanized" antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody. Techniques to humanize antibodies are particularly useful when non-human animal (e.g., marine) antibodies which inhibit marine TLR9 activity are identified.
These non-human animal antibodies can be humanized for use in the treatment of a human subject in the methods according to the invention. Examples of methods for humanizing a marine antibody are provided in U.S. patents 4,816,567, 5,2f5,539, 5,585,089, 5,693,762 and 5,859,205. Other antibodies, including fragments of intact antibodies with antigen-binding ability, are often referred to as "chimeric" antibodies.
Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides for F(ab')2 and Fab fragments of an anti-marine TLR9 monoclonal antibody;

chimeric antibodies in which the Fc and/or FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions of an anti-marine TLR9 antibody have been replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR
and/or CDRI
and/or CDR2 and/or light chain CDR3 regions of an anti-marine TLR9 antibody have been replaced by homologous human or non-human sequences; and chimeric Fab fragment antibodies in which the FR and/or CDRl and/or CDRZ and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences.
According to the invention marine TLR9 inhibitors also include "dominant negative"
polypeptides derived from SEQ ll~ N0:3. A dominant negative polypeptide is an inactive l0 variant of a polypeptide, which, by interacting with the cellular machinery, displaces an active polypeptide from its interaction with the cellular machinery or competes with the active polypeptide, thereby reducing the effect of the active polypeptide. For example, a dominant negative receptor which binds a ligand but does not transmit a signal in response to binding of the ligand can reduce the biological effect of expression of the receptor. As shown in the Examples below, TLR9 polypeptides which incorporate the substitution of histidine for proline at as 915 (P915H mutation) are functionally inactive and are dominant negative with respect to the native TLR9 polypeptide.
The end result of the expression of a dominant negative marine TLR9 polypeptide of the invention in a cell is a reduction in TLR9 activity such as signaling through the TIR
pathway. One of ordinary skill in the art can assess the potential for a dominant negative variant of a marine TLR9 polypeptide and, using standard mutagenesis techniques, create one or more dominant negative variant polypeptides. For example, given the teachings contained herein of a marine TLR9 polypeptide, one of ordinary skill in the art can modify the sequence of the marine TLR9 polypeptide by site-specific mutagenesis, scanning mutagenesis, partial gene deletion or truncation, and the like. See, e.g., U.S. Patent No.
5,580,723 and Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. The skilled artisan then can test the population of mutagenized polypeptides for diminution in marine TLR9 activity and/or for retention of such an activity. Other similar methods for creating and testing 3o dominant negative variants of a marine TLR9 polypeptide will be apparent to one of ordinary skill in the art.

Each of the compositions according to this aspect of the invention is useful for a variety of therapeutic and non-therapeutic purposes. For example, the marine TLR9 nucleic acids of the invention are useful as oligonucleotide probes. Such oligonucleotide probes can be used herein to identify genomic or cDNA library clones possessing an identical or substantially similar nucleic acid sequence. A suitable oligonucleotide or set of oligonucleotides, which is capable of hybridizing under stringent hybridization conditions to the desired sequence, a variant or fragment thereof, or an anti-sense complement of such an oligonucleotide or set of oligonucleotides, can be synthesized by means well known in the art (see, for example, Synthesis and Application of DNA and RNA, S.A. Narang, ed., 1987, to Academic Press, San Diego, CA) and employed as a probe to identify and isolate the desired sequence, variant or fragment thereof by techniques known in the art.
Techniques of nucleic acid hybridization and clone identification are disclosed by Sambrook, et al., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989.
To facilitate the detection of a desired nucleic acid sequence, or variant or fragment thereof, whether for cloning purposes or for the mere detection of the presence of the sequence, the above-described probes may be labeled with a detectable group.
Such a detectable group may be any material having a detectable physical or chemical property.
Such materials have been well developed in the field of nucleic acid hybridization and, in 2o general, many labels useful in such methods can be applied to the present invention.
Particularly useful are radioactive labels. Any radioactive label may be employed which provides for an adequate signal and has a sufficient half life. If single stranded, the oligonucleotide may be radioactively labeled using kinase reactions.
Alternatively, oligonucleotides are also useful as nucleic acid hybridization probes when labeled with a non-radioactive marker such as biotin, an enzyme or a fluorescent group. See, for example, Leary JJ et al., Proc Natl Acad Sci ZISA 80:4045 (1983); Renz M et al., Nucleic Acids Res 12:3435 (1984); and Renz M, EMBO J6:817 (1983).
Additionally, complements of the marine TLR9 nucleic acids can be useful as antisense oligonucleotides, e.g., by delivering the antisense oligonucleotide to an animal to 3o induce a marine TLR9 "knockout" phenotype. The administration of antisense RNA probes to block gene expression is discussed in Lichtenstein C, Nature 333:801-802 (1988).

Alternatively, the marine TLR9 nucleic acid of the invention can be used to prepare a non-human transgenic animal. A "transgenic animal" is an animal having cells that contain DNA which has been artificially inserted into a cell, which DNA becomes part of the genome of the animal which develops from that cell. Preferred transgenic animals are primates, mice, rats, cows, pigs, horses, goats, sheep, dogs and cats. Animals suitable for transgenic experiments can be obtained from standard commercial sources such as Charles River (Wilmington, MA), Taconic (Germantown, NY), Harlan (Indianapolis, IN), etc.
Transgenic animals having a particular property associated with a particular disease can be used to study the effects of a variety of drugs and treatment methods on the disease, and thus serve as 1o genetic models for the study of a number of human diseases. The invention, therefore, contemplates the use of marine TLR9 knockout and transgenic animals as models for the study of disorders involving TLR9-mediated signaling. A variety of methods known to one of ordinary skill in the art are available for the production of transgenic animals associated with this invention.
Inactivation or replacement of the endogenous TLR9 gene can be achieved by a homologous recombination system using embryonic stem cells. The resultant transgenic non-human mammals having a TLR9--~-- knockout phenotype may be made transgenic for the marine TLR9 and used as a model for screening compounds as modulators (agonists or antagonists/inhibitors) of the marine TLR9. In this manner, such therapeutic drugs can be 2o identified.
Additionally, a normal or mutant version of marine TLR9 can be inserted into the germ line to produce transgenic animals which constitutively or inducibly express the normal or mutant form of marine TLR9. These animals are useful in studies to define the role and function of marine TLR9 in cells.
Generally, doses of active compounds would be from about 0.01 mg/kg per day to 1000 mg/kg per day. It is expected that doses ranging from 50-500 mg/kg will be suitable and in one or several administrations per day. Lower doses will result from other forms of administration, such as intravenous administration. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a 3o different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of -5~-compound, although fewer doses typically will be given when compounds are prepared as slow release or sustained release medications.
The antagonists, agonists, nucleic acids, and polypeptides of marine TLR9 useful according to the invention may be combined, optionally, with a pharmaceutically acceptable carrier. Thus the invention also provides pharmaceutical compositions and a method for preparing the pharmaceutical compositions which contain compositions of this aspect of the invention. The pharmaceutical compositions include any one or combination of the antagonists, agonists, nucleic acids and polypeptides of marine TLR9 useful according to the invention and, optionally, a pharmaceutically acceptable carrier. Each pharmaceutical composition is prepared by selecting an antagonist, agonist, nucleic acid or polypeptide of marine TLR9 useful according to the invention, as well as any combination thereof, and, optionally, combining it with a pharmaceutically acceptable carrier.
The term "pharmaceutically acceptable carrier" as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
The components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
2o The pharmaceutical compositions may contain suitable buffering agents, including, without limitation: acetic acid in~a salt; citric acid in a salt; and phosphoric acid in a salt.
The pharmaceutical compositions also may contain, optionally, suitable preservatives, such as benzalkonium chloride, chlorobutanol, parabens, and thimerosal.
When administered, the pharmaceutical preparations of the invention are applied in pharmaceutically acceptable amounts and in pharmaceutically acceptable compositions. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the-salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, malefic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.
A variety of administration routes are available. The particular mode selected will depend, of course, upon the particular compound selected, the severity of the condition being treated, and the dosage required for therapeutic efficacy. The methods of the invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects. Such modes of administration include oral, l0 rectal, topical, nasal, intradermal, or parenteral routes. The term "parenteral" includes, without limitation, subcutaneous, transdermal, intravenous, infra-arterial, intrathecal, intramuscular, intraperitoneal, mucosal (apart from gastrointestinal mucosa), pulmonary, intralesional, and infusion.
The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy.
All methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the active compound. Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir or an emulsion.
Compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the antagonists, agonists, nucleic acids, or polypeptides of marine TLR9, which is preferably isotonic with the blood of the recipient. This aqueous preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid maybe used in the preparation of injectables. Carrier formulation suitable for oral, subcutaneous, intravenous, intrathecal, intramuscular, etc.
administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.
Other delivery systems can include time-release, delayed release or sustained release delivery systems such as the biological/chemical vectors is discussed above.
Such systems can avoid repeated administrations of the active compound, increasing convenience to the 1o subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. Use of a long-term sustained release implant may be desirable. Long-term release, are used herein, means that the implant is constructed and arranged to delivery therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days. Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.
In another aspect the invention involves the identification of cDNAs encoding mouse TLR7 and mouse TLRB, referred to herein as marine TLR7 and marine TLR8 and, equivalently, mTLR7 and mTLRB, respectively. The nucleotide sequence of the cDNA for marine TLR7 is presented as SEQ ID N0:173, the coding region of the cDNA for marine 2o TLR7 is presented as SEQ ID N0:174, and the amino acid sequence of the marine TLR7 is presented as SEQ ID N0:175. The closely related human TLR7 (equivalently, hTLR7) was previously deposited in GenBank under accession numbers AF245702 and AF240467.
The nucleotide sequence of the cDNA for marine TLR7 presented as SEQ ID N0:173 is nucleotides long and includes the ORF spanning bases 117-3266, presented as SEQ ID
N0:174, which spans 3150 nucleotides (excluding the stop codon). The amino acid sequence of the marine TLR7 presented as SEQ m N0:175 is 1050 amino acids long.
The nucleotide sequence of the cDNA for marine TLR8 is presented as SEQ ID
N0:190, the coding region of the cDNA for marine TLRB is presented as SEQ ID
N0:191, and the amino acid sequence of the marine TLR8 is presented as SEQ m N0:192.
The 3o closely related human TLR8 (equivalently, hTLRB) was previously deposited in GenBank under accession numbers AF245703 and AF246971.

Like both human and marine TLR9, human TLR7 and human TLR8 each contains one CXXC motif and one MBD motif. The hTLR7 CXXC motif contains amino acids 273, and the hTLR8 CXXC motif contains amino acids 255-270.
CXXC motif GNCXXCXXXXXXCXXC SEQ m N0:196 hTLR9: GNCRRCDHAPNPCMEC SEQ ID NO:197 mTLR9: GNCRRCDHAPNPCMIC SEQ ID N0:198 hTLR7: GNCPRCYNAPFPCAPC SEQ ID N0:199 mTLR7: GNCPRCYNVPYPCTPC SEQ ID N0:200 hTLRB: GNCPRCFNAPFPCVPC SEQ ID N0:201 mTLRB: GNCPRCYNAPFPCTPC SEQ ID N0:202 Also like human and marine TLR9, human TLR7 and TLR8 also have a single MBD
motif. The the hTLR7 MBD motif spans amino acids 545-575, and the hTLR8 MBD
motif amino acids spans 533-563.
MBD motif MBD-1 R-XXXXXXX-R-X-D-X-Y-XXXXXXXXX-R-S-XXXXXX-Y SEQ ~ NO:12S
2o hTLR9 Q-XXXXXXX-K-X-D-X-Y-XXXXXXXXX-R-L-XXXXXX-Y SEQ ID N0:126 mTLR9 Q-XXXXXXX-K-X-D-X-Y-XXXXXXXXX-Q-L-XXXXXX-Y SEQ ID N0:127 hTLR7 R-XXXXXXX-R-X-D-X-L-XXXXXXXXX-K-L-XXXXXX-S SEQ ll~ N0:203 mTLR7 R-XXXXXXX-R-X-D-X-L-XXXXXXXXX-S-L-XXXXXX-S SEQ ID N0:204 hTLRB K-XXXXXXX-R-X-D-X-D-XXXXXXXXX-D-L-XXXXXX-Y SEQ ID NO:2OS
mTLR8 K-XXXXXXX-R-X-D-X-D-XXXXXXXXX-D-L-XXXXXX-H SEQ ID N0:206 hTLR7 R-YLDFSNN-R-L-D-L-L-HSTAFEELH-K-L-EVLDIS-S SEQ ID N0:212 mTLR7 R-YLDFSNN-R-L-D-L-L-YSTAFEELQ-S-L-EVLDLS-S SEQ ID N0:213 hTLRB K-YLDLTNN-R-L-D-F-D-NASALTELS-D-L-EVLDLS-Y SEQ m N0:214 mTLRB K-YLDLTNN-R-L-D-F-D-DNNAFSDLH-D-L-EVLDLS-H SEQ ID NO:21S

The core D-X-Y in the MBD motif is involved in CpG binding of the MBD-1 protein and is conserved in TLR9 but only partially conserved in TLR8 and TLR7 (Y to D
or L). The other mismatches are highly or moderately conserved; example R to I~, Q, or D.
These changes could explain MBD-1 as a methyl-CpG binder and TLR9 as a binder for CpG-DNA.
The modification in the core sequence (D-X-Y) in hTLR7 (D-X-L) and TLR8 (D-X-D) is likely a structural basis for the recognition of different nucleic acid motifs. Combined with the presence of a CXXC domain TLR7 and TLR8 appear certainly to be nucleic acid binding receptors relevant to the innate immune system and thus clinical value.
The invention involves in one aspect marine TLR7 and marine TLR8 nucleic acids and polypeptides, as well as therapeutics relating thereto. The invention also embraces isolated functionally equivalent variants, useful analogs and fragments of the foregoing marine TLR7 and marine TLR8 nucleic acids and polypeptides; complements of the foregoing marine TLR7 and marine TLR8 nucleic acids; and molecules which selectively bind the foregoing marine TLR7 and marine TLR8 nucleic acids and polypeptides.
The marine TLR7 and marine TLR8 nucleic acids and polypeptides of the invention are isolated. The term "isolated," with respect to marine TLR7 and marine TLRB
nucleic acids and polypepetides, has the same meaning as used elsewhere herein.
As used herein a marine TLR7 nucleic acid refers to an isolated nucleic acid molecule which codes for a marine TLR7 polypeptide. Such nucleic acid molecules code for marine TLR7 polypeptides which include the sequence of SEQ m N0:175 and fragments thereof.
The nucleic acid molecules include the nucleotide sequences of SEQ m N0:173, SEQ ID
N0:174, and nucleotide sequences which differ from the sequences of SEQ ID
N0:173 and SEQ ID NO:174 in codon sequence due to the degeneracy of the genetic code.
Also as used herein a marine TLRB nucleic acid refers to an isolated nucleic acid molecule which codes for a marine TLR8 polypeptide. Such nucleic acid molecules code for marine TLR8 polypeptides which include the sequences of SEQ m N0:193, and fragments thereof. The nucleic acid molecules include the nucleotide sequences of SEQ m N0:190, SEQ m N0:191, and nucleotide sequences which differ from the sequences of SEQ
m N0:190 and SEQ ID N0:191 in codon sequence due to the degeneracy of the genetic code.
The marine TLR7 and marine TLRS nucleic acids of the invention also include alleles as well as fragments of the foregoing nucleic acids. Such fragments can be used, for example, as probes in hybridization assays and as primers in a polymerase chain reaction.
Preferred marine TLR7 nucleic acids include the nucleic acid sequence of SEQ
ID N0:173 and SEQ ID N0:174. Preferred marine TLR8 nucleic acids include the nucleic acid sequence of SEQ ID N0:190 and SEQ ID N0:191. Complements of the foregoing nucleic acids also are embraced by the invention.
As used herein a marine TLR7 nucleic acid or marine TLR7 polypeptide also embraces homologues and alleles of marine TLR7. Likewise, as used herein a marine TLRB
nucleic acid or marine TLRB polypeptide also embraces homologues and alleles of marine TLRB. Homologues and alleles of marine TLR7 and marine TLR8 comply with the degrees of nucleotide and amino acid identity as previously set forth herein in reference to homologues and alleles of marine TLR9.
Alleles of the marine TLR7 and marine TLRB nucleic acids of the invention can be identified by conventional techniques. For example, alleles of marine TLR7 can be isolated by hybridizing a probe which includes at least a fragment of SEQ ID N0:173 or SEQ ID
NO:174 under stringent conditions with a cDNA library and selecting positive clones. Thus, an aspect of the invention is those nucleic acid sequences which code for marine TLR7 polypeptides and which hybridize to a nucleic acid molecule consisting of SEQ
ID N0:173 or SEQ ID N0:174 under stringent conditions. Likewise, an aspect of the invention is those nucleic acid sequences which code for marine TLR8 polypeptides and which hybridize to a nucleic acid molecule consisting of SEQ ID N0:190 or SEQ ID N0:191 under stringent conditions. Stringent conditions in this context has the same meaning as described elsewhere herein, including the use of a suitable hybridization buffer and a temperature of about 65°C.
In screening for marine TLR7 or marine TLRB nucleic acids, a Southern blot may be performed using the stringent conditions previously described herein, together with a radioactive probe. After washing the membrane to which the DNA is finally transferred, the membrane can be placed against X-ray film to detect the radioactive signal.
Corresponding non-radioactive methods are also well known in the art and can be used to similar effect.
The marine TLR7 and marine TLRB nucleic acids of the invention also include degenerate nucleic acids which include alternative codons to those present in the native materials, as previously described herein.

The invention also provides modified nucleic acid molecules which include additions, substitutions and deletions of one or more nucleotides. The modified nucleic acid molecules according to this aspect of the invention exclude fully native human TLR7 (SEQ
m N0:168, SEQ m N0:169, GenBank Accession No. AF245702, and GenBank Accession No.
AF240467) and fully native human TLR8 nucleic acid molecules (SEQ m N0:182, SEQ m N0:183, GenBank Accession No. AF245703, and GenBank Accession No.AF246971). In preferred embodiments, these modified nucleic acid molecules and/or the polypeptides they encode retain at least one activity or function of the unmodified nucleic acid molecule and/or l0 the polypeptides, such as signaling activity, etc. In certain embodiments, the modified nucleic acid molecules encode modified polypeptides, preferably polypeptides having conservative amino acid substitutions as are described elsewhere herein. The modified nucleic acid molecules are structurally related to the unmodified nucleic acid molecules and in preferred embodiments are sufficiently structurally related to the unmodified nucleic acid molecules so that the modified and unmodified nucleic acid molecules hybridize under stringent conditions known to one of skill in the art.
The invention also provides isolated fragments of nucleotide sequences for marine TLR7 (SEQ m NO:173 and SEQ m N0:174) and for marine TLR8 (SEQ m N0:190 and SEQ m N0:191). The fragments can be used as probes in Southern blot assays to identify 2o such nucleic acids, or can be used in amplification assays such as those employing PCR.
Smaller fragments are those comprising 12, 13, 14, 15, 16, 17, 18, 20, 22, 25, 30, 40, 50, or 75 nucleotides, and every integer therebetween, and are useful, e.g., as primers for nucleic acid amplification procedures. As known to those skilled in the art, larger probes such as 200, 250, 300, 400 or more nucleotides are preferred for certain uses such as Southern blots, while smaller fragments will be preferred for uses such as PCR. Fragments also can be used to produce fusion proteins for generating antibodies or determining binding of the polypeptide fragments. Likewise, fragments can be employed to produce non-fused fragments of the marine TLR7 and marine TLRB polypeptides, useful, for example, in the preparation of antibodies, in immunoassays, and the like. The foregoing nucleic acid fragments further can 3o be used as antisense molecules to inhibit the expression of marine TLR7 and marine TLR8 nucleic acids and polypeptides.

The invention also includes functionally equivalent variants of the marine TLR7 and marine TLRB, which include variant nucleic acids and polypeptides which retain one or more of the functional properties of the marine TLR7 and marine TLRB. Preferably such variants include the marine-specific N-terminal domain.
Functionally equivalent variants also include a marine TLR7 or marine TLRB
which has had a portion (e.g., of the N-terminus) removed or replaced by a similar domain from another TLR (e.g., a "domain-swapping" variant). Examples of such domain-swapping variants include those involving swapping a TLR7 domain from another species and swapping a TLR domain from another TLR.
l0 Other functionally equivalent variants will be known to one of ordinary skill in the art, as will be methods for preparing such variants. The activity of a functionally equivalent variant can be determined using the methods provided herein, and in references that have described assays using other TLRs and TLRs of other species. Such variants are useful, inter alia, for evaluating bioavailability of drugs, in assays for identification of compounds which 15 bind to and/or regulate the signaling function of the marine TLR7 and marine TLRB, and for determining the portions of the marine TLR7 and marine TLR8 which are required for signaling activity.
Variants which are non-functional also can be prepared as described above.
Such variants are useful, for example, as negative controls in experiments testing TLR7 and TLRB
20 signaling activity. Examples of non-functional variants include those incorporating a truncation or mutation of amino acids deemed critical to ligand binding or signaling activity.
In certain embodiments a marine TLR7 or marine TLR8 nucleic acid is operably linked to a gene expression sequence which can direct the expression of the marine TLR7 or marine TLR8 nucleic acid within a eukaryotic or prokaryotic cell. The terms "gene 25 expression sequence" and "operably linked" are as previously described herein.
The marine TLR7 and marine TLR8 nucleic acid molecules and the marine TLR7 and marine TLR8 polypeptides of the invention can be delivered to a eukaryotic or prokaryotic cell alone or in association with a vector. As applied to marine TLR7 and marine TLR8 nucleic acid molecules, a "vector" is any vehicle capable of facilitating: (1) delivery of a 30 marine TLR7 or marine TLRB nucleic acid or polypeptide to a target cell, (2) uptake of a marine TLR7 or marine TLRB nucleic acid or polypeptide by a target cell, or (3) expression of a marine TLR7~or marine TLR8 nucleic acid molecule or polypeptide in a target cell.
In addition to the biological vectors, chemical/physical vectors may be used to deliver a marine TLR7 or marine TLRB nucleic acid or polypeptide to a target cell and facilitate uptake thereby. As used herein with respect to a marine TLR7 or marine TLR8 nucleic acid or polypeptide, a "chemical/physical vector"~ refers to a natural or synthetic molecule, other than those derived from bacteriological or viral sources, capable of delivering the isolated marine TLR7 or marine TLRB nucleic acid or polypeptide to a cell.
Other exemplary compositions that can be used to facilitate uptake by a target cell of the marine TLR7 or marine TLR8 nucleic acids include calcium phosphate and other to chemical mediators of intracellular transport, microinjection compositions, electroporation and homologous recombination compositions (e.g., for integrating a marine TLR7 or marine TLRB nucleic acid into a preselected location within a target cell chromosome).
It will also be recognized that the invention embraces the use of the marine TLR7 and marine TLR8 cDNA sequences in expression vectors to transfect host cells and cell lines, be these prokaryotic (e.g., E. coli), or eukaryotic (e.g., 293 fibroblast cells (ATCC, CRL-1573), MonoMac-6, THP-1, U927, CHO cells, COS cells, yeast expression systems and recombinant baculovirus expression in insect cells). Especially useful are mammalian cells such as human, pig, goat, primate, rodent, guinea pig, etc. They may be of a wide variety of tissue types, and include primary cells and cell lines. The expression vectors require that the 2o pertinent sequence, i.e., those nucleic acids described supra, be operably linked to a promoter.
The invention also provides isolated marine TLR7 and isolated marine TLR8 polypeptides which include the amino acid sequences of SEQ ID N0:175, SEQ ll~
N0:192, and fragments thereof, encoded by the marine TLR7 and marine TLR8 nucleic acids described above. Marine TLR7 and marine TLR8 polypeptides also embrace alleles, functionally equivalent variants and analogs (those non-allelic polypeptides which vary in amino acid sequence from the disclosed marine TLR7 and marine TLR8 polypeptides by l, 2, 3, 4, 5, or more amino acids) provided that such polypeptides retain marine TLR7 or marine TLRB activity. Non-functional variants also are embraced by the invention;
these are useful as antagonists of TLR7 and TLRB signaling function, as negative controls in assays, and the like. Such alleles, variants, analogs and fragments are useful, for example, alone or as fusion proteins for a variety of purposes including as a component of assays.

The invention also embraces variants of the marine TLR7 and marine TLRB
polypeptides described above. Modifications which create a marine TLR7 variant or marine TLR8 variant can be made to a marine TLR7 or marine TLRB polypeptide for a variety of reasons, including 1) to reduce or eliminate an activity of a marine TLR7 or marine TLRB
polypeptide, such as signaling; 2) to enhance a property of a marine TLR7 or marine TLR8 polypeptide, such as signaling, binding affinity for nucleic acid ligand or other liga~nd molecule, protein stability in an expression system, or the stability of protein-protein binding;
3) to provide a novel activity or property to a marine TLR7 or rnurine TLR8 polypeptide, such as addition of an antigenic epitope or addition of a detectable moiety, e.g., luciferase, to FLAG peptide, GFP; 4) to establish that an amino acid substitution does or does not affect molecular signaling activity; or 5) reduce immunogenicity. Modifications to a marine TLR7 or marine TLR8 polypeptide are typically made to the nucleic acid which encodes the marine TLR7 or marine TLRB polypeptide, and can include deletions, point mutations, truncations, amino acid substitutions and additions of amino acids or non-amino acid moieties.
Alternatively, modifications can be made directly to the polypeptide, such as.by cleavage, addition of a linker molecule, addition of a detectable moiety (for example, biotin, fluorophore, radioisotope, enzyme, or peptide), addition of a fatty acid, and the like.
Modifications also embrace fusion proteins comprising all or part of the marine TLR7 or marine TLR8 amino acid sequence.
2o Variants include marine TLR7 and marine TLR8 polypeptides which are modified specifically to alter a feature of each polypeptide unrelated to its physiological activity. For example, cysteine residues can be substituted or deleted to prevent unwanted disulfide linkages. Similarly, certain amino acids can be changed to enhance expression of a marine TLR7 or marine TLR8 polypeptide by eliminating proteolysis by proteases in an expression system (e.g., dibasic amino acid residues in yeast expression systems in which KEX2 protease activity is present).
Mutations of a nucleic acid which encode a marine TLR7 or marine TLRB
polypeptide preferably preserve the amino acid reading frame of the coding sequence, and preferably do not create regions in the nucleic acid which are likely to hybridize to form secondary structures, such as hairpins or loops, which can be deleterious to expression of the variant polypeptide. Methods of making mutations of marine TLR7 or marine TLR8 are as described elsewhere herein with reference to making mutations of marine TLR9.
The activity of variants of marine TLR7 and marine TLRB polypeptides can be tested by cloning the gene encoding the variant marine TLR7 or marine TLR8 polypeptide into a prokaryotic or eukaryotic (e.g., mammalian) expression vector, introducing the vector into an appropriate host cell, expressing the variant marine TLR7 or marine TLR8 polypeptide, and testing for a functional capability of the marine TLR7 or marine TLR8 polypeptides as disclosed herein.
The skilled artisan will also realize that conservative amino acid substitutions may be made in marine TLR7 and marine TLR8 polypeptides to provide functionally equivalent to variants of the foregoing polypeptides, i.e., variants which retain the functional capabilities of the marine TLR7 and marine TLR8 polypeptides.
A variety of methodologies well known to the skilled practitioner can be utilized to obtain isolated marine TLR7 and marine TLR8 polypeptide molecules, as previously described in reference to marine TLR9 polypeptides.
The invention as described herein has a number of uses, some of which are described elsewhere herein. For example, the invention permits isolation of the marine TLR7 and the marine TLR8 polypeptide molecules by, e.g., expression of a recombinant nucleic acid to produce large quantities of polypeptide which may be isolated using standard protocols. As another example, the isolation of the marine TLR7 gene makes it possible for marine TLR7 to be used in methods for assaying molecular interactions involving TLR7.
The invention also embraces agents which bind selectively to the marine TLR7 or marine TLR8 nucleic acid molecules or polypeptides as well as agents which bind to variants and fragments of the polypeptides and nucleic acids as described herein. The agents include polypeptides which bind to marine TLR7 or marine TLRB, and antisense nucleic acids, both of which are described in greater detail below. Some agents can inhibit or increase marine TLR7-mediated signaling activity (antagonists and agonists, respectively), and some can inhibit or increase marine TLRB-mediated signaling activity.
In one embodiment the marine TLR7 inhibitor is an antisense oligonucleotide that selectively binds to a marine TLR7 nucleic acid molecule, to reduce the expression of marine 3o TLR7 (or TLR7 of another species) in a cell. This is desirable in virtually any medical condition wherein a reduction of TLR7 signaling activity is desirable. Based upon SEQ m N0:173 and SEQ m N0:174, or upon allelic or homologous genomic and/or cDNA
sequences, one of skill in the art can easily choose and synthesize any of a number of appropriate antisense molecules for use in accordance with the present invention.
In one embodiment the marine TLR8 inhibitor is an antisense oligonucleotide that selectively binds to a marine TLR8 nucleic acid molecule, to reduce the expression of marine TLR8 (or TLR8 of another species) in a cell. This is desirable in virtually any medical condition wherein a reduction of TLRB signaling activity is desirable. Based upon SEQ m N0:190 and SEQ ID N0:191, or upon allelic or homologous genomic and/or cDNA
sequences, one of skill in the art can easily choose and synthesize any of a number of 1o appropriate antisense molecules for use in accordance with the present invention.
Antisense oligonucleotides for marine TLR7 or marine TLR8 can include "natural"
and "modified" oligonucleotides as previously described herein.
Agents which bind marine TLR7 or marine TLR8 also include binding peptides and other molecules which bind to the marine TLR7 or marine TLR8 polypeptide and complexes containing the marine TLR7 or marine TLR8 polypeptide, respectively. When the binding molecules are inhibitors, the molecules bind to and inhibit the activity of marine TLR7 or marine TLRB. When the binding molecules are activators, the molecules bind to and increase the activity of marine TLR7 or marine TLRB. To determine whether a marine TLR7 or marine TLR8 binding agent binds to marine TLR7 or marine TLRB, any known binding 2o assay may be employed. For example, the binding agent may be immobilized on a surface and then contacted with a labeled marine TLR7 or marine TLR8 polypeptide. The amount of marine TLR7 or marine TLR8 which interacts with the marine TLR7 or marine TLR8 binding agent, or the amount which does not bind to the marine TLR7 or marine binding agent, may then be quantitated to determine whether the marine TLR7 or marine TLR8 binding agent binds to marine TLR7 or marine TLRB.
The marine TLR7 or marine TLRB binding agents include molecules of numerous size and type that bind selectively or preferentially to marine TLR7 or marine polypeptides, and to complexes involving marine TLR7 or marine TLRB
polypeptides and their binding partners. These molecules may be derived from a variety of sources. For 3o example, marine TLR7 or marine TLR8 binding agents can be provided by screening degenerate peptide libraries which can be readily prepared in solution, in immobilized form or as phage display libraries. Combinatorial libraries also can be synthesized of peptides containing one or more amino acids. Libraries further can be synthesized of peptoids and non-peptide synthetic moieties.
Exemplary methods useful for identifying marine TLR7 and marine TLR8 binding peptides are analogous to those described herein with reference to methods for identifying marine TLR9 binding peptides marine, and thus are not repeated here.
Therefore the invention generally provides efficient methods of identifying pharmacological agents or lead compounds for agents useful in the treatment of conditions associated with TLR7 and TLRB activity, and the compounds and agents so identified.
to Generally, the screening methods involve assaying for compounds which inhibit or enhance the expression of or signaling through marine TLR7 or marine TLRB. Such methods are adaptable to automated, high throughput screening of compounds.
A variety of assays for pharmacological agents are provided, including labeled in vitro protein binding assays, signaling assays using detectable molecules, etc. For example, protein binding screens are used to rapidly examine the binding of candidate pharmacological agents to a marine TLR7 or marine TLRB. The candidate pharmacological agents can be derived from, for example, combinatorial peptide or nucleic acid libraries. Convenient reagents for such assays are known in the art. An exemplary cell-based assay of signaling involves contacting a cell having a marine TLR7 or marine TLR8 with a candidate pharmacological 2o agent under conditions whereby the induction of a detectable molecule can occur. A reduced degree of induction of the detectable molecule in the presence of the candidate pharmacological agent indicates that the candidate pharmacological agent reduces the signaling activity of marine TLR7 or marine TLRB. An increased degree of induction of the detectable molecule in the presence of the candidate pharmacological agent indicates that the candidate pharmacological agent increases the signaling activity of marine TLR7 or marine TLRB.
Marine TLR7 and marine TLR8 used in the methods of the invention can be added to an assay mixture as an isolated polypeptide (where binding of a candidate pharmaceutical agent is to be measured) or as a cell or other membrane-encapsulated space which includes a 3o marine TLR7 or marine TLR8 polypeptide. In the latter assay configuration, the cell or other membrane-encapsulated space can contain the marine TLR7 or marine TLR8 as a polypeptide or as a nucleic acid (e.g., a cell transfected with an expression vector containing a nucleic acid molecule encoding marine TLR7). In the assays described herein, the marine TLR7 or marine TLR8 polypeptide can be produced recombinantly, isolated from biological extracts, or synthesized in vitro. Marine TLR7 or marine TLRB polypeptides encompass chimeric proteins comprising a fusion of a marine TLR7 or marine TLR8 polypeptide with another polypeptide, e.g., a polypeptide capable of providing or enhancing protein-protein binding, enhancing signaling capability, facilitating detection, or enhancing stability of the marine TLR7 or marine TLR8 polypeptide under assay conditions. A polypeptide fused to a marine TLR7 or marine TLR8 polypeptide or fragment thereof may also provide means of to readily detecting the fusion protein, e.g., by immunological recognition or by fluorescent labeling.
The assay mixture also comprises a candidate pharmacological agent, as previously described in reference to marine TLR9. Candidate pharmacologic agents are obtained from a wide variety of sources, including libraries of natural, synthetic, or semisynthetic compounds, or any combination thereof. Presently, natural ligands of marine TLR7 and marine TLRB are unknown, but they appear not to include CpG-ODN.
A variety of other reagents also can be included in the assay mixture. These include reagents such as salts, buffers, neutral proteins (e.g., albumin), detergents, etc. which may be used to facilitate optimal protein-protein and/or protein-nucleic acid binding. Such a reagent may also reduce non-specific or background interactions of the reaction components. Other reagents that improve the efficiency of the assay such as protease inhibitors, nuclease inhibitors, antimicrobial agents, and the like may also be used.
The mixture of the foregoing assay materials is incubated under conditions whereby, but for the presence of the candidate pharmacological agent, the marine TLR7 or marine TLR8 mediates TLR7-mediated or TLRB-mediated signaling, preferably TLR/IL-1R
signaling. For determining the binding of a candidate pharmaceutical agent to a marine TLR7 or marine TLRB, the mixture is incubated under conditions which permit binding. The order of addition of components, incubation temperature, time of incubation, and other parameters of the assay may be readily determined. Such experimentation merely involves optimization of the assay parameters, not the fundamental composition of the assay.
Incubation temperatures typically are between 4°C and 40°C. Incubation times preferably are minimized to facilitate rapid, high throughput screening; and typically are between 1 minute and 10 hours.
After incubation, the level of signaling or the level of specific binding between the marine TLR7 or marine TLR8 polypeptide and the candidate pharmaceutical agent is detected by any convenient method available to the user, as described elsewhere herein.
The marine TLR7 or marine. TLR8 binding agent may also be an antibody or a functionally active antibody fragment. Antibodies, including monoclonal antibodies and antibody fragments, are well known to those of ordinary skill in the science of immunology and are as described elsewhere herein. Monoclonal antibodies may be made by any of the to methods known in the art utilizing marine TLR7 or marine TLRB, or a fragment thereof, as an immunogen. Alternatively the antibody may be a polyclonal antibody specific for marine TLR7 or marine TLR8 which inhibits marine TLR7 or marine TLRB activity. The preparation and use of polyclonal antibodies are also known to one of ordinary skill in the art.
The sequences of the antigen-binding Fab' portion of the anti-marine TLR7 or anti-marine TLR8 monoclonal antibodies identified as being useful according to the invention in the assays provided above, as well as the relevant FR and CDR regions, can be determined using amino acid sequencing methods that are routine in the art. Such sequence information can be used to generate humanized and chimeric antibodies, as well as various fusion proteins and binding fragments, as described elsewhere herein.
2o Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides for F(ab')2 and Fab fragments of an anti-marine TLR7 or anti-marine TLRB
monoclonal antibody; chimeric antibodies in which the Fc and/or FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions of an anti-marine TLR7 or anti-marine antibody have been replaced by homologous human or non-human sequences;
chimeric F(ab')2 fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions of an anti-marine TLR7 or anti-marine TLR8 antibody have been replaced by homologous human or non-human sequences; and chimeric Fab fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences.
3o According to the invention marine TLR7 and marine TLRB inhibitors also include "dominant negative" polypeptides derived from SEQ m N0:175 or SEQ )D N0:192, respectively. The end result of the expression of a dominant negative marine TLR7 or dominant negative marine TLR8 polypeptide of the invention in a cell is a reduction in TLR7 or marine TLR8 activity such as signaling through the TIR pathway. One of ordinary skill in the art can assess the potential for a dominant negative variant of a marine TLR7 or dominant negative marine TLR8 polypeptide and, using standard mutagenesis techniques, create one or more dominant negative variant polypeptides.
Each of the compositions according to this aspect of the invention is useful for a variety of therapeutic and non-therapeutic purposes. For example, the marine TLR7 and marine TLR8 nucleic acids of the invention are useful as oligonucleotide probes. Such to oligonucleotide probes can be used herein to identify genomic or cDNA
library clones possessing an identical or substantially similar nucleic acid sequence.
Methods of hybridization, synthesis of probes, and detection are generally as described elsewhere herein.
Additionally, complements of the marine TLR7 and marine TLRB nucleic acids can be useful as antisense oligonucleotides, e.g., by delivering the antisense oligonucleotide to an 15 animal to induce a marine TLR7 or marine TLR8 "knockout" phenotype.
Alternatively, the marine TLR7 and marine TLR8 nucleic acids of the invention can be used to prepare a non-human transgenic animal. The invention, therefore, contemplates the use of marine TLR7 and marine TLR8 knockout and transgenic animals as models for the study of disorders involving TLR7- and marine TLRB-mediated signaling. A
variety of 2o methods known to one of ordinary skill in the art are available for the production of transgenic animals associated with this invention.
Inactivation or replacement of the endogenous TLR7 or TLR8 gene can be achieved by a homologous recombination system using embryonic stem cells. The resultant transgenic non-human mammals having a TLR7-~- or TLRB-~- knockout phenotype may be made 25 transgenic for the marine TLR7 or marine TLR8 and used as a model for screening compounds as modulators (agonists or antagonists/inhibitors) of the marine TLR7 or marine TLRB. In this manner, such therapeutic drugs can be identified.
Additionally, a normal or mutant version of marine TLR7 or marine TLR8 can be inserted into the germ line to produce transgenic animals which constitutively or inducibly 3o express the normal or mutant form of marine TLR7 or marine TLRB. These animals are useful in studies to define the role and function of marine TLR7 or marine TLR8 in cells.

