WO2007053428A2 - Method to identify polypeptide-toll-like receptor (tlr) ligands - Google Patents

Abstract

The present invention provides methods to identify phage populations enriched for specific binding to a Toll-like Receptor (TLR), such as TLR2, TLR4 and TLR5. The present invention also provides methods to identify polypeptide ligands for Toll-like Receptors (TLRs), such as TLR2, TLR4 and TLR5. The methods of the invention involve the use of phage display technology in a two phase, iterative screening procedure. The polypeptide TLR ligands so identified modulate TLR signaling and thereby regulate the Innate Immune Response. The invention provides polypeptide TLR ligands identified by the methods of the invention, as well as methods of modulating TLR signaling using the identified polypeptide ligands. The invention also provides vaccines comprising a polypeptide TLR ligand identified by the methods of the invention and an antigen. The invention further provides methods to stimulate an immune response using a polypeptide TLR ligand identified by the methods of the invention, or using a vaccine comprising a polypeptide TLR ligand identified by the method of the invention of the invention.

Description

METHOD TO IDENTIFY POLYPEPTIDE TOLL-LIKE RECEPTOR (TLR)

LIGANDS

RELATED APPLICATIONS This application claims priority to U.S. Provisional Application Serial

No. 60/731,594, filed on October 28, 2005. The contents of which is expressly incorporated herein by reference.

STATEMENT OF GOVERNMENT SUPPORT

The research leading to this invention was supported, in part, by contract # NIH-NIAID-D AIT-B AA-03 -41 and NO1-AI-40043 awarded by the National Institutes of Health. Accordingly, the United States government may have certain rights to this invention.

FIELD OF THE INVENTION

The present invention provides methods to identify phage populations enriched for specific binding to a Toll-like Receptor (TLR), such as TLR2, TLR4 and

TLR5. The present invention also provides methods to identify polypeptide ligands for Toll-like Receptors (TLRs), such as TLR2, TLR4 and TLR5. The methods of the invention involve the use of phage display technology in a two phase, iterative screening procedure. The polypeptide TLR ligands so identified modulate TLR signaling and thereby regulate the Innate Immune Response. The invention provides polypeptide TLR ligands identified by the methods of the invention, as well as methods of modulating TLR signaling using the identified polypeptide ligands. The invention also provides vaccines comprising a polypeptide TLR ligand identified by the methods of the invention and an antigen. The invention further provides methods to stimulate an immune response using a polypeptide TLR ligand identified by the methods of the invention, or using a vaccine comprising a polypeptide TLR ligand identified by the method of the invention of the invention. BACKGROUND OF THE INVENTION

Multicellular organisms have developed two general systems of immunity to infectious agents. The two systems are innate or natural immunity (usually referred to as "innate immunity") and adaptive (acquired) or specific immunity. The major difference between the two systems is the mechanism by which they recognize infectious agents. Recent studies have demonstrated that the innate immune system plays a crucial role in the control of initiation of the adaptive immune response and in the induction of appropriate cell effector responses (Fearon et al. Science 1996;272:50-53 and Medzhitov et al. Cell 1997;91:295-298). The innate immune system uses a set of germline-encoded receptors for the recognition of conserved molecular patterns present in microorganisms. These molecular patterns occur in certain constituents of microorganisms including: lipopolysaccharides, peptidoglycans, lipoteichoic acids, phosphatidyl cholines, bacterial proteins, including lipoproteins, bacterial DNAs, viral single and double- stranded RNAs, unmethylated CpG-DNAs, mannans, and a variety of other bacterial and fungal cell wall components. Such molecular patterns can also occur in other molecules such as plant alkaloids. These targets of innate immune recognition are called Pathogen Associated Molecular Patterns (PAMPs) since they are produced by microorganisms and not by the infected host organism (Janeway Qt al. Cold Spring Harb. Symp. Quant. Biol. 1989;54:1-13 and Medzhitov et al Curr. Opin Immunol. 1997;94:4-9). PAMPs are discrete molecular structures that are shared by a large group of microorganisms. They are conserved products of microbial metabolism, which are not subject to antigenic variability (Medzhitov et al. Cur Op Immun 1997;9:4). The receptors of the innate immune system that recognize PAMPs are called Pattern Recognition Receptors (PRRs) (Janeway et al. Cold Spring Harb. Symp. Quant. Biol. 1989;54:1-13 and Medzhitov et al. Curr. Opin. Immunol. 1997;94:4-9). These receptors vary in structure and belong to several different protein families. Some of these receptors recognize PAMPs directly (e.g., CD14, DEC205, collectins), while others (e.g. , complement receptors) recognize the products generated by PAMP recognition.

Cellular PRRs are expressed on effector cells of the innate immune system, including cells that function as professional antigen-presenting cells (APC) in

_9_ adaptive immunity. Such effector cells include, but are not limited to, macrophages, dendritic cells, B lymphocytes, and epithelial cells. This expression profile allows PRRs to directly induce innate effector mechanisms, and also to alert the host organism to the presence of infectious agents by inducing the expression of a set of endogenous signals, such as inflammatory cytokines and chemokines. This latter function allows efficient mobilization of effector forces to combat the invaders.

The best characterized class of cellular PRRs are members of the family of Toll-like receptors (TLRs)₃ so called because they are homologous to the Drosophila Toll protein which is involved both in dorsoventral patterning in Drosophila embryos and in the immune response in adult flies (Lemaitre et al Cell 1996;86:973-83). At least 12 mammalian TLRs, TLRs 1 through 11 and TLR13, have been identified to date (see, for example, Medzhitov et al Nature 1997;388:394- 397; Rock et al Proc Nail Acad Sci USA 1998,95:588-593; Takeuchi et al Gene 1999;231 :59-65; and Chuang and Ulevitch. Biochim Biophys Acta. 2001 :1518:157- 61).

In mammalian organisms, such TLRs have been shown to recognize PAMPs such as the bacterial products LPS (Schwandner et al J. Biol. Chem. 1999;274: 17406-9 and Hoshino et al J. Immunol 1999; 162:3749-3752), lipoteichoic acid (Schwandner et al J. Biol Chem. 1999;274: 17406-9), peptidoglycan (Yoshimura et al J. Immunol 1999; 163: 1-5), lipoprotein (Aliprantis et al Science 1999;285:736- 9), CpG-DNA (Hemmi et al Nature 2000;408:740-745), and flagellin (Hayashi et al. Nature 2001;410:1099-1103), as well as the viral product double stranded RNA (Alexopoulou el al Nature 2001 ;413 :732-738) and the yeast product zymosan (Underhill. J Endotoxin Res. 2003 ;9: 176-80). TLR2 is essential for the recognition of a variety of PAMPs, including bacterial lipoproteins, peptidoglycan, and lipoteichoic acids. TLR3 is implicated in virus-derived double-stranded RNA. TLR4 is predominantly activated by lipopolysaccharide. TLR5 detects bacterial flagellin and TLR9 is required for response to unmethylated CpG DNA. Recently, TLR7 and TLR8 have been shown to recognize small synthetic antiviral molecules (Jurk M. et al Nat Immunol 2002;3:499). Furthermore, in many instances, TLRs require the presence of a co- receptor to initiate the signaling cascade. One example is TLR4 which interacts with MD2 and CD 14, a protein that exists both in soluble form and as a GPI-anchored protein, to induce NF-κB in response to LPS stimulation (Takeuchi and Akira. Microbes Infect 2002;4:887~95). Figure 1 illustrates some of the known interactions between PAMPs and TLRs (reviewed in Janeway and Medzhitov. Annu Rev Immunol 2002;20:197-216). TLR2 is involved in the recognition of, e.g., multiple products of

Gram-positive bacteria, mycobacteria and yeast, including LPS and lipoproteins. TLR2 is known to heterodimerize with other TLRs, a property believed to extend the range of PAMPs that TLR2 can recognize. For example, TLR2 cooperates with TLR6 in the response to peptidoglycan (Ozinsky et a Proc Natl Acad Sci U S A 2000;97: 13766-71) and diacylated mycoplasmal lipopeptide (Takeuchi et a Int Immunol 2001;13:933-40), and associates with TLRl to recognize triacylated lipopeptides (Takeuchi et a J Immunol 2002; 169: 10-4). Pathogen recognition by TLR2 is strongly enhanced by CD14. A pentapeptide, ALTTE (SEQ ID NO: 1), derived from fimbrial subunit protein was shown to activate monocytes and epithelial cells via TLR2 signaling (Ogawa et a FEMS Immunol Med Microbiol 1995;11 :197- 206; Asai et a Infect Immtm 2001 ;69:7378-7395; and Ogawa et a Eur J Immunol 2002;32:2543-2550). A single amino acid substitution (A to G) in the peptide (GLTTE, SEQ ID NO: 2) was shown to antagonize the activity of the wild-type peptide and full-length protein (Ogawa et ah FEMS Immunol Med Microbiol 1995;11 :197-206).