The antagonists, agonists, nucleic acids, and polypeptides of marine TLR7 and marine TLR8 useful according to the invention may be combined, optionally, with a pharmaceutically acceptable carrier. Thus the invention also provides pharmaceutical compositions and a method for preparing the pharmaceutical compositions which contain compositions of this aspect of the invention. The pharmaceutical compositions include one or any combination of the antagonists, agonists, nucleic acids and polypeptides of marine TLR7 and marine TLR8 useful according to the invention and, optionally, a pharmaceutically acceptable carrier. Each pharmaceutical composition is prepared by selecting an antagonist, agonist, nucleic acid or polypeptide of marine TLR7 and marine TLR8 useful according to l0 the invention, as well as any combination thereof, and, optionally, combining it with a.
pharmaceutically acceptable carrier.
A variety of administration routes are available, as described previously herein. The particular mode selected will depend, of course, upon the particular compound selected, the severity of the condition being treated, and the dosage required for therapeutic efficacy.
Likewise, a variety of formulations are contemplated, including, by analogy those discussed above in reference to marine TLR9, unit dose solids, liquids, extended release formulations, etc.
Screening Assays 2o In another aspect the invention provides methods for screening candidate compounds that act as ISNA mimics, agonists or antagonists in ISNA-induced immunomodulation via TLR7, TLR8, and TLR9. Preferably the screening method can be adapted to accommodate high throughput screening assays, as can be achieved, for example, through the use of multiwell arrays of samples in conjunction with robotic or automated array handling devices.
Immunostimulatory nucleic acids include but are not limited to CpG nucleic acids.
A "CpG nucleic acid" or a "CpG immunostimulatory nucleic acid" as used herein is a nucleic acid containing at least one unmethylated CpG dinucleotide (cytosine-guanine dinucleotide sequence, i.e. "CpG DNA" or DNA containing a 5' cytosine followed by 3' guanine and linked by a phosphate bond) and activates a component of the immune system.
3o The entire CpG nucleic acid can be unmethylated or portions may be unmethylated but at least the C of the 5' CG 3' must be unmethylated.

In one embodiment a CpG nucleic acid is represented by at least the formula:
5'-N1X1CGX2N2-3' wherein Xl and Xz are nucleotides, N is any nucleotide, and Nl and N2 are nucleic acid sequences composed of from about 0-25 N's each. In some embodiments Xl is adenine, guanine, or thymine and/or X2 is cytosine, adenine, or thymine. In other embodiments Xl is cytosine and/or X2 is guanine.
In other embodiments the CpG nucleic acid is represented by at least the formula:
5'-N1X1X2CGX3X4N2-3.
wherein Xl, XZ, X3, and X4 are nucleotides; N is any nucleotide; and Nl and NZ
are nucleic 1o acid sequences composed of from about 0-25 N's each. In some embodiments, X1X2 are nucleotides selected from the group consisting of GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT, and TpG; and X3X4 are nucleotides selected from the group consisting of TpT, CpT, ApT, TpG, ApG, CpG, TpC, ApC, CpC, TpA, ApA, and CpA. In some embodiments, X1X2 are GpA or GpT and X3X4 are TpT. In other embodiments Xl or X2 ox 15 both are purines and X3 or X4 or both are pyrimidines or X1X2 are GpA and X3 or X4 or both are pyrimidines.
In another embodiment the CpG nucleic acid is represented by at least the formula:
5'-TCN~TX1XZCGX3X4-3' wherein Xl, X2, X3, and X4 are nucleotides; N is any nucleotide; and Nl and N2 are nucleic 2o acid sequences composed of from about 0-25 N's each. In some embodiments, XIXz are nucleotides selected from the group consisting of GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT, and TpG; and X3X4 are nucleotides selected from the group consisting of TpT, CpT, ApT, TpG, ApG, CpG, TpC, ApC, CpC, TpA, ApA, and CpA. In some embodiments, X1X2 are GpA or GpT and X3X4 are TpT. In other embodiments Xl or X~ or 25 both are purines and X3 or X4 or both are pyrimidines or X1X2 are GpA and X3 or X4 or both are pyrimidines.
Examples of CpG nucleic acids according to the invention include but are not limited to those listed in Table 1, such as SEQ m NOs:21-29, 31-42, 44, 46-50, 52-62, 64-75, 77-88, 90-117, 119-124.

Table 1. Exemplary CpG nucleic acids AA_CGTTCT SEQ IDN0:21 CGAAAATGAAATTGACT SEQ IDN0:22 AAG

_ SEQ IDN0:23 ACCATGGA_CGAACTGTTTCCCCTC

CGACCTGTTTCCCCTC SEQ IDN0:24 ACCATGGA

_ SEQ IDN0:25 ACCATGGA_CGAGCTGTTTCCCCTC

ACCATGGA_CGATCTGTTTCCCCTC SEQ IDN0:26 ACCATGGA_CGGTCTGTTTCCCCTC SEQ IDN0:27 CGTACTGTTTCCCCTC SEQ IDN0:28 ACCATGGA

_ SEQ IDN0:29 ACCATGGA_CGTTCTGTTTCCCCTC

AGATTTCTAGGAATTCAATC SEQ IDN0:30 CGAG_CGGGGG SEQ IDN0:31 CG
AG
CGGGGG

_ SEQ IDN0:32 _ _ AGCTATGA_CGTTCCAAGG

AT_CGACTCT_CGAG_CGTTCTC SEQ IDN0:33 ATGA SEQ IDN0:34 CGTTCCTGA
CGTT

_ SEQ IDN0:35 _ ATGGAAGGTCCAA
CGTTCTC

_ SEQ IDN0:36 ATGGAAGGTCCAG
CGTTCTC

_ SEQ IDN0:37 ATGGACTCTCCAG
CGTTCTC

_ SEQ IDN0:38 ATGGAGGCTCCAT
CGTTCTC

_ SEQ IDN0:39 CAA_CGTT

CA SEQ IDN0:40 CGTTGAGGGGCAT

_ SEQ IDN0:41 CAGGCATAA
CGGTTC
CGTAG

_ SEQ IDN0:42 _ CCAA_CGTT

CTCCTAGTGGGGGTGTCCTAT SEQ IDN0:43 CGAAATGATG SEQ IDN0:44 CTGATTTCCC

_ SEQ IDN0:45 CTGCTGAGACTGGAG

CGATGGACCTTCCAT SEQ IDN0:46 GAGAA

_ SEQ IDN0:47 GAGAA_CGCTCCAGCACTGAT

GAGAA SEQ IDN0:48 CGCT
CGACCTTCCAT

_ SEQ IDN0:49 _ GAGAA_CGCT_CGACCTT_CGAT

GAGAA SEQ IDN0:50 CGCTGGACCTTCCAT

_ SEQ IDN0:51 GAGCAAGCTGGACCTTCCAT

GATTGCCTGA_CGTCAGAGAG SEQ IDN0:52 GCATGA SEQ IDN0:53 CGTTGAGCT

_ SEQ IDN0:54 G_CGG_CGGG_CGGCGCGCGCCC

G_CGTG_CGTTGT_CGTTGT_CGTT SEQ IDN0:55 GCTAGA_CGTTAG_CGT SEQ IDN0:56 GCTAGA_CGTTAGTGT SEQ IDN0:57 GCTAGATGTTAG SEQ IDN0:58 CGT

_ SEQ IDN0:59 GCTTGATGACTCAGC_CGGAA

GGAATGA_CGTTCCCTGTG SEQ IDN0:60 GGGGTCAA_CGTTGA_CGGGG SEQ IDN0:61 GGGGTCAGTCTTGA_CGGGG SEQ IDN0:62 GTATTTCCCAGAAAAGGAAC SEQ IDN0:63 GTCCATTTCC SEQ IDN0:64 CGTAAATCTT

_ SEQ IDN0:65 GT
CGCT

_ SEQ IDN0:66 GT_CGTT

TACCGCGTG_CGACCCTCT SEQ IDN0:67 TATGCATATTCCTGTAAGTG SEQ IDN0:68 TCAA_CGTC SEQ IDN0:69 TCAA_CGTT SEQ IDN0:70 TCAAGCTT SEQ IDN0:71 TCAG SEQ IDN0:72 CGCT

_ SEQ IDN0:73 TCAG
CGTG
CGCC

_ SEQ IDN0:74 _ TCAT_CGAT

CGA_CGTT SEQ IDN0:75 TCCA
CGTTTT
CGA

_ ~ SEQ IDN0:76 _ _ TCCAGGACTTCTCTCAGGTT

CGTTCCTGATGCT SEQ IDN0:77 TCCATAA

_ SEQ IDN0:78 CGTT , TCCATAG
CGTTCCTAG

_ SEQ IDN0:79 _ TCCATCA_CGTGCCTGATGCT

TCCATGA_CGGTCCTGATGCT SEQ IDN0:80 TCCATGACGTCCCTGATGCT SEQ IDN0:81 _77_ TCCATGA_CGTGCCTGATGCT SEQ IDN0:82 TCCATGA_CGTTCCTGA_CGTT SEQ IDN0:83 TCCATGA_CGTTCCTGATGCT SEQ IDN0:84 TCCATGAGCTTCCTGATGCT SEQ IDN0:85 TCCATGC_CGGTCCTGATGCT SEQ IDN0:86 TCCATG_CGTG_CGTG_CGTTTT SEQ IDN0:87 TCCATG_CGTTG_CGTTG_CGTT SEQ IDN0:88 TCCATGCTGGTCCTGATGCT SEQ IDN0:89 TCCATGG SEQ IDN0:90 CGGTCCTGATGCT

_ SEQ IDN0:91 TCCATGT_CGATCCTGATGCT

TCCATGT_CGCTCCTGATGCT SEQ IDN0:92 TCCATGT_CGGTCCTGATGCT SEQ IDN0:93 TCCATGT_CGGTCCTGCTGAT SEQ IDN0:94 TCCATGT SEQ IDN0:95 CGTCCCTGATGCT

_ SEQ IDN0:96 TCCATGT_CGTTCCTGATGCT

TCCATGT_CGTTCCTGT_CGTT SEQ IDN0:97 TCCATGT_CGTTTTTGT_CGTT SEQ IDN0:98 TCCTGA_CGTTCCTGA_CGTT SEQ IDN0:99 TCCTGT SEQ IDN0:100 CGTTCCTGT
CGTT

_ SEQ IDNO:101 _ TCCTGT_CGTTCCTTGT
CGTT

_ SEQ IDN0:102 TCCTGT_CGTTTTTTGT_CGTT

TCCTTGT_CGTTCCTGT_CGTT SEQ IDN0:103 T_CGAT_CGGGG_CGGGG_CGAGC SEQ IDN0:104 T SEQ IDN0:105 CGT
CGCTGTCTC
CGCTTCTT

_ SEQ IDN0:106 _ _ T_CGT_CGCTGTCTC_CGCTTCTTCTTGCC

T_CGT_CGCTGTCTGCCCTTCTT SEQ IDN0:107 T_CGT_CGCTGTTGT_CGTTTCTT SEQ IDN0:108 T_CGT_CGT_CGT SEQ IDN0:109 CGTT

_ SEQ IDN0:110 T
CGT
CGTTGT
CGTTGT
CGTT

_ SEQ IDN0:111 _ _ _ T_CGT_CGTTGT_CGTTTTGT_CGTT

T_CGT_CGTTTTGT_CGTTTTGT_CGTTSEQ IDN0:112 TCTCCCAGCGCGCGCCAT SEQ IDN0:113 TCTCCCAG_CGGG_CGCAT SEQ IDN0:114 TCTCCCAG SEQ IDN0:115 CGTG
CGCCAT

_ SEQ IDN0:116 _ TCTT_CGAA

TGCAGATTG_CGCAATCTGCA SEQ IDN0:117 TGCTGCTTTTGTGCTTTTGTGCTT SEQ IDN0:118 TGT_CGCT SEQ IDN0:119 TGT SEQ IDN0:120 CGTT

_ SEQ IDN0:121 TGT_CGTTGT_CGTT ' TGT_CGTTGT_CGTTGT_CGTT SEQ IDN0:122 TGT_CGTTGT_CGTTGT_CGTTGT_CGTTSEQ IDN0:123 TGTCGTTTGTCGTTTGTCGTT SEQ IDN0:124 Other ISNAs include but are not limited to T-rich nucleic acids, poly G
nucleic acids, and nucleic acids having phosphate modified backbones, such as phosphorothioate backbones.
A "T rich nucleic acid" or "T rich immunostimulatory nucleic acid" is a nucleic acid which includes at least one poly T sequence and/or which has a nucleotide composition of 5o greater than 25% T nucleotide residues and which activates a component of the immune system. A nucleic acid having a poly-T sequence includes at least four Ts in a row, such as 5'TTTT3'. Preferably the T rich nucleic acid includes more than one poly T
sequence. In preferred embodiments the T rich nucleic acid may have 2, 3, 4, etc poly T
sequences. One of _78_ the most highly immunostimulatory T rich oligonucleotides discovered according to the invention is a nucleic acid composed entirely of T nucleotide residues. Other T rich nucleic acids have a nucleotide composition of greater than 25% T nucleotide residues, but do not necessarily include a poly T sequence. In these T rich nucleic acids the T
nucleotide resides may be separated from one another by other types of nucleotide residues, i.e., G, C, and A. In some embodiments the T rich nucleic acids have a nucleotide composition of greater than 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 99%, T nucleotide residues and every integer in between. Preferably the T rich nucleic acids have at least one poly T
sequence and a nucleotide composition of greater than 25% T nucleotide residues.
to In one embodiment the T rich nucleic acid is represented by at least the formula:
5' X1XZTTTTX3X4 3' wherein Xl, X2, X3, and X4 are nucleotides. In one embodiment X1X2 is TT
and/or X3X4 is TT. In another embodiment X1X2 are any one of the following nucleotides TA, TG, TC, AT, AA, AG, AC, CT, CC, CA, CG, GT, GG, GA, and GC; and X3X4 are any one of the following nucleotides TA, TG, TC, AT, AA, AG, AC, CT, CC, CA, CG, GT, GG, GA, and GC. .
In some embodiments it is preferred that the T-rich nucleic acid does not contain poly C (CCCC), poly A (AAAA), poly G (GGGG), CpG motifs, or multiple GGs. In other embodiments the T-rich nucleic acid includes these motifs. Thus in some embodiments of 2o the invention the T rich nucleic acids include CpG dinucleotides and in other embodiments the T rich nucleic acids are free of CpG dinucleotides. The CpG dinucleotides may be methylated or unmethylated.
Poly G containing nucleic acids are also immunostimulatory. A variety of references, including Pisetsky and Reich, 1993 Mol. Biol. Reports, 18:217-221; Krieger and Herz, 1994, Anna. Rev. Biochem., 63:601-637; Macaya et al., 1993, PNAS, 90:3745-3749;
Wyatt et al., 1994, PNAS, 91:1356-1360; Rando and Hogan, 1998, In Applied Antisense Oligonucleotide Technology, ed. Krieg and Stein, p. 335-352; and Kimura et al., 1994, J.
BioclZem. 116, 991-994 also describe the immunostimulatory properties of poly G nucleic acids.
Poly G nucleic acids preferably are nucleic acids having the following formulas:
5' X1XZGGGX3X4 3' .

wherein Xl, X2, X3, and X4 are nucleotides. In preferred embodiments at least one of X3 and X4 are a G. In other embodiments both of X3 and X4 are a G. In yet other embodiments the preferred formula is 5' GGGNGGG 3', or 5' GGGNGGGNGGG 3' wherein N represents between 0 and 20 nucleotides. In other embodiments the Poly G nucleic acid is free of unmethylated CG dinucleotides. In other embodiments the poly G nucleic acid includes at least one unmethylated CG dinucleotide.
Nucleic acids having modified backbones, such as phosphorothioate backbones, also fall within the class of immunostimulatory nucleic acids. U.S. Patents Nos.
5,723,335 and 5,663,153 issued to Hutcherson, et al. and related PCT publication W095/26204 describe to immune stimulation using phosphorothioate oligonucleotide analogues. These patents describe the ability of the phosphorothioate backbone to stimulate an immune response in a non-sequence specific manner.
The ISNAs may be double-stranded or single-stranded. Generally, double-stranded molecules may be more stable in vivo, while single-stranded molecules may have increased activity. The terms "nucleic acid" and "oligonucleotide" refer to multiple nucleotides (i.e.
molecules comprising a sugar (e.g. ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g.
cytosine (C), thyrnine (T) or uracil (I~) or a substituted purine (e.g., adenine (A) or guanine (G)) or a modified base. As used herein, the terms refer to oligoribonucleotides as well as oligodeoxyribonucleotides. The terms shall also include polynucleosides (i.e., a polynucleotide minus the phosphate) and any other organic base-containing polymer. The terms "nucleic acid" and "oligonucleotide" also encompass nucleic acids or oligonucleotides with a covalently modified base and/or sugar. For example, they include nucleic acids having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position.
Thus modified nucleic acids may include a 2'-O-alkylated ribose group. In addition, modified nucleic acids may include sugars such as arabinose instead of ribose. Thus the nucleic acids may be heterogeneous in backbone composition thereby containing any possible combination of polymer units linked together such as peptide- nucleic acids (which have amino acid 3o backbone with nucleic acid bases). In some embodiments the nucleic acids are homogeneous in backbone composition.

The substituted purines and pyrimidines of the ISNAs include standard purines and pyrimidines such as cytosine as well as base analogs such as C-5 propyne substituted bases.
Wagner RW et al., Nat Biotechnol 14:840-844 (1996). Purines and pyrimidines include but are not limited to adenine, cytosine, guanine, thymine, 5-methylcytosine, 2-aminopurine, 2 amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, and other naturally and non naturally occurring nucleobases, substituted and unsubstituted aromatic moieties.
The ISNA is a linked polymer of bases or nucleotides. As used herein with respect to linked units of a nucleic acid, "linked" or "linkage" means two entities are bound to one another by any physicochemical means. Any linkage known to those of ordinary skill in the l0 art, covalent or non-covalent, is embraced. Such linkages are well known to those of ordinary skill in the art. Natural linkages, which are those ordinarily found in nature connecting the individual units of a nucleic acid, are most common. The individual units of a nucleic acid may be linked, however, by synthetic or modified linkages.
Whenever a nucleic acid is represented by a sequence of letters it will be understood that the nucleotides are in 5' to 3' order from left to right and that "A"
denotes adenine, "C"
denotes cytosine, "G" denotes guanine, "T" denotes thymidine, and "U" denotes uracil unless otherwise noted.
Immunostimulatory nucleic acid molecules useful according to the invention can be obtained from natural nucleic acid sources (e.g., genomic nuclear or mitochondrial DNA or cDNA), or are synthetic (e.g., produced by oligonucleotide synthesis). Nucleic acids isolated from existing nucleic acid sources are referred to herein as native, natural, or isolated nucleic acids. The nucleic acids useful according to the invention may be isolated from any source, including eukaryotic sources, prokaryotic sources, nuclear DNA, mitochondria) DNA, etc.
Thus, the term nucleic acid encompasses both synthetic and isolated nucleic acids.
The term "isolated" as used herein with reference to an ISNA means substantially free of or separated from components which it is normally associated with in nature, e.g., nucleic acids, proteins, lipids, carbohydrates or in vivo systems to an extent practical and appropriate for its intended use. In particular, the nucleic acids are sufficiently pure and are sufficiently free from other biological constituents of host cells so as to be useful in, for example, 3o producing pharmaceutical preparations. Because an isolated nucleic acid of the invention may be admixed with a pharmaceutically-acceptable carrier in a pharmaceutical preparation, the nucleic acid may comprise only a small percentage by weight of the preparation. The nucleic acid is nonetheless substantially pure in that it has been substantially separated from the substances with which it may be associated in living systems.
The ISNAs can be produced on a large scale in plasmids, (see Molecular Cloning: A
Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989) and separated into smaller pieces or administered whole. After being administered to a subj ect the plasmid can be degraded into oligonucleotides. One skilled in the art can purify viral, bacterial, eukaryotic, etc. nucleic acids using standard techniques, such as those employing restriction enzymes, exonucleases to or endonucleases.
For use in the instant invention, the ISNAs can be synthesized de novo using any of a number of procedures well known in the art. For example, the (3-cyanoethyl phosphoramidite method (Beaucage SL and Caruthers MH, Tetrahedron Let 22:1859 (1981));
nucleoside H-phosphonate method (Garegg et al., Tetrahedron Let 27:4051-4054 (1986);
Froehler et al., 15 Nucl Acid Res 14:5399-5407 (1986); Garegg et al., Tetrahedron Let 27:4055-4058 (1986);
Gaffney et al., Tetrahedron Let 29:2619-2622 (1988)). These chemistries can be performed by a variety of automated oligonucleotide synthesizers available in the market.
ISNAs having modified backbones, such as phosphorothioate backbones, also fall within the class of immunostimulatory nucleic acids. U.S. Patents Nos.
5,723,335 and 20 5,663,153 issued to Hutcherson, et al. and related PCT publication W095/26204 describe immune stimulation using phosphorothioate oligonucleotide analogues. These patents describe the ability of the phosphorothioate backbone to stimulate an immune response in a non-sequence specific manner.
The ISNA may be any size of at least 6 nucleotides but in some embodiments are in 25 the range of between 6 and 100 or in some embodiments between 8 and 35 nucleotides in size. Immunostimulatory nucleic acids can be produced on a large scale in plasmids. These may be administered in plasmid form or alternatively they can be degraded into oligonucleotides before administration.
"Palindromic sequence" shall mean an inverted repeat (i.e., a sequence such as 3o ABCDEE'D'C'B'A' in which A and A', B and B', etc., are bases capable of forming the usual Watson-Crick base pairs and which includes at least 6 nucleotides in the palindrome. In vivo, such sequences may form double-stranded structures. In one embodiment the nucleic acid contains a palindromic sequence. In some embodiments when the nucleic acid is a CpG
nucleic acid, a palindromic sequence used in this context refers to a palindrome in which the CpG is part of the palindrome, and optionally is the center of the palindrome.
In another embodiment the nucleic acid is free of a palindrome. A nucleic acid that is free of a palindrome does not have any regions of 6 nucleotides or greater in length which are palindromic. A nucleic acid that is free of a palindrome can include a region of less than 6 nucleotides which are palindromic.
A "stabilized ISNA" shall mean a nucleic acid molecule that is relatively resistant to to in vivo degradation (e.g. via an exo- or endo-nuclease). Stabilization can be a function of length or secondary structure. Nucleic acids that are tens to hundreds of kbs long are relatively resistant to in vivo degradation. For shorter nucleic acids, secondary structure can stabilize and increase their effect. For example, if the 3' end of an oligonucleotide has self complementarity to an upstream region, so that it can fold back and form a sort of stem loop structure, then the oligonucleotide becomes stabilized and therefore exhibits more activity.
Some stabilized ISNAs of the instant invention have a modified backbone. It has been demonstrated that modification of the oligonucleotide backbone provides enhanced activity of the ISNAs when administered in vivo. Nucleic acids, including at least two phosphorothioate linkages at the 5' end of the oligonucleotide and multiple phosphorothioate linkages at the 3' 2o end, preferably 5, may provide maximal activity and protect the oligonucleotide from degradation by intracellular exo- and endo-nucleases. Other modified oligonucleotides include phosphodiester modified oligonucleotide, combinations of phosphodiester and phosphorothioate oligonucleotide, methylphosphonate, methylphosphorothioate, phosphorodithioate, and combinations thereof. Each of these combinations and their particular effects on immune cells is discussed in more detail in U.S. Patent Nos. 6,194,388 and 6,207,646, the entire contents of which is hereby incorporated by reference. It is believed that these modified oligonuclebtides may show more stimulatory activity due to enhanced nuclease resistance, increased cellular uptake, increased protein binding, and/or altered intracellular localization. Both phosphorothioate and phosphodiester nucleic acids axe active 3o in immune cells.
Other stabilized ISNAs include: nonionic DNA analogs, such as alkyl- and aryl--~3-phosphates (in which the charged phosphonate oxygen is replaced by an alkyl or aryl group), phosphodiester and alkylphosphotriesters, in which the charged oxygen moiety is alkylated.
Oligonucleotides which contain diol, such as tetraethyleneglycol or hexaethyleneglycol, at either or both termini have also been shown to be substantially resistant to nuclease degradation.
For use ira vivo, ISNAs are preferably relatively resistant to degradation (e.g., via endo- and exo-nucleases). Secondary structures, such as stem loops; can stabilize nucleic acids against degradation. Alternatively, nucleic acid stabilization can be accomplished via phosphate backbone modifications. One type of stabilized nucleic acid has at least a partial to phosphorothioate modified backbone. Phosphorothioates may be synthesized using automated techniques employing either phosphoramidate or H-phosphonate chemistries.
Aryl- and alkyl-phosphonates can be made, e.g., as described in U.S. Patent No. 4,469,63;
and alkylphosphotriesters (in which the charged oxygen moiety is alkylated as described in U.S. Patent No. 5,023,243 and European Patent No. 092,574) can be prepared by automated solid phase synthesis using commercially available reagents. Methods for making other DNA
backbone modifications and substitutions have been described. Uhlmann E and Peyman A, Chefra Rev 90:544 (1990); Goodchild J, Biocohjugate Chem 1:165 (1990).
Other sources of immunostimulatory nucleic acids useful according to the invention include standard viral and bacterial vectors, many of which are commercially available. In its broadest sense, a "vector" is any nucleic acid material which is ordinarily used to deliver and facilitate the transfer of nucleic acids to cells. The vector as used herein may be an empty vector or a vector carrying a gene which can be expressed. In the case when the vector is carrying a gene the vector generally transports the gene to the target cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
In this case the vector optionally includes gene expression sequences to enhance expression of the gene in target cells such as immune cells, but it is not required that the gene be expressed in the cell.
A basis for certain of the screening assays is the presence of a functional TLR 7, TLR
~, or TLR9 in a cell. The functional TLR in some instances is naturally expressed by the cell. .
In other instances, expression of the functional TLR can involve introduction or reconstitution of a species-specific TLR9 into a cell or cell line that otherwise lacks the TLR

or lacks responsiveness to ISNA, resulting in a cell or cell line capable of activating the TLR/IL-1R signaling pathway in response to contact with an ISNA. Examples of cell lines lacking TLR9 or ISNA responsiveness include, but are not limited to, 293 fibroblasts (ATCC
CRL-1573), MonoMac-6, THP-1, U937, CHO, and any TLR9 knock-out. The introduction of the species-specific TLR into the cell or cell line is preferably accomplished by transient or stable transfection of the cell or cell line with a TLR-encoding nucleic acid sequence operatively linked to a gene expression sequence (as described above).
The species-specific TLR, including TLR7, TLRB, and TLR9, is not limited to a marine TLR, but rather can include a TLR derived from marine or non-marine sources.
Examples of non-marine sources include, but are not limited to, human, bovine, canine, feline, ovine, porcine, and equine. Other species include chicken and fish, e.g., aquaculture species.
The species-specific TLR, including TLR7, TLRB, and TLR9, also is not limited to native TLR polypeptides. In certain embodiments the TLR can be, e.g., a chimeric TLR in which the extracellular domain and the cytoplasmic domains are derived from TLR
polypeptides from different species. Such chimeric TLR polypeptides, as described above, can include, for example, a human TLR extracellular domain and a marine TLR
cytoplasmic domain, each domain derived from the corresponding TLR7, TLRB, or TLR9 of each species.
In alternative embodiments, such chimeric TLR polypeptides can include chimeras created°
with different TLR splice variants or allotypes. Other chimeric TLR
polypeptides useful for the purposes of screening ISNA mimics, agonists and antagonists can include chimeric polypeptides created with a TLR of a first type, e.g., TLR9, and another TLR, e.g., TLR7 or TLRB, of the same or another species as the TLR of the first type. Also contemplated are chimeric polypeptides which incorporate sequences derived from more than two polypeptides, e.g., an extracellular domain, a transmembrane domain, and a cytoplasmic domain all derived from different polypeptide sources, provided at least one such domain derives from a TLR7, TLRB, or TLR9 polypeptide~ As a further example, also contemplated are constructs such as include an extracellular domain of one TLR9, an intracellular domain of another TLR9, and a non-TLR reporter such as luciferase, GFP, etc. Those of skill in the art will recognize how to 3o design and generate DNA sequences coding for such chimeric TLR
polypeptides.
The screening assays can have any of a number of possible readout systems based upon either TLR/IL-1R signaling pathway or other assays useful for assaying response to ISNAs. It has been reported that immune cell activation by CpG
immunostimulatory sequences is dependent in some way on endosomal processing. It is not yet known whether TLR9 is directly involved in this endosomal pathway, or if there is some intermediary between TLR9 and the endosome.
In preferred embodiments, the readout for the screening assay is based on the use of native genes or, alternatively, cotransfected or otherwise co-introduced reporter genie constructs which are responsive to the TLR/IL-1R signal transduction pathway involving MyD88, TRAF6, p38, and/or ERIC. Hacker H et al., EMBO J 18:6973-6982 (1999).
These to pathways activate kinases including xB kinase complex and c-Jun N-terminal kinases. Thus reporter genes and reporter gene constructs particularly useful for the assays can include a reporter gene operatively linked to a promoter sensitive to NF-xB. Examples of such promoters include, without limitation, those for NF-~cB, IL-1 [3, IL-6, IL-8, IL-12 p40, CD80, CD86, and TNF-a. The reporter gene operatively linked to the TLR7-, TLRB-, or sensitive promoter can include, without limitation, an enzyme (e.g., luciferase, alkaline phosphatase, (3-galactosidase, chloramphenicol acetyltransferase (CAT), etc.), a bioluminescence marker (e.g., green-fluorescent protein (GFP, U.S. patent 5,491,084), etc.), a surface-expressed molecule (e.g., CD25), and a secreted molecule (e.g., IL-8, IL-12 p40, TNF-a). In preferred embodiments the reporter is selected from IL-8, TNF-a, NF-~cB-luciferase (NF-xB-luc; Hacker H et al., EMBO J 18:6973-6982 (1999)), IL-12 p40-luc (Murphy TL et al., Mol Cell Biol 15:5258-5267 (1995)), and TNF-luc (Hacker H
et al., EMBO J 18:6973-6982 (1999)). In assays relying on enzyme activity readout, substrate can be supplied as part of the assay, and detection can involve measurement of chemiluminescence, fluorescence, color development, incorporation of radioactive label, drug resistance, or other marker of enzyme activity. For assays relying on surface expression of a molecule, detection can be accomplished using FACS analysis or functional assays. Secreted molecules can be assayed using enzyme-linked immunosorbent assay (ELISA) or bioassays.
Many such readout systems are well known in the art and are commercially available.
In another aspect the invention provides a screening method for identifying an 3o immunostimulatory nucleic acid molecule (ISNA). The method entails contacting a functional TLR selected from the group consisting of TLR7, TLRB, and TLR9 with a test nucleic acid molecule; detecting the presence or absence of a response mediated by a TLR
signal transduction pathway in the presence of the test nucleic acid molecule arising as a result of an interaction between the functional TLR and the test nucleic acid molecule; and determining the test nucleic acid molecule is an ISNA when the presence of a response mediated by the TLR signal transduction pathway is detected. "Functional TLR"
and a "cell expressing functional TLR" are as described elsewhere herein. A response mediated by a TLR signal transduction pathway includes induction of a gene under control of a promoter responsive to the TLR/IL-1R signaling pathway, including but not limited to promoters responsive to NF-oB. The biological response thus can include, e.g., secretion of IL-.8 and to luciferase activity in a cell transfected with NF-xB-luc, IL-12 p40-luc, or TNF-luc. A test nucleic acid molecule can include a DNA, RNA, or modified nucleic acid molecule as described herein. In some embodiments the test nucleic acid molecule is a CpG
nucleic acid.
Preferably, the test nucleic acid molecule is a sequence variant of a reference ISNA, containing at least one alternative base, at least one alternative internucleotide backbone linkage, or at least one alternative sugar moiety as compared to the particular reference ISNA.
In a preferred embodiment the test nucleic acid molecule is a member of a library of such test nucleic acid molecules.
According to one embodiment of this method, comparison can be made to a reference ISNA. The reference ISNA may be any ISNA, including a CpG nucleic acid. In preferred 2o embodiments the screening method is performed using a plurality of test nucleic acids.
Preferably comparison of test and reference responses is based on comparison of quantitative measurements of responses in each instance.
The method can be used to select a subset of test nucleic acid molecules based on their ability to induce a similar specific response mediated by the TLR signal transduction pathway. For instance, the method can be used to classify test CpG nucleic acids as predominantly B-cell activating CpG nucleic acids, or as predominantly IFN-a inducing CpG
nucleic acids. Other new classes of ISNAs may be identified and characterized using the method.
Application of this method permits the identification of ISNAs, delineation of 3o sequence specificity of a given TLR, and also optimization of ISNA
sequences. Identification of ISNAs involves screening candidate ISNAs as above and selecting any ISNA
that induces _87_ a response as defined. Delineation of sequence specificity involves screening candidate ISNAs as above with reference to a particular TLR9, selecting any ISNAs that induce a response as defined, and categorizing ISNAs that do and do not induce a response on the basis of their sequence. Optimization of ISNA sequences involves an iterative application of the method as described, further including the steps of selecting the best sequence at any given stage or round in the screening and substituting it as a benchmark or reference in a subsequent round of screening. This latter process can further include selection of parameters to modify in choosing and generating candidate ISNAs to screen.
In another aspect the invention provides screening method for identifying species 1o specificity of an ISNA. The method involves contacting a functional TLR
selected from the group consisting of TLR7, TLRB, and TLR9 of a first species with a test ISNA;
contacting a functional TLR selected from the group consisting of TLR7, TLRB, and TLR9 of a second species with the test ISNA; measuring a response mediated by a TLR signal transduction pathway associated with the contacting the functional TLR of the first species with the test ISNA; measuring a response mediated by the TLR signal transduction pathway associated with the contacting the functional TLR of the second species with the test ISNA; and comparing (a) the response mediated by a TLR signal transduction pathway associated with the contacting the functional TLR of the first species with the test ISNA with (b) the response mediated by the TLR signal transduction pathway associated with the contacting the functional TLR of the second species with the test ISNA. The functional TLR
may be expressed by a cell or it may be part of a cell-free system. The functional TLR may be part of a complex, with either another TLR or with another protein, e.g., MyD88, IRAK, TRAF6, IoB, NF-~cB, or functional homologues and derivatives thereof. Thus for example a given ODN can be tested against a panel of 293 fibroblast cells transfected with TLR7, TLRB, or TLR9 from various species and optionally cotransfected with a reporter construct (e.g., NF-xB-luc) sensitive to TLR/Ih-1R activation pathways. Thus in another aspect, the invention provides a method for screening species selectivity with respect to a given nucleic acid sequence.
As mentioned above, the invention in one aspect provides a screening method for 3o comparing TLR signaling activity or a test compound against corresponding TLR signaling activity of a reference ISNA. The methods generally involve contacting a functional TLR

_88_ selected from the group consisting of TLR7, TLRB, and TLR9 with a reference ISNA and detecting a reference response mediated by a TLR signal transduction pathway;
contacting a functional TLR selected from the group consisting of TLR7, TLRB, and TLR9 with a test compound and detecting a test response mediated by a TLR signal transduction pathway; and comparing the test response with the reference response to compare the TLR
signaling activity of the test compound with the ISNA. Assays in which the test compound and the reference ISNA contact the TLR independently may be used to identify test compounds that are ISNA mimics. Assays in which the test compound and the reference ISNA
contact the TLR concurrently may be used to identify test compounds that are ISNA agonists and ISNA
1o antagonists.
An ISNA mimic as used herein is a compound which causes a response mediated by a TLR signal transduction pathway. As used herein the term "response mediated by a TLR
signal transduction pathway" refers to a response which is characteristic of an ISNA-TLR
interaction. As demonstrated herein responses which are characteristic of ISNA-TLR
interactions include the induction of a gene under control of an ISNA-specific promoter such as a NF-oB promoter, increases in Thl cytokine levels, etc. The gene under the control of the .
NF-xB promoter may be a gene which naturally includes an NF-xB promoter or it may be a gene in a construct in which an NF-xB promoter has been inserted. Genes which naturally include the NF-~cB promoter include but are not limited to IL-8, Ih-12 p40, NF-~cB-luc, IL-12 p40-luc, and TNF-luc. Tncreases in Thl cytokine levels is another measure characteristic of an ISNA-TLR interaction. Increases in Thl cytokine levels may result from increased production or increased stability or increased secretion of the Thl cytokines in response to the ISNA-TLR interaction. Thl cytokines include but are not limited to IL-2, IFN-y, and IL-12.
Other responses which are characteristic of an ISNA-TLR interaction include but are not limited to a reduction in Th2 cytokine levels. Th2 cytokines include but are not limited to 1L-4, IL,-5, and IL,-10.
The response which is characteristic of an ISNA-TLR interaction may be a direct response or an indirect response. A direct response is a response that arises directly as a result of the ISNA-TLR interaction. An indirect response is a response which involves the modulation of other parameters prior to its occurrence.
An ISNA agonist as used herein is a compound which causes an enhanced response to an ISNA mediated by a TLR signal transduction pathway. Thus an ISNA agonist as used herein is a compound which causes an increase in at least one aspect of an immune response that is ordinarily induced by the reference ISNA. For example, an immune response that is ordinarily induced by ari ISNA can specifically include TLR7-, TLRB-, or TLR9-mediated signal transduction in response to immunostimulatory CpG nucleic acid. An ISNA
agonist will in some embodiments compete with ISNA for binding to TLR7, TLRB, or TLR9.
In other embodiments an ISNA agonist will bind to a site on TLR7, TLRB, or TLR9 that is distinct from the site for binding ISNA. In yet other embodiments an ISNA
agonist will act via another molecule or pathway distinct from TLR7, TLRB, or TLR9.
l0 An ISNA antagonist as used herein is a compound which causes a decreased response to an ISNA mediated by a TLR signal transduction pathway. Thus an ISNA
antagonist as used herein is a compound which causes a decrease in at least one aspect of an immune response that is ordinarily induced by the reference ISNA. For example, an immune response that is ordinarily induced by an ISNA can specifically include TLR7-, TLRB-, or TLR9-mediated signal transduction in response to immunostimulatory CpG nucleic acid. An ISNA
antagonist will in some embodiments compete with ISNA for binding to TLR7, TLRB, or TLR9. In other embodiments an ISNA antagonist will bind to a site on TLR7, TLRB, or TLR9 that is distinct from the site for binding ISNA. In yet other embodiments an ISNA
antagonist will act via another molecule or pathway distinct from TLR7, TLRB, or TLR9.
2o The screening methods for comparing TLR signaling activity of a test compound with signaling activity of an ISNA involve contacting at least one test compound with a functional TLR selected from TLR7, TLRB, and TLR9 under conditions which, in the absence of a test compound, permit a reference ISNA to induce at least one aspect of an immune response.
The functional TLR may be expressed by a cell or it may be part of a cell-free system. A cell expressing a functional TLR is a cell that either naturally expresses the TLR, or is a cell into which has been introduced a TLR expression vector, or is a cell manipulated to express TLR
in a manner that allows the TLR to be expressed by the cell and to transduce a signal under conditions which normally permit signal transduction by the signal transducing portion of the TLR. The TLR can be a native TLR or it can be a fragment or variant thereof, as described 3o above. According to these methods, the test compound is contacted with a functional TLR or TLR-expressing cell before, after, or simultaneously with contacting a reference ISNA with the functional TLR or TLR-expressing cell. A response of the functional TLR or TLR-expressing cell is measured and compared with the corresponding response that results or would result under the same conditions in the absence of the test compound.
Where it is appropriate, the response in the absence of the test compound can be determined as a concurrent or historical control. Examples of such responses include, without limitation, a response mediated through the TLR signal transduction pathway, secretion of a cytokine, cell proliferation, and cell activation. In a preferred embodiment, the measurement of a response involves the detection of IL-8 secretion (e.g., by ELISA). In another preferred embodiment, the measurement of the response involves the detection of luciferase activity (e.g., NF-xB-to luc, IL-12 p40-luc, or TNF-luc).
Examples of reference ISNAs include;°without limitation, those listed in Table 1 (above). In some preferred embodiments the reference ISNA is a CpG nucleic acid.
Test compounds can~include but are not limited to peptide nucleic acids (PNAs), antibodies, polypeptides, carbohydrates, lipids, hormones, and small molecules. Test compounds can further include variants of a reference ISNA incorporating any one or combination of the substitutions described above. Test compounds can be generated as members of a combinatorial library of compounds.
In preferred embodiments, the methods for screening test compounds, test nucleic acid molecules, test ISNAs, and candidate pharmacological agents can be performed on a large 2o scale and with high throughput by incorporating, e.g., an array-based assay system and at least one automated or semi-automated step. For example, the assays can be set up using multiple-well plates in which cells are dispensed in individual wells and reagents are added in a systematic manner using a multiwell delivery device suited to the geometry of the multiwell plate. Manual and robotic multiwell delivery devices suitable for use in a high throughput screening assay are well known by those skilled in the art. Each well or array element can be mapped in a one-to-one manner to a particular test condition, such as the test compound.
Readouts can also be performed in this multiwell array, preferably using a multiwell plate reader device or the like. Examples of such devices are well known in the art and are available through commercial sources. Sample and reagent handling can be automated to 3o further enhance the throughput capacity of the screening assay, such that dozens, hundreds, thousands, or even millions of parallel assays can be performed in a day or in a week. Fully robotic systems are known in the art for applications such as generation and analysis of combinatorial libraries of synthetic compounds. See, for example, U.S. patents 5,443,791 and 5,708,158.
The invention will be more fully understood by reference to the following examples.
These examples, however, are merely intended to illustrate the embodiments of the invention and are not to be construed to limit the scope of the invention.
Examples 1o Example 1. Method of cloning the mouse TLR9 Alignment of human TLR9 protein sequence with mouse EST database using tfasta yielded 7 hits with mouse EST sequences aa197442, ai451215, aa162495, aw048117, ai463056, aw048548, and aa273731. Two primers were designed that bind to aa197442 EST
sequence for use in a RACE-PCR to amplify 5' and 3' ends of the mouse TLR9 cDNA. The library used for the RACE PCR was a mouse spleen marathon-ready cDNA
commercially available from Clonetech. A 5' fragment with a length of 1800 by obtained by this method was cloned into Promega pGEM-T Easy vector. After sequencing of the 5' end, additional primers were designed for amplification of the complete mouse TLR9 cDNA. The primer for the 5' end was obtained from the sequence of the 5' RACE product whereas the primer for the 3' end was selected from the mouse EST sequence aa273731.
Three independent PCR reactions were set up using a murine macrophage RAW264.7 (ATCC TIB-71) cDNA as a template, and the resulting amplification products were cloned into the pGEM-T Easy vector. The inserts were fully sequenced, translated into protein and aligned to the human protein sequence. One out of three clones was error-free based on alignment comparison (clone mt1r932e.pep). The cDNA sequence for mTLR9 is SEQ
ID
NO:l, is presented in Table 2. The ATG start codon occurs at base 40, and a TAG
termination codon occurs at base 3136. SEQ m N0:2 (Table 3), corresponding to bases 40-3135 of SEQ ID NO:1, is the coding region for the polypeptide of SEQ m N0:3.
3o Table 2. cDNA Sequence for Murine TLR9 (5' to 3'; SEQ ID NO:1) tgtcagaggg agcctcggga gaatcctcca tctcccaaca tggttctccg tcgaaggact 60 ctgcacccct tgtccctcct ggtacaggct gcagtgctgg ctgagactct ggccctgggt 120 accctgcctg ccttcctaccctgtgagctgaagcctcatggcctggtggactgcaattgg180 ctgttcctga agtctgtaccccgtttctctgcggcagcatcctgctccaacatcacccgc240 ctctccttga tctccaaccgtatccaccacctgcacaactccgacttcgtccacctgtcc300 aacctgcggc agctgaacctcaagtggaactgtccacccactggccttagccccctgcac360 ttctcttgcc acatgaccattgagcccagaaccttcctggctatgcgtacactggaggag420 ctgaacctga gctataatggtatcaccactgtgccccgactgcccagctccctggtgaat480 ctgagcctga gccacaccaacatcctggttctagatgctaacagcctcgccggcctatac540 agcctgcgcg ttctcttcatggacgggaactgetactacaagaacccctgcacaggagcg600 gtgaaggtga ccccaggcgccctcctgggcctgagcaatctcacccatctgtctctgaag660 tataacaacc tcacaaaggtgccccgccaactgccccccagcctggagtacctcctggtg720 tcctataacc tcattgtcaagctggggcctgaagacctggccaatctgacctcccttcga780 gtacttgatg tgggtgggaattgccgtcgctgcgaccatgcccccaatccctgtatagaa840 tgtggccaaa agtccctccacctgcaccctgagaccttccatcacctgagccatctggaa900 ggcctggtgc tgaaggacagctctctccatacactgaactcttcctggttccaaggtctg960 gtcaacctct cggtgctggacctaagcgagaactttctctatgaaagcatcaaccacacc1020 aatgcctttc agaacctaacccgcctgcgcaagctcaacctgtccttcaattaccgcaag1080 aaggtatcct ttgcccgcctccacctggcaagttccttcaagaacctggtgtcactgcag1140 gagctgaaca tgaacggcatcttcttccgctcgctcaacaagtacacgctcagatggctg1200 gccgatctgc ccaaactccacactctgcatcttcaaatgaacttcatcaaccaggcacag1260 ctcagcatct ttggtaccttccgagcccttcgctttgtggacttgtcagacaatcgcatc1320 agtgggcctt caacgctgtcagaagccacccctgaagaggcagatgatgcagagcaggag1380 gagctgttgt ctgcggatcctcacccagctccactgagcacccctgcttctaagaacttc1440 atggacaggt gtaagaacttcaagttcaccatggacctgtctcggaacaacctggtgact1500 atcaagccag agatgtttgtcaatctctcacgcctccagtgtcttagcctgagccacaac1560 tccattgcac aggctgtcaatggctctcagttcctgccgctgactaatctgcaggtgctg1620 gacctgtccc ataacaaactggacttgtaccactggaaatcgttcagtgagctaccacag1680 ttgcaggccc tggacctgagctacaacagccagccctttagcatgaagggtataggccac1740 aatttcagtt ttgtggcccatctgtccatgctacacagccttagcctggcacacaatgac1800 attcataccc gtgtgtcctcacatctcaacagcaactcagtgaggtttcttgacttcagc1860 ggcaacggta tgggccgcatgtgggatgaggggggcctttatctccatttcttccaaggc1920 ctgagtggcc tgctgaagctggacctgtctcaaaataacctgcatatcctccggccccag1980 aaccttgaca acctccccaagagcctgaagctgctgagcctccgagacaactacctatct2040 ttctttaact ggaccagtctgtccttcctgCCCaaCCtggaagtcctagacctggcaggc2100 aaccagctaa aggccctgaccaatggcaccctgcctaatggcaccctcctccagaaactg2160 gatgtcagca gcaacagtatcgtctctgtggtcccagccttcttcgctctggcggtcgag2220 ctgaaagagg tcaacctcagccacaacattctcaagacggtggatcgctc-ctggtttggg2280 cccattgtga tgaacctgacagttctagacgtgagaagcaaccctctgcactgtgcctgt2340 ggggcagcct tcgtagacttactgttggaggtgcagaccaaggtgcctggcctggctaat2400 ggtgtgaagt gtggcagccccggccagctgcagggccgtagcatcttcgcacaggacctg2460 cggctgtgcc tggatgaggtcctctcttgggactgctttggcctttcactcttggctgtg2520 gccgtgggca tggtggtgcctatactgcaccatctctgcggctgggacgtctggtactgt2580 tttcatctgt gcctggcatggctacctttgCtggCCCgCagccgacgcagcgcccaagct2640 ctcccctatg atgccttcgt ggtgttcgat aaggcacaga gcgcagttgc ggactgggtg 2700 tataacgagc tgcgggtgcg gctggaggag cggcgcggtc gccgagccct acgcttgtgt 2760 ctggaggacc gagattggct gcctggccag acgctcttcg agaacctctg ggcttccatc 2820 tatgggagcc gcaagactct atttgtgctg gcccacacgg accgcgtcag tggcctcctg 2880 cgcaccagct tcctgctggc tcagcagcgc ctgttggaag accgcaagga cgtggtggtg 2940 ttggtgatcc tgcgtccgga tgcccaccgc tcccgctatg tgcgactgcg ccagcgtctc 3000 tgccgccaga gtgtgctctt ctggccccag cagcccaacg ggcagggggg cttctgggcc 3060 cagctgagta cagccctgac tagggacaac cgccacttct ataaccagaa cttctgccgg 3120 ggacctacag cagaatagct cagagcaaca gctggaaaca gctgcatctt catgcctggt 3180 tcccgagttg CtCtgCCtgC 3200 Table 3. Coding region for murine TLR9 (SEQ ID N0:2) atggttctcc gtcgaaggactCtgCaCCCCttgtccctcctggtacaggctgcagtgctg60 gctgagactc tggCCCtgggtaccctgcctgCCttCCtaCCCtgtgagCtgaagcctcat120 ggcctggtgg actgcaattggctgttcctgaagtctgtaccccgtttctctgcggcagca180 tcctgctcca acatcacccgcctctccttgatctccaaccgtatccaccacctgcacaac240 tCCgaCttCg tCCaCCtgtCCaaCCtgCggcagctgaacctcaagtggaactgtccaccc300 actggcctta gccccctgcacttctcttgccacatgaccattgagcccagaaccttcctg360 gctatgcgta cactggaggagctgaacctgagctataatggtatcaccactgtgccccga420 ctgcccagct ccctggtgaatctgagcctgagccacaccaacatcctggttctagatgct480 aacagcctcg CCggCCtataCagCCtgCgCgttctcttcatggacgggaactgctactac540 aagaacccct gcacaggagcggtgaaggtgaccccaggcgccctcctgggcctgagcaat600 ctcacccatc tgtctctgaagtataacaacctcacaaaggtgccccgccaactgcccccc660 agcctggagt acctcctggtgtcctataacctcattgtcaagctggggcctgaagacctg720 gccaatctga cctcccttcgagtacttgatgtgggtgggaattgccgtcgctgcgaccat780 gcccccaatc cctgtatagaatgtggccaaaagtccctccacctgcaccctgagaccttc840 catcacctga gccatctggaaggcctggtgctgaaggacagctctctccatacactgaac900 tcttcctggt tccaaggtctggtcaacctctcggtgctggacctaagcgagaactttctc960 tatgaaagca tcaaccacaccaatgcctttcagaacctaacccgcctgcgcaagctcaac1020 ctgtccttca attaccgcaagaaggtatcctttgcccgcctCCaCCtggCaagttCCttC1080 aagaacctgg tgtcactgcaggagctgaacatgaacggcatcttcttccgctcgctcaac1140 aagtacacgc tcagatggctggccgatctgcccaaactccacactctgcatcttcaaatg1200 aacttcatca accaggcacagctcagcatctttggtaCCttCCgagCCCttCgCtttgtg1260 gacttgtcag acaatcgcatcagtgggccttcaacgctgtcagaagccacccctgaagag1320 gcagatgatg cagagcaggaggagctgttgtctgcggatcctcacccagctccactgagc1380 acccctgctt ctaagaacttcatggacaggtgtaagaacttcaagttcaccatggacctg1440 tctcggaaca acctggtgactatcaagccagagatgtttgtcaatctctcacgcctccag1500 tgtcttagcc tgagccacaactccattgcacaggctgtcaatggctctcagttcctgccg1560 ctgactaatc tgcaggtgctggacctgtcccataacaaactggacttgtaccactggaaa1620 tcgttcagtg agctaccacagttgcaggccctggacctgagctacaacagccagcccttt1680 agcatgaagg gtataggccacaatttcagttttgtggcccatctgtccatgctacacagc1740 cttagcctgg cacacaatga cattcatacc cgtgtgtcct cacatctcaa cagcaactca 1800 gtgaggtttc ttgacttcag cggcaacggt atgggccgca tgtgggatga ggggggcctt 1860 tatctccatt tcttccaagg cctgagtggc ctgctgaagc tggacctgtc tcaaaataac 1920 ctgcatatcc tccggcccca gaaccttgac aacctcccca agagcctgaa gctgctgagc 1980 ctccgagaca actacctatc tttctttaac tggaccagtc tgtccttcct gcccaacctg 2040 gaagtcctag acctggcagg caaccagcta aaggccctga ccaatggcac cctgcctaat 2100 ggcaccctcc tccagaaact ggatgtcagc agcaacagta tcgtctctgt ggtcccagcc 2160 ttcttcgctc tggcggtcga gctgaaagag gtcaacctca gccacaacat tctcaagacg 2220 gtggatcgct cctggtttgg gcccattgtg atgaacctga cagttctaga cgtgagaagc 2280 aaccctctgc actgtgcctg tggggcagcc ttcgtagact tactgttgga ggtgcagacc 2340 aaggtgcctg gcctggctaa tggtgtgaag tgtggcagcc ccggccagct gcagggccgt 2400 agcatcttcg cacaggacct gcggctgtgc ctggatgagg tcctctcttg ggactgcttt 2460 ggcctttcac tcttggctgt ggccgtgggc atggtggtgc ctatactgca ccatctctgc 2520 ggctgggacg tctggtactg ttttcatctg tgcctggcat ggctaccttt gctggcccgc 2580 agccgacgca gcgcccaagc tctcccctat gatgccttcg tggtgttcga taaggcacag 2640 agcgcagttg cggactgggt gtataacgag ctgcgggtgc ggctggagga gcggcgcggt 2700 CgCCgagCCC tacgcttgtg tctggaggac c.gagattggc tgcctggcca gacgctcttc 2760 gagaacctct gggcttccat ctatgggagc cgcaagactc tatttgtgct ggcccacacg 2820 gaccgcgtca gtggcctcct gcgcaccagc ttcctgctgg ctcagcagcg cctgttggaa 2880 gaccgcaagg acgtggtggt gttggtgatc ctgcgtccgg atgcccaccg ctcccgctat 2940 gtgcgactgc gccagcgtct ctgcegccag agtgtgctet tetggcccca gcagcccaac 3000 gggcaggggg gcttctgggc ccagctgagt acagccctga ctagggacaa ccgccacttc 3060 tataaccaga acttctgccg gggacctaca gcagaa 3096 The deduced amino acid sequence for marine TLR9 (SEQ ID N0:3), comprising 1032 amino acid residues, is shown in Table 4 below in the aligned sequence comparison as mt1r932e.pep. The deduced amino acid sequence for human TLR9 (SEQ )D N0:6), comprising 1032 amino acid residues, is shown in Table 4 below in the aligned sequence comparison as htlr9.pro.
Table 4. Amino Acid Sequence of Marine and Human TLR.9 htlr9.pro MGFCRSALHPLSLLVQAIMLAMTLALGTLPAFLPCELQPHGLVNCNWLFLKSVPHFSMAA 60 mt1r932e.pep MVLRRRTLHPLSLLVQAAVLAETLALGTLPAFLPCELKPHGLVDCNWLFLKSVPRFSAAA 60 . . . . . . . 120 htlr9.pro PRGNVTSLSLSSNRIHHLHDSDFAHLPSLRHLNLKWNCPPVGLSPMHFPCHMTIEPSTFL 120 mt1r932e.pep SCSNITRLSLISNRIHHLHNSDFVHLSNLRQLNLKWNCPPTGLSPLHFSCHMTIEPRTFL 120 . . . . . . . . . . . . 180 htlr9.pro AVPTLEELNLSYNNIMTVPALPKSLISLSLSHTNILMLDSASLAGLHALRFLFMDGNCYY 180 mt1r932e.pep AMRTLEELNLSYNGITTVPRLPSSLVNLSLSHTNILVLDANSLAGLYSLRVLFMDGNCYY 180 htlr9.pro KNPCRQALEVAPGALLGLGNLTHLSLKYNNLTVVPRNLPSSLEYLLLSYNRIVKLAPEDL 240 mt1r932e.pep KNPCTGAVKVTPGALLGLSNLTHLSLKYNNLTKVPRQLPPSLEYLLVSYNLIVKLGPEDL 240 htlr9.pro ANLTALRVLDVGGNCRRCDHAPNPCMECPRHFPQLHPDTFSHLSRLEGLVLKDSSLSWLN 300 mt1r932e.pep ANLTSLRVLDVGGNCRRCDHAPNPCIECGQKSLHLHPETFHHLSHLEGLVLKDSSLHTLN 300 aa197442.pep LNLSFNYRKKVSFARLHLASSF 22 htlr9.pro ASWFRGLGNLRVLDLSENFLYKCITKTKAFQGLTQLRKLNLSFNYQKRVSFAHLSLAPSF 360 mt1r932e.pep SSWFQGLVNLSVLDLSENFLYESINHTNAFQNLTRLRKLNLSFNYRKKVSFARLHLASSF 360 mousepepl C 1 aa197442.pep KNLVSLQELNMNGIFFRLLNKYTLRWLADLPKLHTLHLQMNFINQAQLSIFGTFRALRFV 82 htlr9.pro GSLVALKELDMHGIFFRSLDETTLRPLARLPMLQTLRLQMNFINQAQLGIFRAFPGLRYV 420 mt1r932e.pep KNLVSLQELNMNGIFFRSLNKYTLRWLADLPKLHTLHLQMNFINQAQLSIFGTFRALRFV 420 mousepepl DLSDNRISGPSTLSEA 17 humanpepl PAPVDTPSSEDFRPNC 16 aa197442.pep DLSDNRISGPSTLSEATPEEADDAEQEELLSADPHPAPLSTPASKNFMDRCKNFKFNMDL142 htlr9.pro DLSDNRISGASELT-ATMGEADGGEKVWLQPGDLAPAPVDTPSSEDFRPNCSTLNFTLDL479 mt1r932e.pep DLSDNRISGPSTLSEATPEEADDAEQEELLSADPHPAPLSTPASKNFMDRCKNFKFTMDL480 . . . . . . . . . . . . 540 aa197442.pep SRNNLVTITAEMFVNLSRLQCLSLSHNSIAQAVNGS 178 htlr9.pro SRNNLVTVQPEMFAQLSHLQCLRLSHNCISQAVNGSQFLPLTGLQVLDLSRNKLDLYHEH 539 mt1r932e.pep SRNNLVTTKPEMFVNLSRLQCLSLSHNSIAQAVNGSQFLPLTNLQVLDLSHNKLDLYHWK 540 . . . . . . . . . . . . 600 aa162495.pep YNSQPFSMKGIGHNFSFVTHLSMLQSLSLAHNDIHTRVSSHLNSNS 46 htlr9.pro SFTELPRLEALDLSYNSQPFGMQGVGHNFSFVAHLRTLRHLSLAHNNIHSQVSQQLCSTS 599 mt1r932e.pep SFSELPQLQALDLSYNSQPFSMKGIGHNFSFVAHLSMLHSLSLAHNDIHTRVSSHLNSNS 600 . . . . . . . . . . . . 660 aa162495.pep VRFLDFSGNGMGRMWDEGGLYLHFFQGLSGVLKLDLSQNNLHILRPQNLDNLPKSLKLLS 106 htlr9.pro LRALDFSGNALGHMWAEGDLYLHFFQGLSGLIWLDLSQNRLHTLLPQTI,RNLPKSZ,QVLR 659 mt1r932e.pep VRFLDFSGNGMGRMWDEGGLYLHFFQGLSGLLKLDLSQNNLHILRPQNLDNLPKSLKLLS 660 . . . . . . . . . . . . 720 aa162495.pep LRDNYLSFFNWTSLSFLPNLEVLDLAGNQLKALTNGTLPNGTLLQKLDVSSNSIVS 162 htlr9.pro LRDNYLAFFKWWSLHFLPICLEVLDLAGNRLKALTNGSLPAGTRLRRLDVSCNSISFVAPG 719 mt1r932e.pep LRDNYLSFFNWTSLSFLPNLEVLDLAGNQLKALTNGTLPNGTLLQKLDVSSNSIVSWPA 720 ai451215.pep PIVMNLTVLDVRSNPLHCACGAAFVDLLLEVQT 33 htlr9.pro FFSKAKELRELNLSANALKTVDHSWFGPLASALQILDVSANPLHCACGAAFMDFLLEVQA 779 mt1r932e.pep FFALAVELKEVNLSHNILKTVDRSWFGPIVMNLTVLDVRSNPLHCACGAAFVDLLLEVQT 780 ai451215.pep KVPGLANGVKCGSPGQLQGRSIFAQDLRLCLDEVLSWDCFGLSLLAVAVGMWPILHHLC 93 htlr9.pro AVPGLPSRVKCGSPGQLQGLSIFAQDLRLCLDEALSWDCFALSLLAVALGLGVPMLHHLC 839 mt1r932e.pep KVPGLANGVKCGSPGQLQGRSIFAQDLRLCLDEVLSWDCFGLSLLAVAVGMWPILHHLC 840 ai451215.pep GWDVWYCFHLCLAWLPLLAR-SRRSAQTLPYDAFWFDKAQSAVADWVYNELRVRLEERR 152 htlr9.pro GWDLWYCFHLCLAWLPWRGRQSGRDEDALPYDAFWFDKTQSAVADWVYNELRGQLEECR 899 mt1r932e.pep GWDVWYCFHLCLAWLPLLAR-SRRSAQALPYDAFWFDKAQSAVADWVYNELRVRLEERR 899 aa273731.pep AHTDRVSGLLRTSFLLAQQRLL 22 ai463056.pep EDRDWLPGQTLFENLWASIYGSRKTLFVLAHTDRVSGLLRTSFLLAQQRLL 51 ai451215.pep GR 154 htlr9.pro GRWALRLCLEERDWLPGKTLFENLWASVYGSRKTLFVLAHTDRVSGLLRASFLLAQQRLL 959 mt1r932e.pep GRRALRLCLEDRDWLPGQTLFENLWASIYGSRKTLFVLAHTDRVSGLLRTSFLLAQQRLL 959 humanpep2 H 1 mousepep2 H 1 aa273731.pep EDRKDVWLVILRPDAXPSRYVRLRQRLCRQSVLFWPQRPNGQGGFWAQLSTALTRDNRH 82 ai463056.pep EDRKDVWLVILRPDAHRSRYVRLRQRLCRQSVLFWPQQPNGQGGFWAQLSTALTRDNRH 111 htlr9.pro EDRKDVWLVILSPDGRRSRYVRLRQRLCRQSVLLWPHQPSGQRSFWAQLGMALTRDNHH 1019 mt1r932e.pep EDRKDVWLVILRPDAHRSRYVRLRQRLCRQSVLFWPQQPNGQGGFWAQLSTALTRDNRH 1019 humanpep2 FYNRNFCQGPTAE 14 mousepep2 FYNQNFCRGPTAE 14 aa273731.pep FXNQNFCRGPTAE 95 ai463056.pep FYNQNFCRGPTA 123 htlr9.pro FYNRNFCQGPTAE 1032 mt1r932e.pep FYNQNFCRGPTAE 1032 The following SEQ m NOs correspond to the sequences as shown in Table 4:
htlr9.pro: SEQ ID N0:6; mt1r932e.pep: SEQ m N0:3; aa197442.pep: SEQ ID N0:8;
mousepepl: SEQ >D N0:17; humanpepl: SEQ m N0:19; aa162495.pep: SEQ m N0:14;

ai451215.pep: SEQ ID N0:16; aa273731.pep: SEQ ID NO:10; ai463056.pep: SEQ ID
N0:12; humanpep2: SEQ 1D N0:20; and mousepep2: SEQ ID N0:18.
Example 2. Reconstitution of TLR9 signaling in 293 fibroblasts The cloned mouse TLR9 cDNA (see above) and human TLR9 cDNA (gift from B.
Beutler, Howard Hughes Medical Institute, Dallas, TX) in pT-Adv vector (from Clonetech) were cloned into the expression vector pcDNA3.l (-) from Invitrogen using the EcoRI site.
Utilizing a "gain of function" assay it was possible to reconstitute human TLR9 (hTLR9) and marine TLR9 (mTLR9) signaling in CpG DNA non-responsive human 293 fibroblasts to (ATCC, CRL-1573). The expression vectors mentioned above were transfected into 293 fibroblast cells using the calcium phosphate method.
Since NF-xB activation is central to the IL-1/TLR signal transduction pathway (Medzhitov R et al., Mol Cell 2:253-258 (1998); Muzio M et al., JExp Med 187:2097-2101 (1998)), cells were transfected with hTLR9 or co-transfected with hTLR9 and a NF-xB-driven luciferase reporter construct. Human fibroblast 293 cells were transiently transfected with (Figure 1A) hTLR9 and a six-times NF-xB-luciferase reporter plasmid (NF-xB-luc, kindly provided by Patrick Baeuerle, Munich, Germany) or (Figure 1B) with hTLR9 alone.
After stimulus with CpG-ODN (2006, 2~,M, TCGTCGTTTTGTCGTTTTGTCGTT, SEQ ID
NO:l 12), GpC-ODN (2006-GC, 2pM, TGCTGCTTTTGTGCTTTTGTGCTT, SEQ ID
NO:l 18), LPS (100 ng/ml) or media, NF-~cB activation by luciferase readout (8h, Figure 1A) or IL-8 production by ELISA (48h, Figure 1B) were monitored. Results are representative of three independent experiments. Figure 1 shows that cells expressing hTLR9 responded to CpG-DNA but not to LPS.
Figure 2 demonstrates the same principle for the transfection of mTLR9. Human fibroblast 293 cells were transiently transfected with mTLR9 and the NF-~cB-luc construct (Figure 2). Similar data was obtained for 1L-8 production (not shown). Thus expression of TLR9 (human or mouse) in 293 cells results in a gain of function for CpG-DNA
stimulation similar to hTLR4 reconstitution of LPS responses.
To generate stable clones expressing human TLR9, marine TLR9, or either TLR9 with the NF-xB-luc reporter plasmid, 293 cells were transfected in 10 cm plates (2x106 cells/plate) with 16 pg of DNA and selected with 0.7 mg/ml 6418 (PAA
Laboratories GmbH, Colbe, Germany). Clones were tested for TLR9 expression by RT-PCR, for example as shown in Figure 3. The clones were also screened for IL-8 production or NF-xB-luciferase activity after stimulation with ODN. Four different types of clones were generated.
293-hTLR9-luc: expressing human TLR9 and 6-fold NF-~c.B-luciferase reporter 293-mTLR9-luc: expressing marine TLR9 and 6-fold NF-KB-luciferase reporter 293-hTLR9: expressing human TLR9 293-mTLR9: expressing marine TLR9 to Figure 4 demonstrates the responsiveness of a stable 293-hTLR9-luc clone after stimulation with CpG-ODN (2006, 2~,M), GpC-ODN (2006-GC, 2~,M), Me-CpG-ODN
(2006 methylated, 2wM; TZGTZGTTTTGTZGTTTTGTZGTT, Z = 5-methylcytidine, SEQ
ID N0:128), LPS (100 ng/ml) or media, as measured by monitoring NF-xB
activation.
Similar results were obtained utilizing 1L-8 production with the stable clone 293-hTLR9.
15 293-mTLR9-luc were also stimulated with CpG-ODN (1668, 2~M;
TCCATGACGTTCCTGATGCT, SEQ ID NO:84), GpC-ODN (1668-GC, 2wM;
TCCATGAGCTTCCTGATGCT, SEQ ID N0:85), Me-CpG-ODN (1668 methylated, 2p,M;
TCCATGAZGTTCCTGATGCT, Z = 5-methylcytidine, SEQ ID N0:207), LPS (100 ng/ml) or media, as measured by monitoring NF-~cB activation (Figure 5). Similar results were 20 obtained utilizing IL-8 production with the stable clone 293-mTLR9. Results are representative of at least two independent experiments. These results demonstrate that CpG-DNA non-responsive cell lies can be stably genetically complemented with TLR9 to become responsive to CpG DNA in a motif specific manner. These cells can be used for screening of optimal ligands for innate immune responses driven by TLR9 in multiple species.
Example 3. Expression of soluble recombinant human TLR9 in yeast cells (Piclaia pastoris) Human TLR9 cDNA coding for amino acids 1 to 811 was amplified by PCR using the primers 5'-ATAGAATTCAATAATGGGTTTCTGCCGCAGCGCCCT-3' (SEQ ID N0:194) 3o and 5'-ATATCTAGATCCAGGCAGAGGCGCAGGTC-3' (SEQ 1D N0:195), digested with EcoRI and XbaI, cloned into the yeast expression vector pPICZB (Invitrogen, Groningen, Netherlands) and transfected into yeast cells (Pichia pastoris). Clones were selected with the antibiotic zeozin and protein production of soluble human TLR9 was induced with methanol (see Figure 6: SDS-PAGE, Coomassie stained, arrow marks hTLR9; lane l:
supernatant of culture induced with methanol; lane 2: supernatant of culture not induced).
Thus TLR9 protein can be isolated from transfectants and further utilized for protein studies and vaccination purposes.
Example 4. hTLR9 expression correlates with CpG-DNA responsiveness.
Bacterial DNA has been described as a mitogen for both marine and human B
cells.
Although LPS is also mitogenic for marine B cells, it is generally accepted that LPS is not a mitogen for human B cells. Figure 7 demonstrates that human B cells proliferate after stimulation with E. coli DNA or a CpG-ODN but not Dnase-digested E. coli DNA
or a control GpC-ODN. Purified human B cells were stimulated with SO~g/ml E. coli DNA, a comparable amount of DNase I-digested E. coli DNA, 2wM CpG-ODN (2006), 2~M GpC-ODN (2006-GC) or 100 ng/ml LPS. B cell proliferation was monitored at day two by 3H-thymidine uptake. These data demonstrate that it was DNA within the E. coli DNA
preparation that was mitogenic and that a CpG-motif within the ODN was required.
Human dendritic cells (DC) have been claimed to be responsive to CpG-DNA.
While analyzing human dendritic cell responses to CpG-DNA, we noted that plasmacytoid DC
(CD123+DC) produced IFN-a, TNF, GM-CSF, and IL-8 upon exposure to CpG-DNA but not to LPS (Figure 8 and unpublished data). The converse was true for stimulation of monocyte-derived dendritic cells (MDDC) (Figure 8 and unpublished data). Purified CD123+DC or MDDC were stimulated with 50~,g/ml E. coli DNA, a comparable amount of DNase I-digested E. coli DNA, 2~,M CpG-ODN (2006), 2pM GpC-ODN (2006-GC) or 100 ng/ml LPS (Figure 8). IL-8 and TNF concentration was determined by enzyme-linked immunosorbent assay (ELISA). The CD123+DC response was DNA- and CpG-motif restricted. Monocyte-derived dendritic cells (MDDC) however demonstrated the converse response pattern, a response to LPS but not CpG-DNA. Due to this segregated response we analyzed TLR expression.
We have shown that CpG-DNA utilizes the Toll/1L-1R (TIR) signal transduction pathway implying the need for a TIR domain in the CpG-DNA signaling receptor.
Hacker H