TLR4, the first human TLR identified, is involved in the recognition of, for example, products of Gram negative bacteria such as lipopolysaccharide (LPS), products of Gram positive bacteria such as lipoteichoic acid, and the F protein of Respiratory Syncytial Virus (RSV F protein) (reviewed in Janeway and Medzhitov. Annu Rev Immunol 2002;20: 197-216). The envelope protein of Mouse Mammary Tumor Virus (MMTV env protein) has been shown to activate B-cells via TLR4 (Rassa et a Proc Natl Acad Sci USA 2002;99:2281-2286). The Tlr4 gene is mutated in C3H/HeJ and C57BL/1 OScCr mice, both of which are low responders to LPS (Poltorak et a Science 1998;282:2085-2088). In many instances, TLR4 requires the presence of accessory molecules to initiate the signaling cascade. For example, TLR4 interacts with MD2 and CD 14, a protein that exists both in soluble form and as a GPI- anchored protein, to induce NF-κB in response to LPS stimulation (Shimazu et a J Exp Med 1999;189:1777-1782 and Takeuchi and Akira. Microbes Infect 2002;4:887- 95). TLR4 is known to homodimerize in a multisubunit cell surface protein complex containing two monomers of TLR4, a MD2 monomer, and a CD 14 monomer. TLR4 signaling is mediated through the adapter protein MyD88 but also through a MyD88- independent pathway that involves the TIR domain containing adapter protein (TIRAP) (Horng et al Nat Immunol 2001 ;2:835-41).

TLR5 is the Toll-like receptor that recognizes flagellin from both Gram-positive and Gram-negative bacteria. Activation of the receptor stimulates the production of proinflammatory cytokines, such as TNFα, through signaling via the adaptor protein MyD88 and the serine kinase IRAK (Gewirtz et al. J Immunol 2001;167:1882-5 and Hayashi et al. Nature 2001;410:1099-103). TLR5 can generate a proinflammatory signal as a homodimer suggesting that it might be the only TLR required for flagellin recognition (Hayashi et al. Nature 2001;410:1099-103).

Activation of signal transduction pathways by TLRs leads to the induction of various genes including inflammatory cytokines, chemokines, major histocompatability complex, and co-stimulatory molecules (e.g., B7). The intracellular signaling pathways initiated by activated TLRs vary slightly from TLR to TLR, with some signaling pathways being common to all TLRs (shared pathways), and some being specific to particular TLRs (specific pathways).

In one of the shared pathways, the cytoplasmic adaptor proteins myeloid differentiation factor 88 (MyD88) and TOLLIP (Toll-interacting protein) independently associate with the cytoplasmic tail of the TLR. Each of these adaptors recruits the serine/threonine kinase IRAK to the receptor complex, each with different kinetics. Recruitment of IRAK to the receptor complex results in auto- phosphorylation of IRAK. Phosphorylated IRAK then associates with another adaptor protein, TRAF6. TRAF6, in turn, associates with and activates the MAP kinase kinases TAK-I and MKK6. Activation of TAK-I leads, via one or more intermediate steps, to the activations of the IKB kinase (IKK), whose activity directs the degradation of IKB and the activation of NF-κB. Activation of MKK6 leads to the activation of JNK (c-Jun N-terminal kinase) and the MAP kinase p38 (Medzbitov and Janeway. Trends in Microbiology) 2000;8:452-456; and Medzhitov. Nature Reviews 2001;l :135-145). Other cytoplasmic proteins implicated in TLR signaling include the RHO family GTPase RACl and protein kinase B (PKB), as well as the adapter protein TIRAP and its associated proteins protein kinase PKR and the PKR regulatory proteins PACT and p58 (Medzhitov. Nature Reviews 2001;l :135-145). Cytoplasmic proteins specifically implicated in TLR-signaling by mutational studies include MyD88 (Schnare et al Nature Immunol 2001;2:947-950), TIRAP (Horng et al Nature Immunol 2001;2:835-842), IRAK and TRAF6 (Medzhitov et al MoI Cell 1998;2:253-258), RICK/Rip2/CARDIAK (Kobayashi et al. Nature 2002:416:194- 199), IRAK-4 (Suzuki et al. Nature 2002;416:750~746), and MaI (MyD88-adapter like) (Fitzgerald et al Nature 2001;413:78-83).

Due to TLR signaling through shared pathways (e.g., NF-κB, see above), some biological responses will likely be globally induced by any TLR signaling event. However, an emerging body of evidence demonstrates divergent responses induced by the specific pathways of individual TLRs. For example, TLR2 and TLR4 activate different immunological programs in human and murine cells, manifested in divergent patterns of cytokine expression (Hirschfeld et al Infect Immun 2001 ;69: 1477-1482 and Re and Strominger. J Biol Chem 2001;276:37692- 37699). These divergent phenotypes could be detected in an antigen-specific response, when lipopolysaccharides that signal through TLR2 or TLR4 were used to guide the response (Pulendran et al J Immun 2001 ;167:5067-5076). TLR4 and TLR2 signaling requires the adaptor TIRAP/Mal, which is involved in the MyD88- dependent pathway (Horng et al. Nature 2002;420:329-33). TLR3 triggers the production of IFNβ in response to double-stranded RNA, in an MyD 88 -independent manner. This response is mediated by the adaptor TRIF/TICAM-1 (Yamamoto et al. J Immunol 2002; 169:6668-72). TRAM/TICAM2 is another adaptor molecule involved in the MyD 88 -independent pathway (Miyake. Int Immiinopharmacol 2003 ;3:119-28) which function is restricted to the TLR4 pathway (Yamamoto et al. Nat Immunol. 2003 ;4: 1144-50).

Thus, different TLR "switches" turn on different immune response "circuits", where activation of a particular TLR determines the type of antigen- specific response that is triggered. Depending upon the cell type exposed to a PAMP and the particular TLR that binds to that PAMP, the profile of cytokines produced and secreted can vary. This variation in TLR signaling response can influence, for example, whether the resultant adaptive immune response will be predominantly T- cell- or B-cell-mediated, as well as the degree of inflammation accompanying the response. As discussed above, the innate immune system plays a crucial role in the control of initiation of the adaptive immune response and in the induction of appropriate cell effector responses. Recent evidence demonstrates that fusing a polypeptide ligand specific for a Toll-like receptor (TLR) to an antigen of interest generates a vaccine that is more potent and selective than the antigen alone. The inventors have previously shown that immunization with recombinant TLR- ligand:antigen fusion proteins: a) induces antigen-specific T-cell and B-cell responses comparable to those induced by the use of conventional adjuvant, b) results in significantly reduced non-specific inflammation; and c) results in CD 8 T-cell- mediated protection that is specific for the fused antigen epitopes (see, for example US published patent applications 2002/0061312 and 2003/0232055 to Medzhitov, and US published patent application 2003/0175287 to Medzhitov and Kopp, all incorporated herein by reference). Mice immunized with a fusion protein consisting of the polypeptide PAMP BLP linked to Leishmania major antigens mounted a Type 1 immune response characterized by antigen-induced production of γ-interferon and antigen-specific IgG_2a (Cote-Sierra et al. Infect Immun 2002;70:240-248). The response was protective, as demonstrated in experiments in which immunized mice developed smaller lesions than control mice did following challenge with live L. major. Thus, the binding of PAMPs to TLRs activates immune pathways that can be mobilized for the development of more potent vaccines. Ideally, a vaccine design should ensure that every cell that is exposed to pathogen-derived antigen also receives a TLR receptor innate immune signal and vice versa. This can be effectively achieved by designing the vaccine to contain a chimeric macromolecule of antigen plus PAMP, e.g., a fusion protein of PAMP and antigen(s). Such molecules trigger signal transduction pathways in their target cells that result in the display of co- stimulatory molecules on the cell surface, as well as antigenic peptide in the context of major histocompatability context molecules.

Although polypeptide ligands to some TLRs are known (see Figure 1), cognate polypeptide ligands for other TLRs have not been discovered. Furthermore, for many of the known TLR ligands, the particular amino acid residues that contribute to ligand:TLR interaction are not known. Gross deletion studies, alanine-scanning and site directed mutagenesis have been used to delineate the critical amino acids in E. coli flagellin (fliC; Donelly and Steiner. J Biol Chem 2002;277:40456~40461) and Measles Virus hemagglutinin (HA; Bieback et al. J Virol 2002;76: 8729-8736) necessary for PAMP activity. In these protocols, every polypeptide ligand variant construct must be individually expressed and the resulting recombinant protein purified for biological activity assays. Thus, these previously disclosed strategies for characterization of TLR polypeptide ligands are laborious and time-consuming.