et al., JExp Med 192:595-600 (2000). It was further demonstrated that TLR9-deficient mice are non-responsive to CpG-ODN. Hemmi H et al., Nature 408:740-5. By semi-quantitative RT-PCR both B cells and CD 123+ DC yielded positive signals for hTLR9 while MDDC, monocytes and T cells were weak to negative (Figure 9). The cDNAs were prepared from monocyte-derived dendritic cells (MDDC), lane 1; purified CD14+ monocytes, lane 2; B
cells, lane 3; CD123+ DC, lane 4; CD4+ T cells, lane 5; and CD8+ T cells, lane 6. cDNA
amounts were normalized based on GAPDH amount determined by TAG-MAN PCR
(Perkin-Elmer). RT-PCR was performed for 30 cycles on normalized cDNA diluted 1:5 for human TLR2, 4 and 9, while GAPDH was diluted 1:125. We also tested for hTLR2 and hTLR4 1o expression. MDDC and monocytes were positive while B cells, T cells and CD123+DC were weak to negative (Figure 9). Weak signals delivered by PCR could be explained by contaminating cells, however a strong positive signal implies expression.
These data demonstrated a clear correlation between hTLR9 mRNA expression and B cell or CD123+DC responsiveness to CpG-DNA (Figures 7 and 8). A correlation could also be shown for hTLR2 and hTLR4 expression and MDDC responsiveness to LPS (Figure 8).
This data demonstrates that hTLR9 is a relevant receptor for CpG-DNA responses and that its expression determines responsiveness. If TLR9 expression could be modulated, agonism or antagonism of CpG-DNA responses could be achieved.
2o Example 5. Species specificity of TLR9 signaling By iterative examination of the flanking sequences surrounding CG
dinucleotides, CpG-motifs have been identified. Paradoxically, or by twist of nature, the human optimal CpG-motif, GTCGTT (SEQ ID N0:66), is different from the marine motif, GACGTT
(SEQ
ID N0:129). Human peripheral blood mononuclear cells (PBMC) (Figure 10A) and marine splenocytes (Figure 10B) were stimulated with ODN 2006 (filled circle, TCGTCGTTTTGTCGTTTTGTCGTT, SEQ ID N0:112), ODN 2006-GC (open circle), ODN
1668 (filled triangle, TCCATGACGTTCCTGATGCT, SEQ ID N0:84) or ODN 1668-GC
(open triangle, TCCATGAGCTTCCTGATGCT, SEQ ID N0:85) at indicated concentrations and IL-12 production was monitored after 8 hours. Figure 10A shows that titration of the optimal human ODN, 2006, on PBMC induces IL-12 production. The optimal marine sequence, 1668, however was much less effective in eliciting IL-12 from PBMC.
The two control GpC-ODNs were essentially negative. The converse was true for marine splenocytes (Figure 10B), in that the marine sequence induced optimal IL-12 while the human sequence was much less effective. It should also be noted that the Ka~ (concentration of half maximal activation) of marine splenocytes for 1668 was greater than human PBMC for 2006 (compare Fig. 10A to Fig. 10B).
Because stable TLR9 transfectants mirrored primary cell responsiveness to CpG-DNA
(Figures 4 and 5), it was hypothesized that stable transfectants could potentially discern species-specific CpG-motifs through TLR9 receptors. Therefore 293-hTLR9-luc (expressing human TLR9 and 6-fold NF-xB-luc reporter), 293-mTLR9-luc (expressing marine TLR9 and 6-fold NF-~cB-luc reporter), 293-hTLR9 (expressing human TLR9) and 293-mTLR9 (expressing marine TLR9) clones were tested for CpG-DNA motif responsiveness.
Figure 11 shows titration curves for 2006 or 1668 and their controls versus either hTLR9 or mTLR9 cells. Depicted are both NF-xB-driven luciferase and IL-8 production as readout. In both 293 hTLR9-luc and 293-mTLR9-luc cells stimulation with CpG-DNA resulted in NF-~cB
activation, as determined by measurement of the induced expression of firefly luciferase under the control of a minimal promotor containing six tandem NF-~cB-binding sites. After lysis of the cells luciferase can be detected photometrically based on an enzymatic reaction by luciferase which creates photons. IL-8 production was monitored using enzyme-linked immunosorbent assay (ELISA). Figure 11 depicts clones stimulated with ODN 2006 (filled 2o circle), ODN 2006-GC (open circle), ODN 1668 (filled triangle) or ODN 1668-GC (open triangle) at indicated concentrations and NF-xB activation or IL-8 production were measured after 10 and 48 hours, respectively. Results shown in Figure 11 axe representative of three independent experiments. Strikingly, CpG-motif sequence specificity was conferred in a species-specific manner by TLR9. Additionally, the half maximal concentration for either 2006 or 1668 appears nearly the same as those determined on primary cells (compare Figure 10 and Figure 11). These data demonstrate that TLR9 is the CpG-DNA receptor and that exquisite specificity to CpG-DNA sequence is conferred by TLR9.
Example 6. Use of stable TLR9 clones to test responsiveness to substances other than 3o phosphorothioate ODN
As described in the foregoing Examples, the stable TLR9 clones were initially screened for fidelity of phosphorothioate CpG-ODN reactivity. The 293-hTLR9 cells demonstrated reactivity to CpG-DNA and not LPS in a CpG-motif dependent manner (Figures 4 and 5). In the present example the stable TLR9 transfectants were tested for responsiveness to additional DNAs. NF-xB activation was monitored after stimulation with E. coli DNA (black bars). or E. coli DNA digested with DNAse I (gray bars) in 293-hTLR9-luc cells. Figure 12 demonstrates an E. coli DNA dose-dependent induction of NF-~cB-driven luciferase expression to a level comparable to phosphorothioate CpG-ODN
(Figure 11).
Activity was destroyed by DNase I digestion, indicating specificity of response to DNA and not contaminant bacterial products. The stable TLR9 transfectants can be used to screen the l0 activity of DNAs from various species or vector DNAs intended for immune system stimulation. In particular, TLR9 transfectants can be used to screen and compare the immunostimulatory activity of DNAs from various species of pathogens, DNA
constructs, DNAs intended for use as vaccines, gene replacement therapeutics, and nucleic acid vectors.
293-hTLR9-luc cells also were stimulated with the phosphodiester variants of ODN
2006 (filled circle), ODN 2006-GC (open circle), ODN 1668 (filled triangle) or GC (open triangle) at indicated concentrations, and NF-~cB activation was monitored after 12 hours (Figure 13A). Likewise, 293-mTLR9-luc cells were stimulated with the phosphodiester variants of ODN 2006 (filled circle), ODN 2006-GC (open circle), ODN 1668 (filled triangle) or ODN 1668-GC (open triangle) at indicated concentrations, and NF-xB
activation was monitored after 12 hours (Figure 13B). These assays show that the stable TLR9 transfectants responded to DNAs other than phosphorothioate-modified ODN.
These data demonstrate the utility of stable TLR transfectants for screening for agonists of theTLR9 receptor.
Example 7. TLR9 determines CpG-ODN activity Although 2006 and 1668 are discussed in terms of CpG-motif differences, they are very different in several aspects (see Table 5 for comparison). The lengths are different, 24 versus 20 nucleotides, and 2006 has four CG dinucleotides compared to one in 1668.
Additional differences are the CG position relative to the 5' and 3' ends and also 5' sequence differences. In order to determine if motif specificity is a quality of the motif and not the global sequence environment, for this experiment several sequences were produced holding these variables constant. As a starting point, the 1668 sequence was modified by converting the central C to T and the distal TG to CG, thereby creating a second CG in the resulting sequence 5000 (SEQ m N0:130, Table 5). Then point nucleotide changes were made, progressing toward a 2006-like sequence, 5007 (SEQ 1D N0:98). The ODN 5002 (SEQ )D
N0:132) is most like 1668 with the exception that C's at positions 12 and 19 have been converted to T's. The last 16 nucleotides of ODN 5007 are the same as the lastly nucleotides of 2006 with the exception of an additional T. The ODN concentration of half maximal activation (Kay) was determined by producing ODN titration curves using either 293-hTLR9-luc or 293-mTLR9-luc cells and NF-xB-driven luciferase expression as a readout. Example 1o curves are given in Figure 14. Stable transfectants 293-hTLR9-luc and 293-mTLR9-luc were stimulated with ODN 5002 (filled circle) or ODN 5007 (open circle) at indicated concentrations and NF-~cB activation was monitored after 12 hours. Results shown in Figure 14 are representative of three independent experiments. Values for Ka~ for multiple ODN are given in Table 5. Similar results were obtained for those ODN tested with 293-hTLR9 and 293-mTLR9 cells utilizing IL-8 as readout.
Table 5. CpG-DNA sequence specificity of human and murine TLR9 signaling activity CpG-DNA Sequence 293-hTLR9 293-mTLR9 SEQ m NO:

Kac (~) Kac (~) 1668 TCCATGACGTTCCTGATGCT >10,000 70 84 1668-GC TCCATGAGCTTCCTGATGCT >10,000 >10,000 85 2006 TCGTCGTTTTGTCGTTTTGTCGTT 400 >10,000 112 2006-GC TGCTGCTTTTGTGCTTTTGTGCTT >10,000 >10,000 118 5000 TCCATGACGTTCTTGACGCT 10,000 82 130 5001 TCCATGACGTTCTTGACGTT 7,000 55 131 5002 TCCATGACGTTCTTGATGTT 7,000 30 132 5003 TCCATGACGTTTTTGATGTT 10,000 30 133 5004 TCCATGTCGTTCTTGATGTT 5,000 400 134 5005 TCCATGTCGTTTTTGATGTT 3,000 2,000 135 5006 TCCATGTCGTTTTTGTTGTT 3,000 650 136 5007 TCCATGTCGTTTTTGTCGTT 700 1,000 98 5002 TCCATGACGTTCTTGATGTT ~ 30 132 5008 TCCATGACGTTATTGATGTT j~ 40 137 5009 TCCATGACGTCCTTGATGTT ND >10,000 138 5010 TCCATGACGTCATTGATGTT ND >10,000 139 In previous unpublished work by the inventors, it had been noted that a CA
substitution converting the mouse CpG-motif from GACGTTC to GACGTCA was deleterious. To extend our examination of the motif, three more ODN were created to dissect this effect (5008-5010, SEQ ID NOs:l37-139, Table 5).
The activity displayed by the 293-hTLR9-luc clone increased with progressive nucleotide substitutions converting the mouse sequence toward the human sequence (Table 5, sequences 5000-5007). The converse was true for the 293-mTLR9-luc clone, which showed highest activity for the mouse sequences. The originally hypothesized CpG-motif was purine-purine-CG-pyrimidine-pyrimidine. Most notable to motif definition as determined by TLR9' to genetic complementation was the non-conservative pyrimidine for purine change A to T
immediately 5' of the CG (Table 5). These changes improved 293-hTLR9-luc responsiveness but diminished 293-mTLR9-luc responsiveness. These results support the notion that the preferred mouse motif contains ACG while the preferred human sequence contains TCG.
The conservative pyrimidine for pyrimidine change T to C in the mouse motif, ACGTT
15 versus ACGTC (5002 versus 5009), completely destroyed 293-mTLR9 responsiveness.
Although not a complete iterative analysis of the CpG-motif, the data refine our understanding of the motif. More importantly these data strongly support direct CpG-motif engagement by TLR9.
2o Example 8. Antagonist definition It has been demonstrated that DNA uptake and endosomal maturation are required for signal initiation by CpG-DNA. It has been hypothesized that in order for DNA
to enter the endosomal/lysosomal compartment a non-CpG dependent uptake receptor may be required.
293 cells were transiently transfected with mTLR9 treated with either medium only or 1.0 ~,M
25 CpG-ODN 1668 (Figure 15). Additionally the 1668-treated TLR9 transfectants were simultaneously exposed to various doses of a non-CpG ODN (PZ2;
S'-CTCCTAGTGGGGGTGTCCTAT-3', SEQ ID N0:43). IL-8 production was monitored after 48h by ELISA. Figure 15 shows that PZ2, in a dose-dependent manner, was able to antagonize the activation of TLR9-transfected cells stimulated with a CpG ODN.
3o Figure 16 demonstrates that the stable TLR9 transfectants, 293-hTLR9-luc cells, are sensitive to non-CpG-ODN blockade. 293-hTLR9-luc cells were incubated with CpG-ODN

(0.5 ~,M) (black bars) or TNF-a (10 ng/ml) (gray bars) and increasing concentrations of a blocking ODN (5'-~THHHHHHHHHTi~IHHWGGGGG-3', SEQ ID N0:140; H = A, T, C; W
= A, T) as indicated. NF-xB activation was monitored after 12 hours and is presented as percent yields. Thus both mTLR9 and hTLR9 activity can be blocked by non-stimulatory ODN. The blockade is specific to blocking ODN since the TNF-driven NF-xB
signal was not diminished. Antagonism of CpG-DNA responses could thus be defined in stable TLR9 cells and therefore high throughput screening can be done for TLR9 antagonist.
Bafilomycin A poisons the proton pump needed for H'- transport into endosomes, which is required for endosomal maturation. Figure 17 shows that blockade of endosomal l0 maturation in 293-hTLR9-luc cells fully blocks CpG-ODN induction of NF-xB.
293-hTLR9-luc cells were preincubated with 10 nM Bafilomycin A (gray bars) or dimethylsulfoxide (DMSO) control (black bars) for 30 min and stimulated with CpG-ODN (2006, 0.5 ~,M), IL-1 (10 ng/ml) or TNF-oc (10 ng/ml) as indicated. NF-xB activation was monitored after 12 hours and is presented as percent yields. The blockade was specific to CpG-DNA
generated signal because both IL-1 and TNF induction of NF-~cB was unaffected. These data demonstrate that 293 cells stably complemented with hTLR9 behave in a manner similar to primary CpG-DNA
responsive cells, in that cellular uptake and endosomal maturation are required for induction of signal by CpG-DNA. Thus the stable transfectants can be used as indicator for TLR9 drug antagonist.
CpG-DNA signaling appears to occur via a Toll/IL-1R-like pathway. It was shown in the mouse that CpG-DNA signaling is dependent on MyD88, TR A_K_ and TRAF6.
Hacker H
et al., JExp Med 192:595-600 (2000). Hemmi et al. demonstrated that mTLR9-deficient mice lack activation of IRAN upon CpG-ODN stimulation. Hemmi H et al., Nature 408:740-5 (2000). Figure 18 shows that CpG-DNA signaling via human TLR9 was MyD88 dependent. hTLR9 (293-hTLR9) was co-transfected with a six-times NF-~cB
luciferase reporter plasmid and increasing concentrations of the dominant negative human MyD88 expression vector. Cells were not stimulated (filled circles), stimulated with CpG-ODN
(2006, 2wM) (open circles) or TNF-a (10 ng/ml) (filled triangles) and NF-xB
activation was monitored after 12 hours. Results are representative of at least two independent experiments.
Figure 18 demonstrates that dominant negative MyD88 blocks NF-xB induction in hTLR9 cells following CpG-DNA stimulation. The blockade of MyD88 did not affect NF-xB induction via TNF induced signal transduction. In general these data confirm the central role of MyD88 to TLR signaling and specifically the role of MyD88 in CpG-DNA
initiation of signal. Thus human cells transfected with TLR9 can be used as indicators to find molecules to antagonize CpG-DNA via genetic mechanisms.
Example 9. Antibody production Peptides for human and mouse TLR9 were designed for coupling to a carrier protein and injected into rabbits to obtain anti-peptide polyclonal antisera. Mouse peptide 1 (mousepepl, see Table 4) can be found in EST aa197442 and peptide 2 (mousepep2, see to Table 4) in EST aa273731 and ai463056. Human peptide 1 (humanpepl, see Table 4) and peptide 2 (humanpep2, see Table 4) were taken from the published human sequence.
Three rabbit antisera were generated by this method: anti-mousepep 1, specific for the extracellular domain of marine TLR9; anti-humanpepl, specific for the extracellular domain of hTLR9; and antisera against a combination of mousepep2 and humanpep2, specific for the 15 cytoplasmic domain of both marine and human TLR9. hnmunoprecipitates with anti-FLAG
antibody were electrophoresed by PAGE and, using standard Western blotting techniques, transferred to membrane and probed with the various antisera. Figure 19 shows the response to hTLR9-FLAG and mTLR9-FLAG. The TLR9 in these blots are indicated with arrows, while the lower molecular weight bands represent anti-FLAG antibody.
Example 10. Mutation adjacent to the CXXC-domain (hTLR9-CXXCm, mTLR9-CXXCh) The CXXC motif resembles a zinc finger motif and is found in DNA-binding proteins and in certain specific CpG binding proteins, e.g. methyl-CpG binding protein-1 (MBD-1).
Fujita N et al., Mol Cell Biol 20:5107-5118 (2000). Human and marine TLR9 contain two CXXC motifs. The CXXC domain is highly conserved between human and marine TLR9 but followed by 6 amino acids (aa) which differ quite substantially in polaxity and size. By the use of a site-specific mutagenesis kit (Stratagene, La Jolla, CA, USA) these six amino acid .
residues (human: PRHFPQ 269-274); mouse: GQKSLH 269-274) were interchanged between 3o human and marine TLR9. These mutations were generated by the use of the primers 5'-CTGCATGGAGTGCGGCCAAAAGTCCCTCCACCTACATCCCGATAC-3' (SEQ m N0:141) and 5'-GTATCGGGATGTAGGTGGAGGGACTTTTGGCCGCACTCCATGCAG-3' (SEQ ID
N0:142) for human TLR9 and the primers 5'-CTGTATAGAATGTCCTCGTCACTTCCCCCAGCTGCACCCTGAGAC-3' (SEQ ID
N0:143) and 5'-GTCTCAGGGTGCAGCTGGGGGAAGTGACGAGGACATTCTATACAG-3' (SEQ ID
N0:144) for marine TLR9 according to the manufacturer's protocol.
CXXC motif CXXCXXXXXXCXXC SEQ ID N0:145 1o Wildtype hTLR9:CRRCDHAPNPCMECPRHFPQ as 255-274 SEQ ID N0:146 hTLR9-CXXCm: CRRCDHAPNPCMECGQKSLH as 255-274 SEQ ID N0:147 Wildtype mTLR9: CRRCDHAPNPCMI CGQKSLH as 255-274 SEQ ID N0:148 .

mTLR9-CXXCh: CRRCDHAPNPCMICPRHFPQ as 255-274 SEQ ID N0:149 For the stimulation of the hTLR9 variant hTLR9-CXXCm, 293 cells were transiently transfected with hTLR9 or hTLR9-CXXCm and stimulated after 16 hours with ODN

and ODN 1668 at concentrations indicated (Figure 20). 48 hours after stimulation supernatant was harvested and IL-8 production was measured by ELISA. The data show that hTLR9 can be improved by converting the human CXXC domain to the marine CXXC
2o domain. For the stimulation of the mTLR9 variant mTLR9-CXXCh, 293 cells were transiently transfected with mTLR9 or mTLR9-CXXCh and stimulated after 16 hours with ODN 2006 and ODN 1668 at concentrations indicated (Figure 21). 48 hours after stimulation supernatant was harvested and IL-8 production was measured by ELISA. It appears that the human CXXC domain may diminish mTLR9-CXXCh activity relative to the wild type mTLR9.
Example 11. Mutation in the MBD motif (hTLR9-MBDmut, mTLR9-MBDmut) The MBD motif is a domain recently described for CpG binding in the protein MBD-1. Fujita N et al., Mol Cell Biol 20:5107-5118 (2000); Ohki I et al., EMBO J
18:6653-6661 (1999). Human and marine TLR9 contain this motif at position 524-554 and 525-555, respectively.

MBD-1 R-XXXXXXX-R-X-D-X-Y-XXXXXXXXX-R-S-XXXXXX-YSEQ ID NO:125 hTLR9 Q-XXXXXXX-K-X-D-X-Y-XXXXXXXXX-R-L-XXXXXX-YSEQID N0:126 mTLR9 Q-XXXXXXX-K-X-D-X-Y-XXXXXXXXX-Q-L-XXXXXX-YSEQID N0:127 The core of this domain consists of D-L-Y in human TLR9 (aa 534-536) and mouse TLR9 (aa 535-537). Through site-specific mutagenesis D534 and Y536 in human TLR9, and D535 and Y537 in marine TLR9, were mutated to alanines creating the sequence A-L-A for human (aa 534-536) and marine TLR9 (aa 535-537). These mutations were generated by the use of the 1o primers 5'-CACAATAAGCTGGCCCTCGCCCACGAGCACTC-3' (SEQ ID NO:150) and 5'-GAGTGCTCGTGGGCGAGGGCCAGCTTATTGTG-3' (SEQ ID N0:151) for human TLR9 and the primers 5'-CATAACAAACTGGCCTTGGCCCACTGGAAATC-3' (SEQ m N0:152) and 5'-GATTTCCAGTGGGCCAAGGCCAGTTTGTTATG-3' (SEQ ID N0:153) for marine TLR9 according to the manufacturer's protocol.
15 For the stimulation of mTLR9 variant, mTLR9-MBDmut, 293 cells were transiently transfected with mTLR9 or mTLR9-MBD-mat and stimulated after 16 hours with ODN

and ODN 1668 at concentrations indicated (Figure 22). 48 hours after stimulation supernatant was harvested and IL-8 production was measured by ELISA. For the stimulation of hTLR9 variant, hTLR9-MBDmut, 293 cells were transiently transfected with hTLR9 or 2o hTLR9-MBD-mat and stimulated after 16 hours with ODN 2006 and ODN 1668 at concentrations indicated (Figure 23). 48 hours after stimulation supernatant was harvested and IL-8 production was measured by ELISA. The disruption of the putative CpG
binding domain DXY in TLR9 destroyed receptor activity. These data demonstrate that the MBD
motif is most likely involved in CpG-DNA binding and can be thus be manipulated to better 25 understand CpG-DNA binding and efficacy.
Example 12. Proline to Histidine mutation in the TIR-domain (hTLR9-PHmut, mTLR9-PHmut) Toll-like receptors have a cytoplasmic Toll/IL-1 receptor (TIR) homology domain 3o which initiates signaling after binding of the adapter molecule MyD88.
Medzhitov R et al., Mol Cell 2:253-8 (1998); Kopp EB et al., Curr Opin Immunol 11:15-8 (1999).
Reports by others have shown that a single-point mutation in the signaling TIR-domain in marine TLR4 (Pro712 to His) or human TLR2 (Pro681 to His) abolishes host immune response to lipopolysaccharide or gram-positive bacteria, respectively. Poltorak A et aL, Science 282:2085-8 (1998); Underhill DM et al., Nature 401:811-5 (1999). Through site-specific mutagenesis the equivalent Proline at position 915 of human and marine TLR9 were mutated to Histidine (Pro915 to His). These mutations were generated by the use of the primers 5'-GCGACTGGCTGCATGGCAAAACCCTCTTTG-3' (SEQ ID N0:154) and 5'-CAAAGAGGGTTTTGCCATGCAGCCAGTCGC-3' (SEQ ID NO:155) for human TLR9 and the primers 5'-CGAGATTGGCTGCATGGCCAGACGCTCTTC-3' (SEQ 1D N0:156) to and 5'-GAAGAGCGTCTGGCCATGCAGCCAATCTCG-3' (SEQ ID N0:157) for marine TLR9 according to the manufacturer's protocol.
For the stimulation of mTLR9 variant, mTLR9-PHmut, 293 cells were transiently transfected with mTLR9 or mTLR9-PHmut and stimulated after 16 hours with ODN

and ODN 1668 at concentrations indicated (Figure 22). 48 hours after stimulation is supernatant was harvested and IL-8 production was measured by ELISA. For the stimulation of hTLR9 variant, hTLR9-PHmut, 293 cells were transiently transfected with hTLR9 or hTLR9-PHmut and stimulated after 16 hours with ODN 2006 and ODN 1668 at concentrations indicated (Figure 23). 48 hours after stimulation supernatant was harvested and IL-8 production was measured by ELISA. These data demonstrate that TLR9 activity can 20 be destroyed by the Pro to His mutation. This mutation has the potential to be used as a dominant negative to block TLR9 activity thus a genetic variant could compete for ligand or signaling partners and disrupt signaling.
Example 13. Exchange of the TIR-domain between marine and human TLR9 (hTLR9-25 TIRm, mTLR9-TIRh) Toll-like receptors have a cytoplasmic Toll/IL-1 receptor (TIR) homology domain that initiates signaling after binding of the adapter molecule MyD88. Medzhitov R
et al., Mol Cell 2:253-8 (1998); Kopp EB et al., Curr Opin Immunol 11:15-8 (1999). This is also true for TLR9. To generate molecules consisting of human extracellular TLR9 and marine TIR
30 domain (hTLR9-TIRm) or marine extracellular TLR9 and human TIR domain (mTLR9-TIRh), the following approach was chosen. Through site-specific mutagenesis a ClaI

restriction site was introduced in human and marine TLR9. For human TLR9 the DNA
sequence 5'-GGCCTCAGCATCTTT-3' (3026-3040, SEQ 1D N0:158) was mutated to 5'-GGCCTATCGATTTTT-3' (SEQ ID NO:I59), introducing a CIaI site (underlined in the sequence) but leaving the amino acid sequence (GLSIF, as 798-802) unchanged.
For marine TLR9 the DNA sequence 5'-GGCCGTAGCATCTTC-3' (2434-2447, SEQ ID N0:160) was mutated to 5'-GGCCTATCGATTTTT-3' (SEQ 1D N0:161), introducing a CIaI site and creating the amino acid sequence (GLSIF, as 799-803) which differs in one position (aa 800) from the wil'dtype marine TLR9 sequence (GRS1F, as 799-803) but is identical to the human sequence.
l0 hTLR9-TIRm. The primers used for human TLR9 were 5'-CAGCTCCAGGGCCTATCGATTTTTGCACAGGACC-3' (SEQ ID N0:162) and 5'-GGTCCTGTGCAAAAATCGATAGGCCCTGGAGCTG-3' (SEQ ID N0:163). For creating an expression vector containing the extracellular portion of human TLR9 connected to the marine TIR domain, the human expression vector was cut with CIaI and limiting amounts of i5 EcoRI and the fragment coding for the marine TIR domain generated by a ClaI
and EcoRI
digestion of marine TLR9 expression vector was ligated in the vector fragment containing the extracellular portion of hTLR9. Transfection into E. coli yielded the expression vector hTLR9-TlRm (human extracellular TLR9-marine TIR-domain).
mTLR9-TIRh. The primers used for marine TLR9 were 5'-2o CAGCTGCAGGGCCTATCGATTTTCGCACAGGACC-3' (SEQ ID N0:164) and 5'-GGTCCTGTGCGAAAATCGATAGGCCCTGCAGCTG-3' (SEQ ID N0:165). For creating an expression vector containing the extracellular portion of marine TLR9 connected to the human T1R domain, the marine expression vector was cut with CIaI and limiting amounts of EcoRI and the fragment coding for the human T1R domain generated by a CIaI and EcoRI
25 digestion of human TLR9 expression vector was ligated in the vector fragment containing the extracellular portion of mTLR9. Transfection into E. coli yielded the expression vector mTLR9-TIRh (marine extracellular TLR9-human TlR-domain).
For the stimulation of the mTLR9 variant, mTLR9-TIRh, 293 cells were transiently transfected with mTLR9 or mTLR9-TIRh and stimulated after 16 hours with ODN
2006 and 3o ODN 1668 at concentrations indicated (Figure 24). 48 hours after stimulation supernatant was harvested and IL-8 production was measured by ELISA. For the stimulation of the hTLR9 variant, hTLR9-TIRm, 293 cells were transiently transfected with hTLR9 or hTLR9-TIRm and stimulated after 16 hours with ODN 2006 and ODN 1668 at concentrations indicated (Figure 25). 48 hours after stimulation supernatant was harvested and IL-8 production was measured by ELISA. Replacement of the marine TLR9-TIR domain with human does not significantly affect mTLR9 activity. Replacement of the human with marine however appears to have a negative effect on hTLR9. These data demonstrate that manipulations could be made to influence TLR9 activities.
Example 14. TLR9-fusion protein with green-fluorescent-protein (hTLR9-GFP, mTLR9-GFP) Human and marine TLR9 were individually cloned into the vector pEGFP-Nl (Clontech, Palo Alto, CA, USA) to create expression vectors encoding human and marine fusion proteins consisting of an N-terminal TLR9 protein fused to C-terminal green-fluorescent protein (GFP). These constructs can be used to trace TLR9 localization and expression. Such detections can be used for staining in FACS analysis, confocal microscopy and Western blot, or for purification of polypeptides and subsequent antibody production.
Example 15. TLR9-fusion protein with FLAG-peptide (hTLR9-FLAG, mTLR9-FLAG) Human and marine TLR9 were individually cloned into the vector pFLAG-CMV-1 (Sigma, St. Louis, MO, USA) to create expression vectors encoding human and marine fusion proteins consisting of an N-terminal leader peptide (preprotrypsin, which is cleaved intracellularly during processing of the protein), FLAG-peptide (DYKDDDDI~) and TLR9 protein which does not contain its own signal peptide. These constructs can be used to trace TLR9 localization and expression, e.g., using anti-FLAG antibodies. Such detections can be used for staining in FACS analysis, confocal microscopy and Western blot, or for purification of polypeptides and subsequent antibody production.
Example 16. Method of cloning human TLR7 Two accession numbers in the GenBanl~ database, AF245702 and AF240467, describe the DNA sequence for human TLR7. To create an expression vector for human TLR7, human TLR7 cDNA was amplified from a cDNA made from human peripheral mononuclear blood cells (PBMC) using the primers 5'-CACCTCTCATGCTCTGCTCTCTTC-3' (SEQ ID
N0:166) and 5'-GCTAGACCGTTTCCTTGAACACCTG-3' (SEQ ID N0:167). The fragment was cloned into pGEM-T Easy vector (Promega), cut with the restriction enzyme NotI and ligated into a NotI-digested pCDNA3.1 expression vector (Invitrogen).
The insert was fully sequenced and translated into protein. The cDNA sequence for hTLR7 is SEQ ID
N0:168, is presented in Table 6. The open reading frame starts at base 124, ends at base 3273, and codes for a protein of 1049 amino acids. SEQ ID N0:169 (Table 7), corresponding to bases 124-3273 of SEQ ID N0:168 (Table 6), is the coding region for the polypeptide of SEQ ID N0:170 (Table 8).
1o The protein sequence of the cloned hTLR7 cDNA matches the sequence described under the GenBank accession number AF240467. The sequence deposited under GenBank accession number AF245702 contains two amino acid changes at position 725 (L
to H) and 738 (L to P). ' Table 6. cDNA Sequence for Human TLR7 (5' to 3'; SEQ ID N0:168) agctggctag cgtttaaacgggccctctagactcgagcggccgcgaattcactagtgatt60 CaCCtCtCat gCtCtgCtCtCttCaaCCagaCCtCtaCattCCattttggaagaagacta120 aaaatggtgt ttccaatgtggacactgaagagacaaattcttatcctttttaacataatc180 ctaatttcca aactccttggggctagatggtttcctaaaactctgccctgtgatgtcact240 ctggatgttc caaagaaccatgtgatcgtggactgcacagacaagcatttgacagaaatt300 cctggaggta ttcccacgaacaccacgaacctcaccctcaccattaaccacataccagac360 atctccccag cgtcctttcacagactggaccatctggtagagatcgatttcagatgcaac420 tgtgtaccta ttccactggggtcaaaaaacaacatgtgcatcaagaggctgcagattaaa480 cccagaagct ttagtggactcacttatttaaaatccctttacctggatggaaaccagcta540 ctagagatac cgcagggcctcccgcctagcttacagcttctcagccttgaggccaacaac600 atcttttcca tcagaaaagagaatctaacagaactggccaacatagaaatactctacctg660 ggccaaaact gttattatcgaaatccttgttatgtttcatattcaatagagaaagatgcc720 ttcctaaact tgacaaagttaaaagtgctctccctgaaagataacaatgtcacagccgtc780 cctactgttt tgccatctactttaacagaactatatctctacaacaacatgattgcaaaa840 atccaagaag atgattttaataacctcaaccaattacaaattcttgacctaagtggaaat900 tgccctcgtt gttataatgccccatttccttgtgcgccgtgtaaaaataattctccccta960 cagatccctg taaatgcttttgatgcgctgacagaattaaaagttttacgtctacacagt1020 aactctcttc agcatgtgcccccaagatggtttaagaacatcaacaaactccaggaactg1080 gatctgtccc aaaacttcttggccaaagaaattggggatgctaaatttctgcattttctc1140 cccagcctca tccaattggatctgtctttcaattttgaacttcaggtcta~tcgtgcatct1200 atgaatctat cacaagcattttcttcactgaaaagcctgaaaattctgcggatcagagga1260 tatgtcttta aagagttgaaaagctttaacctctcgccattacataatcttcaaaatctt1320 gaagttcttg atcttggcactaactttataaaaattgctaacctcagcatgtttaaacaa1380 tttaaaagac tgaaagtcatagatctttcagtgaataaaatatcaccttcaggagattca1440 agtgaagttg gcttctgctcaaatgccagaacttctgtagaaagttatgaaccccaggtc1500 ctggaacaat tacattatttcagatatgataagtatgcaaggagttgcagattcaaaaac1560 aaagaggctt ctttcatgtctgttaatgaaagctgctacaagtatgggcagaccttggat1620 ctaagtaaaa atagtatattttttgtcaagtcctctgattttcagcatctttctttcctc1680 aaatgcctga atctgtcaggaaatctcattagccaaactcttaatggcagtgaattccaa1740 cctttagcag agctgagatatttggacttctccaacaaccggcttgatttactccattca1800 acagcatttg aagagcttcacaaactggaagttctggatataagcagtaatagccattat1860 tttcaatcag aaggaattactcatatgctaaactttaccaagaacctaaaggttctgcag1920 aaactgatga tgaacgacaatgacatctcttcctccaccagcaggaccatggagagtgag1980 tctcttagaa ctctggaattcagaggaaatcacttagatgttttatggagagaaggtgat2040 aacagatact tacaattattcaagaatctgctaaaattagaggaattagacatctctaaa2100 aattccctaa gtttcttgccttctggagtttttgatggtatgcctccaaatctaaagaat2160 ctctctttgg ccaaaaatgggctcaaatctttcagttggaagaaactccagtgtctaaag2220 aacctggaaa ctttggacctcagccacaaccaactgaccactgtccctgagagattatcc2280 aactgttcca gaagcctcaagaatctgattcttaagaataatcaaatcaggagtctgacg2340 aagtattttc tacaagatgccttccagttgcgatatctggatctcagctcaaataaaatc2400 cagatgatcc aaaagaccagcttcccagaaaatgtcctcaacaatctgaagat~'ttgctt2460 ttgcatcata atcggtttctgtgcacctgtgatgctgtgtggtttgtctggtgggttaac2520 catacggagg tgactattccttacctggccacagatgtgacttgtgtggggccaggagca2580 cacaagggcc aaagtgtgatctccctggat.ctgtacacctgtgagttagatctgactaac2640 ctgattctgt tctcactttccatatctgtatctctctttctcatggtgatgatgacagca2700 agtcacctct atttctgggatgtgtggtatatttaccatttctgtaaggccaagataaag2760 gggtatcagc gtctaatatcaccagactgttgctatgatgcttttattgtgtatgacact2820 aaagacccag ctgtgaccgagtgggttttggctgagctggtggccaaactggaagaccca2880 agagagaaac attttaatttatgtctcgaggaaagggactggttaccagggcagccagtt2940 ctggaaaacc tttcccagagcatacagcttagcaaaaagacagtgtttgtgatgacagac3000 aagtatgcaa agactgaaaattttaagatagcattttacttgtcccatcagaggctcatg3060 gatgaaaaag ttgatgtgattatcttgatatttcttgagaagccttttcagaagtccaag3120 ttcctccagc tccggaaaaggctctgtgggagttctgtccttgagtggccaacaaacccg3180 caagctcacc catacttctggcagtgtctaaagaacgccctggccacagacaatcatgtg3240 gcctatagtc aggtgttcaaggaaacggtctagaatcgaattcccgcggccgccactgtg3300 ctggatatct gcagaattccaccacactggactagtggatccgagctcggtaccaagctt3360 aagtttaaac cgc 3373 Table 7. Coding Region for Human TLR7 (5' to 3'; SEQ ID N0:169) atggtgtttc caatgtggac actgaagaga caaattctta tcctttttaa cataatccta 60 atttccaaac tccttggggc tagatggttt cctaaaactc tgccctgtga tgtcactctg 120 gatgttccaa agaaccatgt gatcgtggac tgcacagaca agcatttgac agaaattcct 180 ggaggtattc ccacgaacac cacgaacctc accctcacca ttaaccacat accagacatc 240 tccccagcgt cctttcacagactggaccatctggtagagatcgatttcagatgcaactgt300 gtacctattc cactggggtcaaaaaacaacatgtgcatcaagaggctgcagattaaaccc360 agaagcttta gtggactcacttatttaaaatccctttacctggatggaaaccagctacta420 gagataccgc agggcctcccgcctagcttacagcttctcagccttgaggccaacaacatc480 ttttccatca gaaaagagaatctaacagaactggccaacatagaaatactctacctgggc540 caaaactgtt attatcgaaatccttgttatgtttcatattcaatagagaaagatgccttc600 ctaaacttga caaagttaaaagtgctctccctgaaagataacaatgtcacagccgtccct660 actgttttgc catctactttaacagaactatatctctacaacaacatgattgcaaaaatc720 caagaagatg attttaataacctcaaccaattacaaattcttgacctaagtggaaattgc780 cctcgttgtt ataatgccccatttccttgtgcgccgtgtaaaaataattctcccctacag840 atccctgtaa atgcttttgatgcgctgacagaattaaaagttttacgtctacacagtaac900 tctcttcagc atgtgcccccaagatggtttaagaacatcaacaaactccaggaactggat960 ctgtcccaaa acttcttggccaaagaaattggggatgctaaatttctgcattttctcccc1020 agcctcatcc aattggatctgtctttcaattttgaacttcaggtctatcgtgcatctatg1080 aatctatcac aagcattttcttcactgaaaagcctgaaaattctgcggatcagaggatat1140 gtctttaaag agttgaaaagctttaacctctcgccattacataatcttcaaaatcttgaa1200 gttcttgatc ttggcactaactttataaaaattgctaacctcagcatgtttaaacaattt1260 aaaagactga aagtcatagatctttcagtgaataaaatatcaccttcaggagattcaagt1320 gaagttggct tctgctcaaatgccagaacttctgtagaaagttatgaaccccaggtcctg1380 gaacaattac attatttcagatatgataagtatgcaaggagttgcagattcaaaaacaaa1440 gaggcttctt tcatgtctgttaatgaaagctgctacaagtatgggcagaccttggatcta1500 agtaaaaata gtatattttttgtcaagtcctctgattttcagcatctttctttcctcaaa1560 tgcctgaatc tgtcaggaaatctcattagccaaactcttaatggcagtgaattccaacct1620 ttagcagagc tgagatatttggacttctccaacaaccggcttgatttactccattcaaca1680 gcatttgaag agcttcacaaactggaagttctggatataagcagtaatagccattatttt1720 caatcagaag gaattactcatatgctaaactttaccaagaacctaaaggttctgcagaaa1800 ctgatgatga acgacaatgacatctcttcctccaccagcaggaccatggagagtgagtct1860 cttagaactc tggaattcagaggaaatcacttagatgttttatggagagaaggtgataac1920 agatacttac aattattcaagaatctgctaaaattagaggaattagacatctctaaaaat1980 tccctaagtt tcttgccttctggagtttttgatggtatgcctccaaatctaaagaatctc2040 tctttggcca aaaatgggctcaaatctttcagttggaagaaactccagtgtctaaagaac2100 ctggaaactt tggacctcagccacaaccaactgaccactgtccctgagagattatccaac2160 tgttccagaa gcctcaagaatctgattcttaagaataatcaaatcaggagtctgacgaag2220 tattttctac aagatgccttccagttgcgatatctggatctcagctcaaataaaatccag2280 atgatccaaa agaccagcttcccagaaaatgtcctcaacaatctgaagatgttgcttttg2340 catcataatc ggtttctgtgcacctgtgatgctgtgtggtttgtctggtgggttaaccat2400 acggaggtga ctattccttacctggccacagatgtgacttgtgtggggccaggagcacac2460 aagggccaaa gtgtgatctccctggatctgtacacctgtgagttagatctgactaacctg2520 attctgttct cactttccatatctgtatctctctttctcatggtgatgatgacagcaagt2580 cacctctatt tctgggatgtgtggtatatttaccatttctgtaaggccaagataaagggg2640 tatcagcgtc taatatcaccagactgttgctatgatgcttttattgtgtatgacactaaa2700 gacccagctg tgaccgagtgggttttggctgagctggtggccaaactggaagacccaaga2760 gagaaacatt ttaatttatg tctcgaggaa agggactggt taccagggca gccagttctg 2820 gaaaaccttt cccagagcat acagcttagc aaaaagacag tgtttgtgat gacagacaag 2880 tatgcaaaga ctgaaaattt taagatagca ttttacttgt cccatcagag gctcatggat 2940 gaaaaagttg atgtgattat cttgatattt cttgagaagc cttttcagaa gtccaagttc 3000 ctccagctcc ggaaaaggct ctgtgggagt tctgtccttg agtggccaac aaacccgcaa 3060 gCtC3CCCat acttctggca gtgtctaaag aacgccctgg ccacagacaa tcatgtggcc 3120 tatagtcagg tgttcaagga aacggtc 3147 Table 8. Amino Acid Sequence of Human TLR7 . . . . . . . . . . . . 60 AF240467.pep MVFPMWTLKRQILILFNIILISKLLGARWFPKTLPCDVTLDVPKNHVIVDCTDKHLTEIP 60 hTLR7.pep MVFPMWTLKRQILILFNIILISKLLGARWFPKTLPCDVTLDVPKNHVIVDCTDKHLTEIP 60 AF245702.pep MVFPMWTLKRQTLILFNIILISKLLGARWFPKTLPCDVTLDVPKNHVIVDCTDKHLTEIP 60 . . . . . . . . . . . . 120 AF240467.pep GGIPTNTTNLTLTTNHIPDTSPASFHRLDHLVEIDFRCNCVPIPLGSKNNMCIKRLQIKP 120 hTLR7.pep GGIPTNTTNLTLTINHIPDISPASFHRLDHLVEIDFRCNCVPIPLGSKNNMCIKRLQIKP 120 AF245702.pep GGIPTNTTNLTLTINHIPDISPASFHRLDHLVEIDFRCNCVPIPLGSKNNMCIKRLQIKP 120 . . . . . . . . . . . . 180 AF240467.pep RSFSGLTYLKSLYLDGNQLLEIPQGLPPSLQLLSLEANNIFSIRKENLTELANIEILYLG 180 hTLR7.pep RSFSGLTYLKSLYLDGNQLLEIPQGLPPSLQLLSLEANNIFSIRKENLTELANIEILYLG l80 AF245702.pep RSFSGLTYLKSLYLDGNQLLEIPQGLPPSLQLLSLEANNIFSIRKENLTELANIEILYLG 180 2$ . . . . . . . . . . . . 240 AF240467.pep QNCYYRNPCYVSYSIEKDAFLNLTKLKVLSLKDNNVTAVPTVLPSTLTELYLYNNMIAKI 240 hTLR7.pep QNCYYRNPCYVSYSIEKDAFLNLTKLKVLSLKDNNVTAVPTVLPSTLTELYLYNNMIAKI 240 AF245702.pep QNCYYRNPCYVSYSIEKDAFLNLTKLKVLSLKDNNVTAVPTVLPSTLTELYLYNNMIAKI 240 . . . . . . . . . . . . 300 AF240467.pep QEDDFNNLNQLQILDLSGNCPRCYNAPFPCAPCKNNSPLQIPVNAFDALTELKVLRLHSN 300 hTLR7.pep QEDDFNNLNQLQILDLSGNCPRCYNAPFPCAPCKNNSPLQIPVNAFDALTELKVLRLHSN 300 AF245702.pep QEDDFNNLNQLQILDLSGNCPRCYNAPFPCAPCKNNSPLQIPVNAFDALTELKVLRLHSN 300 . . . . . . . . . . . . 360 AF240467.pep SLQHVPPRWFKNINKLQELDLSQNFLAKEIGDAKFLHFLPSLIQLDLSFNFELQVYRASM 360 hTLR7.pep SLQHVPPRWFKNINKLQELDLSQNFLAKEIGDAKFLHFLPSLIQLDLSFNFELQVYRASM 360 AF245702.pep SLQHVPPRWFKNINKLQELDLSQNFLAKEIGDAKFLHFLPSLIQLDLSFNFELQVYRASM 360 4~ . . . . . . , . . . . . 420 AF240467.pep NLSQAFSSLKSLKILRIRGYVFKELKSFNLSPLHNLQNLEVLDLGTNFIKIANLSMFKQF 420 hTLR7.pep NLSQAFSSLKSLKILRIRGYVFKELKSFNLSPLHNLQNLEVLDLGTNFIKIANLSMFKQF 420 AF245702.pep NLSQAFSSLKSLKILRIRGYVFKELKSFNLSPLHNLQNLEVLDLGTNFIKIANLSMFKQF 420 . . . . . . . . . . . . 4so AF240467.pep KRLKVIDLSVNKISPSGDSSEVGFCSNARTSVESYEPQVLEQLHYFRYDKYARSCRFKNK 480 hTLR7.pep KRLKVIDLSVNKISPSGDSSEVGFCSNARTSVESYEPQVLEQLHYFRYDKYARSCRFKNK 480 AF245702.pep KRLKVIDLSVNKISPSGDSSEVGFCSNARTSVESYEPQVLEQLHYFRYDKYARSCRFKNK 480 . . . . . . . . . . . . 540 AF240467.pep EASFMSVNESCYKYGQTLDLSKNSIFFVKSSDFQHLSFLKCLNLSGNLISQTLNGSEFQP 540 hTLR7.pep EASFMSVNESCYKYGQTLDLSKNSIFFVKSSDFQHLSFLKCLNLSGNLISQTLNGSEFQP 540 AF245702.pep EASFMSVNESCYKYGQTLDLSKNSIFFVKSSDFQHLSFLKCLNLSGNLISQTLNGSEFQP 540 . . . . . . . . . . . . 600 AF240467.pep LAELRYLDFSNNRLDLLHSTAFEELHKLEVLDISSNSHYFQSEGITHMLNFTKNLKVLQK600 hTLR7.pep LAELRYLDFSNNRLDLLHSTAFEELHKLEVLDISSNSHYFQSEGITHMLNFTKNLKVLQK600 AF245702.pep LAELRYLDFSNNRLDLLHSTAFEELHKLEVLDISSNSHYFQSEGITHMLNFTKNLKVLQK600 . . . . . . . . . . . . 660 AF240467.pep LMMNDNDISSSTSRTMESESLRTLEFRGNHLDVLWREGDNRYLQLFKNLLKLEELDISKN 660 hTLR7.pep LMMNDNDISSSTSRTMESESLRTLEFRGNHLDVLWREGDNRYLQLFKNLLKLEELDISKN 660 AF245702.pep LMMNDNDISSSTSRTMESESLRTLEFRGNHLDVLWREGDNRYLQLFKNLLKLEELDISKN 660 . . . . . . . . . . . . 720 AF240467.pep SLSFLPSGVFDGMPPNLKNLSLAKNGLKSFSWKKLQCLKNLETLDLSHNQLTTVPERLSN720 hTLR7.pep SLSFLPSGVFDGMPPNLKNLSLAKNGLKSFSWKKLQCLKNLETLDLSHNQLTTVPERLSN720 AF245702.pep SLSFLPSGVFDGMPPNLKNLSLAKNGLKSFSWKKLQCLKNLETLDLSHNQLTTVPERLSN720 . . . . . . . . . . . . 7so AF240467.pep CSRSLKNLILKNNQIRSLTKYFLQDAFQLRYLDLSSNKIQMIQKTSFPENVLNNLKMLLL 780 hTLR7.pep CSRSLKNLILKNNQIRSLTKYFLQDAFQLRYLDLSSNKIQMIQKTSFPENVLNNLKMLLL 780 AF245702.pep CSRSHKNLILKNNQIRSPTKYFLQDAFQLRYLDLSSNKIQMIQKTSFPENVLNNLKMLLL 780 . . . . . . . . . . . . e4o AF240467.pep HHNRFLCTCDAVWFVWWVNHTEVTIPYLATDVTCVGPGAHKGQSVISLDLYTCELDLTNL 840 hTLR7.pep HHNRFLCTCDAVWFVWWVNHTEVTIPYLATDVTCVGPGAHKGQSVISLDLYTCELDLTNL 840 AF245702.pep HHNRFLCTCDAVWFVWWVNHTEVTIPYLATDVTCVGPGAHKGQSVISLDLYTCELDLTNL 840 . . . . . . . . . . . . 900 AF240467.pep ILFSLSISVSLFLMVMMTASHLYFWDVWYIYHFCKAKIKGYQRLISPDCCYDAFIVYDTK 900 hTLR7.pep ILFSLSISVSLFLMVMMTASHLYFWDVWYIYHFCKAKIKGYQRLISPDCCYDAFIVYDTK 900 AF245702.pep ILFSLSISVSLFLMVMMTASHLYFWDVWYIYHFCKAKIKGYQRLISPDCCYDAFIVYDTK 900 . . . . . . . . . . . . 960 AF240467.pep DPAVTEWVLAELVAKLEDPREKHFNLCLEERDWLPGQPVLENLSQSIQLSKKTVFVMTDK 960 hTLR7.pep DPAVTEWVLAELVAKLEDPREKHFNLCLEERDWLPGQPVLENLSQSIQLSKKTVFVMTDK 960 AF245702.pep DPAVTEWVLAELVAKLEDPREKHFNLCLEERDWLPGQPVLENLSQSIQLSKKTVFVMTDK 960 . . . . . . . . . . . . 1020 AF240467.pep YAKTENFKIAFYLSHQRLMDEKVDVIILIFLEKPFQKSKFLQLRKRLCGSSVLEWPTNPQ 1020 hTLR7.pep YAKTENFKIAFYLSHQRLMDEKVDVIILIFLEKPFQKSKFLQLRKRLCGSSVLEWPTNPQ 1020 AF245702.pep YAKTENFKIAFYLSHQRLNmEKVDVTILIFLEKPFQKSKFLQLRKRLCGSSVLEWPTNPQ 1020 AF240467.pep AHPYFWQCLKNALATDNHVAYSQVFKETV 1049 hTLR7.pep AHPYFWQCLKNALATDNHVAYSQVFKETV 1049 AF245702.pep AHPYFWQCLKNALATDNHVAYSQVFKETV 1049 In Table 8 the sequences are assigned as follows: hTLR7.pep, SEQ ID N0:170;
to AF240467.pep, SEQ ID N0:171; AF245702.pep, SEQ ID N0:172.
Example 17. Method of cloning the marine TLR7 Alignment of human TLR7 protein sequence with mouse EST database using tfasta yielded 4 hits with mouse EST sequences bb116163, aa266744, bb210780 and aa276879.
Two primers were designed that bind to aa266744 sequence for use in a RACE-PCR
to amplify 5' and 3' ends of the marine TLR7 cDNA. The library used for the RACE
PCR was a mouse spleen marathon-ready cDNA commercially available from Clontech. A 5' fragment with a length of 3000 by obtained by this method was cloned into Promega pGEM-T Easy vector. After sequencing of the 5' end, additional primers were designed for amplification of the complete marine TLR7 cDNA. The primer for the 5' end was obtained from the sequence of the 5' RACE product whereas the primer for the 3' end was selected from the mouse EST
sequence aa266744.
Three independent PCR reactions were set up using a marine macrophage RAW264.7 (ATCC TIB-71) cDNA as a template with the primers 5'-CTCCTCCACCAGACCTCTTGATTCC-3' (SEQ ID NO:208) and 5'-CAAGGCATGTCCTAGGTGGTGACATTC-3' (SEQ ID N0:209). The resulting amplification products were cloned into pGEM-T Easy vector and fully sequenced (SEQ ID
N0:173; Table 9). The open reading frame of mTLR7 (SEQ ID NO:174; Table 10) starts at base 49, ends at base 3201 and codes for a protein of 1050 amino acids (SEQ ID
NO:175;
Tablell). To create an expression vector for marine TLR7 cDNA, pGEM-T Easy vector plus mTLR7 insert was cut with NotI, the fragment isolated and ligated into a NotI
digested pCDNA3.1 expression vector (Invitrogen).
Table 9. cDNA Sequence for Marine TLR7 (5' to 3'; SEQ ID N0:173) - 11~ -AACTGAAAAT

TTCCGTAGGC TGAACCATCTGGAAGAAATC~GATTTAAGATGCAATTGTGTACCTGTTCTA360 AATGTCCCAT ATCCGTGTACACCGTGTGAAAATAATTCCQ'CCTTACAGATCCATGACAAT900 m s GTGACAGAAT GGGTTTTGCA GGAGCTGGTG GCAAAATTGG AAGATCCAAG AGAAAAACAC 2820 1s Table 10. Coding Region for Murine TLR7 (5' to 3'; SEQ ID N0:174) TATGCTCTTA

2s CAAAACTGTT ATTATCGAAATCCTTGCAATGTTTCCTATTCTATTGAAAAAGATGCTTTC600 3s GTCTTTAAAG AGCTGAAAAACTCCAGTCTTTCTGTATTGCACAAGCTTCCCAGGCTGGAA1200 AAACACCATT