A need exists in the art for methods to identify novel polypeptide ligands for TLRs. In particular, the need exists for the identification of polypeptide ligands specific for individual TLR receptors, which can be used to specifically tune the innate immune system response. The present invention fulfills these needs in the art by providing a method for identifying novel polypeptide ligands of TLRs based upon screening of phage display libraries for the ability to bind live cells expressing a TLR of interest. The methods of the invention can be applied to identify novel peptides that interact specifically with individual TLRs. Polypeptide TLR ligands identified according to the methods of the invention have the potential to be powerful and selective activators of the innate immune system, and may be engineered into vaccines to generate vigorous antigen-specific immune responses with minimal inflammation. Such TLR-specific polypeptide ligands can be incorporated into polypeptide TLR ligand: antigen conjugate vaccines, whereby the polypeptide TLR ligand will provide for an enhanced antigen-specific immune response as regulated by signaling through a particular TLR.

Furthermore, a need exists in the art for efficient methods to further characterize known polypeptide TLR ligands. The invention further provides methods to optimize the polypeptide sequence of known polpeptide TLR ligands. These novel and optimized polypeptide TLR ligands may be incorporated into vaccines, e.g., for use against infectious diseases that pose a public health and national defense threat.

Phage display is a selection technique in which a peptide or protein is genetically fused to a coat protein of a bacteriophage (Smith. Science 1985;228:1315~ 1317). The fusion protein is displayed on the exterior of the phage virion, while the DNA encoding the fusion protein resides within the virion. This physical linkage between the displayed protein and the DNA encoding it allows screening of vast numbers of variants of the protein by a simple in vitro selection procedure termed "biopanning". Phage display technology offers a very powerful tool for the isolation of new ligands from large collections of potential ligands including short peptides, antibody fragments and randomly modified physiological ligands to receptors (Scott and Smith. Science 1990;249:386-390; Smith and Scott. Meth Em 1993;217:228-257; and Smith and Petrenko. Chem Rev 1997;97:391-410). These systems have been effectively employed in studies of structural and functional aspects of receptor-ligand interactions using either purified receptors immobilized on a polymer surface (Smith and Petrenko. Chem Rev 1997;97:391-41O), or the receptors in their natural environment on the surface of living cells (Fong. et al. Drug Dev Res 199433:64-70; Doorbar and Winter. JMoI Biol 1994;244:361369; Goodson et al. Proc Natl Acad Sci USA 1994;91 :7129-7133; and Szardenings et al. J Biol Chem 1997;272:27943- 27948).

SUMMARY OF THE INVENTION

The present invention is directed to a method to identify a phage population enriched for specific binding to a TLR comprising: i) providing a multiplicity of test phage in the form of a phage display library, wherein each test phage comprises a nucleic acid insert encoding a polypeptide; ii) performing a first phase of screening comprising the steps of: a) contacting a TLR¹⁰ cell with the multiplicity of test phage; b) retaining the test phage that do not bind to the TLR¹⁰ cell; and c) optionally, repeating steps a) and b); iii) dividing the test phage retained in step ii) into at least a first phage portion and a second phage portion; iv) performing a second phase of screening comprising the steps of: d) contacting a TLR ' cell with the first phage portion, and contacting a

TLR¹⁰ cell with the second phage portion, wherein each TLR is the same TLR as in step ii); e) retaining the test phage of the first phage portion that bind to the TLR^hl cell and retaining the test phage of the second phage portion that bind to the TLR¹⁰ cell; f) optionally, determining the number of retained test phage of the first phage portion and determining the number of retained test phage of the second phage portion; g) amplifying the retained test phage of the first phage portion and amplifying the retained test phage of the second phage portion; h) optionally, determining the number of test phage in the amplified first phage portion and determining the number of test phage in the amplified second phage portion; and i) optionally, repeating steps d) through h); wherein step f) is performed at least once or step h) is performed at least once; and v) performing at least one of steps j) or k): wherein step j) comprises comparing the number of retained test phage of the first phage portion determined in step iv) with the number of retained test phage of the second phage portion determined in step iv); and wherein step k) comprises comparing the number of test phage in the amplified first phage portion determined in step iv) with the number of test phage in the amplified second phage portion determined in step iv), wherein: if the number of retained test phage of the first phage portion determined in step iv) is greater that the number of retained test phage of the second phage portion determined in step iv), or if the number of test phage in the amplified first phage portion determined in step iv) is greater than the number of test phage in the amplified second phage portion determined in step iv), the test phage of the first phage portion amplified in step iv) are identified as a phage population enriched for specific binding to a TLR.

In certain embodiments, steps a) and b) are performed at least two times. In certain embodiments, steps d) through h) are performed at least four times.

In certain other embodiment, TLR¹⁰ cells are not tested in parallel with TLR^hl cells. Rather, isolates that test positive on TLR^hl cells are later retested on TLR¹⁰ cells (see Example 18).

In some embodiments the method to identify a phage population enriched for specific binding to a TLR comprises: i) providing a multiplicity of test phage in the form of a phage display library, wherein each test phage comprises a nucleic acid insert encoding a polypeptide; ii) performing a first phase of screening comprising the steps of: a) contacting a TLR¹⁰ cell with the multiplicity of test phage; b) retaining the test phage that do not bind to the TLR¹⁰ cell; and c) repeating steps a) and b) once; iϋ) dividing the test phage retained in step ii) into a first phage portion and a second phage portion, wherein the number of test phage in the first phage portion is approximately equal to the number of test phage in the second phage portion; iv) performing a second phase of screening comprising the steps of: d) contacting a TLR" cell with the first phage portion, and contacting a TLR'° cell with the second phage portion, wherein each TLR is the same TLR as in step ii); e) retaining the test phage of the first phage portion that bind to the TLR¹¹ cell and retaining the test phage of the second phage portion that bind to the

TLR¹⁰ cell; g) amplifying the retained test phage of the first phage portion and amplifying the retained test phage of the second phage portion; h) determining the number of test phage in the amplified first phage portion and determining the number of test phage in the amplified second phage portion; and i) repeating steps d) through g) three times; and v) comparing the number of test phage in the amplified first phage portion determined in step iv) with the number of test phage in the amplified second phage portion determined in step iv), wherein if the number of test phage in the amplified first phage portion determined in step iv) is greater than the number of test phage in the amplified second phage portion determined in step iv) the test phage of the amplified first phage portion of step iv) are identified as a phage population enriched for specific binding to a TLR. In certain embodiments, TLR¹⁰ cells are not tested in parallel with

TLR^hl cells. Rather, isolates that test positive on TLR^hl cells are later retested on TLR¹⁰ cells (see Example 18). In preferred embodiments, the TLR is a mammalian TLR. In preferred embodiments the TLR is a TLR2, a TLR4, or a TLR5. In particularly preferred embodiments, the TLR is a mammalian TLR2, a mammalian TLR4, or a mammalian TLR5. In preferred embodiments, the TLR¹⁰ cell of step ii) and the TLR¹⁰ cell of step iv) are the same cell type. In preferred embodiments, the TLR¹⁰ cell of step ii), the TLR¹⁰ cell of step iv), and the TLR^hl cell of step iv) are the same cell type. In preferred embodiments, the TLR¹⁰ cell of step ii), the TLR¹⁰ cell of step iv), and the TLR^hl cell of step iv) are the same cell type. In certain embodiments, the TLR¹⁰ cell of step ii), the TLR¹⁰ cell of step iv), and the TLR^hi cell of step iv) are each a HEK293 cell or an NIH3T3 cell.

The invention is further directed to a method to identify a polypeptide TLR ligand comprising: i) identifying a phage population enriched for specific binding to a TLR according to the method of the invention; and ii) characterizing the polypeptide encoded by the nucleic acid insert of a test phage of the phage population enriched for specific binding to a TLR.

In some embodiments, characterizing the polypeptide encoded by the nucleic acid insert of a test phage of the phage population enriched for specific binding to a TLR comprises: i) determining the nucleic acid sequence of the nucleic acid insert; and ii) using the nucleic acid sequence from step i) to deduce the amino acid sequence of the polypeptide encoded by the nucleic acid insert.

In particular embodiments, characterizing the polypeptide encoded by the nucleic acid insert of a test phage of the phage population enriched for specific binding to a TLR comprises: i) translating the nucleic acid insert to generate the polypeptide encoded by the nucleic acid insert; and ii) characterizing said polypeptide, for example by determining the amino acid sequence of the polypeptide or measuring the ability of the polypeptide to modulate TLR signaling.

In some embodiments, characterizing the polypeptide encoded by the nucleic acid insert of a test phage of the phage population enriched for specific binding to a TLR comprises measuring the ability of the test phage to modulate TLR signaling. The invention is further directed to a phage population enriched for specific binding to a TLR identified by the method to identify a phage population enriched for specific binding.

The invention is also directed to a polypeptide TLR ligand identified by the methods described herein.

The invention is further directed to a polypeptide comprising: i) at least one polypeptide TLR ligand identified by the method to identify a polypeptide TLR ligand; and ii) at least one polypeptide antigen. In particular embodiments, the at least one antigen is a pathogen-related antigen, a tumor-associated antigen, or an allergen- related antigen. In preferred embodiments, the pathogen-related antigen is an Influenza antigen, a Listeria monocytogenes antigen, or a West Nile Virus antigen. The invention is further directed to a vaccine comprising such a polypeptide and, optionally, a pharmaceutically acceptable carrier.

In one embodiment, the invention is directed to a method to stimulate an immune response in a subject comprising administering to a subject in need thereof a polypeptide of the invention that has agonist activity. In another embodiment, the invention is also directed to a method to inhibit an immune response in a subject comprising administering to a subject in need thereof a polypeptide of the invention that has antagonist activity. In preferred embodiments, the subject is a mammal. The invention is further directed to a method of modulating, e.g., agonizing or antagonizing, TLR signaling in a cell comprising contacting a cell, wherein the cell comprises the TLR, with a polypeptide of the invention. In preferred embodiments, the cell is a mammalian cell.

The invention is further directed to a vaccine comprising: i) at least one polypeptide TLR ligand of the invention; ii) at least one antigen; and iii) optionally, a pharmaceutically acceptable carrier. In some embodiments, the at least one polypeptide TLR ligand and the at least one antigen are covalently linked. In certain embodiments, the at least one antigen is a polypeptide, a lipoprotein, a glycoprotein, a mucoprotein, a lipid, a saccharide, a lipopolysaccharide, or a nucleic acid. In particular embodiments, the at least one antigen is a pathogen-related antigen, a tumor-associated antigen, or an allergen-related antigen. In preferred embodiments, the pathogen-related antigen is an Influenza antigen, a Listeria monocytogenes antigen, or a West Nile Virus antigen. The present invention is also directed to a method to stimulate an immune response in a subject comprising administering to a subject in need thereof a vaccine of the invention. In preferred embodiments, the subject is a mammal.

DESCRIPTION OF THE DRAWINGS Figure 1 depicts known interactions of PAMPs with various Toll-like

Receptors (TLRs). (G+) = Gram-positive. (G-) = Gram-negative.

Figure 2 (Figure 2A and Figure 2B) depicts an amino acid sequence alignment of amino acid sequences for human TLR4 (hTLR4) isoforms A (SEQ ID NO: 106), B (SEQ ID NO: 108), C (SEQ ID NO: 110), and D (SEQ ID NO: 112). "^•*" indicates that the amino acid residue at the indicated position is common to all four isoforms.

Figure 3 is a graph depicting secretion of interleukin 8 (1L-8, in pg/ml) by HEK293-null cells (Invivogen; cat. # 293-null) cells ("HEK293", -♦-) versus HEK293:hTLR4A/MD2-CD14 cells ("HEK293:TLR4", -■-) upon exposure to various indicate concentrations of lipopolysaccharide (LPS, in ng/ml).

Figure 4 is a schematic depicting the extension strategy used to generate the random peptide inserts for construction of cyclic lθ~mer and 7-mer random peptide phage display libraries. "NNK" represents nucleotides that comprise the random peptide, where N is A/T/G/C and K is G/T. Bold lowercase letters denote restriction enzyme sites, and "xxxxxxx" depicts additional nucleotides within the oligonucleotides (SEQ ID NO: 123).

Figure 5 is a schematic depicting a method of screening of phage display libraries to identify a phage population enriched for specific binding to a TLR, and to identify polypeptide TLR ligands. Figure 6 is a graph depicting the phage titer of retained, cell-bound phage (Recovered Phage Titer, in units of 10⁴ phage/ml) for each round of positive screening ("Rounds of Biopanning"). "TLR4+/S-Tag" = S-Tag phage portion on TLR4 expressing cells. "TLR4+/10mer" = 10-mer phage display library phage portion on TLR4 expressing cells. "TLR4-/S-Tag" - S-Tag phage portion on cells not expressing TLR4. "TLR4-/10mer" = 10-mer phage display library phage portion on cells not expressing TLR4. Figure 7 depicts a schematic of exemplary plasmid vector T7.LIST. T7.LIST is designed to express a recombinant LLO-p60 (SEQ ID NO: 98) fusion protein with a V5 epitope (GKP1PNPLLGLDST; SEQ ID NO: 3) and a polyhistidine tag (6xHis)(SEQ ID NO: 4). T7 - T7 promoter. Rbs = ribosome binding site. Figure 8 depicts the amino acid sequence of human TLR2 (SEQ ID

NO: 102).

Figure 9 is a bar graph showing activation of NF-κB-dependent luciferase activity in 293 ("293") and 293.hTLR5 ("293/hTLR5") cells exposed to T7 phage displaying the fliC protein ("Phage", black bar) or to medium alone ("Medium", striped bar); and in 293.hTLR5 cells exposed to the T7 phage displaying the S-tag polypeptide ("S-Tag", "Phage", black bar) or to medium alone ("S-Tag", "Medium", striped bar). "RLU" = relative luciferase units.

Figure 10 depicts activity of synthetic peptides on HEK293:TLR4 cells. Serial 5-fold dilutions of peptide were added to HEK293:TLR4 cells beginning at a concentration of 500 μM and ending at 0.00064 μM (x-axis). After 24 hours, cell supernatants were tested for IL-8 by ELISA (y-axis).

Figure 11 depicts activity of synthetic peptides on RAW264.7 cells.

Serial 5-fold dilutions of peptides were added to RAW264.7 cells beginning at a concentration of 20 μM and ending at 0.0064 μM (x-axis) in the presence of protease inhibitors (Sigma) and polymyxin B (Invivogen). After 24 hours, cell supernatants were harvested and tested for TNF by ELISA.

Figure 12 depicts TLR4 bioactivity of new synthetic peptides identified by phage display. (A) The response of TLR4+ HEK cells to the novel six peptides in Formulation 121a as well as to D2 in Formulation 121a and Formulation 121a alone was measured by IL-8 production. (B) LPS is shown to have similar activity on TLR4+ HEK cells when resuspended in either PBS or Formulation 121a.

Figure 13 depicts activation of BMDC by synthetic peptides,

C3H/HeJ (left) and C3H/HeN (right) BMDC were cultured with the indicated peptides or known TLR ligands for 18 hours. The concentrations of TNF, MCP-I₅ and IL-6 in the cell supernatants were determined by CBA (BDBiosciences). Values have been normalized with an unstimulated or "blank" control culture. Figure 14 illustrates D2 Activation of Human DCs. DCs differentiated from CD 14+ monocytes were cultured with either D2 at 10 or 50 μM, F3 at 10 or 50 μM, LPS at 10 ng/mL or 100 ng/mL. Supernatant samples were collected at 24 and 48 hours after stimulation and cytokines were detected by CBA.

DETAILED DESCRIPTION

The present invention provides a method to identify a phage population enriched for specific binding to a TLR. This method of the invention comprises the steps of: i) providing a multiplicity of test phage in the form of a phage display library, wherein each test phage comprises a nucleic acid insert encoding a polypeptide; ii) performing a first phase of screening comprising the steps of: a) contacting a TLR¹⁰ cell with the multiplicity of test phage; b) retaining the test phage that do not bind to the TLR¹⁰ cell; and c) optionally, repeating steps a) and b); iii) dividing the test phage retained in step ii) into at least a first phage portion and a second phage portion; iv) performing a second phase of screening comprising the steps of: d) contacting a TLR^hl cell with the first phage portion, and contacting a TLR¹⁰ cell with the second phage portion, wherein each TLR is the same TLR as in step ii); e) retaining the test phage of the first phage portion that bind to the TLR^hl cell and retaining the test phage of the second phage portion that bind to the TLR¹⁰ cell; f) optionally, determining the number of retained test phage of the first phage portion and determining the number of retained test phage of the second phage portion; g) amplifying the retained test phage of the first phage portion and amplifying the retained test phage of the second phage portion; h) optionally, determining the number of test phage in the amplified first phage portion and determining the number of test phage in the amplified second phage portion; and i) optionally, repeating steps d) through h); wherein step f) is performed at least once or step h) is performed at least once; and v) performing at least one of steps j) or k): wherein step j) comprises comparing the number of retained test phage of the first phage portion determined in step iv) with the number of retained test phage of the second phage portion determined in step iv); and wherein step k) comprises comparing the number of test phage in the amplified first phage portion determined in step iv) with the number of test phage in the amplified second phage portion determined in step iv), wherein: if the number of retained test phage of the first phage portion determined in step iv) is greater that the number of retained test phage of the second phage portion determined in step iv), or if the number of test phage in the amplified first phage portion determined in step iv) is greater than the number of test phage in the amplified second phage portion determined in step iv), the test phage of the amplified first phage portion of step iv) are identified as a phage population enriched for specific binding to a TLR.

In the method of the invention, the phage display library provided in step i) is subjected to a screen comprising a first phase of screening and second phase of screening. The first phase of screening represents a negative selection phase, while the second phase of screening represents a positive selection phase. In order to maximize cell viability, it is preferable that both the first phase of screening and the second phase of screening be performed at 4⁰C.