Table 11. Amino Acid Sequences of Murine TLR7 and Human TLR7 . . . . . . . . . . . . 60 hTLR7.pep MVFPMWTLKRQILILFNIILISKLLGARWFPKTLPCDVTLDVPKNHVIVDCTDKHLTEIP 60 mTLR7.pep MVFSMWTRKRQILIFLNMLLVSRVFGFRWFPKTLPCEVKVNIPEAHVIVDCTDKHLTEIP 60 hTLR7.pep GGIPTNTTNLTLTINHhPDISPASFHRLDHLVEIDFRCNCVPIPLGSKNNMCIKRLQIKP 120 mTLR7.pep EGIPTNTTNLTLTINHIPSISPDSFRRLNHLEEIDLRCNCVPVLLGSKANVCTKRLQIRP 120 hTLR7.pep RSFSGLTYLKSLYLDGNQLLEIPQGLPPSLQLLSLEANNIFSIRKENLTELANIEILYLG 180 mTLR7.pep GSFSGLSDLKALYLDGNQLLEIPQDLPSSLHLLSLEANNIFSITKENLTELVNIETLYLG 180 hTLR7.pep QNCYYRNPCYVSYSIEKDAFLNLTKLKVLSLKDNNVTAVPTVLPSTLTELYLYNNMIAKI 240 mTLR7.pep QNCYYRNPCNVSYSIEKDAFLVMRNLKVLSLKDNNVTAVPTTLPPNLLELYLYNNIIKKI 240 hTLR7.pep QEDDFNNLNQLQILDLSGNCPRCYNAPFPCAPCKNNSPLQIPVNAFDALTELKVLRLHSN 300 mTLR7.pep QENDFNNLNELQVLDLSGNCPRCYNVPYPCTPCENNSPLQIHDNAFNSLTELKVLRLHSN 300 hTLR7.pep SLQHVPPRWFKNINKLQELDLSQNFLAKEIGDAKFLHFLPSLIQLDLSFNFELQVYRASM 360 mTLR7.pep SLQHVPPTWFKNMRNLQELDLSQNYLAREIEEAKFLHFLPNLVELDFSFNYELQVYHASI 360 hTLR7.pep NLSQAFSSLKSLKILRIRGYVFKELKSFNLSPLHNLQNLEVLDLGTNFIKIANLSMFKQF 420 mTLR7.pep TLPHSLSSLENLKILRVKGYVFKELKNSSLSVLHKLPRLEVLDLGTNFIKIADLNIFKHF 420 . . . . . . . . . . . . 480 hTLR7.pep KRLKVIDLSVNKISPSGDSSEVGFCSNARTSVESYEPQVLEQLHYFRYDKYARSCRFKNK 480 mTLR7.pep ENLKLIDLSVNKISPSEESREVGFCPNAQTSVDRHGPQVLEALHYFRYDEYARSCRFKNK 480 hTLR7.pep EA-SFMSVNESCYKYGQTLDLSKNSIFFVKSSDFQHLSFLKCLNLSGNLISQTLNGSEFQ 539 mTLR7.pep EPPSFLPLNADCHIYGQTLDLSRNNIFFIKPSDFQHLSFLKCLNLSGNTIGQTLNGSELW 540 hTLR7.pep PLAELRYLDFSNNRLDLLHSTAFEELHKLEVLDISSNSHYFQSEGITHMLNFTKNLKVLQ 599 mTLR7.pep PLRELRYLDFSNNRLDLLYSTAFEELQSLEVLDLSSNSHYFQAEGITHMLNFTKKLRLLD 600 hTLR7.pep KLMMNDNDISSSTSRTMESESLRTLEFRGNHLDVLWREGDNRYLQLFKNLLKLEELDISK 659 mTLR7.pep KLMMNDNDISTSASRTMESDSLRILEFRGNHLDVLWRAGDNRYLDFFKNLFNLEVLDISR 660 hTLR7.pep NSLSFLPSGVFDGMPPNLKNLSLAKNGLKSFSWKKLQCLKNLETLDLSHNQLTTVPERLS 719 mTLR7.pep NSLNSLPPEVFEGMPPNLKNLSLAKNGLKSFFWDRLQLLKHLEILDLSHNQLTKVPERLA 720 . . . . . . . . . . . . 7so hTLR7.pep NCSRSLKNLILKNNQIRSLTKYFLQDAFQLRYLDLSSNKIQMIQKTSFPENVLNNLKMLL 779 mTLR7.pep NCSKSLTTLILKHNQIRQLTKYFLEDALQLRYLDISSNKIQVIQKTSFPENVLNNLEMLV 780 hTLR7.pep LHHNRFLCTCDAVWFVWWVNHTEVTIPYLATDVTCVGPGAHKGQSVISLDLYTCELDLTN 839 mTLR7.pep LHHNRFLCNCDAVWFVWWVNHTDVTIPYLATDVTCVGPGAHKGQSVISLDLYTCELDLTN 840 hTLR7.pep LILFSLSISVSLFLMVMMTASHLYFWDVWYIYHFCKAKIKGYQRLISPDCCYDAFIVYDT 899 mTLR7.pep LILFSVSISSVLFLMVVMTTSHLFFWDMWYIYYFWKAKIKGYQHLQSMESCYDAFIVYDT 900 hTLR7.pep KDPAVTEWVLAELVAKLEDPREKHFNLCLEERDWLPGQPVLENLSQSIQLSKKTVFVMTD959 mTLR7.pep KNSAVTEWVLQELVAKLEDPREKHFNLCLEERDWLPGQPVLENLSQSIQLSKKTVFVMTQ960 $ . . . . . . . . . . . . 1020 bb210788.pep VDVIILIFLVKPFQKFNFL*LRKRISRSSVLECPPNP 37 aa276879.pep QKSKFLQLRKRLCRSSVLEWPANP 24 aa266744.pep LGKPLQKSKFLQLRKRLCRSSVLEWPANP 29 bb116163.pep IETFQMPSFLSIQRLLDDKVDVIILIFLE*PL*KSKFLQLRKRFCRSSVLEWPANP56 hTLR7.pep KYAKTENFKIAFYLSHQRLMDEKVDVTTLIFLEKPFQKSKFLQLRKRLCGSSVLEWPTNP1019 mTLR7.pep KYAKTESFKMAFYLSHQRLLDEKVDVIILIFLEKPLQKSKFLQLRKRLCRSSVLEWPANP1020 bb210788.pep QAHPYFCQCLKNALTTDNHVAYSQMFKETV 67 aa276879.pepQAHPYFWQCLKL~ALTTDNHVAYSQMFKETV 54 aa266744.pep QAHPYFWQCLKNALTTDNHVAYSQMFKETV 59 bb116163.pep QAHPYFWQCLKNALTTDNHVAYSQMFKETV 86 hTLR7.pep QAHPYFWQCLKNALATDNHVAYSQVFKETV 1049 mTLR7.pep QAHPYFWQCLKNALTTDNHVAYSQMFKETV 1050 In Table 11 the sequences are assigned as follows: mTLR7.pep, SEQ ID N0:175;
hTLR7.pep, SEQ 117 N0:170; bb210788.pep, SEQ ID N0:176; aa276879.pep, SEQ ID
NO:177; aa266744.pep, SEQ m N0:178; and bb 116163.pep, SEQ ID N0:179.
Example 18. Method of cloning human TLR8 Two accession numbers in the GenBank database, AF245703 and AF246971, describe the DNA sequence for human TLRB. To create an expression vector for human TLRB, human TLR8 cDNA was amplified from a cDNA made from human peripheral mononuclear blood cells (PBMC) using the primers 5'-CTGCGCTGCTGCAAGTTACGGAATG-3' (SEQ
ID N0:180) and 5'-GCGCGAAATCATGACTTAACGTCAG-3 (SEQ ID N0:181). The fragment was cloned into pGEM-T Easy vector (Promega), cut with the restriction enzyme NotI and ligated into a NotI-digested pCDNA3.1 expression vector (Invitrogen).
The insert was fully sequenced and translated into protein. The cDNA sequence for hTLR8 is SEQ ID
N0:182, is presented in Table 12. The open reading frame starts at base 83, ends at base 3208, and codes for a protein of 1041 amino acids. SEQ ID N0:183 (Table 13), corresponding to bases 83-3205 of SEQ ID N0:182 (Table 12), is the coding region for the polypeptide of SEQ ID N0:184 (Table 14).
The protein sequence of the cloned hTLRB cDNA matches the sequence described under the GenBank accession number AF245703. The sequence deposited under GenBank accession number AF246971 contains an insertion at the N-terminus of 15 amino acids (MI~EESSLQNSSCSLGKETI~KK; SEQ m N0:185) and three single amino acid changes at positions 217 (P to S), 266 (L to P) and 867 (V to 1).
Table 12. cDNA Sequence for Human TLRB (5' to 3'; SEQ ID N0~:182) gCtCCCggCC gccatggcggccgcgggaattcgattctgcgctgctgcaagttacggaat60 gaaaaattag aacaacagaaacatggaaaacatgttccttcagtcgtcaatgctgacctg120 cattttcctg ctaatatctggttcctgtgagttatgcgccgaagaaaatttttctagaag180 ctatccttgt gatgagaaaaagcaaaatgactcagttattgcagagtgcagcaatcgtcg240 actacaggaa gttccccaaacggtgggcaaatatgtgacagaactagacctgtctgataa300 tttcatcaca cacataacgaatgaatcatttcaagggctgcaaaatctcactaaaataaa360 tctaaaccac aaccccaatgtacagcaccagaacggaaatcccggtatacaatcaaatgg420 cttgaatatc acagacggggcattcctcaacctaaaaaacctaagggagttactgcttga480 agacaaccag ttaccccaaataccctctggtttgccagagtctttgacagaacttagtct540 aattcaaaac aatatatacaacataactaaagagggcatttcaagacttataaacttgaa600 aaatctctat ttggcctggaactgctattttaacaaagtttgcgagaaaactaacataga660 agatggagta tttgaaacgctgacaaatttggagttgctatcactatctttcaattctct720 ttcacacgtg ccacccaaactgccaagctccctacgcaaactttttctgagcaacaccca780 gatcaaatac attagtgaagaagatttcaagggattgataaatttaacattactagattt840 aagcgggaac tgtccgaggtgcttcaatgccccatttccatgcgtgccttgtgatggtgg900 tgcttcaatt aatatagatcgttttgcttttcaaaacttgacccaacttcgatacctaaa960 CCtCtCtagC aCttCCCtCaggaagattaatgctgcctggtttaaaaatatgcctcatct1020 gaaggtgctg gatcttgaattcaactatttagtgggagaaatagcctctggggcattttt1080 aacgatgctg CCCCgCttagaaatacttgacttgtcttttaactatataaaggggagtta1140 tccacagcat attaatatttccagaaacttctctaaacttttgtctctacgggcattgca1200 tttaagaggt tatgtgttccaggaactcagagaagatgatttccagcccctgatgcagct1260 tccaaactta tcgactatcaacttgggtattaattttattaagcaaatcgatttcaaact1320 tttccaaaat ttctccaatctggaaattatttacttgtcagaaaacagaatatcaccgtt1380 ggtaaaagat acccggcagagttatgcaaatagttcctcttttcaacgtcatatccggaa1440 acgacgctca acagattttgagtttgacccacattcgaacttttatCatttCaCCCgtCC1500 tttaataaag ccacaatgtgctgcttatggaaaagccttagatttaagcctcaacagtat1560 tttcttcatt gggccaaaccaatttgaaaatcttcctgacattgcctgtttaaatctgtc1620 tgcaaatagc aatgctcaagtgttaagtggaactgaattttcagccattcctcatgtcaa1680 atatttggat ttgacaaacaatagactagactttgataatgctagtgctcttactgaatt1740 gtccgacttg gaagttctagatctcagctataattcacactatttcagaatagcaggcgt1800 aacacatcat ctagaatttattcaaaatttcacaaatctaaaagttttaaacttgagcca1860 caacaacatt tatactttaacagataagtataacctggaaagcaagtccctggtagaatt1920 agttttcagt ggcaatcgccttgacattttgtggaatgatgatgacaacaggtatatctc1980 cattttcaaa ggtctcaagaatctgacacgtctggatttatcccttaataggctgaagca2040 catcccaaat gaagcattcc ttaatttgcc agcgagtctc actgaactac atataaatga 2100 taatatgtta aagtttttta actggacatt actccagcag tttcctcgtc tcgagttgct 2160 tgacttacgt ggaaacaaac tactcttttt aactgatagc ctatctgact ttacatcttc 2220 ccttcggaca ctgctgctga gtcataacag gatttcccac ctaccctctg gctttctttc 2280 tgaagtcagt agtctgaagc acctcgattt aagttccaat ctgctaaaaa caatcaacaa 2340 atccgcactt gaaactaaga ccaccaccaa attatctatg ttggaactac acggaaaccc 2400 ctttgaatgc acctgtgaca ttggagattt ccgaagatgg atggatgaac atctgaatgt 2460 caaaattccc agactggtag atgtcatttg tgccagtcct ggggatcaaa gagggaagag 2520 tattgtgagt ctggagctaa caacttgtgt ttcagatgtc actgcagtga tattattttt 2580 cttcacgttc tttatcacca ccatggttat gttggctgcc ctggctcacc atttgtttta 2640 ctgggatgtt tggtttatat ataatgtgtg tttagctaag gtaaaaggct acaggtctct 2700 ttccacatcc caaactttct atgatgctta catttcttat gacaccaaag acgcctctgt 2760 tactgactgg gtgataaatg agctgcgcta ccaccttgaa gagagccgag acaaaaacgt 2820 tctcctttgt ctagaggaga gggattggga cccgggattg gccatcatcg acaacctcat 2880 gcagagcatc aaccaaagca agaaaacagt atttgtttta accaaaaaat atgcaaaaag 2940 ctggaacttt aaaacagctt tttacttggc tttgcagagg ctaatggatg agaacatgga 3000 tgtgattata tttatcctgc tggagccagt gttacagcat tctcagtatt tgaggctacg 3060 gcagcggatc tgtaagagct CCatCCtcca gtggcctgac aacccgaagg cagaaggctt 3120 gttttggcaa actctgagaa atgtggtctt gactgaaaat gattcacggt ataacaatat 3180 gtatgtcgat tccattaagc aatactaact gacgttaagt catgatttcg cgcaatcact 3240 agtgaattcg cggccgcctg caggtcgacc atatgggaga gctcccaacg cgttggatgc 3300 atagcttgag 3310 Table 13..
Coding Region for Human TLR8 (5' to 3'; SEQ
ID N0:183) 25atggaaaaca tgttccttcagtcgtcaatgctgacctgcattttcctgctaatatctggt60 tcctgtgagt tatgcgccgaagaaaatttttctagaagctatccttgtgatgagaaaaag120 caaaatgact cagttattgcagagtgcagcaatcgtcgactacaggaagttccccaaacg180 gtgggcaaat atgtgacagaactagacctgtctgataatttcatcacacacataacgaat240 gaatcatttc aagggctgcaaaatctcactaaaataaatctaaaccacaaccccaatgta300 30cagcaccaga acggaaatcccggtatacaatcaaatggcttgaatatcacagacggggca360 ttcctcaacc taaaaaacctaagggagttactgcttgaagacaaccagttaccccaaata420 ccctctggtt tgccagagtctttgacagaacttagtctaattcaaaacaatatatacaac480 ataactaaag agggcatttcaagacttataaacttgaaaaatctctatttggcctggaac540 tgctatttta acaaagtttgcgagaaaactaacatagaagatggagtatttgaaacgctg600 35acaaatttgg agttgctatcactatctttcaattctctttcacacgtgccacccaaactg660 ccaagctccc tacgcaaactttttctgagcaacacccagatcaaatacattagtgaagaa720 gatttcaagg gattgataaatttaacattactagatttaagcgggaactgtccgaggtgc780 ttcaatgccc catttccatgcgtgcettgtgatggtggtgcttcaattaatatagatcgt840 tttgcttttc aaaacttgacccaacttcgatacctaaacctctctagcacttccctcagg900 40aagattaatg ctgcctggtttaaaaatatgcctcatctgaaggtgctggatcttgaattc960 aactatttag tgggagaaatagcctctggggcatttttaacgatgctgccccgcttagaa1020 atacttgact tgtcttttaactatataaaggggagttatccacagcatattaatatttcc1080 agaaacttct ctaaacttttgtctctacgggcattgcatttaagaggttatgtgttccag1140 gaactcagag aagatgatttccagcccctgatgcagcttccaaacttatcgactatcaac1200 ttgggtatta attttattaagcaaatcgatttcaaacttttccaaaatttctccaatctg1260 gaaattattt acttgtcagaaaacagaatatcaccgttggtaaaagatacccggcagagt1320 tatgcaaata gttcctcttttcaacgtcatatccggaaacgacgctcaacagattttgag1380 tttgacccac attcgaacttttatcatttcacccgtcctttaataaagccacaatgtgct1440 gcttatggaa aagccttagatttaagcctcaacagtattttcttcattgggccaaaccaa1500 tttgaaaatc ttcctgacattgcctgtttaaatctgtctgcaaatagcaatgctcaagtg1560 ttaagtggaa ctgaattttcagccattcctcatgtcaaatatttggatttgacaaacaat1620 agactagact ttgataatgctagtgctcttactgaattgtccgacttggaagttctagat1680 ctcagctata attcacactatttcagaatagcaggcgtaacacatcatctagaatttatt1740 caaaatttca caaatctaaaagttttaaacttgagccacaacaacatttatactttaaca1800 gataagtata acctggaaagcaagtccctggtagaattagttttcagtggcaatcgcctt1860 gacattttgt ggaatgatgatgacaacaggtatatctccattttcaaaggtctcaagaat1920 ctgacacgtc tggatttatcccttaataggctgaagcacatcccaaatgaagcattcctt1980 aatttgccag cgagtctcactgaactacatataaatgataatatgttaaagttttttaac2040 tggacattac tccagcagtttcctcgtctcgagttgcttgacttacgtggaaacaaacta2100 ctctttttaa ctgatagcctatctgactttacatcttcccttcggacactgctgctgagt2160 cataacagga tttcccacctaccctctggctttctttctgaagtcagtagtctgaagcac2220 ctcgatttaa gttccaatctgctaaaaacaatcaacaaatccgcacttgaaactaagacc2280 accaccaaat tatctatgttggaactacacggaaacccctttgaatgcacctgtgacatt2340 ggagatttcc gaagatggatggatgaacatctgaatgtcaaaattcccagactggtagat2400 gtcatttgtg ccagtcctggggatcaaagagggaagagtattgtgagtctggagctaaca2460 acttgtgttt cagatgtcactgcagtgatattatttttcttcacgttctttatcaccacc2520 atggttatgt tggctgccctggctcaccatttgttttactgggatgtttggtttatatat2580 aatgtgtgtt tagctaaggtaaaaggctacaggtctctttccacatcccaaactttctat2640 gatgcttaca tttcttatgacaccaaagacgcctctgttactgactgggtgataaatgag2700 ctgcgctacc accttgaagagagccgagacaaaaacgttctcctttgtctagaggagagg2760 gattgggacc cgggattggccatcatcgacaacctcatgcagagcatcaaccaaagcaag2820 aaaacagtat ttgttttaaccaaaaaatatgcaaaaagctggaactttaaaacagctttt2880 tacttggctt tgcagaggctaatggatgagaacatggatgtgattatatttatcctgctg2940 gagccagtgt tacagcattctcagtatttgaggctacggcagcggatctgtaagagctcc3000 atcctccagt ggcctgacaacccgaaggcagaaggcttgttttggcaaactctgagaaat3060 gtggtcttga ctgaaaatgattcacggtataacaatatgtatgtcgattccattaagcaa3120 tac 3123 Table 14. Amino Acid Sequence of Human TLR8 40 AF245703.pep MENMFLQSSMLTCIFLLISGSCELCAEENFSRSYPCDEKKQN 42 hTLR8.pep MENMFLQSSMLTCIFLLISGSCELCAEENFSRSYPCDEKKQN 42 AF246971.pep t4ZCESSLQNSSCSLGKETKKENMFLQSSMLTCIFLLISGSCELCAEENFSRSYPCDEKKQN 60 AF245703.pep DSVIAECSNRRLQEVPQTVGKYVTELDLSDNFITHITNESFQGLQNLTKINLNHNPNVQH 102 hTLR8.pep DSVIAECSNRRLQEVPQTVGKYVTELDLSDNFITHITNESFQGLQNLTKINLNHNPNVQH 102 AF246971.pep DSVTAECSNRRLQEVPQTVGKYVTELDLSDNFITHITNESFQGLQNLTKINLNHNPNVQH 120 AF245703.pep QNGNPGIQSNGLNITDGAFLNLKNLRELLLEDNQLPQIPSGLPESLTELSLIQNNIYNIT 162 hTLR8.pep QNGNPGIQSNGLNITDGAFLNLKNLRELLLEDNQLPQIPSGLPESLTELSLIQNNIYNIT 162 AF246971.pep QNGNPGIQSNGLNITDGAFLNLKNLRELLLEDNQLPQIPSGLPESLTELSLIQNNIYNIT 180 AF245703.pep KEGISRLINLKNLYLAWNCYFNKVCEKTNIEDGVFETLTNLELLSLSFNSLSHVPPKLPS222 hTLR8.pep KEGISRLINLKNLYLAWNCYFNKVCEKTNIEDGVFETLTNLELLSLSFNSLSHVPPKLPS222 AF246971.pep KEGISRLINLKL~LYLAWNCYFNKVCEKTNIEDGVFETLTNLELLSLSFNSLSHVSPKLPS240 AF245703.pep SLRKLFLSNTQIKYISEEDFKGLINLTLLDLSGNCPRCFNAPFPCVPCDGGASINIDRFA 282 hTLR8.pep SLRKLFLSNTQIKYISEEDFKGLINLTLLDLSGNCPRCFNAPFPCVPCDGGASINIDRFA 282 AF246971.pep SLRKLFLSNTQIKYISEEDFKGLINLTLLDLSGNCPRCFNAPFPCVPCDGGASINIDRFA 300 AF245703.pep FQNLTQLRYLNLSSTSLRKINAAWFKNMPHLKVLDLEFNYLVGEIASGAFLTMLPRLEIL 342 hTLR8.pep FQNLTQLRYLNLSSTSLRKINAAWFKNMPHLKVLDLEFNYLVGEIASGAFLTMLPRLEIL 342 AF246971.pep FQNLTQLRYLNLSSTSLRKINAAWFKNMPHLKVLDLEFNYLVGEIASGAFLTMLPRLEIL 360 AF245703.pep DLSFNYIKGSYPQHINISRNFSKLLSLRALHLRGYVFQELREDDFQPLMQLPNLSTINLG 402 hTLR8.pep DLSFNYIKGSYPQHINISRNFSKLLSLRALHLRGYVFQELREDDFQPLMQLPNLSTINLG 402 AF246971.pep DLSFNYIKGSYPQHINISRNFSKPLSLRALHLRGYVFQELREDDFQPLMQLPNLSTINLG 420 AF245703.pep INFIKQIDFKLFQNFSNLETIYLSENRISPLVKDTRQSYANSSSFQRHIRKRRSTDFEFD 462 hTLR8.pep INFIKQIDFKLFQNFSNLEIIYLSENRISPLVKDTRQSYANSSSFQRHIRKRRSTDFEFD 462 AF246971.pep INFIKQIDFKLFQNFSNLEIIYLSENRISPLVKDTRQSYANSSSFQRHIRKRRSTDFEFD 480 AF245703.pep PHSNFYHFTRPLIKPQCAAYGKALDLSLNSIFFIGPNQFENLPDIACLNLSANSNAQVLS 522 hTLR8.pep PHSNFYHFTRPLIKPQCAAYGKALDLSLNSIFFIGPNQFENLPDIACLNLSANSNAQVLS 522 AF246971.pep PHSNFYHFTRPLIKPQCAAYGKALDLSLNSIFFIGPNQFENLPDIACLNLSANSNAQVLS 540 AF245703.pep GTEFSAIPHVKYLDLTNNRLDFDNASALTELSDLEVLDLSYNSHYFRIAGVTHHLEFIQN 582 hTLR8.pep GTEFSAIPHVKYLDLTNNRLDFDNASALTELSDLEVLDLSYNSHYFRIAGVTHHLEFIQN 582 AF246971.pep GTEFSAIPHVKYLDLTNNRLDFDNASALTELSDLEVLDLSYNSHYFRIAGVTHHLEFIQN 600 AF245703.pep FTNLKVLNLSHNNIYTLTDKYNLESKSLVELVFSGNRLDILWNDDDNRYISIFKGLKNLT 642 hTLR8.pep FTNLKVLNLSHNNIYTLTDKYNLESKSLVELVFSGNRLDILWNDDDNRYISIFKGLKNLT 642 AF246971.pep FTNLKVLNLSHNNIYTLTDKYNLESKSLVELVFSGNRLDILWNDDDNRYISIFKGLKNLT 660 AF245703.pep RLDLSLNRLKHIPNEAFLNLPASLTELHINDNMLKFFNWTLLQQFPRLELLDLRGNKLLF 702 hTLRB.pep RLDLSLNRLKHIPNEAFLNLPASLTELHINDNMLKFFNWTLLQQFPRLELLDLRGNKLLF 702 AF246971.pep RLDLSLNRLKHIPNEAFLNLPASLTELHINDNMLKFFNWTLLQQFPRLELLDLRGNKLLF 720 AF245703.pep LTDSLSDFTSSLRTLLLSHNRISHLPSGFLSEVSSLKHLDLSSNLLKTINKSALETKTTT762 hTLR8.pep LTDSLSDFTSSLRTLLLSHNRISHLPSGFLSEVSSLKHLDLSSNLLKTINKSALETKTTT762 AF246971.pep LTDSLSDFTSSLRTLLLSHNRISHLPSGFLSEVSSLKHLDLSSNLLKTINKSALETKTTT780 AF245703.pep KLSMLELHGNPFECTCDIGDFRRWMDEHLNVKIPRLVDVICASPGDQRGKSIVSLELTTC 822 hTLR8.pep KLSMLELHGNPFECTCDIGDFRRWMDEHLNVKIPRLVDVICASPGDQRGKSIVSLELTTC 822 AF246971.pep KLSMLELHGNPFECTCDIGDFRRWMDEHLNVKIPRLVDVICASPGDQRGKSIVSLELTTC 840 90,0 AF245703.pep VSDVTAVILFFFTFFITTMVMLAALAHHLFYWDVWFIYNVCLAKVKGYRSLSTSQTFYDA 882 hTLR8.pep VSDVTAVILFFFTFFITTMVMLAALAHHLFYWDVWFIYNVCLAKVKGYRSLSTSQTFYDA 882 AF246971.pep VSDVTAVILFFFTFFITTMVMLAALAHHLFYWDVWFIYNVCLAKIKGYRSLSTSQTFYDA 900 AF245703.pep YISYDTKDASVTDWVINELRYHLEESRDKNVLLCLEERDWDPGLAIIDNLMQSINQSKKT 942 hTLRB.pep YTSYDTKDASVTDWVINELRYHLEESRDKNVLLCLEERDWDPGLAIIDNLMQSINQSKKT 942 AF246971.pep YISYDTKDASVTDWVINELRYHLEESRDKNVLLCLEERDWDPGLAIIDNLMQSINQSKKT 960 - . . . . . . . . . . 1020 AF245703.pep VFVLTKKYAKSWNFKTAFYLALQRLMDENMDVIIFILLEPVLQHSQYLRLRQRICKSSIL 1002 hTLR8.pep VFVLTKKYAKSWNFKTAFYLALQRLMDENMDVIIFILLEPVLQHSQYLRLRQRICKSSIL 1002 AF246971.pep VFVLTKKYAKSWNFKTAFYLALQRLMDENMDVIIFILLEPVLQHSQYLRLRQRICKSSIL 1020 AF245703.pep QWPDNPKAEGLFWQTLRNWLTENDSRYNNMYVDSIKQY 1041 hTLR8.pep QWPDNPKAEGLFWQTLRNWLTENDSRYNNMYVDSIKQY 1041 AF246971.pep QWPDNPKAEGLFWQTLRNWLTENDSRYNNMYVDSIKQY 1059 In Table 14 the sequences are assigned as follows: hTLRB.pep, SEQ m N0:184;
AF245703.pep, SEQ m N0:186; and AF246971.pep, SEQ m N0:187.
Example 19. Method of cloning the murine TLR8 Alignment of human TLRB protein sequence with mouse EST database using tfasta yielded 1 hit with mouse EST sequence bf135656. Two primers were designed that bind to bf135656 sequence for use in a RACE-PCR to amplify 5' and 3' ends of the marine TLRB
cDNA. The library used for the RACE PCR was a mouse spleen marathon-ready cDNA
commercially available from Clontech. A 5' fragment with a length of 2900 by and a 3' fragment with a length of 2900 by obtained by this method were cloned into Promega pGEM-T Easy vector. After sequencing of the 5' end and 3' end of each fragment, partial sequences of mTLR8 were obtained and allowed the design of primers for amplification of the complete marine TLR8 cDNA.
Three independent PCR reactions were set up using a spleen marine cDNA from Clontech as a template with the primers 5'-GAGAGAAACAAACGTTTTACCTTC-3' (SEQ.
ID N0:188) and 5'-GATGGCAGAGTCGTGACTTCCC-3' (SEQ ID N0:189). The resulting amplification products were cloned into pGEM-T Easy vector, fully sequenced, translated into protein, and aligned to the human TLRB protein sequence (GenBank accession number AF245703). The cDNA sequence for mTLR8 is SEQ ID N0:190, presented in Table 15.
The open reading frame of mTLR8 starts at base 59, ends at base 3157, and codes for a protein of 1032 amino acids. SEQ ID N0:191 (Table 16), corresponding to bases 59-3154 of SEQ ID N0:190 (Table 15), is the coding region for the polypeptide of SEQ ID
NO:192 (Table 17). To create an expression vector for marine TLRB, cDNA pGEM-T Easy vector with the mTLRB insert was cut with NotI, the fragment isolated, and ligated into a NotI-digested pCDNA3.1 expression vector (Invitrogen).
Table 15. cDNA Sequence for Marine TLR8 (5' to 3'; SEQ ID N0:190) attcagagtt ggatgttaagagagaaacaaacgttttaccttcctttgtctatagaacat60 ggaaaacatg ccccctcagtcatggattctgacgtgcttttgtctgctgtcctctggaac120 cagtgccatc ttccataaagcgaactattccagaagctatccttgtgacgagataaggca180 caactccctt gtgattgcagaatgcaaccatcgtcaactgcatgaagttccccaaactat240 aggcaagtat gtgacaaacatagacttgtcagacaatgccattacacatataacgaaaga300 gtcctttcaa aagctgcaaaacctcactaaaatcgatctgaaccacaatgccaaacaaca360 gcacccaaat gaaaataaaaatggtatgaatattacagaaggggcacttctcagcctaag420 aaatctaaca gttttactgctggaagacaaccagttatatactatacctgctgggttgcc480 tgagtctttg aaagaacttagcctaattcaaaacaatatatttcaggtaactaaaaacaa540 cacttttggg cttaggaacttggaaagactctatttgggctggaactgctattttaaatg600 taatcaaacc tttaaggtagaagatggggcatttaaaaatcttatacacttgaaggtact660 ctcattatct ttcaataaccttttctatgtgccccccaaactaccaagttctctaaggaa720 actttttctg agtaatgccaaaatcatgaacatcactcaggaagacttcaaaggactgga780 aaatttaaca ttactagatctgagtggaaactgtccaaggtgttacaatgctccatttcc840 ttgcacacct tgcaaggaaaactcatccatccacatacatcctctggcttttcaaagtct900 CdCCCaaCtt CtCtatCtaaacctttccagCaCttCCCtCaggacgattcCttCtaCCtg960 gtttgaaaat ctgtcaaatctgaaggaactccatcttgaattcaactatttagttcaaga1020 aattgcctcg ggggcatttttaacaaaactacccagtttacaaatccttgatttgtcctt1080 caactttcaa tataaggaatatttacaatttattaatatttcctcaaatttctctaagct1140 tcgttctctc aagaagttgcacttaagaggctatgtgttccgagaacttaaaaagaagca1200 tttcgagcat ctccagagtcttccaaacttggcaaccatcaacttgggcattaactttat1260 tgagaaaatt gatttcaaagctttccagaatttttccaaactcgacgttatctatttatc1320 aggaaatcgc atagcatctgtattagatggtacagattattcctcttggcgaaatcgtct1380 tcggaaacct ctctcaacagacgatgatgagtttgatccacacgtgaatttttaccatag1440 caccaaacct ttaataaagccacagtgtactgcttatggcaaggccttggatttaagttt1500 gaacaatatt ttcattattgggaaaagccaatttgaaggttttcaggatatcgcctgctt1560 aaatctgtcc ttcaatgccaatactcaagtgtttaatggcacagaattctcctccatgcc1620 ccacattaaa tatttggatttaaccaacaacagactagactttgatgataacaatgcttt1680 cagtgatctt cacgatctagaagtgctggacctgagccacaatgcacactatttcagtat1740 agcaggggta acgcaccgtctaggatttatccagaacttaataaacctcagggtgttaaa1800 cctgagccac aatggcatttacaccctcacagaggaaagtgagctgaaaagcatctcact1860 gaaagaattg gttttcagtggaaatcgtcttgaccatttgtggaatgcaaatgatggcaa1920 atactggtcc atttttaaaagtctccagaatttgatacgcctggacttatcatacaataa1980 ccttcaacaa atcccaaatggagcattcctcaatttgcctcagagcctccaagagttact2040 tatcagtggt aacaaattacgtttctttaattggacattactccagtattttcctcacct2100 tcacttgctg gatttatcgagaaatgagctgtattttctacccaattgcctatctaagtt2160 tgcacattcc ctggagacactgctactgagccataatcatttctctcacctaccctctgg2220 cttcctctcc gaagccaggaatctggtgcacctggatctaagtttcaacacaataaagat2280 gatcaataaa tcctccctgcaaaccaagatgaaaacgaacttgtctattctggagctaca2340 tgggaactat tttgactgcacgtgtgacataagtgattttcgaagctggctagatgaaaa2400 tctgaatatc acaattcctaaattggtaaatgttatatgttccaatcctggggatcaaaa2460 atcaaagagt atcatgagcctagatctcacgacttgtgtatcggataccactgcagctgt'2520 CCtgtttttC CtCaCattCCttaCCaCCtCcatggttatgttggctgctctggttcacca2580 cctgttttac tgggatgtttggtttatctatcacatgtgctctgctaagttaaaaggcta2640 caggacttca tccacatcccaaactttctatgatgcttatatttcttatgacaccaaaga2700 tgcatctgtt actgactgggtaatcaatgaactgcgctaccaccttgaagagagtgaaga2760 caaaagtgtc ctcctttgtttagaggagagggattgggatccaggattacccatcattga2820 taacctcatg cagagcataaaccagagcaagaaaacaatctttgttttaaccaagaaata2880 tgccaagagc tggaactttaaaacagctttctacttggccttgcagaggctaatggatga2940 gaacatggat gtgattattttcatcctcctggaaccagtgttacagtactcacagtacct3000 gaggcttcgg cagaggatctgtaagagctccatcctccagtggcccaacaatcccaaagc3060 agaaaacttg ttttggcaaagtctgaaaaatgtggtcttgactgaaaatgattcacggta3120 tgacgatttg tacattgattccattaggcaatactagtgatgggaagtcacgactctgcc3180 atcataaaaa cacacagctt ctccttacaa tgaaccgaat 3220 Table 16. Coding Region for Murine TLR8 (5' to 3'; SEQ ID N0:191) atggaaaaca tgccccctcagtcatggattctgacgtgcttttgtctgctgtcctctgga60 accagtgcca tcttccataaagcgaactattccagaagctatccttgtgacgagataagg120 cacaactccc ttgtgattgcagaatgcaaccatcgtcaactgcatgaagttccccaaact180 ataggcaagt atgtgacaaacatagacttgtcagacaatgccattacacatataacgaaa240 gagtcctttc aaaagctgcaaaacctcactaaaatcgatctgaaccacaatgccaaacaa300 cagcacccaa atgaaaataaaaatggtatgaatattacagaaggggcacttctcagccta360 agaaatctaa cagttttactgctggaagacaaccagttatatactatacctgctgggttg420 cctgagtctt tgaaagaacttagcctaattcaaaacaatatatttcaggtaactaaaaac480 aacacttttg ggcttaggaacttggaaagactctatttgggctggaactgctattttaaa540 tgtaatcaaa cctttaaggtagaagatggggcatttaaaaatcttatacacttgaaggta600 ctctcattat ctttcaataaccttttctatgtgccccccaaactaccaagttctctaagg660 aaactttttc tgagtaatgccaaaatcatgaacatcactcaggaagacttcaaaggactg720 gaaaatttaa cattactagatctgagtggaaactgtccaaggtgttacaatgctccattt780 ccttgcacac cttgcaaggaaaactcatccatccacatacatcctctggcttttcaaagt840 ctcacccaac ttctctatctaaacctttccagcacttccctcaggacgattccttctacc900 tggtttgaaa atctgtcaaatctgaaggaactccatcttgaattcaactatttagttcaa960 gaaattgcct cgggggcatttttaacaaaactacccagtttacaaatccttgatttgtcc1020 ttcaactttc aatataaggaatatttacaatttattaatatttcctcaaatttctctaag1080 cttcgttctc tcaagaagttgcacttaagaggctatgtgttccgagaacttaaaaagaag1140 catttcgagc atctccagagtcttccaaacttggcaaccatcaacttgggcattaacttt1200 attgagaaaa ttgatttcaaagctttccagaatttttccaaactcgacgttatctattta1260 tcaggaaatc gcatagcatctgtattagatggtacagattattcctcttggcgaaatcgt1320 cttcggaaac ctctctcaacagacgatgatgagtttgatccacacgtgaatttttaccat1380 agcaccaaac ctttaataaagccacagtgtactgcttatggcaaggccttggatttaagt1440 ttgaacaata ttttcattattgggaaaagccaatttgaaggttttcaggatatcgcctgc1500 ttaaatctgt ccttcaatgccaatactcaagtgtttaatggcacagaattctcctccatg1560 ccccacatta aatatttggatttaaccaacaacagactagactttgatgataacaatgct1620 ttcagtgatc ttcacgatctagaagtgctggacctgagccacaatgcacactatttcagt1680 atagcagggg taacgcaccgtctaggatttatccagaacttaataaacctcagggtgtta1740 aacctgagcc acaatggcatttacaccctcacagaggaaagtgagctgaaaagcatctca1800 ctgaaagaat tggttttcagtggaaatcgtcttgaccatttgtggaatgcaaatgatggc1860 aaatactggt ccatttttaaaagtctccagaatttgatacgcctggacttatcatacaat1920 aaccttcaac aaatcccaaatggagcattcctcaatttgcctcagagcctccaagagtta1980 cttatcagtg gtaacaaattacgtttctttaattggacattactccagtattttcctcac2040 cttcacttgc tggatttatcgagaaatgagctgtattttctacccaattgcctatctaag2100 tttgcacatt ccctggagacactgctactgagccataatcatttctctcacctaccctct2160 ggcttcctct ccgaagccaggaatctggtgcacctggatctaagtttcaacacaataaag2220 atgatcaata aatcctccctgcaaaccaagatgaaaacgaacttgtctattctggagcta2280 catgggaact attttgactg cacgtgtgac ataagtgatt ttcgaagctg gctagatgaa 2340 aatctgaata tcacaattcc taaattggta aatgttatat gttccaatcc tggggatcaa 2400 aaatcaaaga gtatcatgag cctagatctc acgacttgtg tatcggatac cactgcagct 2460 gtcctgtttt tcctcacatt ccttaccacc tccatggtta tgttggctgc tctggttcac 2520 cacctgtttt actgggatgt ttggtttatc tatcacatgt gctctgctaa gttaaaaggc 2580 tacaggactt catccacatc ccaaactttc tatgatgctt atatttctta tgacaccaaa 2640 gatgcatctg ttactgactg ggtaatcaat gaactgcgct accaccttga agagagtgaa 2700 gacaaaagtg tcctcctttg tttagaggag agggattggg atccaggatt acccatcatt 2760 gataacctca tgcagagcat aaaccagagc aagaaaacaa tctttgtttt aaccaagaaa 2820 tatgccaaga gctggaactt taaaacagct ttctacttgg ccttgcagag gctaatggat 2880 gagaacatgg atgtgattat tttcatcctc ctggaaccag tgttacagta ctcacagtac 2940 ctgaggcttc ggcagaggat ctgtaagagc tccatcctcc agtggcccaa caatcccaaa 3000 gcagaaaact tgttttggca aagtctgaaa aatgtggtct tgactgaaaa tgattcacgg 3060 tatgacgatt tgtacattga ttccattagg caatac 3096 Table 17. Amino Acid Sequences of Murine TLR8 and Human TLR8 . . . . . . . . . . . . 60 mTLR8.pep MENMPPQSWILTCFCLLSSGTSAIFHKANYSRSYPCDEIRHNSLVIAECNHRQLHEVPQT 60 hTLRB.pep MENMFLQSSMLTCIFLLISGSCELCAEENFSRSYPCDEKKQNDSVIAECSNRRLQEVPQT 60 mTLR8.pep IGKYVTNiDLSDNAITHITKESFQKLQNLTKIDLNHNAKQQH----PNENKNGMNITEGA 116 hTLR8.pep VGKYVTELDLSDNFITHITNESFQGLQNLTKINLNHNPNVQHQNGNPGIQSNGLNITDGA 120 . . . . . . . . . . . . 180 mTLR8.pep LLSLRNLTVLLLEDNQLYTIPAGLPESLKELSLIQNNIFQVTKNNTFGLRNLERLYLGWN l76 hTLR8.pep FLNLKNLRELLLEDNQLPQIPSGLPESLTELSLIQNNIYNITKEGISRLINLKNLYLAWN 180 mTLR8.pep CYFK--CNQTFKVEDGAFKNLIHLKVLSLSFNNLFYVPPKLPSSLRKLFLSNAKIMNITQ 234 hTLR8.pep CYFNKVCEKT-NIEDGVFETLTNLELLSLSFNSLSHVPPKLPSSLRKLFLSNTQIKYISE 239 mTLR8.pep EDFKGLENLTLLDLSGNCPRCYNAPFPCTPCKENSSIHIHPLAFQSLTQLLYLNLSSTSL 294 hTLR8.pep EDFKGLINLTLLDLSGNCPRCFNAPFPCVPCDGGASINIDRFAFQNLTQLRYLNLSSTSL 299 mTLRB.pep RTIPSTWFENLSNLKELHLEFNYLVQEIASGAFLTKLPSLQTLDLSFNFQYKEYLQFINI 354 hTLR8.pep RKINAAWFKNMPHLKVLDLEFNYLVGETASGAFLTMLPRLEZLDLSFNYIKGSYPQHINI 359 mTLR8.pep SSNFSKLRSLKKLHLRGYVFRELKKKHFEHLQSLPNLATINLGINFIEKIDFKAFQNFSK 414 hTLR8.pep SRNFSKLLSLRALHLRGYVFQELREDDFQPLMQLPNLSTINLGINFIKQIDFKLFQNFSN 419 mTLR8.pep LDVIYLSGNRIASVLDGT--DY---SSWRNRLRKPLSTDDDEFDPHVNFYHSTKPLIKPQ 469 hTLR8.pep LEIIYLSENRISPLVKDTRQSYANSSSFQRHIRKRRSTDF-EFDPHSNFYHFTRPLIKPQ 478 . . . . . . . . . . . . 540 mTLR8.pep CTAYGKALDLSLNNIFIIGKSQFEGFQDIACLNLSFNANTQVFNGTEFSSMPHIKYLDLT 529 hTLR8.pep CAAYGKALDLSLNSIFFIGPNQFENLPDIACLNLSANSNAQVLSGTEFSAIPHVKYLDLT 538 mTLR8.pep NNRLDFDDNNAFSDLHDLEVLDLSHNAHYFSIAGVTHRLGFIQNLINLRVLNLSHNGIYT 589 hTLR8.pep NNRLDFDNASALTELSDLEVLDLSYNSHYFRIAGVTHHLEFIQNFTNLKVLNLSHNNIYT 598 mTLR8.pep LTEESELKSISLKELVFSGNRLDHLWNANDGKYWSTFKSLQNLIRLDLSYNNLQQTPNGA 649 hTLR8.pep LTDKYNLESKSLVELVFSGNRLDILWNDDDNRYISIFKGLKNLTRLDLSLNRLKHIPNEA 658 mTLR8.pep FLNLPQSLQELLISGNKLRFFNWTLLQYFPHLHLLDLSRNELYFLPNCLSKFAHSLETLL 709 hTLRB.pep FLNLPASLTELHINDNMLKFFNWTLLQQFPRLELLDLRGNKLLFLTDSLSDFTSSLRTLL 718 bf135656.pep NHFSHLPSGFLSEARNLVHLDLSFNTIKMINKSSLQTKMKTNLSILELHGNYFDCTC 57 mTLR8.pep LSHNHFSHLPSGFLSEARNLVHLDLSFNTIKMINKSSLQTKMKTNLSILELHGNYFDCTC 769 hTLR8.pep LSHNRISHLPSGFLSEVSSLKHLDLSSNLLKTINKSALETKTTTKLSMLELHGNPFECTC 778 bf135656.pep DISDFRSWLDENLNITIPKLVNVICSNPGDQKSKSIMSLDLTTCVSDTTAAVLFFLTFLT 117 mTLR8.pep DISDFRSWLDENLNITIPKLVNVICSNPGDQKSKSIMSLDLTTCVSDTTAAVLFFLTFLT 829 hTLR8.pep DIGDFRRWMDEHLNVKIPRLVDVICASPGDQRGKSIVSLELTTCVSDVTAVILFFFTFFI 838 bf135656.pep TSMVMLAALVHHLFYWDVWFIYHMCSAKLKGYRTSSTSQTFYDAYISYDTKDASVTDWVI 177 mTLR8.pep TSMVMLAALVHHLFYWDVWFIYHMCSAKLKGYRTSSTSQTFYDAYISYDTKDASVTDWVI 889 hTLR8.pep TTMVMLAALAHHLFYWDVWFIYNVCLAKVKGYRSLSTSQTFYDAYISYDTKDASVTDWVT 898 bf135656.pep NELRYHLE 185 mTLR8.pep NELRYHLEESEDKSVLLCLEERDWDPGLPIIDNLMQSINQSKKTIFVLTKKYAKSWNFKT 949 hTLR8.pep NELRYHLEESRDKNVLLCLEERDWDPGLAIIDNLMQSINQSKKTVFVLTKKYAKSWNFKT 958 . . . . . . . . . . . 1020 mTLR8.pep AFYLALQRLMDENMDVIIFILLEPVLQYSQYLRLRQRICKSSILQWPNNPKAENLFWQSL 1009 hTLR8.pep AFYLALQRLMDENMDVIIFILLEPVLQHSQYLRLRQRICKSSILQWPDNPKAEGLFWQTL 1018 . . . . . . . . . . . . 1080 mTLR8.pep KNWLTENDSRYDDLYIDSIRQY 1032 hTLRB.pep RNWLTENDSRYNNMYVDSIKQY 1041 In Table 17 the sequences are assigned as follows: mTLRB.pep, SEQ ID N0:192;
hTLRB.pep, SEQ ID N0:184; and bf135656.pep, SEQ ID N0:193.
Example 20. Transient transfectants expressing TLR8 and TLR7 The cloned human TLR7 and human TLRB cDNA (our result) were cloned into the expression vector pCDNA3.1 (-) from Invitrogen using the NotI site. Utilizing a "gain of function" assay, hTLR7 and hTLR8 expression vectors were transiently expressed in human l0 293 fibroblasts (ATCC, CRL-1573) using the calcium phosphate method.
Activation was monitored by IL-8 production after stimulus with CpG-ODN (2006 or 1668, 2~M) or LPS
(100 ng/ml). None of the stimuli used activated 293 cells transfected with either hTLR7 or hTLRB. ' 15 Example 21. Screening for TLR9, 8 and 7 modulators Human TLR receptors 9, 8 and 7 are expressed differentially among tissues which may be due to differences in promoter structure. Du X et al., Eur Cytokine Netw 11:362-71 (2000); Chuang TH et al., Eur Cytokine Netw 11:372-8 (2000). For the human Toll-like receptors 9, 8 and 7 the genomic locus has been defined and sequenced. TLR9 is located on 2o chromosome 3 (GenBank accession numbers NT 005985, AC006252), TLR7 on chromosome X (GenBank accession numbers NT 011774, AC005859, AC003046) and TLR8 close~to TLR7 also on chromosome X (GenBank accession numbers NT 011774, AC005859). To verify differences in the promoter regions the putative promoter region of each gene are cloned in reporter vectors like pGL2-Basic (Promega, Madison, WI, USA) 25 which contains the luciferase gene (luc) adjacent to a multiple cloning site. After transient transfection of these constructs in various cell lines, different stimuli can be tested for the activation of the inserted promoter region which is detected by luciferase activity. The promoter regions defined by the cloning of mTLR9, mTLR8 and mTLR7 can be utilized in the same manner. Definition of compounds that agonize or antagonize TLR9, 8, or 7 3o expression can be used to enhance or dampen responses to nucleic acid ligands or to any TLR9, 8 or 7 ligand defined by screening. These constructs can be adapted to high throughput screening after stable transfection similar to the use of TLR9 stable transfectants.

Each of the foregoing patents, patent applications and references is hereby incorporated by reference. While the invention has been described with respect to certain embodiments, it should be appreciated that many modifications and changes may be made by those of ordinary shill in the art without departing from the spirit of the invention. It is intended that such modification, changes and equivalents fall within the scope of the following claims.
Example 22. Method cloning the marine and human extracellular TLR9 domain fused to human IgGl Fc 1o Human IgGl Fc was amplified from human B cell cDNA using the sense and antisense primers 5' TATGGATCCTCTTGTGACAAAACTCACACATGC (SEQ ID
N0:216) and 5' ATA AAGCTTTCATTTACCCGGAGACAGGGAGAG (SEQ ID NO:217) and ligated into pCDNA3.1(-) (Invitrogen) after digestion with the restriction endonucleases BamHI and HindIlI creating the vector pcDNA-IgGFc. The extracellular domain of human 15 TLR9 (amino acids 1 to 815) was amplified with the sense and antisense primers 5' TATGAATTCCCACCATGGGTTTCTGCCGCAG (SEQ ID N0:218) and 5'ATAGGATCCCCGGGGCACCAGGCCGCCGCCGCGGCCGCCGGAGAGGGCCTCAT
CCAGGC (SEQ ID N0:219). The primers amplify the extracellular domain of human and create adjacent to amino acid 815 an additional NotI restriction site, a glycine linker and 2o thrombin protease recognition site. The translated sequence of this region starting at amino acid 812 is DEALSGGRGGGLVPRGS (SEQ ID N0:220). The fragment was cut with EcoRI and BamHI and cloned into pcDNA-IgGFc, creating the vector coding for the fusion protein of the extracellular domain of human TLR9 fused to the Fc part of human IgGl (pcDNAhTLR9IgGFc). Expressed extracellular TLR9 protein can be separated from the 25 IgGl Fc fragment by digestion with Thrombin (see figure).
The extracellular part of marine TLR9 (amino acids 1 to 816) was amplified with the sense and antisense primers 5' TATATGCGGCCGCCCACCATGGTTCTCCGTCGAAG
(SEQ ID N0:221) and 5' TATATGCGGCCGCCAGAGAGGACCTCATCCAGGC (SEQ ID
N0:222) and cloned into pcDNAhTLR9IgGFc after NotI digestion of PCR fragment and 30 vector. This procedure exchanged the human extracellular part of TLR9 with the marine counterpart.

Example 23. Method of expression and purification of the extracellular domain of TLR9 fused to human IgG1 Fc Vector DNA coding for the human or marine TLR9 human IgGFc fusion protein was transfected by CaZP04 method into 293 fibroblast cells. Transfected cells were selected with 0.7 mg/ml 6418 and cloned. Expression of fusion protein was monitored by enzyme-linked immunosorbent assay (ELISA). Cells were lysed in lysis buffer (PBS, 1% Triton X-100) and supernatant was applied to ELISA plates coated with polyclonal antibody against human IgG
Fc. Bound fusion protein was detected by incubation with biotinylated polyclonal antibodies 1o against human IgG-Fc and streptavidin-horseradish peroxidase conjugate.
For purification of the fusion protein cell lysates from 109 cells were produced and incubated with Protein A sepharose which binds tightly to human IgG-Fc.
Incubation with the protease thrombin releases the soluble extracellular domain of human TLR9.
Figure 27 shows an example of the TLR9 fusion protein visualized by a silver stained SDS-gel. Figure 27 demonstrates that lysates of transfected cells included a strong band travelling between 100 and 150 kD which was not present either in lysates of mock-transfected cells or in supernatants transfected or mock-transfected cells. The apparent molecular weight of the band decreased following thrombin treatment, consistent with cleavage at the thrombin protease recognition site interposed between the extracellular TLR9 domain and the Fc fragment.
Example 24. Method of cloning the marine and human extracellular TLR7 and TLR8 domain fused to human IgGl Fc and its expression in 293 cells The extracellular domains of marine TLR7 (amino acids 1 to 837), human TLR7 (amino acids 1 to 836), marine TLR8 (amino acids 1 to 816) and human TLR8 (amino acids 1 to 825) were amplified with the primer pairs 5' TATATGCGGCCGCCCACCATGGTGTTTTCGATGTGGACACG (SEQ ID N0:223) and 5' TATATGCGGCCGCCATCTAACTCACACGTATACAGATC (SEQ ID N0:224);
5' TATATGCGGCCGCCCACCATGGTGTTTCCAATGTGGACACTG (SEQ ID N0:225) 3o and 5' TATATGCGGCCGCCATCTAACTCACAGGTGTACAGATC (SEQ ID N0:226);
5' TATATGCGGCCGCCCACCATGGAAAACATGCCCCCTCAG (SEQ ID N0:227) and 5' TATATGCGGCCGCCATCCGATACACAAGTCGTGAGATC (SEQ ID N0:228); and 5' TATATGCGGCCGCCCACCATGGAAAACATGTTCCTTCAGTC (SEQ ID N0:229) and 5' TATATGCGGCCGCCATCTGAAACACAAGTTGTTAGCTC (SEQ ID N0:230), respectively. Fragments were cloned into pcDNA-IgGFc after NotI digestion.
Vector DNA coding for the extracellular domain of human or marine TLR7 or TLR8 fused to human IgGFc fusion protein was transfected by Ca2P04 method into 293 fibroblast cells. Transfected cells were selected with 0.7 mg/ml 6418 and cloned.
Expression of fusion protein was monitored by ELISA. Cells were lysed in lysis buffer (PBS, 1 %
Triton X-100) and supernatant was applied to ELISA plates coated with polyclonal antibody against human 1o IgG-Fc. Bound fusion protein was detected by incubation with biotinylated polyclonal antibodies against human IgG-Fc and Streptavidin-horseradish peroxidase conjugate.
Example 25. Method of antibody production against marine and human TLR9 and characterization of activity 15 C57B6 mice were immunized three times by intraperitoneal administration of 20 ~g of the extracellular domain of human TLR9 mixed with 10 nmol of the CpG-ODN
1668.
B cells taken from immunized mice were fused with a non antibody producing B-cell hybridoma P3XAG8 using standard protocols. Hybridoma supernatants were screened for reactivity in ELISA using marine and human TLR9 fusion proteins. For identification of 2o positive hybridomas ELISA plates were coated with polyclonal antibody against human IgG-Fc and incubated with lysate containing marine or human TLR9 IgG-Fc fusion protein.
Plates were then incubated with individual hybridoma supernatants, and bound TLR9-specific antibodies were detected by incubation with biotinylated polyclonal antibodies against marine IgG and Streptavidin-horseradish peroxidase conjugate.
25 Ten antibodies have been isolated which are of IgGl, IgG2a and IgG2b isotype. They have been tested for reactivity against human and marine TLR9 and their performance in western blotting or intracellular staining. Table 18 shows themames (ID), isotypes, reactivity and performance in western blotting and intracellular staining.
All isolated antibodies were readily purified using standard protein A
affinity 3o chromatography.

Table 18. Monclonal Antibodies Raised Against Marine and Human TLR9 # Reactivit Western Intracellular in ELISA

ID Isotype mTLR9 hTLR9 BlottingStaining 1 1-3A11 Gl YES YES YES NO

3 1-2A9 G2a NO YES YES YES

2-lE2 G2a NO YES YES YES

6 1-5G5 G2a YES YES YES YES

8 1-SF12 G2b NO YES NO NO

9 1-3C9 G2a NO YES YES YES

1-3F5 G2b ~ NO ~ YES ~ NO NO
~ ~

Example 26. Method for Intracellular Staining Mock transfected 293 cells and human TLR9 transfected 293 cells were seeded on cover slips and cultured overnight. The following day cells were washed in PBS
and fixed with 2% formalin for 10 minutes at room temperature. Cells were permeabilized with 0.2%
saponin in PBS and incubated with 2~g/ml anti human TLR9-specific antibody 2-lE2 for 1h.
After two wash steps cells were incubated with Alexis488-conjugated goat anti-mouse IgG
antibody and TLR9 was visualized utilizing confocal microscopy on a Zeiss to microscope. Results indicated that cytoplasms of human TLR9 transfected 293 cells, but not mock transfected 293 cells, stained positive for human TLR9.
Example 27. Method for Western Blotting Lysates of 293 cells transfected with marine TLR9, human TLR9 or marine TLR2 IgGl-Fc fusion protein were separated by SDS-PAGE. Proteins were transferred to a nylon membrane utilizing a BioRad semi dry blotter according to the manufacturer's protocol. The membrane was incubated with 2~,g/ml of the human TLR9-specific antibody 2-1E2, and human TLR9 was detected by polyclonal goat anti-mouse peroxidase conjugate.
Peroxidase activity was monitored with ECL reagent (Amersham) and incubation of the membrane on film (see Figure 29).
What is claimed is:

SEQUENCE LISTING
<110> Coley Pharmaceutical GmbH
<120> PROCESS FOR HIGH THROUGHPUT SCREENING OF
CpG-BASED IMMUNO-AGONIST/ANTAGONIST
<130> C1041/7016W0 (AWS) <150> US 60/233,035 <151> 2000-09-15 <150> US 60/263,657 <151> 2001-01-23 <150> US 60/291,726 <151> 2001-05-17 <150> US 60/300,210 <151> 2001-06-22 <160> 16 <170> FastSEQ for Windows Version 3.0 <210> 1 <211> 3200 <212> DNA
<213> unknown <400> 1 tgtcagagggagcctcgggagaatcctccatctcccaacatggttctccgtcgaaggact60 ctgcaccccttgtccctcctggtacaggctgcagtgctggctgagactctggccctgggt120 accctgcctgccttcctaccctgtgagctgaagcctcatggcctggtggactgcaattgg180 ctgttcctgaagtctgtaccccgtttctctgcggcagcatcctgctccaacatcacccgc240 ctctccttgatctccaaccgtatccaccacctgcacaactccgacttcgtccacctgtcc300 aacctgcggcagctgaacctcaagtggaactgtccacccactggccttagccccctgcac360 ttctcttgccacatgaccattgagcccagaaccttcctggctatgcgtacactggaggag420 ctgaacctgagctataatggtatcaccactgtgccccgactgcccagctccctggtgaat480 ctgagcctgagccacaccaacatcctggttctagatgctaacagcctcgccggcctatac540 agcctgcgcgttctcttcatggacgggaactgctactacaagaacccctgcacaggagcg600 gtgaaggtgaccccaggcgccctcctgggcctgagcaatctcacccatctgtctctgaag660 tataacaacctcacaaaggtgccccgccaactgccccccagcctggagtacctcctggtg720 tcctataacctcattgtcaagctggggcctgaagacctggccaatctgacctcccttcga780 gtacttgatgtgggtgggaattgccgtcgctgcgaccatgcccccaatccctgtatagaa840 tgtggccaaaagtccctccacctgcaccctgagaccttccatcacctgagccatctggaa900 ggcctggtgctgaaggacagctctctccatacactgaactcttcctggttccaaggtctg960 gtcaacctctcggtgctggacctaagcgagaactttctctatgaaagcatcaaccacacc1020 aatgcctttcagaacctaacccgcctgcgcaagctcaacctgtccttcaattaccgcaag1080 aaggtatcctttgcccgcctccacctggcaagttccttcaagaacctggtgtcactgcag1140 gagctgaacatgaacggcatcttcttccgctcgctcaacaagtacacgctcagatggctg1200 gccgatctgcccaaactccacactctgcatcttcaaatgaacttcatcaaccaggcacag1260 ctcagcatctttggtaccttccgagcccttcgctttgtggacttgtcagacaatcgcatc1320 agtgggccttcaacgctgtcagaagccacccctgaagaggcagatgatgcagagcaggag1380 gagctgttgtctgcggatcctcacccagctccactgagcacccctgcttctaagaacttc1440 atggacaggtgtaagaacttcaagttcaccatggacctgtctcggaacaacctggtgact1500 atcaagccagagatgtttgtcaatctctcacgcctccagtgtcttagcctgagccacaac1560 tccattgcacaggctgtcaatggctctcagttcctgccgctgactaatctgcaggtgctg 1620 gacctgtcccataacaaactggacttgtaccactggaaatcgttcagtgagctaccacag 1680 ttgcaggccctggacctgagctacaacagccagccctttagcatgaagggtataggccac 1740 aatttcagttttgtggcccatctgtccatgctacacagccttagcctggcacacaatgac 1800 attcatacccgtgtgtcctcacatctcaacagcaactcagtgaggtttcttgacttcagc 1860 ggcaacggtatgggccgcatgtgggatgaggggggcctttatctccatttcttccaaggc 1920 ctgagtggcctgctgaagctggacctgtctcaaaataacctgcatatcctccggccccag 1980 aaccttgacaacctccccaagagcctgaagctgctgagcctccgagacaactacctatct 2040 ttctttaactggaccagtctgtccttcctgcccaacctggaagtcctagacctggcaggc 2100 aaccagctaaaggccctgaccaatggcaccctgcctaatggcaccctcctccagaaactg 2160 gatgtcagcagcaacagtatcgtctctgtggtcccagccttcttcgctctggcggtcgag 2220 ctgaaagaggtcaacctcagccacaacattctcaagacggtggatcgctcctggtttggg 2280 cccattgtgatgaacctgacagttctagacgtgagaagcaaccctctgcactgtgcctgt 2340 ggggcagccttcgtagacttactgttggaggtgcagaccaaggtgcctggcctggctaat 2400 ggtgtgaagtgtggcagccccggccagctgcagggccgtagcatcttcgcacaggacctg 2460 cggctgtgcctggatgaggtcctctcttgggactgctttggcctttcactcttggctgtg 2520 gccgtgggcatggtggtgcctatactgcaccatctctgcggctgggacgtctggtactgt 2580 tttcatctgtgcctggcatggctacctttgctggcccgcagccgacgcagcgcccaagct 2640 ctcccctatgatgccttcgtggtgttcgataaggcacagagcgcagttgcggactgggtg 2700 tataacgagctgcgggtgcggctggaggagcggcgcggtcgccgagccctacgcttgtgt 2760 ctggaggaccgagattggctgcctggccagacgctcttcgagaacctctgggcttccatc 2820 tatgggagccgcaagactctatttgtgctggcccacacggaccgcgtcagtggcctcctg 2880 cgcaccagcttcctgctggctcagcagcgcctgttggaagaccgcaaggacgtggtggtg 2940 ttggtgatcctgcgtccggatgcccaccgctcccgctatgtgcgactgcgCCagCgtCtC 3000 tgccgccagagtgtgctcttctggccccagcagcccaacgggcaggggggcttctgggcc 3060 cagctgagtacagccctgactagggacaaccgccacttctataaccagaacttctgccgg 3120 ggacctacagcagaatagctcagagcaacagctggaaacagctgcatcttcatgcctggt 3180 tcccgagttgctctgcctgc 3200 <210> 2 <211> 3096 <212> DNA
<213> unknown <400> 2 atggttctccgtcgaaggactctgcaccccttgtccctcctggtacaggctgcagtgctg 60 gctgagactctggCCCtgggtaCCCtgCCtgCCttCCtaCCCtgtgagCtgaagCCtCat 120 ggcctggtggactgcaattggctgttcctgaagtctgtaccccgtttctctgcggcagca 180 tCCtgCtCCaaCatCaCCCgcctctccttgatCtCCaaCCgtatccaccacctgcacaac 240 tccgacttcgtccacctgtccaacctgcggcagctgaacctcaagtggaactgtccaccc 300 actggccttagccccctgcacttctcttgccacatgaccattgagcccagaaccttcctg 360 gctatgcgtacactggaggagctgaacctgagctataatggtatcaccactgtgccccga 420 ctgcccagctccctggtgaatctgagcctgagccacaccaacatcctggttctagatgct 480 aacagcctcgccggcctatacagcctgcgcgttctcttcatggacgggaactgctactac 540 aagaacccctgcacaggagcggtgaaggtgaccccaggcgccctcctgggcctgagcaat 600 ctcacccatctgtctctgaagtataaCaaCCtCaCaaaggtgCCCCgCCaaCtgCCCCCC 660 agcctggagtaCCtCCtggtgtCCtataaCCtCattgtCaagCtggggCCtgaagacctg 720 gccaatctgacctcccttcgagtacttgatgtgggtgggaattgccgtcgctgcgaccat 780 gcccccaatccctgtatagaatgtggccaaaagtccctccacctgcaccctgagaccttc 840 catcacctgagccatctggaaggcctggtgctgaaggacagctctctccatacactgaac 900 tcttcctggttccaaggtctggtcaacctctcggtgctggacctaagcgagaactttctc 960 tatgaaagcatcaac.cacaccaatgcctttcagaacctaacccgcctgcgcaagctcaac 1020 ctgtccttcaattaccgcaagaaggtatcctttgcccgcctccacctggcaagttccttc 1080 aagaacctggtgtcactgcaggagctgaacatgaacggcatcttcttccgctcgctcaac 1140 aagtacacgctcagatggctggccgatctgcccaaactccacactctgcatcttcaaatg 1200 aacttcatcaaccaggcacagctcagcatctttggtaccttccgagcccttcgctttgtg 1260 gacttgtcagacaatcgcatcagtgggccttcaacgctgtcagaagccacccctgaagag 1320 gcagatgatgcagagcaggaggagctgttgtctgcggatcctcacccagctccactgagc 1380 acccctgcttctaagaacttcatggacaggtgtaagaacttcaagttcaccatggacctg 1440 tctcggaacaacctggtgactatcaagccagagatgtttgtcaatctctcacgcctccag 1500 tgtcttagcctgagccacaactccattgcacaggctgtcaatggctctcagttcctgccg 1560 ctgactaatctgcaggtgctggacctgtcccataacaaactggacttgtaccactggaaa 1620 tcgttcagtgagctaccacagttgcaggccctggacctgagctacaacagccagcccttt 1680 agcatgaagggtataggccacaatttcagttttgtggcccatctgtccatgctacacagc 1740 cttagcctggcacacaatgacattcatacccgtgtgtcctcacatctcaacagcaactca 1800 gtgaggtttcttgacttcagcggcaacggtatgggccgcatgtgggatgaggggggcctt 1860 tatctccatttcttccaaggcctgagtggcctgctgaagctggacctgtctcaaaataac 1920 ctgcatatcctccggccccagaaccttgacaacctccccaagagcctgaagctgctgagc 1980 ctccgagacaactacctatctttctttaactggaccagtctgtCCttCCtgCCCaaCCtg 2040 gaagtcctagacctggcaggcaaccagctaaaggccctgaccaatggcaccctgcctaat 2100 ggcaccctcctccagaaactggatgtcagcagcaacagtatcgtotctgtggtcccagcc 2160 ttcttcgctctggcggtcgagctgaaagaggtcaacctcagccacaacattctcaagacg 2220 gtggatcgctcctggtttgggcccattgtgatgaacctgacagttctagacgtgagaagc 2280 aaccctctgcactgtgcctgtggggcagccttcgtagacttactgttggaggtgcagacc 2340 aaggtgcctggcctggctaatggtgtgaagtgtggcagccccggccagctgcagggccgt 2400 agcatcttcgcacaggacctgcggctgtgcctggatgaggtcctctcttgggactgcttt 2460 ggcctttcactcttggctgtggccgtgggcatggtggtgcctatactgcaccatctctgc 2520 ggctgggacgtctggtactgttttcatctgtgcctggcatggctacctttgctggcccgc 2580 agccgacgcagcgcccaagctctcccctatgatgccttcgtggtgttcgataaggcacag 2640 agcgcagttgcggactgggtgtataacgagctgcgggtgcggctggaggagcggcgcggt 2700 cgccgagccctacgcttgtgtctggaggaccgagattggctgcctggccagacgctcttc 2760 gagaacctctgggcttccatctatgggagccgcaagactctatttgtgctggcccacacg 2820 gaccgcgtcagtggcctcctgcgcaccagcttcctgctggctcagcagcgcctgttggaa 2880 gaccgcaaggacgtggtggtgttggtgatcctgcgtccggatgcccaccgctcccgctat 2940 gtgcgactgcgccagcgtctctgccgccagagtgtgctcttctggececagcagcccaac 3000 gggcaggggggcttctgggcccagctgagtacagccctgactagggacaaccgccacttc 3060 tataaccagaacttctgccggggacctacagcagaa 3096 <210> 3 <211> 3352 <212> DNA
<213> unknown <400> 3 aggctggtataaaaatcttacttcctctattctctgagccgctgctgcccctgtgggaag 60 ggacctcgagtgtgaagcatccttccctgtagctgctgtccagtctgcccgccagaccct 120 ctggagaagcccctgccccccagcatgggtttctgccgcagcgccctgcacccgctgtct 180 ctcctggtgcaggccatcatgctggccatgaccctggccctgggtaccttgcctgccttc 240 ctaccctgtgagctccagccccacggcctggtgaactgcaactggctgttcctgaagtct 300 gtgCCCCaCttCtCCatggCagCaCCCCgtggCaatgtCaCCagCCtttCCttgtCCtCC 360 aaccgcatccaccacctccatgattctgactttgcccacctgcccagcctgcggcatctc 420 aacctcaagtggaactgcccgccggttggcctcagccccatgcacttcccctgccacatg 480 accatcgagcccagcaccttcttggctgtgcccaccctggaagagctaaacctgagctac 540 aacaacatcatgactgtgcctgcgctgcccaaatccctcatatccctgtccctcagccat 600 accaacatcctgatgctagactctgccagcctcgccggcctgcatgccctgcgcttccta 660 ttcatggacggcaactgttattacaagaacccctgcaggcaggcactggaggtggccccg 720 ggtgccctccttggcctgggCaaCCtC3CCCaCCtgtCaCtCaagtaCaaCaaCCtC3Ct 780 gtggtgccccgcaacctgccttccagcctggagtatctgctgttgtcctacaaccgcatc 840 gtcaaactggcgcctgaggacctggccaatctgaccgccctgcgtgtgctcgatgtgggc 900 ggaaattgccgCCgCtgCgaCCdCgCtCCCaaCCCCtgCatggagtgccctcgtcacttc 960 ccccagctacatcccgataccttcagccacctgagccgtcttgaaggcctggtgttgaag 1020 gacagttctctctcctggctgaatgccagttggttccgtgggctgggaaacctccgagtg 1080 ctggacctgagtgagaacttcctctacaaatgcatcactaaaaccaaggccttccagggc 1140 ctaacacagctgcgcaagcttaacctgtccttcaattaccaaaagagggtgtcctttgcc 1200 cacctgtctctggccccttccttcgggagcctggtcgccctgaaggagctggacatgcac 1260 ggcatcttcttccgctcactcgatgagaccacgctccggccactggcccgcctgcccatg 1320 ctccagactctgcgtctgcagatgaacttcatcaaccaggcccagctcggcatcttcagg 1380 gccttccctggcctgcgctacgtggacctgtcggacaaccgcatcagcggagcttcggag 1440 ctgacagccaccatgggggaggcagatggaggggagaaggtctggctgcagcctggggac 1500 cttgctccggccccagtggacactcccagctctgaagacttcaggcccaactgcagcacc 1560 ctcaacttcaccttggatctgtcacggaacaacctggtgaccgtgcagccggagatgttt 1620 gcccagctctcgcacctgcagtgcctgcgcctgagccacaactgcatctcgcaggcagtc 1680 aatggctcccagttcctgccgctgaccggtctgcaggtgctagacctgtcccgcaataag 1740 ctggacctctaccacgagcactcattcacggagctaccgcgactggaggccctggacctc 1800 agctacaacagccagccctttggcatgcagggcgtgggccacaacttcagcttcgtggct 1860 cacctgcgcaccctgcgccacctcagcctggcccacaacaacatccacagccaagtgtcc 1920 cagcagctctgcagtacgtcgctgcgggccctggacttcagcggcaatgcactgggccat 1980 atgtgggccgagggagacctctatctgcacttcttccaaggcctgagcggtttgatctgg 2040 ctggacttgtcccagaaccgcctgcacaccctcctgccccaaaccctgcgcaacctcccc 2100 aagagcctacaggtgctgcgtctccgtgacaattacctggccttctttaagtggtggagc 2160 ctccacttcctgcccaaactggaagtcctcgacctggcaggaaaccggctgaaggccctg 2220 accaatggcagcctgcctgctggcacccggctccggaggctggatgtcagctgcaacagc 2280 atcagcttcgtggcccccggcttcttttccaaggccaaggagctgcgagagctcaacctt 2340 agcgccaacgccctcaagacagtggaccactcctggtttgggcccctggcgagtgccctg 2400 caaatactagatgtaagcgccaaccctctgcactgcgcctgtggggcggcctttatggac 2460 ttcctgctggaggtgcaggctgccgtgcccggtctgcccagccgggtgaagtgtggcagt 2520 ccgggccagctccagggcctcagcatctttgcacaggacctgcgcctctgcctggatgag 2580 gccctctcctgggactgtttcgccctctcgctgctggctgtggctctgggcctgggtgtg 2640 cccatgctgcatcacctctgtggctgggacctctggtactgcttccacctgtgcctggcc 2700 tggcttccctggcgggggcggcaaagtgggcgagatgaggatgccctgccctacgatgcc 2760 ttcgtggtcttcgacaaaacgcagagcgcagtggcagactgggtgtacaacgagcttcgg 2820 gggcagctggaggagtgccgtgggcgctgggcactccgcctgtgcctggaggaacgcgac 2880 tggctgcctggcaaaaccctctttgagaacctgtgggcctcggtctatggcagccgcaag 2940 acgctgtttgtgctggcccacacggaccgggtcagtggtctcttgcgcgccagcttcctg 3000 ctggcccagcagcgcctgctggaggaccgcaaggacgtcgtggtgctggtgatcctgagc 3060 cctgacggccgccgctcccgctacgtgcggctgcgccagcgcctctgccgccagagtgtc 3120 C'tCCtCtggCCCCaCCagCCCagtggtCagcgcagcttctgggcccagctgggcatggcc 3180 ctgaccagggacaaccaccacttctataaccggaacttctgccagggacccacggccgaa 3240 tagccgtgagccggaatcctgcacggtgccacctccacactcacctcacctctgcctgcc 3300 tggtctgaccC'tCCCCtgCtCgCCtCCCtCaccccacacctgacacagagCa 3352 <210> 4 <211> 3868 <212> DNA
<213> unknown <400~> 4 ggaggtcttgtttccggaagatgttgcaaggctgtggtgaaggcaggtgcagcctagcct 60 cctgctcaagctacaccctggccctccacgcatgaggccctgcagaactctggagatggt 120 gcctacaagggcagaaaaggacaagtcggcagccgctgtcctgagggcaccagctgtggt 180 gcaggagccaagacctgagggtggaagtgtcctcttagaatggggagtgcccagcaaggt 240 gtacccgctactggtgctatccagaattcccatctctccctgctctctgcctgagctctg 300 ggccttagctcctccctgggcttggtagaggacaggtgtgaggccctcatgggatgtagg 360 ctgtctgagaggggagtggaaagaggaaggggtgaaggagctgtctgccatttgactatg 420 caaatggcctttgactcatgggaccctgtcctcctcactgggggcagggtggagtggagg 480 gggagctactaggctggtataaaaatcttacttcctctattctctgagccgctgctgccc 540 ctgtgggaagggacctcgagtgtgaagcatccttccctgtagctgctgtccagtctgccc 600 gccagaccctctggagaagcccctgccccccagcatgggtttctgccgcagcgccctgca 660 cccgctgtctctcctggtgcaggccatcatgctggccatgaccctggccctgggtacctt 720 gcctgccttcctaccctgtgagctccagccccacggcctggtgaactgcaactggctgtt 780 cctgaagtctgtgccccacttctccatggcagcaccccgtggcaatgtcaccagcctttc 840 cttgtcctccaaccgcatccaccacctccatgattctgactttgcccacctgcccagcct 900 gcggcatctcaacctcaagtggaactgcccgccggttggcctcagccccatgcacttccc 960 ctgccacatgaccatcgagcccagcaccttcttggctgtgcccaccctggaagagctaaa 1020 cctgagctacaacaacatcatgactgtgcctgcgctgcccaaatccctcatatccctgtc 1080 cctcagccataccaacatcctgatgctagactctgccagcctcgccggcctgcatgccct 1140 gcgcttcctattcatggacggcaactgttattacaagaacccctgcaggcaggcactgga 1200 ggtggccccgggtgccctccttggcctgggcaacctcacccacctgtcactcaagtacaa1260 caacctcactgtggtgccccgcaacctgccttccagcctggagtatctgctgttgtccta1320 caaccgcatcgtcaaactggcgcctgaggacctggccaatctgaccgccctgcgtgtgct1380 cgatgtgggcggaaattgccgccgetgcgaccacgctcccaacccctgcatggagtgccc1440 tCgtCa.CttCCCCCagCtaCatcccgatacCttCagCCaCCtgagCCgtCttgaaggcct1500 ggtgttgaaggacagttctctctcctggctgaatgccagttggttccgtgggctgggaaa1560 cctccgagtgctggacctgagtgagaacttcctctacaaatgcatcactaaaaccaaggc1620 cttccagggcctaacacagctgcgcaagcttaacctgtccttcaattaccaaaagagggt1680 gtcctttgcccacctgtctctggccccttccttcgggagcctggtcgccctgaaggagct1740 ggacatgcacggCatCttCttCCgCtCa.Ctcgatgagaccacgctccggccactggcccg1800 CCtgCCCatgCtCCagaC'tCtgcgtctgcagatgaacttcatcaaccaggCCCagCtCgg1860 catcttcagggccttccctggcctgcgctacgtggacctgtcggacaaccgcatcagcgg1920 agcttcggagctgacagccaccatgggggaggcagatggaggggagaaggtctggctgca1980 gcctggggaccttgctccggccccagtggacactcccagctctgaagacttcaggcccaa2040 ctgcagcaccctcaacttcaccttggatctgtcacggaacaacctggtgaccgtgcagcc2100 ggagatgtttgcccagctctcgcacctgcagtgcctgcgcctgagccacaactgcatctc2160 gcaggcagtcaatggctcccagttcctgccgctgaccggtctgcaggtgctagacctgtc2220 ccacaataagctggacctctaccacgagcactcattcacggagctaccacgactggaggc2280 cctggacctcagctacaacagccagccctttggcatgcagggcgtgggccacaacttcag2340 cttcgtggctcacctgcgcaccctgcgccacctcagcctggcccacaacaacatccacag2400 ccaagtgtcccagcagctctgcagtacgtcgctgcgggccctggacttcagcggcaatgc2460 actgggccatatgtgggccgagggagacctctatctgcacttcttccaaggcctgagcgg2520 tttgatctggctggacttgtcccagaaccgCCtgCaCaCCCtCCtgCCCCaaaCCCtgCg2580 caacctccccaagagcctacaggtgctgcgtctccgtgacaattacctggccttctttaa2640 gtggtggagcctccacttcctgcccaaactggaagtcctcgacctggcaggaaaccagct2700 gaaggccctgaccaatggcagcctgcctgctggcacccggctccggaggctggatgtcag2760 ctgcaacagcatcagcttcgtggcccccggcttcttttccaaggccaaggagctgcgaga2820 gctcaaccttagcgccaacgccctcaagacagtggaccactcctggtttgggcccctggc2880 gagtgccctgcaaatactagatgtaagcgccaaccctctgcactgcgcctgtggggcggc2940 ctttatggacttcctgctggaggtgcaggctgccgtgcccggtctgcccagccgggtgaa3000 gtgtggcagtccgggccagctccagggcctcagcatctttgcacaggacctgcgcctctg3060 cctggatgaggccctctcctgggactgtttcgccctctcgctgctggctgtggctctggg3120 cctgggtgtgcccatgctgcatcacctctgtggctgggacctctggtactgcttccacct3180 gtgcctggcctggcttccctggcgggggcggcaaagtgggcgagatgaggatgCCCtgCC3240 ctacgatgccttcgtggtcttcgacaaaacgcagagcgcagtggcagactgggtgtacaa3300 cgagcttcgggggcagctggaggagtgccgtgggcgctgggcactccgcctgtgcctgga3360 ggaacgcgactggCtgCCtggCaaaaCCCtctttgagaacctgtgggcctcggtctatgg3420 cagccgcaagacgctgtttgtgctggcccacacggaccgggtcagtggtctcttgcgcgc3480 cagcttcctgctggcccagcagcgcctgctggaggaccgcaaggacgtcgtggtgctggt3540 gatCCtgagCCCtgaCggCCgccgctcccgctacgtgcggctgcgccagcgcctctgccg3600 ccagagtgtcctcctctggccccaccagcccagtggtcagcgcagcttctgggcccagct3660 gggcatggccctgaccagggacaaccaccacttctataaccggaacttctgccagggacc3720 cacggccgaatagccgtgagccggaatcctgcacggtgccacctccacactcacctcacc3780 tctgcctgcctggtctgaccctcccctgctCgCCtCCCtCaCCCCaCdCCtgacacagag3840 caggcactcaataaatgctaccgaaggc 3868 <210> 5 <211> 557 <212> DNA
<213> unknown <400>

ggctttcaacctaaccgctggcactcaacctgtccttcaattaccgcaagaaggtatcct 60 ttgcccgcctccacctggcaagttcctttaagaacctggtgtcactgcaggagctgaaca 120 tgaacggcatcttcttccgottgctcaacaagtacacgctcagatggctggccgatctgc 180 ccaaactccacactctgcatcttcaaatgaacttcatcaaccaggcacagctcagcatct 240 ttggtaccttCCgagCCCttcgctttgtggacttgtcagacaatcgcatcagtgggcctt 300 caacgctgtcagaagccacccctgaagaggcagatgatgcagagcaggaggagctgttgt 360 ctgcggatcctcacccagctccgctgagcacccctgcttctaagaacttcatggacaggt 420 gtaagaactt caagttcaac atggacctgt ctcggaacaa cctggtgact atcacagcag 480 agatgtttgt aaatctctca cgcctccagt gtcttagcct gagccacaac tcaattgcac 540 aggctgtcaa tggctct 557 <210> 6 <211> 497 <212> DNA
<213> unknown <400> .