In the first phase of screening, steps a) through c) of step ii) serve to deplete the phage population of test phage that bind non-specifically to the TLR'⁰ cell

(i.e., test phage whose binding to the cell is not mediated by the target TLR). In particular, where TLRs other than the target TLR are expressed by the TLR¹⁰ cell, this phase of screening serves to deplete the phage population of test phage that bind to

TLRs other than the target TLR. Upon iteration of steps a) through c) of step ii), the phage population is dramatically depleted of phage that bind non-specifically to the

TLR¹⁰ cell. In certain embodiments, steps a) through c) of step ii) are repeated once

(for a total of two cycles in the first phase of screening). In this first phase of screening, the retained test phage represent a phage population depleted of test phage that bind non-specifically to the TLR¹⁰ cell (i.e., test phage whose binding to the cell is not mediated by the target TLR).

In step iii), these retained test phage are divided into at least a first phage portion and a second phage portion. In some embodiments, these retained test phage are divided into a first phage portion and a second phage portion.

In preferred embodiments, the number of test phage in the first phage portion is approximately equal to the number of phage in the second phage portion.

However, embodiments wherein the number of test phage in the first phage portion is not approximately equal to the number of phage in the second phage portion are also contemplated.

In preferred embodiments these retained test phage are divided into a first phage portion and a second phage portion, and the number of test phage in the first phage portion is approximately equal to the number of phage in the second phage portion.

The first and second phage portions are then subjected to a second phase of screening. In the second phase of screening, comprising steps d) though i) of step iv), the first and second phage portions are screened in parallel on TLR^hl and TLR¹⁰ cells, respectively. Upon iteration of steps d) through i) of step iv), the first and second phage portions are enriched for phage that bind to TLR^hl and TLR¹⁰ cells, respectively. In certain embodiments, steps d) through i) of step iv) are repeated three times (for a total of four cycles in the second phase of screening).

In certain embodiments, steps e) and g) of step iv) are performed simultaneously. For example, cell bound phage may be simultaneously retained and amplified by direct liquid amplification in E.coli (strain 5615). In such embodiments, step h) is performed at least once, and step v) comprises performing step k).

In steps d) through i) of step iv), the retained test phage of the first phage portion are test phage that bind to the TLR^hl cell. This population of retained test phage may contain test phage that bind non-specifically to the TLR^hl cell (i.e., test phage whose binding to the cell is not mediated by the target TLR), as well as test phage that bind specifically to the TLR^hl cell (i.e., test phage whose binding to the cell is mediated by the target TLR). Accordingly, the amplified test phage of the first phage portion may contain test phage that bind non-specifically to the TLR^hl cell, as well as test phage that bind specifically to the TLR^hl cell. Therefore, enrichment of the first phage portion obtained upon iteration of steps d) through i) of step iv) may represent enrichment for non-specific binding, as well as enrichment for specific binding. In steps d) through i) of step iv), the retained test phage of the second phage portion are test phage that bind to the TLR¹⁰ cell. These test phage bind non- specifically to the TLR¹⁰ cell (i.e., binding to the cell is not mediated by the target TLR). Accordingly, the amplified test phage of the second phage portion bind non- specifically to the TLR cell. Therefore, any enrichment of the second phage portion obtained upon iteration of steps d) through i) of step iv) represents enrichment for non-specific binding.

In the second phase of screening, parallel screening of the second phage portion on TLR cells provides a control by which to gauge the contribution of enrichment for non-specific binding to the enrichment of the test phage of the first phage portion obtained upon iteration of steps d) through i) of step iv). Thus, where the enrichment of the test phage of the first phage portion obtained upon iteration of steps d) through i) of step iv) is greater than the enrichment of the test phage of the second phage portion obtained upon iteration of steps d) through i) of step iv), the difference represents enrichment for specific binding in the amplified first phage portion. Accordingly, such an amplified first phage portion represents a phage population enriched for specific binding to the target TLR.

In order for enrichment of the test phage of the first phage portion obtained upon iteration of steps d) through i) of step iv) to be compared to enrichment of the test phage of the second phage portion obtained upon iteration of steps d) through i) of step iv), it is necessary that at least one measurement of enrichment of the first phage portion and at least one measurement of enrichment of the second phage portion be provided.

In the context of the present invention, enrichment of the first and second phage portions is measured by determining the number of test phage in the retained test phage of the first and second phage portions of step iv) or by determining the number of test phage in the amplified first and second phage portions of step iv). Thus in the context of the present invention, step f) is performed at least once or step h) is performed at least once. In some embodiments step f) is performed at least once and step h) is performed at least once,

In certain embodiments, steps d) through i) of step iv) are repeated and at least one of step f) or step h) is performed during each cycle of steps d) through i). For example, in one embodiment, steps d) through i) are repeated two times (for a total of three cycles), and step h) is performed during each of the three cycles.

In other embodiments, steps d) through i) of step iv) are repeated and at least one of step f) or step h) is performed during at least the final cycle of steps d) though i). For example, in one embodiment, steps d) though i) are repeated four times (for a total of five cycles) and step f) and step h) are performed during the fifth cycle of steps d) through i). For example, in another embodiment, steps d) though i) are repeated five times (for a total of six cycles) and step f) is performed during the fifth and sixth cycles of steps d) through i).

In particular embodiments, steps d) through i) of step iv) are repeated three times (for a total of four cycles), and step f) is performed during each of the four cycles of steps d) through i).

In step v) these measurements of enrichment are then compared to determine if the test phage of the amplified first test phage portion of step iv) represent a phage population enriched for specific binding to the target TLR. Thus, in step v), at least one of steps j) or k) is performed.

Step j) of step v) comprises comparing the number of retained test phage of the first phage portion determined in step iv) with the number of retained test phage of the second phage portion determined in step iv). For step j), the numbers of retained test phage of the first and second phage portions as determined in step f) of a given cycle of step iv) are compared. For example, where steps d) through i) of step iv) are repeated once (for a total of two cycles) and step f) is performed in each cycle, the number of retained test phage of the first phage portion as determined in step f) of the first cycle is compared to the number of retained test phage of the second phage portion as determined in step f) of the first cycle and/or the number of retained test phage of the first phage portion as determined in step f) of the second cycle is compared to the number of retained test phage of the second phage portion as determined in step, f) of the second cycle. Step k) of step v) comprises comparing the number of test phage in the amplified first phage portion determined in step iv) with the number of test phage in the amplified second phage portion determined in step iv). For step k), the numbers of test phage in the amplified first and second phage portions as determined in step h) of a given cycle of step iv) are compared. For example, where steps d) through i) of step iv) are repeated once (for a total of two cycles) and step h) is performed in each cycle, the number of test phage in the amplified first phage portion as determined in step h) of the first cycle is compared to the number of test phage in the amplified second phage portion as determined in step h) of the first cycle and/or the number of test phage in the amplified first phage portion as determined in step h) of the second cycle is compared to the number of test phage in the amplified second phage portion as determined in step h) of the second cycle in step iv).

In some embodiments, step f) is performed at least once, step h) is performed at least once, and both step j) and step k) are performed. In embodiments wherein steps d) through i) of step iv) are repeated, and step f) or step h) is performed more than once, it is preferable that step j) or k) comprises comparing the sets of numbers obtained in the final cycle of steps d) through i) of step iv). For example, where steps d) through i) of step iv) are repeated twice (for a total of three cycles) and step f) is performed in each cycle, the number of retained test phage of the first phage portion as determined in step f) of the third cycle is compared to the number of retained test phage of the second phage portion as determined in step f) of the third cycle.

If in the comparison of step j) the number of retained test phage of the first phage portion determined in step iv) is greater that the number of retained test phage of the second phage portion determined in step iv), the test phage of the first phage portion amplified in step iv) are identified as a phage population enriched for specific binding to the target TLR.

Similarly, if in the comparison of step k) the number of test phage in the amplified first phage portion determined in step iv) is greater than the number of test phage in the amplified second phage portion determined in step iv), the test phage of the first phage portion amplified in step iv) are identified as a phage population enriched for specific binding to the target TLR. In embodiments wherein multiple data pairs for number of retained test phage are available [i.e., step f) was performed more than once], the test phage of the first phage portion amplified in step iv) are identified as a phage population enriched for specific binding to the target TLR, if the number of retained test phage of the first phage portion determined in step iv) is greater that the number of retained test phage of the second phage portion determined in step iv) for at least one data pair [i.e., the numbers determined in at least one step f)]. Where multiple data pairs for number of retained test phage are available [i.e., step f) was performed more than once], it is preferable that the number of retained test phage of the first phage portion determined in step iv) be greater that the number of retained test phage of the second phage portion determined in step iv) for all data pairs [i.e., the numbers determined for all repetitions of step f)].

In embodiments wherein multiple data pairs for number of test phage in an amplified phage portion are available [i.e., step h) was performed more than once], the test phage of the first phage portion amplified in step iv) are identified as a phage population enriched for specific binding to the target TLR, if the number of test phage in the amplified first phage portion determined in step iv) is greater that the number of test phage in the amplified second phage portion determined in step iv) for at least one data pair [i.e., the numbers determined in at least one step h)]. Where multiple data pairs for number of test phage in an amplified phage portion are available [i.e., step h) was performed more than once], it is preferable that the number of test phage in the amplified first phage portion determined in step iv) be greater that the number of test phage in the amplified second phage portion determined in step iv) for all data pairs [i.e., the numbers determined for all repetitions of step h)]. In embodiments wherein both step j) and step k) are performed, the test phage of the first phage portion amplified in step iv) are identified as a phage population enriched for specific binding to the target TLR if the number of retained test phage of the first phage portion determined in step iv) is greater that the number of retained test phage of the second phage portion determined in step iv) or if the number of test phage in the amplified first phage portion determined in step iv) is greater than the number of test phage in the amplified second phage portion determined in step iv). In some embodiments wherein both step j) and step k) are performed, the test phage of the first phage portion amplified in step iv) are identified as a phage population enriched for specific binding to the target TLR if the number of retained test phage of the first phage portion determined in step iv) is greater that the number of retained test phage of the second phage portion determined in step iv) and if the number of test phage in the amplified first phage portion determined in step iv) is greater than the number of test phage in the amplified second phage portion determined in step iv).

In certain embodiments, TLR¹⁰ cells are not tested in parallel with TLR^h) cells. Rather, isolates that test positive on TLR^hl cells are later retested on TLR¹⁰ cells. This allows rapid screening of phage isolates. In certain other embodiments, the method to identify a phage population enriched for specific binding to a TLR comprises the steps of: i) providing a multiplicity of test phage in the form of a phage display library, wherein each test phage comprises a nucleic acid insert encoding a polypeptide; ii) performing a first phase of screening comprising the steps of: a) contacting a TLR¹⁰ cell with the multiplicity of test phage; b) retaining the test phage that do not bind to the TLR¹⁰ cell; and c) repeating steps a) and b) once; iii) dividing the test phage retained in step ii) into a first phage portion and a second phage portion, wherein the number of test phage in the first phage portion is approximately equal to the number of test phage in the second phage portion; iv) performing a second phase of screening comprising the steps of: d) contacting a TLR^hl cell with the first phage portion, and contacting a TLR ° cell with the second phage portion, wherein each TLR is the same TLR as in step ii); e) retaining the test phage of the first phage portion that bind to the

TLR^hl cell and retaining the test phage of the second phage portion that bind to the TLR¹⁰ cell; g) amplifying the retained test phage of the first phage portion and amplifying the retained test phage of the second phage portion; h) determining the number of test phage in the amplified first phage portion and determining the number of test phage in the amplified second phage portion; and i) repeating steps d) through g) three times; and v) comparing the number of test phage in the amplified first phage portion determined in step iv) with the number of test phage in the amplified second phage portion determined in step iv), wherein if the number of test phage in the amplified first phage portion determined in step iv) is greater than the number of test phage in the amplified second phage portion determined in step iv) the test phage of the amplified first phage portion of step iv) are identified as a phage population enriched for specific binding to a TLR. In these embodiments, steps e) and g) of step iv) may be performed simultaneously. For example, cell bound phage may be simultaneously retained and amplified by direct liquid amplification in £.coli (strain 5615).

The present invention also provides phage populations enriched for specific binding to a TLR, where said phage populations are identified according to the method of the invention. The present invention also provides a method to identify a polypeptide

TLR ligand comprising the steps of i) identifying a phage population enriched for specific binding to a TLR according the to method of the invention; and ii) characterizing the polypeptide encoded by the nucleic acid insert of a test phage of the phage population enriched for specific binding to a TLR. The present invention also provides polypeptide TLR ligands, where said polypeptide TLR ligands are identified according to the method of the invention.

Toll-like Receptors (TLRs)

As used herein, the term "Toll-like receptor" or "TLR" refers to any of a family of pattern recognition receptor (PRR) proteins that are homologous to the

Drosophila melanogaster Toll protein. TLRs are type I transmembrane signaling receptor proteins that are characterized by an extracellular leucine-rich repeat domain and an intracellular domain homologous to that of the interleukin 1 receptor. The

TLR family includes, but is not limited to, mammalian TLRs 1 through 11 and 13, including mouse and human TLRs 1-11 and 13. In preferred embodiments, the TLR is a mammalian TLR. In preferred embodiments, the TLR is TLR2, TLR4 or TLR5.

Toll-like receptor 2 (TLR2) is involved in the recognition of, e.g., multiple products of Gram-positive bacteria, mycobacteria and yeast, including LPS and lipoproteins. TLR2 is known to heterodimerize with other TLRs, a property believed to extend the range of PAMPs that TLR2 can recognize. For example, TLR2 cooperates with TLR6 in the response to peptidoglycan and diacylated mycoplasmal lipopeptide, and associates with TLRl to recognize triacylated lipopeptides. Pathogen recognition by TLR2 is strongly enhanced by CD 14. The nucleotide and amino acid sequence for TLR2 has been reported for a variety of species, including, mouse, human, Rhesus monkey, rat, zebrafish, dog, pig and chicken. In preferred embodiments, TLR2 is a mammalian TLR2. In particularly preferred embodiments, TLR2 is mouse TLR2 (mTLR2) or human TLR2 (hTLR2). Exemplary nucleotide and amino acid sequences for mouse TLR2 are set forth in SEQ ID NOs 99 and 100 respectively. Exemplary nucleotide and amino acid sequences for human TLR2 are set forth in SEQ ID NOs 101 and 102, respectively. The exemplary amino acid sequence for human TLR2 is shown in Figure 8.

TLR4, the first human TLR identified, is involved in the recognition of, for example, products of Gram-negative bacteria, such as lipopolysaccharide (LPS), products of Gram-positive bacteria such as lipoteichoic acid, the F protein of Respiratory Syncytial Virus (RSV F protein), and the envelope protein of Mouse Mammary Tumor Virus (MMTV env protein). The Tlr4 gene is mutated in C3H/HeJ and C57BL/1 OScCr mice, both of which are low responders to LPS. In many instances, TLR4 requires the presence of accessory molecules to initiate the signaling cascade. For example, TLR4 interacts with MD2 and CD 14, a protein that exists both in soluble form and as a GPI-anchored protein, to induce NF-κB in response to LPS stimulation. TLR4 is known to homodimerize in a multisubunit cell surface protein complex containing two monomers of TLR4, a MD2 monomer, and a CD 14 monomer. TLR4 signaling is mediated through the adapter protein MyD88 but also through a MyD88-independent pathway that involves the TIR domain containing adapter protein (TIRAP). The nucleotide and amino acid sequences for TLR4 have been reported for a variety of species, including, mouse, human, chimpanzee, baboon, Rhesus monkey, dog, cat, pig, cow, rabbit, rat, chicken, and zebrafish. In preferred embodiments, TLR4 is a mammalian TLR4. In particularly preferred embodiments, TLR4 is a mouse TLR4 (mTLR4) or a human TLR4 (hTLR4). Exemplary nucleotide and amino acids sequences for mouse TLR4 are set forth in SEQ ID NOs 103 and 104, respectively. At least four different protein isoforms of TLR4 (isoforms A, B, C, and D) have been identified in humans. These protein isoforms, which vary in their N-terminal sequence, are the result of alternative splicing of transcripts produced from a single human TLR4 gene. Exemplary nucleotide and amino acid sequences for human TLR4 isoform A are set forth in SEQ ID NOs 105 and 106, respectively. Exemplary nucleotide and amino acid sequences for human TLR4 isoform B are set forth in SEQ ID NOs 107 and 108, respectively. Exemplary nucleotide and amino acid sequences for human TLR4 isoform C are set forth in SEQ ID NOs 109 and 110, respectively. Exemplary nucleotide and amino acid sequences for human TLR4 isoform D are set forth in SEQ ID NOs 111 and 112, respectively. An amino acid sequence alignment of the exemplary amino acid sequences for human TLR4 isoforms A, B₅ C, and D is shown in Figure 2.

TLR5 is the Toll-like receptor that recognizes flagellin from both Gram-positive and Gram-negative bacteria. Activation of the receptor stimulates the production of proinflammatory cytokines, such as TNFα, through signaling via the adaptor protein MyD 88 and the serine kinase IRAK. TLR5 can generate a proinflammatory signal as a homodimer suggesting that it might be the only TLR required for flagellin recognition. The nucleotide and amino acid sequence for TLR5 has been reported for a variety of species, including, mouse, human, rat, dog, Xenopus, rainbow trout, chimpanzee, cat, cow, and zebrafish. Exemplary nucleotide and amino acid sequences for mouse TLR5 are set forth in SEQ ID NOs 113 and 114 respectively. Exemplary nucleotide and amino acid sequences for human TLR5 are set forth in SEQ ID NOs 115 and 116, respectively. In preferred embodiments, TLR5 is a mammalian TLR5. In particularly preferred embodiments, TLR5 is mouse TLR5 (mTLR5) or human TLR5 (hTLR5).