gtgggtttggtgtctatcttcactctcctgaaagatgcatgggaagaaaactacccttta 60 cagccaacctttgctccgtgggcctggtggcttggtagcatatattgcgcacttgccaaa 120 tagcggtgtagtaagacagagcaaggcaggcagagcaactcgggaaccagacatgaagat 180 gcagctgtttccagctgttgctctgagctattctgctgtaggtccccggcagaagttctg 240 gttatagaagtggcggttgtccctagtcagggctgtactcagctgggcccagaagccccc 300 ctgcccgttgggtcgctggggccagaagagcacactctggcggcagagacgctggcgcag 360 tcgcacatagcgggacggtngggcatccggacgcaggatcaccaacaccaccacgtcctt 420 gcggtcttccaacaggcgctgctgagccagcaggaagctggtgcgcaggaggccactgac 480 gcggtccgtgtgggcca 497 <210> 7 <211> 373 <212> DNA
<213> unknown <400> 7 tggaggaccgagattggctgcctggccagacgctcttcgagaacctctgggcttccatct 60 atgggagccgcaagactctatttgtgctggcccacacggaccgcgtcagtggcctcctgc 120 gcaccagcttcctgctggctcagcagcgcctgttggaagaccgcaaggacgtggtggtgt 180 tggtgatcctgcgtccggatgcccaccgctCCCgCtatgtgCgaCtgCgCCagCgtCtCt 240 gccgccagagtgtgctcttctggccccagcagcccaacgggcaggggggcttctgggccc 300 agctgagtacagccctgactagggacaaccgccacttctataaccagaacttctgccggg 360 gacctacagcaga 373 <210> 8 <211> 489 <212> DNA
<213> unknown <400> 8 gctacaacagccagccctttagcatgaagggtataggccacaatttcagttttgtgaccc 60 atctgtccatgctacagagccttagcctggcacacaatgacattcatacccgtgtgtcct 120 cacatctcaacagcaactcagtgaggtttcttgacttcagcggcaacggtatgggccgca 180 tgtgggatgaggggggcctttatctccatttcttccaaggcctgagtggcgtgctgaagc 240 tggacctgtctcaaaataacctgcatatcctccggccccagaaccttgacaacctcccca 300 agagcctgaagctgctgagcctccgagacaactacctatctttctttaa,ctggaccagtc 360 tgtccttcctacccaacctggaagtcctagacctggcaggcaaccagctaaaggccctga 420 ccaatggcaccctgcctaatggcaccctcctccagaaactcgatgtcagtagcaacagta 480 tcgtctctg 489 <210> 9 <211> 462 <212> DNA
<213> unknown <400> 9 gcggccgcgc~egetcctcca gCCgCaCCCg cagctcgtta tacacccagt cggcaactgc 60 gctctgtgcc ttatcgaaca ccacgaaggc atcataaggg agagtttggg cgctgcgtcg 120 gctgcgggct agcaaaggta gccatgccag gcacagatga aaacagtacc agacgtccca 180 _ g _ gccgcagagatggtgcagtataggcaccaccatgcccacggccacagccaagagtgaaag240 gccaaagcagtcccaagagaggacctcatccaggcacagccgcaggtcctgcgcgaagat300 gctacggccctgcagctggccggggctgccacacttcacaccattagccaggccaggcac360 cttggtctgcacctccaacagtaagtctacgaaggctgccccacaggcacagtgcagagg420 gttgcttctcacgtctagaactgtcaggttcatcacaatggg 462 <210> 10 <211> 1032 <212> PRT
<213> unknown <400> 10 Met Val Leu Arg Arg Arg Thr Leu His Pro Leu Ser Leu Leu Val Gln Ala Ala Val Leu Ala Glu Thr Leu Ala Leu Gly Thr Leu Pro Ala Phe Leu Pro Cys Glu Leu Lys Pro His Gly Leu Val Asp Cys Asn Trp Leu Phe Leu Lys Ser Val Pro Arg Phe Ser Ala Ala Ala Ser Cys Ser Asn Ile Thr Arg Leu Ser Leu Ile Ser Asn Arg Ile His His Leu His Asn Ser Asp Phe Val His Leu Ser Asn Leu Arg Gln Leu Asn Leu Lys Trp Asn Cys Pro Pro Thr Gly Leu Ser Pro Leu His Phe Ser Cys His Met Thr Ile Glu Pro Arg Thr Phe Leu Ala Met Arg Thr Leu Glu Glu Leu Asn Leu Ser Tyr Asn Gly Ile Thr Thr Val Pro Arg Leu Pro Ser Ser Leu Val Asn Leu Ser Leu Ser His Thr Asn Ile Leu Val Leu Asp Ala Asn Ser Leu Ala Gly Leu Tyr Ser Leu Arg Val Leu Phe Met Asp Gly Asn Cys Tyr Tyr Lys Asn Pro Cys Thr Gly Ala Val Lys Val Thr Pro Gly Ala Leu Leu Gly Leu Ser Asn Leu Thr His Leu Ser Leu Lys Tyr Asn Asn Leu Thr Lys Val Pro Arg Gln Leu Pro Pro Ser Leu Glu Tyr Leu Leu Val Ser Tyr Asn Leu Ile Val Lys Leu Gly Pro Glu Asp Leu Ala Asn Leu Thr Ser Leu Arg Val Leu Asp Val Gly Gly Asn Cys Arg Arg Cys Asp His Ala Pro Asn Pro Cys Ile Glu Cys Gly Gln Lys Ser Leu His Leu His Pro Glu Thr Phe His His Leu Ser His Leu Glu Gly Leu Val Leu Lys Asp Ser Ser Leu His Thr Leu Asn Ser Ser Trp Phe Gln Gly Leu Val Asn Leu Ser Val Leu Asp Leu Ser Glu Asn Phe Leu Tyr Glu Ser Ile Asn His Thr Asn Ala Phe Gln Asn Leu Thr Arg Leu Arg Lys Leu Asn Leu Ser Phe Asn Tyr Arg Lys Lys Val Ser Phe Ala Arg Leu His Leu Ala Ser Ser Phe Lys Asn Leu Val Ser Leu Gln Glu Leu Asn Met Asn Gly Ile Phe Phe Arg Ser Leu Asn Lys Tyr Thr Leu _ 7 _ Arg Trp Leu Ala Asp Leu Pro Lys Leu His Thr Leu His Leu Gln Met 385 390 ~ 395 400 Asn Phe Ile Asn Gln Ala Gln Leu Ser Ile Phe Gly Thr Phe Arg Ala Leu Arg Phe Val Asp Leu Ser Asp Asn Arg Ile Ser Gly Pro Ser Thr Leu Ser Glu Ala Thr Pro Glu Glu Ala Asp Asp Ala Glu Gln Glu Glu Leu Leu Ser Ala Asp Pro His Pro Ala Pro Leu Ser Thr Pro Ala Ser Lys Asn Phe Met Asp Arg Cys Lys Asn Phe Lys Phe~Thr Met Asp Leu Ser Arg Asn Asn Leu Val Thr Ile Lys Pro Glu Met Phe Val Asn Leu Ser Arg Leu Gln Cys Leu Ser Leu Ser His Asn Ser Ile Ala Gln Ala Val Asn Gly Ser Gln Phe Leu Pro Leu Thr Asn Leu Gln Val Leu Asp Leu Ser His Asn Lys Leu Asp Leu Tyr His Trp Lys Ser Phe Ser Glu Leu Pro Gln Leu Gln Ala Leu Asp Leu Ser Tyr Asn Ser Gln Pro Phe Ser Met Lys Gly Ile Gly His Asn Phe Ser Phe Val Ala His Leu Ser Met Leu His Ser Leu Ser Leu Ala His Asn Asp Ile His Thr Arg Val Ser Ser His Leu Asn Ser Asn Ser Val Arg Phe Leu Asp Phe Ser Gly Asn Gly Met Gly Arg Met Trp Asp Glu Gly Gly Leu Tyr Leu His Phe Phe Gln Gly Leu Ser Gly Leu Leu Lys Leu Asp Leu Ser Gln Asn Asn Leu His Ile Leu Arg Pro Gln Asn Leu Asp Asn Leu Pro Lys Ser Leu Lys Leu Leu Ser Leu Arg Asp Asn Tyr Leu Ser Phe Phe Asn Trp Thr Ser Leu Ser Phe Leu Pro Asn Leu Glu Val Leu Asp Leu Ala Gly Asn Gln Leu Lys Ala Leu Thr Asn Gly Thr Leu Pro Asn Gly Thr Leu Leu Gln Lys Leu Asp Val Ser Ser Asn Ser Ile Val Ser Val Val Pro Ala Phe Phe Ala Leu Ala Val Glu Leu Lys Glu Val Asn Leu Ser~His Asn Ile Leu Lys Thr Val Asp Arg Ser Trp Phe Gly Pro Ile Val Met Asn Leu Thr Val Leu Asp Val Arg Ser Asn Pro Leu His Cys Ala Cys Gly Ala Ala Phe Val Asp Leu Leu Leu Glu Val Gln Thr Lys Val Pro Gly Leu Ala Asn Gly Val Lys Cys Gly Ser Pro Gly Gln Leu Gln Gly Arg Ser Ile Phe Ala Gln Asp Leu Arg Leu Cys Leu Asp Glu Val Leu Ser Trp Asp Cys Phe Gly Leu Ser Leu Leu Ala Val Ala Val Gly Met Val Val Pro Ile Leu His His Leu Cys Gly Trp Asp ~Val Trp Tyr Cys Phe - g _ His Leu Cys Leu Ala Trp Leu Pro Leu Leu Ala Arg Ser Arg Arg Ser Ala Gln Ala Leu Pro Tyr Asp Ala Phe Val Val Phe Asp Lys Ala Gln Ser Ala Val Ala Asp Trp Val Tyr Asn Glu Leu Arg Val Arg Leu Glu Glu Arg Arg Gly Arg Arg Ala Leu Arg Leu Cys Leu Glu Asp Arg Asp Trp Leu Pro Gly Gln Thr Leu Phe Glu Asn Leu Trp Ala Ser Ile Tyr Gly Ser Arg Lys Thr Leu Phe Val Leu Ala His Thr Asp Arg Val Ser Gly Leu Leu Arg Thr Ser Phe Leu Leu Ala Gln Gln Arg Leu Leu Glu Asp Arg Lys Asp Val Val Val Leu Val Ile Leu Arg Pro Asp Ala His Arg Ser Arg Tyr Val Arg Leu Arg Gln Arg Leu Cys Arg Gln Ser Val Leu Phe Trp Pro Gln Gln Pro Asn Gly Gln Gly Gly Phe Trp Ala Gln Leu Ser Thr Ala Leu Thr Arg Asp Asn Arg His Phe Tyr Asn Gln Asn Phe Cys Arg Gly Pro Thr Ala Glu <210> 11 <211> 1032 <212> PRT
<213> unknown <400> 11 Met Gly Phe Cys Arg Ser Ala Leu His Pro Leu Ser Leu Leu Val Gln Ala Ile Met Leu Ala Met Thr Leu Ala Leu Gly Thr Leu Pro Ala Phe Leu Pro Cys Glu Leu Gln Pro His Gly Leu Val Asn Cys Asn Trp Leu Phe Leu Lys Ser Val Pro His Phe Ser Met Ala Ala Pro Arg Gly Asn Val Thr Ser Leu Ser Leu Ser Ser Asn Arg Ile His His Leu His Asp Ser Asp Phe Ala His Leu Pro Ser Leu Arg His Leu Asn Leu Lys Trp Asn Cys Pro Pro Val Gly Leu Ser Pro Met His Phe Pro Cys His Met Thr Ile Glu Pro Ser Thr Phe Leu Ala Val Pro Thr Leu Glu Glu Leu Asn Leu Ser Tyr Asn Asn Ile Met Thr Val Pro Ala Leu Pro Lys Ser Leu Ile Ser Leu Ser Leu Ser His Thr Asn Ile Leu Met Leu Asp Ser Ala Ser Leu Ala Gly Leu His Ala Leu Arg Phe Leu Phe Met Asp Gly Asn Cys Tyr Tyr Lys Asn Pro Cys Arg Gln Ala Leu Glu Val Ala Pro Gly Ala Leu Leu Gly Leu Gly Asn Leu Thr His Leu Ser Leu Lys Tyr Asn Asn Leu Thr Val Val Pro Arg Asn Leu Pro Ser Ser Leu Glu Tyr Leu Leu Leu Ser Tyr Asn Arg Ile Val Lys Leu Ala Pro Glu Asp Leu Ala Asn Leu Thr Ala Leu Arg Val Leu Asp Val Gly Gly Asn Cys Arg Arg Cys Asp His Ala Pro Asn Pro Cys Met Glu Cys Pro Arg His Phe Pro Gln Leu His Pro Asp Thr Phe Ser His Leu Ser Arg Leu Glu Gly Leu Val Leu Lys Asp Ser Ser Leu Ser Trp Leu Asn Ala Ser Trp Phe Arg Gly Leu Gly Asn Leu Arg Val Leu Asp Leu Ser Glu Asn Phe Leu Tyr Lys Cys Ile Thr Lys Thr Lys Ala Phe Gln Gly Leu Thr Gln Leu Arg Lys Leu Asn Leu Ser Phe Asn Tyr Gln Lys Arg Val Ser Phe Ala His Leu Ser Leu Ala Pro Ser Phe Gly Ser Leu Val Ala Leu Lys Glu Leu Asp Met His Gly Ile Phe Phe Arg Ser Leu Asp Glu Thr Thr Leu Arg Pro Leu Ala Arg Leu Pro Met Leu Gln Thr Leu Arg Leu Gln Met Asn Phe Ile Asn Gln Ala Gln Leu Gly Ile Phe Arg Ala Phe Pro Gly Leu Arg Tyr Val Asp Leu Ser Asp Asn Arg Ile Ser Gly Ala Ser Glu Leu Thr Ala Thr Met Gly Glu Ala Asp Gly Gly Glu Lys Val Trp Leu Gln Pro Gly Asp Leu Ala Pro Ala Pro Val Asp Thr Pro Ser Ser Glu Asp Phe Arg Pro Asn Cys Ser Thr Leu Asn Phe Thr Leu Asp Leu Ser Arg Asn Asn Leu Val Thr Val Gln Pro Glu Met Phe Ala Gln Leu Ser His Leu Gln Cys Leu Arg Leu Ser His Asn Cys Ile Ser Gln Ala Val Asn Gly Ser Gln Phe Leu Pro Leu Thr Gly Leu Gln Val Leu Asp Leu Ser Arg Asn Lys Leu Asp Leu Tyr His Glu His Ser Phe Thr Glu Leu Pro Arg Leu Glu Ala Leu Asp Leu Ser Tyr Asn Ser Gln Pro Phe Gly Met Gln Gly Val Gly His Asn Phe Ser Phe Val Ala His Leu Arg Thr Leu Arg His Leu Ser Leu Ala His Asn Asn Ile His Ser Gln Val Ser Gln Gln Leu Cys Ser Thr Ser Leu Arg Ala Leu Asp Phe Ser Gly Asn Ala Leu Gly His Met Trp Ala Glu Gly Asp Leu Tyr Leu His Phe Phe Gln Gly Leu Ser Gly Leu Ile Trp Leu Asp Leu Ser Gln Asn Arg Leu His Thr Leu Leu Pro Gln Thr Leu Arg Asn Leu Pro Lys Ser Leu Gln Val Leu Arg Leu Arg Asp Asn Tyr Leu Ala Phe Phe Lys Trp Trp Ser Leu His Phe Leu Pro Lys Leu Glu Val Leu Asp Leu Ala Gly Asn Arg Leu Lys Ala Leu Thr Asn Gly Ser Leu Pro Ala Gly Thr Arg Leu Arg Arg Leu Asp Val Ser Cys Asn Ser Ile Ser Phe Val Ala Pro Gly Phe Phe Ser Lys Ala Lys Glu Leu Arg Glu Leu Asn Leu Ser Ala Asn Ala Leu Lys Thr Val Asp His Ser Trp Phe Gly Pro Leu Ala Ser Ala Leu Gln Ile Leu Asp Val Ser Ala Asn Pro Leu His Cys Ala Cys Gly Ala Ala Phe Met Asp Phe Leu Leu Glu Val Gln Ala Ala Val Pro Gly Leu Pro Ser Arg Val Lys Cys Gly Ser Pro Gly Gln Leu Gln Gly Leu Ser Ile Phe Ala Gln Asp Leu Arg Leu Cys Leu Asp Glu Ala Leu Ser Trp Asp Cys_Phe Ala Leu Ser Leu Leu Ala Val Ala Leu Gly Leu Gly Val Pro Met Leu His His Leu Cys Gly Trp Asp Leu Trp Tyr Cys Phe His Leu Cys Leu Ala Trp Leu Pro Trp Arg Gly Arg Gln Ser Gly Arg Asp Glu Asp Ala Leu Pro Tyr Asp Ala Phe Val Val Phe Asp Lys Thr Gln Ser Ala Val Ala Asp Trp Val Tyr Asn Glu Leu Arg Gly Gln Leu Glu Glu Cys Arg Gly Arg Trp Ala Leu Arg Leu Cys Leu Glu Glu Arg Asp Trp Leu Pro Gly Lys Thr Leu Phe Glu Asn Leu Trp Ala Ser Val Tyr Gly Ser Arg Lys Thr Leu Phe Val Leu Ala His Thr Asp Arg Val Ser Gly Leu Leu Arg Ala Ser Phe Leu Leu Ala Gln Gln Arg Leu Leu Glu Asp Arg Lys Asp Val Val Val Leu Val Ile Leu Ser Pro Asp Gly Arg Arg Ser Arg Tyr Val Arg Leu Arg Gln Arg Leu Cys Arg Gln Ser Val Leu Leu Trp Pro His Gln Pro Ser Gly Gln Arg Ser Phe Trp Ala Gln Leu Gly Met Ala Leu Thr Arg Asp Asn His His Phe Tyr Asn Arg Asn Phe Cys Gln Gly Pro Thr Ala Glu <210> 12 <211> 178 <212> PRT
<213> unknown <400> 12 Leu Asn Leu Ser Phe Asn Tyr Arg Lys Lys Val Ser Phe Ala Arg Leu His Leu Ala Ser Ser Phe Lys Asn Leu Val Ser Leu Gln Glu Leu Asn Met Asn Gly Ile Phe Phe Arg Leu Leu Asn Lys Tyr Thr Leu Arg Trp Leu Ala Asp Leu Pro Lys Leu His Thr Leu His Leu Gln Met Asn Phe Ile Asn Gln Ala Gln Leu Ser Ile Phe Gly Thr Phe Arg Ala Leu Arg Phe Val Asp Leu Ser Asp Asn Arg Ile Ser Gly Pro Ser Thr Leu Ser Glu Ala Thr Pro Glu Glu Ala Asp Asp Ala Glu Gln Glu Glu Leu Leu Ser Ala Asp Pro His Pro Ala Pro Leu Ser Thr Pro Ala Ser Lys Asn Phe Met Asp Arg Cys Lys Asn Phe Lys Phe Asn Met Asp Leu Ser Arg Asn Asn Leu Val Thr Ile Thr Ala Glu Met Phe Val Asn Leu Ser Arg Leu Gln Cys Leu Ser Leu Ser His Asn Ser Ile Ala Gln Ala Val Asn Gly Ser <210> 13 <211> 95 <212> PRT
<213> unknown <400> 13 Ala His Thr Asp Arg Val Ser Gly Leu Leu Arg Thr Ser Phe Leu Leu Ala Gln Gln Arg Leu Leu Glu Asp Arg Lys Asp Val Val Val Leu Val Ile Leu Arg Pro Asp Ala Xaa Pro Ser Arg Tyr Val Arg Leu Arg Gln Arg Leu Cys Arg Gln Ser Val Leu Phe Trp Pro Gln Arg Pro Asn Gly Gln Gly Gly Phe Trp Ala Gln Leu Ser Thr Ala Leu Thr Arg Asp Asn Arg His Phe Tyr Asn Gln Asn Phe Cys Arg Gly Pro Thr Ala Glu <210> 14 <211> 123 <212> PRT
<213> unknown <400> 14 Glu Asp Arg Asp Trp Leu Pro Gly Gln Thr Leu Phe Glu Asn Leu Trp Ala Ser Ile Tyr Gly Ser Arg Lys Thr Leu Phe Val Leu A1a His Thr Asp Arg Val Ser Gly Leu Leu Arg Thr Ser Phe Leu Leu Ala Gln Gln Arg Leu Leu Glu Asp Arg Lys Asp Val Val Val Leu Val Ile Leu Arg Pro Asp Ala His Arg Ser Arg Tyr Val Arg Leu Arg Gln Arg Leu Cys Arg Gln Ser Val Leu Phe Trp Pro Gln Gln Pro Asn Gly Gln Gly Gly Phe Trp Ala Gln Leu Ser Thr Ala Leu Thr Arg Asp Asn Arg His Phe Tyr Asn Gln Asn Phe Cys Arg Gly Pro Thr Ala <210> 15 <211> 162 <212> PRT
<213> unknown <400> 15 Tyr Asn Ser Gln Pro Phe Ser Met Lys Gly Ile Gly His Asn Phe Ser Phe Val Thr His Leu Ser Met Leu Gln Ser Leu Sex Leu Ala His Asn Asp Ile His Thr Arg Val Ser Ser His Leu Asn Sex Asn Ser Val Arg Phe Leu Asp Phe Ser Gly Asn Gly Met Gly Arg Met Trp Asp Glu Gly Gly Leu Tyr Leu His Phe Phe Gln Gly Leu Ser Gly Val Leu Lys Leu 65 70 75 $0 Asp Leu Ser Gln Asn Asn Leu His Ile Leu Arg Pro Gln Asn Leu Asp $5 90 95 Asn Leu Pro Lys Ser Leu Lys Leu Leu Ser Leu Arg Asp Asn Tyr Leu Ser Phe Phe Asn Trp Thr Ser Leu Ser Phe Leu Pro Asn Leu Glu Val Leu Asp Leu Ala Gly Asn Gln Leu Lys Ala Leu Thr Asn'Gly Thr Leu Pro Asn Gly Thr Leu Leu Gln Lys Leu Asp Val Ser Ser Asn Ser Ile Val Ser <210> 16 <400> 16

Claims (120)

Claims

1. An isolated nucleic acid molecule selected from the group consisting of (a) nucleic acid molecules which hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence set forth as SEQ ID NO:1, and which code for a marine TLR9 having an amino acid sequence set forth as SEQ ID NO:3, (b) nucleic acid molecules that differ from the nucleic acid molecules of (a) in codon sequence due to degeneracy of the genetic code, and (c) complements of (a) or (b).

2. The isolated nucleic acid molecule of claim 1, wherein the isolated nucleic acid molecule codes for SEQ ID NO:3.

3. The isolated nucleic acid molecule of claim 1, wherein the isolated nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO:1.

4. The isolated nucleic acid molecule of claim 1, wherein the isolated nucleic acid molecule comprises a nucleotide sequence set forth as SEQ ID NO:2.

5. An isolated TLR9 polypeptide or fragment thereof comprising at least one amino acid of marine TLR9 selected from the group consisting of amino acids 2, 3, 4, 6, 7, 18, 19, 22, 38, 44, 55, 58, 61, 62, 63, 65, 67, 71, 80, 84, 87, 88, 91, 101, 106, 109, 117, 122, 123, 134, 136, 140, 143, 146, 147, 157, 160, 161, 167, 168, 171, 185, 186, 188, 189, 191, 199, 213, 217, 220, 227, 231, 236, 245, 266, 269, 270, 271, 272, 273, 274, 278, 281, 285, 297, 298, 301, 305, 308, 311, 322, 323, 325, 326, 328, 332, 335, 346, 348, 353, 355, 358, 361, 362, 365, 367, 370, 372, 380, 381, 382, 386, 389, 392, 394, 397, 409, 412, 413, 415, 416, 419, 430, 432, 434, 435, 438, 439, 443, 444, 446, 447, 448, 450, 451, 452, 454, 455, 459, 460, 463, 465, 466, 468, 469, 470, 472, 473, 474, 475, 478, 488, 489; 494, 495, 498, 503;
508, 510, 523, 531, 539, 540, 543, 547, 549, 561, 563, 565, 576, 577, 579, 580, 587, 590, 591, 594, 595, 597, 599, 601, 603, 610, 611, 613, 616, 619, 632, 633, 640, 643, 645, 648, 650, 657, 658, 660, 667, 670, 672, 675, 679, 689, 697, 700, 703, 705, 706, 711, 715, 716, 718, 720, 723, 724, 726, 729, 731, 735, 737, 743, 749, 750, 751, 752, 754, 755, 759, 760, 772, 774, 780, 781, 786, 787, 788, 800, 814, 821, 829, 831, 832, 835, 844, 857, 858, 859, 862, 864, 865, 866, 879, 893, 894, 898, 902, 910, 917, and 927 of SEQ ID NO:3, wherein the TLR9 polypeptide or fragment thereof has an amino acid sequence which is identical to a human TLR9 polypeptide or fragment thereof except for the at least one amino acid of marine TLR9.

6. The isolated TLR9 polypeptide or fragment thereof of claim 5, further comprising at least one amino acid of marine TLR9 selected from the group consisting of amino acids 949, 972, 975, 976, 994, 997, 1000, 1003, 1004, 1010, 1011, 1018, 1023, and 1027 of SEQ ID NO:3.

7. The isolated TLR9 polypeptide or fragment thereof of claim 5, wherein the human TLR9 has an amino acid sequence set forth as SEQ ID NO:6.

8. The isolated TLR9 polypeptide or fragment thereof of claim 5, wherein the isolated TLR9 polypeptide or fragment thereof has an amino acid sequence selected from the group consisting of SEQ ID NO:3 and fragments of SEQ ID NO:3.

9. The isolated TLR9 polypeptide or fragment thereof of claim 5, wherein the isolated TLR9 polypeptide or fragment thereof is an extracytoplasmic domain of TLR9.

10. The isolated TLR9 polypeptide or fragment thereof of claim 5, wherein the isolated TLR9 polypeptide or fragment thereof comprises an MBD motif as set forth as SEQ ID
NO:126 or SEQ ID NO:127.

11. The isolated TLR9 polypeptide or fragment thereof of claim 5, wherein the isolated TLR9 polypeptide or fragment thereof selectively binds to an immunostimulatory nucleic acid (ISNA).

12. The isolated TLR9 polypeptide or fragment thereof of claim 5, wherein the isolated TLR9 polypeptide or fragment thereof selectively binds to a CpG nucleic acid.

13. An isolated nucleic acid molecule which encodes the isolated TLR9 polypeptide or fragment thereof of claim 5.

14. An expression vector comprising the isolated nucleic acid molecule of claim 1 operably linked to a promoter.

15. A host cell comprising the expression vector of claim 14.

16. The host cell of claim 15, further comprising at least one expression vector selected from the group consisting of:
(a) an expression vector comprising a nucleic acid molecule which encodes an isolated TLR7 polypeptide operably linked to a promoter, and (b) an expression vector comprising a nucleic acid molecule which encodes an isolated TLR8 polypeptide operably linked to a promoter.

17. The host cell of claim 15, further comprising a reporter construct capable of interacting with a TIR domain.

18. An expression vector comprising the isolated nucleic acid molecule of claim 13 operably linked to a promoter.

19. A host cell comprising the expression vector of claim 18.

20. The host cell of claim 19, further comprising at least one expression vector selected from the group consisting of:
(a) an expression vector comprising a nucleic acid molecule which encodes an isolated TLR7 polypeptide operably linked to a promoter, and (b) an expression vector comprising a nucleic acid molecule which encodes an isolated TLR8 polypeptide operably linked to a promoter.

21. The host cell of claim 19, further comprising a reporter construct capable of interacting with a TIR domain.

22. An isolated nucleic acid molecule selected from the group consisting of (a) nucleic acid molecules which hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence set forth as SEQ ID NO:173, and which code for a marine TLR7 having an amino acid sequence set forth as SEQ ID NO:175, (b) nucleic acid molecules that differ from the nucleic acid molecules of (a) in codon sequence due to degeneracy of the genetic code, and (c) complements of (a) or (b).

23. The isolated nucleic acid molecule of claim 22, wherein the isolated nucleic acid molecule codes for SEQ ID NO:175.

24. The isolated nucleic acid molecule of claim 22, wherein the isolated nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO:173.

25. The isolated nucleic acid molecule of claim 22, wherein the isolated nucleic acid molecule comprises a nucleotide sequence set forth as SEQ ID NO:174.

26. An isolated TLR7 polypeptide or fragment thereof comprising at least one amino acid of marine TLR7 selected from the group consisting of amino acids 4, 8, 15, 16, 18, 21, 23, 24, 25, 27, 37, 39, 40, 41, 42, 44, 45, 61, 79, 83, 86, 89, 92, 96, 103, 109, 111, 113, 119, 121, 127, 128, 131, 145, 148, 151, 164, 172, 176, 190, 202, 203, 204, 205, 222, 225, 226, 228, 236, 238, 243, 250, 253, 266, 268, 271, 274, 282, 283, 287, 288, 308, 313, 314, 315, 325, 328, 331, 332, 341, 343, 344, 347, 351, 357, 360, 361, 362, 363, 364, 365, 366, 370, 371, 377, 378, 387, 388, 389, 392, 397, 398, 413, 415, 416, 419, 421, 422, 425, 437, 438, 440, 446, 449, 453, 454, 455, 456, 462, 470, 482, 486, 487, 488, 490, 491, 493, 494, 503, 505, 509, 511, 529, 531, 539, 540, 543, 559, 567, 568, 574, 583, 595, 597, 598, 600, 611, 613, 620, 624, 638, 645, 646, 651, 652, 655, 660, 664, 665, 668, 669, 672, 692, 694, 695, 698, 701, 704, 714, 720, 724, 727, 728, 733, 738, 745, 748, 755, 762, 777, 780, 789, 803, 846, 850, 851, 860, 864, 868, 873, 875, 884, 886, 888, 889, 890, 902, 903, 911, 960, 967, 970, 980, 996, 1010, 1018, 1035, and 1045 of SEQ ID NO:175, wherein the polypeptide or fragment thereof has an amino acid sequence which is identical to a human TLR7 polypeptide or fragment thereof except for the at least one amino acid of marine TLR7.

27. The isolated TLR7 polypeptide or fragment thereof of claim 26, wherein the human TLR7 has an amino acid sequence set forth as SEQ ID NO:170.

28. The isolated TLR7 polypeptide or fragment thereof of claim 26, wherein the isolated TLR7 polypeptide or fragment thereof has an amino acid sequence selected from the group consisting of SEQ ID NO:175 and fragments of SEQ ID NO:175.

29. The isolated TLR7 polypeptide or fragment thereof of claim 26, wherein the isolated TLR7 polypeptide or fragment thereof is an extracytoplasmic domain of TLR7.

30. The isolated TLR7 polypeptide or fragment thereof of claim 26, wherein the isolated TLR7 polypeptide or fragment thereof comprises an MBD motif as set forth as any one of SEQ ID NOs: 203, 204, 212, and 213.

31. The isolated TLR7 polypeptide or fragment thereof of claim 26, wherein the isolated TLR7 polypeptide or fragment thereof selectively binds to an ISNA.

32. The isolated TLR7 polypeptide or fragment thereof of claim 26, wherein the isolated TLR7 polypeptide or fragment thereof selectively binds to a CpG nucleic acid.

33. An isolated nucleic acid molecule which encodes the isolated TLR7 polypeptide or fragment thereof of claim 26.

34. An expression vector comprising the isolated nucleic acid molecule of claim 22 operably linked to a promoter.

35. A host cell comprising the expression vector of claim 34.

36. The host cell of claim 35, further comprising a reporter construct capable of interacting with a TIR domain.

37. An expression vector comprising the isolated nucleic acid molecule of claim 33 operably linked to a promoter.

38. A host cell comprising the expression vector of claim 37.

39. The host cell of claim 38, further comprising a reporter construct capable of interacting with a TIR domain.

40. An isolated nucleic acid molecule selected from the group consisting of (a) nucleic acid molecules which hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence set forth as SEQ ID NO:190, and which code for a murine TLR8 having an amino acid sequence set forth as SEQ ID NO:192, (b) nucleic acid molecules that differ from the nucleic acid molecules of (a) in codon sequence due to degeneracy of the genetic code, and (c) complements of (a) or (b).

41. The isolated nucleic acid molecule of claim 40, wherein the isolated nucleic acid molecule codes for SEQ ID NO:192.

42. The isolated nucleic acid molecule of claim 40, wherein the isolated nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO:190.

43. The isolated nucleic acid molecule of claim 40, wherein the isolated nucleic acid molecule comprises a nucleotide sequence set forth as SEQ ID NO:191.

44. An isolated TLR8 polypeptide or fragment thereof comprising at least one amino acid of murine TLR8 selected from the group consisting of amino acids 5, 6, 9, 10, 14, 15, 18, 21, 22, 23, 24, 25, 26, 27, 28, 30, 39, 40, 41, 43, 44, 50, 51, 53, 55, 61, 67, 68, 74, 80, 85, 93, 98, 99, 100, 104, 105, 106, 107, 110, 114, 117, 119, 121, 124, 125, 134, 135, 138, 145, 155, 156, 157, 160, 161, 162, 163, 164, 166, 169, 170, 174, 180, 182, 183, 186, 187, 191, 193, 194, 196, 197, 199, 200, 207, 209, 210, 227, 228, 230, 231, 233, 234, 241, 256, 263, 266, 267, 268, 269, 272, 274, 275, 276, 280, 285, 296, 298, 299, 300, 303, 305, 306, 307, 310, 312, 320, 330, 333, 335, 343, 344, 345, 346, 347, 349, 351, 356, 362, 365, 366, 375, 378, 379, 380, 381, 383, 384, 386, 387, 392, 402, 403, 408, 414, 416, 417, 422, 426, 427, 428, 429, 430, 431, 433, 437, 438, 439, 440, 441, 444, 445, 449, 456, 461, 463, 471, 483, 486, 489, 490, 494, 495, 496, 505, 507, 509, 512, 513, 519, 520, 523, 537, 538, 539, 541, 542, 543, 545, 554, 556, 560, 567, 569, 574, 575, 578, 586, 592, 593, 594, 595, 597, 599, 602, 613, 617, 618, 620, 621, 623, 628, 630, 633, 639, 641, 643, 644, 648, 655, 658, 661, 663, 664, 666, 668, 677, 680, 682, 687, 688, 690, 692, 695, 696, 697, 700, 702, 703, 706, 714, 715, 726, 727, 728, 730, 736, 738, 739, 741, 746, 748, 751, 752, 754, 757, 764, 766, 772, 776, 778, 781, 784, 785, 788, 791, 795, 796, 801, 802, 806, 809, 817, 820, 821, 825, 828, 829, 831, 839, 852, 853, 855, 858, 863, 864, 900, 903, 911, 918, 934, 977, 997, 1003, 1008, 1010, 1022, 1023, 1024, 1026, and 1030 of SEQ ID NO:192, wherein the TLR8 polypeptide or fragment thereof has an amino acid sequence which is identical to a human TLR8 polypeptide or fragment thereof except for the at least one amino acid of marine TLR8.

45. The isolated TLR8 polypeptide or fragment thereof of claim 44, wherein the human TLR8 has an amino acid sequence set forth as SEQ ID NO:184.

46. The isolated TLR8 polypeptide or fragment thereof of claim 44, wherein the isolated TLR8 polypeptide or fragment thereof has an amino acid sequence selected from the group consisting of SEQ ID NO:192 and fragments of SEQ ID NO:192.

47. The isolated TLR8 polypeptide or fragment thereof of claim 44, wherein the isolated TLR8 polypeptide or fragment thereof is an extracytoplasmic domain of TLR8.

48. The isolated TLR8 polypeptide or fragment thereof of claim 44, wherein the isolated TLR8 polypeptide or fragment thereof comprises an MBD motif as set forth as any one of SEQ ID NOs: 205, 206, 214, and 215.

49. The isolated TLR8 polypeptide or fragment thereof of claim 44, wherein the isolated TLR8 polypeptide or fragment thereof selectively binds to an ISNA.

50. The isolated TLR8 polypeptide or fragment thereof of claim 44, wherein the isolated TLRB polypeptide or fragment thereof selectively binds to a CpG nucleic acid.

51. An isolated nucleic acid molecule which encodes the isolated TLR8 polypeptide or fragment thereof of claim 44.

52. An expression vector comprising the isolated nucleic acid molecule of claim 40 operably linked to a promoter.

53. A host cell comprising the expression vector of claim 52.

54. The host cell of claim 53, further comprising a reporter construct capable of interacting with a TIR domain.

55. An expression vector comprising the isolated nucleic acid molecule of claim 51 operably linked to a promoter.

56. A host cell comprising the expression vector of claim 55.

57. The host cell of claim 56, further comprising a reporter construct capable of interacting with a TIR domain.

58. An isolated nucleic acid molecule which hybridizes under stringent conditions to the isolated nucleic acid molecule of claim 1 or claim 13.

59. A method for inhibiting TLR9 signaling activity in a cell, comprising:
contacting the cell with an isolated nucleic acid molecule of claim 58 in an amount effective to inhibit expression of TLR9 polypeptide in the cell.

60. An isolated nucleic acid molecule comprising a nucleotide sequence which is complementary to the nucleotide sequence of the isolated nucleic acid molecule of claim 1 or claim 13.

61. A method for inhibiting TLR9 signaling activity in a cell, comprising:
contacting the cell with an isolated nucleic acid molecule of claim 60 in an amount effective to inhibit expression of TLR9 polypeptide in the cell.

62. A method for identifying nucleic acid molecules which interact with a TLR
polypeptide or a fragment thereof, comprising:
contacting a TLR polypeptide selected from the group consisting of TLR7, TLR8, TLR9, and nucleic acid-binding fragments thereof with a test nucleic acid molecule; and measuring an interaction of the test nucleic acid molecule with the TLR
polypeptide or fragment thereof.

63. The method of claim 62, wherein the TLR polypeptide or fragment thereof is expressed in a cell.

64. The method of claim 62, wherein the TLR polypeptide or fragment thereof is an isolated TLR polypeptide or fragment thereof.

65. The method of claim 64, wherein the isolated TLR polypeptide or fragment thereof is immobilized on a solid support.

66. The method of claim 62, wherein the TLR polypeptide or fragment thereof is fused with an Fc fragment of an antibody.

67. The method of claim 66, wherein the TLR polypeptide or fragment thereof comprises a TLR extracytoplasmic domain.

68. The method of claim 62, wherein the interaction is binding.

69. The method of claim 68, wherein the measuring is accomplished by a method selected from the group consisting of enzyme-linked imunosorbent assay (ELISA), biomolecular interaction assay (BIA), electromobility shift assay (EMSA), radioimmunoassay (RIA), polyacrylamide gel electrophoresis (PAGE), and Western blotting.

70. The method of claim 63, wherein the measuring is accomplished by a method comprising measuring a response mediated by a TLR signal transduction pathway.

71. The method of claim 70, wherein the response mediated by a TLR signal transduction pathway is selected from the group consisting of induction of a gene under control of NF-.kappa.B promoter and secretion of a cytokine.

72. The method of claim 71, wherein the gene under control of NF-.kappa.B
promoter is selected from the group consisting of IL-8, IL-12 p40, NF-.kappa.B-luc, IL-12 p40-luc, and TNF-luc.

73. The method of claim 71, wherein the cytokine is selected from the group consisting of IL-8, TNF-.alpha., and IL-12 p40.

74. The method of claim 70, further comprising:
comparing (a) the response mediated by a TLR signal transduction pathway as measured in presence of the test nucleic acid molecule with (b) a response mediated by a TLR signal transduction pathway as measured in absence of the test nucleic acid molecule; and determining the test nucleic acid molecule is an ISNA when (a) exceeds (b).

75. The method of claim 70, further comprising:
comparing the response to a reference response when the TLR polypeptide is independently contacted with a reference nucleic acid molecule; and determining if the response is stronger or weaker than the reference response.

76. The method of claim 70, further comprising:
comparing the response to a reference response when the TLR polypeptide is concurrently contacted with a reference nucleic acid molecule; and determining if the response is stronger or weaker than the reference response.

77. The method of claim 62, wherein the TLR polypeptide or fragment thereof is TLR7.

78. The method of claim 62, wherein the TLR polypeptide or fragment thereof is TLR8.

79. The method of claim 62, wherein the TLR polypeptide or fragment thereof is TLR9.

80. A screening method for identifying an ISNA, comprising:
contacting a functional TLR selected from the group consisting of TLR7, TLR8, and TLR9 with a test nucleic acid molecule;
detecting presence or absence of a response mediated by a TLR signal transduction pathway in the presence of the test nucleic acid molecule arising as a result of an interaction between the functional TLR and the test nucleic acid molecule; and determining the test nucleic acid molecule is an ISNA when the presence of a response mediated by the TLR signal transduction pathway is detected.

81. The method of claim 80, further comprising comparing the response mediated by the TLR signal transduction pathway arising as a result of an interaction between the functional TLR and the test nucleic acid molecule with a response arising as a result of an interaction between the functional TLR and a reference ISNA.

82. The method of claim 81, wherein the screening method is performed on a plurality of test nucleic acid molecules.

83. The method of claim 82, wherein the response mediated by the TLR signal transduction pathway is measured quantitatively and wherein the response mediated by the TLR signal transduction pathway associated with each of the plurality of test nucleic acid molecules is compared with a response arising as a result of an interaction between the functional TLR and a reference ISNA.

84. The method of claim 83, wherein a subset of the plurality of test nucleic acid molecules is selected based on ability of the subset to produce a specific response mediated by the TLR signal transduction pathway.

85. The method of claim 80, wherein the functional TLR is expressed in a cell.

86. The method of claim 85, wherein the cell is an isolated mammalian cell that naturally expresses the functional TLR.

87. The method of claim 86, wherein the cell comprises an expression vector comprising an isolated nucleic acid which encodes a reporter construct selected from the group consisting of IL-8, IL-12 p40, NF-.kappa.B-luc, IL-12 p40-luc, and TNF-luc.

88. The method of claim 80, wherein the functional TLR is part of a cell-free system.

89. The method of claim 80, wherein the functional TLR is part of a complex with another TLR.

90. The method of claim 89, wherein the complex is a complex of TLR9 and TLR7.

91. The method of claim 89, wherein the complex is a complex of TLR9 and TLR8.

92. The method of claim 80, wherein the functional TLR is part of a complex with a non-TLR protein selected from the group consisting of MyD88, IRAK, TRAF6, I.kappa.B, NF-.kappa.B, and functional homologues and derivatives thereof.

93. The method of claim 80, wherein the reference ISNA is a CpG nucleic acid.

94. The method of claim 80, wherein the test nucleic acid molecule is a CpG
nucleic acid.

95. The method of claim 80, wherein the response mediated by a TLR signal transduction pathway is selected from the group consisting of induction of a gene under control of NF-.kappa.B promoter and secretion of a cytokine.

96. The method of claim 95, wherein the gene under control of NF-.kappa.B
promoter is selected from the group consisting of IL-8, IL-12 p40, NF-.kappa.B-luc, IL-12 p40-luc, and TNF-luc.

97. The method of claim 95, wherein the cytokine is selected from the group consisting of IL-8, TNF-.alpha., and IL-12 p40.

98. A screening method for comparing TLR signaling activity of a test compound with an ISNA, comprising:
contacting a functional TLR selected from the group consisting of TLR7, TLR8, and TLR9 with a reference ISNA and detecting a reference response mediated by a TLR signal transduction pathway;
contacting a functional TLR selected from the group consisting of TLR7, TLR8, and TLR9 with a test compound and detecting a test response mediated by a TLR
signal transduction pathway; and comparing the test response with the reference response to compare the TLR
signaling activity of the test compound with the ISNA.

99. The method of claim 98, wherein the functional TLR is contacted with the reference ISNA and the test compound independently.

100. The method of claim 99, wherein the screening method is a method for identifying an ISNA mimic, and wherein when the test response is similar to the reference response the test compound is an ISNA mimic.

101. The method of claim 98, wherein the functional TLR is contacted with the reference ISNA and the test compound concurrently to produce a test-reference response mediated by a TLR signal transduction pathway and wherein the test-reference response may be compared to the reference response.

102. The method of claim 101, wherein the screening method is a method for identifying an ISNA agonist, and wherein when the test-reference response is greater than the reference response the test compound is an ISNA agonist.

103. The method of claim 101, wherein the screening method is a method for identifying an ISNA antagonist, and wherein when the test-reference response is less than the reference response the test compound is an ISNA antagonist.

104. The method of claim 98, wherein the functional TLR is expressed in a cell.

105. The method of claim 104, wherein the cell is an isolated mammalian cell that naturally expresses the functional TLR9.

106. The method of claim 105, wherein the cell comprises an expression vector comprising an isolated nucleic acid which encodes a reporter construct selected from the group consisting of IL-8, IL-12 p40, NF-.kappa.B-luc, IL-12 p40-luc, and TNF-luc.

107. The method of claim 98, wherein the functional TLR is part of a cell-free system.

108. The method of claim 98, wherein the functional TLR is part of a complex with another TLR.

109. The method of claim 98, wherein the functional TLR is part of a complex with a non-TLR protein selected from the group consisting of MyD88, IRAK, TRAF6, I.kappa.B, NF-.kappa.B, and functional homologues and derivatives thereof.

110. The method of claim 98, wherein the reference ISNA is a CpG nucleic acid.

111. The method of claim 98, wherein the test compound is not a nucleic acid molecule.

112. The method of claim 98, wherein the test compound is a polypeptide.

113. The method of claim 98, wherein the test compound is a part of a combinatorial library of compounds.

114. A screening method for identifying species specificity of an ISNA, comprising:
contacting a functional TLR selected from the group consisting of TLR7, TLR8, and TLR9 of a first species with a test ISNA;
contacting a functional TLR selected from the group consisting of TLR7, TLR8, and TLR9 of a second species with the test ISNA;
measuring a response mediated by a TLR signal transduction pathway associated with the contacting the functional TLR of the first species with the test ISNA;
measuring a response mediated by the TLR signal transduction pathway associated with the contacting the functional TLR of the second species with the test ISNA; and comparing (a) the response mediated by a TLR signal transduction pathway associated with the contacting the functional TLR of the first species with the test ISNA
with (b) the response mediated by the TLR signal transduction pathway associated with the contacting the functional TLR of the second species with the test ISNA.

115. The method of claim 114, wherein the functional TLR is expressed in a cell.

116. The method of claim 115, wherein the cell is an isolated mammalian cell that naturally expresses the functional TLR.

117. The method of claim 114, wherein the functional TLR is part of a cell-free system.

118. The method of claim 114, wherein the functional TLR is part of a complex with~
another TLR.

119. The method of claim 114, wherein the functional TLR is part of a complex with a non-TLR protein selected from the group consisting of MyD88, IRAK, TRAF6, I.kappa.B, NF-.kappa.B, and functional homologues and derivatives thereof.

120. A method for identifying lead compounds for a pharmacological agent useful in treatment of disease associated with TLR9 signaling activity, comprising providing a cell comprising a TLR9 as provided in claim 5;
contacting the cell with a candidate pharmacological agent under conditions which, in the absence of the candidate pharmacological agent, cause a first amount of signaling activity; and determining a second amount of TLR9 signaling activity as a measure of the effect of the pharmacological agent on the TLR9 signaling activity, wherein a second amount of TLR9 signaling activity which is less than the first amount indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which reduces TLR9 signaling activity and wherein a second amount of TLR9 signaling activity which is greater than the first amount indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which increases TLR9 signaling activity.