Polypeptide TLR ligands

The terms "polypeptide ligand for TLR" and "polypeptide TLR ligand" are used interchangeably herein. As used herein, the term "polypeptide" or "protein" refers to a polymer of amino acid monomers that are alpha amino acids joined together through amide bonds. The terms "polypeptide" and "protein" are used interchangeably herein. Polypeptides are therefore at least two amino acid residues in length, and are usually longer. Generally, the term "peptide" refers to a polypeptide that is only a few amino acid residues in length, e.g. from three to 50 amino acid residues. A polypeptide, in contrast with a peptide, may comprise any number of amino acid residues. Hence, the term polypeptide includes peptides as well as longer sequences of amino acids.

Amino acid residues are abbreviated as follows: Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is He or I; Methionine is Met or M; Valine is VaI or V; Serine is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or

A; Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is GIn or Q; Asparagine is

Asn or N; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is GIu or E;

Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg or R; and Glycine is GIy or G.

Polypeptide TLR ligands modulate TLR signaling and thereby regulate the Innate Immune Response. As used herein, the term "TLR signaling" refers to any intracellular signaling pathway initiated by activated TLR, including shared pathways (e.g., activation of NF-κB) and TLR-specific pathways. As used herein the term "modulating TLR signaling" includes both activating (i.e. agonizing) TLR signaling and suppressing (i.e. antagonizing) TLR signaling. Thus, a polypeptide TLR ligand may be a TLR agonist or a TLR antagonist.

The identified polypeptide TLR ligands will find utility in a variety of applications. For example, the identified polypeptide TLR ligands may be used in methods of modulating TLR signaling. The identified polypeptide TLR ligands may also be used in novel polypeptide TLR-ligand:antigen vaccines. The identified polypeptide TLR ligands may also be used to alone to modulate TLR signaling. For example, polypeptide TLR ligands may be administered to a patient to modulate, e.g., agonize or antagonize, an immune response.

TLR'⁰ cells and TLR^hi cells

As used herein the term "TLR¹⁰" and "TLR^hi" are comparative terms referring to the expression level of a given TLR in a cell to be used in the method of the invention. Thus, a TLR ° cell has a relatively low level of expression of a given TLR and a TLR^hl cell has a relatively high level of expression of the same TLR.

In one embodiment, the TLR¹⁰ cell of step ii) and the TLR¹⁰ cell of step iv) are each a cell that does not endogenously express a given TLR and the TLR^hl cell is a cell that does endogenously express the same TLR. In another embodiment, the TLR¹⁰ cell of step ii) and the TLR¹⁰ cell of step iv) are each a cell that endogenously expresses a given TLR and the TLR¹" cell is a cell that endogenously expresses the same TLR to a higher degree. In another embodiment, the TLR¹⁰ cell of step ii) and the TLR¹⁰ cell of step iv) are each a cell that endogenously expresses a given TLR and the TLR^hl cell is a cell that ectopically expresses the same TLR to a higher degree. In another embodiment, the TLR¹⁰ cell of step ii) and the TLR¹⁰ cell of step iv) are each a cell that ectopically expresses a given TLR and the TLR^hl cell is a cell that ectopically expresses the same TLR to a higher degree. In another embodiment, the TLR^hl cell is a cell that endogenously expresses a given TLR and the TLR'⁰ cell of step ii) and the TLR¹⁰ cell of step iv) are each a cell in which endogenous expression of the given TLR has been abrogated (e.g., by mutation).

In preferred embodiments, the level of expression of the target TLR is comparable in the TLR¹⁰ cell of step ii) and the TLR^io cell of step iv). In particularly preferred embodiments, the level of expression of the target TLR and the level of expression of TLRs other than the target TLR are comparable in the TLR¹⁰ cell of step ii) and the TLR¹⁰ cell of step iv).

In preferred embodiments, the level of expression of TLRs other than the target TLR are comparable in the TLR'⁰ cell of step ii), the TLR¹⁰ cell of step iv), and the TLR^hi cell. In one example, the TLR^lci cell of step ii) and the TLR¹⁰ cell of step iv) are each a cell that does not endogenously express TLR2 but which does endogenously express TLR4 and TLR5, while the TLR¹" cell is a cell that endogenously expresses TLR2, TLR4 and TLR5. In another example, the TLR¹⁰ cell of step ii) and the TLR ° cell of step iv) are each a cell that does not endogenously express TLR2 but which does endogenously express TLR5 and TLR6 (e.g., a HEK293 cell, ATCC Accession # CRL-1573), while the TLR^hi cell is a cell that endogenously expresses TLR2, TLR5, and TLR6 (e.g., an NIH3T3 cell, ATCC Accession # CRL-1658). In another example, the TLR¹⁰ cell of step ii) and the TLR¹⁰ cell of step iv) are each a cell of a particular TLR expression profile and the TLR^hl cell is generated by causing ectopic expression of the target TLR in the TLR^io cell of step ii) or the TLR¹⁰ cell of step iv). In this case, the principal difference between the TLR¹⁰ cell of step ii) and the TLR¹⁰ cell of step iv) and the TLR^hl cell is in expression level of the target TLR. In another example, the TLR^hl cell is a cell of a particular TLR expression profile and the TLR'⁰ cell of step ii) and the TLR¹⁰ cell of step iv) are generated by abrogating expression of the target TLR in the TLR^h) cell (e.g., by mutation). In this case, the principal difference between the TLR¹⁰ cell of step ii) and the TLR¹⁰ cell of step iv) and the TLR^hi cell is in expression level of the target TLR.

In preferred embodiments, the TLR¹⁰ cell of step ii) and the TLR¹⁰ cell of step iv) are the same cell type. However, the invention also contemplates embodiments wherein the TLR¹⁰ cell of step ii) and the TLR¹⁰ cell of step iv) are different cell types. In particularly preferred embodiments, the TLR¹⁰ cell of step ii) and the TLR¹⁰ cell of step iv) are each members of a clonal cell population. In such embodiments, the TLR¹⁰ cell of step ii) and the TLR¹⁰ cell of step iv) are considered to be identical.

In particularly preferred embodiments the TLR'⁰ cell of step ii), the TLR¹⁰ cell of step iv), and the TLR¹" cell are the same cell type. However, the invention also contemplates embodiments wherein the TLR¹⁰ cell of step ii), the TLR¹⁰ cell of step iv), and the TLR^hl cell are each different cell types. The invention also contemplates embodiments wherein the TLR¹⁰ cell of step ii) and the TLR ° cell of step iv) are each the same cell type and the TLR ' cell is a different cell type.

Exemplary cells to be used in the methods of the invention include various strains of E. coli., yeast, Drosophila cells (e.g. S-2 cells), and mammalian cells. In preferred embodiments at least one of the cells [i.e., the TLR ° cell of step ii), the TLR¹⁰ cell of step iv), or the TLR^hl cell] is a mammalian cell. In preferred embodiments at least one of the cells [i.e., the TLR⁰ cell of step ii), the TLR⁰ cell of step iv), or the TLR^hi cell] is a HEK293 cell (ATCC Accession # CRL- 1573), a RAW264.7 cell (ATCC Accession # TIB-71), or a NIH3T3 cell (ATCC Accession # CRL-1658). In particularly preferred embodiments the TLR'⁰ cell of step ii), the TLR ° cell of step iv), and the TLR^hl cell are each a mammalian cell. In particularly preferred embodiments the TLR¹⁰ cell of step ii), the TLR¹⁰ cell of step iv), and the TLR^hi cell are each a HEK293 cell, each a RAW264.7 cell, or each a NIH3T3 cell.

The TLR expression profile of a cell may be determined by any of the methods well known in the art, including Western blotting, immunoprecipitation, flow cytometry / FACS, immunohistochemistry/immunocjtochemistry, Northern blotting, RT-PCR, whole mount in situ hybridization, etc. For example, monoclonal and polyclonal antibodies to human or mouse TLR2 are commercially available, e.g., from Active Motif, Bio Vision, IMGENEX, R&D Systems, ProSci, Cellsciences, and eBioscience. Human TLR2 and mouse/rat TLR2 primer pairs are commercially available, e.g., from R&D Systems and Bioscience Corporation. Monoclonal and polyclonal antibodies to human or mouse TLR4 are commercially available, e.g., from BioVision, Cell Sciences, IMGENEX, Novus Biologicals, R&D Systems, Serotec Inc., Stressgen Bioreagents, and Zymed. Mouse TLR4 primer pairs are commercially available, e.g., from Bioscience Corporation. Monoclonal and polyclonal antibodies to human or mouse TLR5 are commercially available, e.g., from BD Biosciences, BioVision, IMGENEX, and Zymed. SuperArray RT-PCR Profiling Kits for simultaneous quantitation of the expression of mouse TLRs 1 through 9 or human TLRs 1 through 10 is available from Bioscience Corporation.

Cells known to endogenously express TLR2 include dendritic cells, macrophages, natural killer cells, B-cells, epithelial cells, NIH3T3 cells, and RAW264.7 cells. Cells known not to endogenously express TLR2 include HEK293 cells. Cells known to endogenously express TLR4 include NIH3T3 cells (ATCC Accession # CRL- 1658), RAW264.7 cells (ATCC Accession # T1B-71), dendritic cells, macrophages, B~cells, and natural killer cells. Cells known not to endogenously express TLR4 include HEK293 cells (ATCC Accession # CRL-1573), HEK293:Null cells (Invivogen Accession #293-null) and 293T/17 cells (ATCC Accession # CRL- 11268). Cells known to endogenously express TLR5 include HEK293 cells, dendritic cells, macrophages, and epithelial cells, especially gut epithelium. Cells known not to endogenously express TLR5 include RAW264.7 cells, and 293T/17 cells (ATCC # CRL-11268).

Cells that ectopically express TLRs may be generated by standard techniques well known in the art. For example, a nucleic acid sequence encoding a TLR may be introduced into a cell. Such nucleic acids may be obtained by any of the synthetic or recombinant DNA methods well known in the art. See, for example, Glover, DNA Cloning: A Practical Approach. 2^nd Edition. Volumes I-IV. (Oxford University Press, 1999); Oligonucleotide Synthesis (Gait ed.:1984); Transcription And Translation (Hames & Higgins, eds.:1984); Perbal. A Practical Guide To Molecular Cloning (1984); Ausubel et al, eds. Current Protocols in Molecular Biology), (John Wiley & Sons, Inc.: 1994); PCR Primer: A Laboratory Manual, Second Edition. Dieffenbach and Dveksler, eds. (Cold Spring Harbor Laboratory Press; 2003); and Sambrook et al. Molecular Cloning: A Laboratory Manual, Third Edition (Cold Spring Harbor Laboratory Press: 2001).

Ectopic expression of a TLR may be achieved, for example, by recombinant expression of an expression construct encoding the TLR. In such an expression construct, a nucleic acid sequence encoding the TLR is operatively associated with expression control sequence elements which provide for the proper transcription and translation of the TLR ligand within the chosen host cells. Such sequence elements may include a promoter, a polyadenylation signal, and optionally internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, and the like. Codon selection, where the target nucleic acid sequence of the construct is engineered or chosen so as to contain codons preferentially used within the desired host call, may be used to minimize premature translation termination and thereby maximize expression.

The nucleic acid sequence may also encode a peptide tag for easy identification and purification of the translated TLR. Preferred peptide tags include GST, myc, His, and FLAG tags. The encoded peptide tag may include recognition sites for site-specific proteolysis or chemical agent cleavage to facilitate removal of the peptide tag. For example a thrombin cleavage site could be incorporated between a TLR and its peptide tag. The promoter sequences may be endogenous or heterologous to the host cell to be modified, and may provide ubiquitous (i.e., expression occurs in the absence of an apparent external stimulus) or inducible (i.e., expression only occurs in presence of particular stimuli) expression. Promoters which may be used to control gene expression include, but are not limited to, cytomegalovirus (CMV) promoter (U.S. Patents No. 5,385,839 and No. 5,168,062), the SV40 early promoter region (Benoist and Chambon. Nature 1981;290:304~310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al. Cell 1980;22:787~797), the herpes thymidine kinase promoter (Wagner et al Proc. Natl. Acad. ScL USA 1981;78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al, Nature 1982;296:39-42); prokaryotic promoters such as the alkaline phosphatase promoter, the trp-lac promoter, the bacteriophage lambda P_L promoter, the T7 promoter, the beta-lactamase promoter (Villa-Komaroff et al. Proc. Natl Acad Set USA 1978;75:3727-3731), or the tac promoter (DeBoer et al. Proc. Natl Acad. Sci. USA 1983;80:21~25); and promoter elements from yeast or other fungi such as the Gal4 promoter, the ADC (alcohol dehydrogenase) promoter, and the PGK (phosphoglycerol kinase) promoter.

The expression constructs may further comprise vector sequences that facilitate the cloning and propagation of the expression constructs. A large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic host cells. Standard vectors useful in the current invention are well known in the art and include (but are not limited to) plasmids, cosmids, phage vectors, viral vectors, and yeast artificial chromosomes. The vector sequences may contain, for example, a replication origin for propagation in E. coli; the S V40 origin of replication; an ampicillin, neomycin, or puromycin resistance gene for selection in host cells; and/or genes (e.g., dihydrofolate reductase gene) that amplify the dominant selectable marker plus the nucleic acid of interest. For example, a plasmid is a common type of vector. A plasmid is generally a self-contained molecule of double-stranded DNA, usually of bacterial origin, that can readily accept additional foreign DNA and that can readily be introduced into a suitable host cell. A plasmid vector generally has one or more unique restriction sites suitable for inserting foreign DNA. Examples of plasmids that may be used for expression in prokaryotic cells include, but are not limited to, pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, pUC -derived plasmids, and pET~LIC-derived plasmids.

Techniques for introduction of nucleic acids to host cells are well established in the art, including, but not limited to, electroporation, microinjection, liposome-mediated transfection, calcium phosphate-mediated transfection, or virus- mediated transfection. See, for example, Artificial self-assembling systems for gene delivery. Feigner et al, eds. (Oxford University Press: 1996); Lebkowski et al. MoI Cell Biol 1988;8:3988-3996; Sambrook et al Molecular Cloning: A Laboratory Manual, 2^nd Edition (Cold Spring Harbor Laboratory: 1989); and Ausubel et al, eds. Current Protocols in Molecular Biolog)> (John Wiley & Sons: 1989). Expression constructs encoding TLRs may be transfected into host cells in vitro. Exemplary host cells include various strains of E. coli., yeast, Drosophila cells (e.g. S-2 cells), and mammalian cells. Preferred in vitro host cells are mammalian cell lines. For example, pUNO-TLR plasraids for TLRs 1 through 11 and TLRl 3 are available from Invivogen. These plasmids provide for high level TLR expression in mammalian host cells (e.g., HEK293 and NIH3T3 cells). Protocols for in vitro culture of mammalian cells are well established in the art. See, for example, Animal Cell Culture: A Practical Approach 3^rd Edition. J. Masters, ed. (Oxford University Press) and Basic Cell Culture 2^nd Edition. Davis, ed. (Oxford University Press:2002).

Phage display libraries As discussed above, phage display is a selection technique in which a peptide or protein is genetically fused to a coat protein of a bacteriophage. The fusion protein is displayed on the exterior of the phage virion, while the DNA encoding the fusion protein resides within the virion. This physical linkage between the displayed protein and the DNA encoding it allows screening of vast numbers of variants of the protein by a simple in vitro selection procedure termed "biopanning". Phage display technology offers a very powerful tool for the isolation of new ligands from large collections of potential ligands including short peptides, antibody fragments and randomly modified physiological ligands to receptors. These systems have been effectively employed in studies of structural and functional aspects of receptor-ligand interactions using either purified receptors immobilized on a polymer surface. The terms "bacteriophage" and "phage" are used interchangeably herein.

As used herein the term "phage display library" refers to a collection of phage wherein each individual phage of the collection comprises a polypeptide genetically fused to a coat protein of the phage such that the fusion protein is displayed on the exterior of the phage virion, while the nucleic encoding the fusion protein resides within the phage. The nucleic acid residing within the phage comprises phage DNA and at least one nucleic acid insert inserted within a portion of the phage DNA encoding a phage coat protein. The size of a phage display library refers to the total number of phage in a library. The complexity of a phage display library refers to the total number of different phage (i.e., number of different nucleic acid inserts encoding different fusion proteins) in a library. For example, a library containing a total of 10³ phage, wherein the phage all comprise the same fusion protein has a size of 10³ and a complexity of 1. Preferably, a phage display library will have high degree of complexity as well as a large size. Techniques for the construction of phage display libraries are well known in the art. See, for example, Smith. Science 1985;228:1315-1317; Scott and Smith. Science 1990;249:386~390; Smith and Scott. Meth Em 1993;217:228-257; Smith and Petrenko. Chem Rev 1997;97:391-410; Hufton et al. J Immunol Methods 1999;231:39-51, and Barbas et ai, eds. Phage Display: A Laboratory Manual (CSHL Press: 2001).

Phage suitable for use in construction of phage display libraries include non-lytic phage (e.g., Ml 3 bacterial filamentous phage) and lytic phage (e.g., lambda- , T7-, and T4-based phage). A variety of phage vectors suitable for use in construction of phage display libraries are commercially available, for example, from Novagen, New England Biolabs, and Spring Bioscience.

For example, one type of phage display library is a biased peptide library (BPL). BPLs include libraries comprised of phage displaying overlapping peptides spanning a known polypeptide of interest. BPLs based on known TLR- binding polypeptides are particularly suitable for use in the methods of the invention. Such BPLs can be used to identify the minimal peptide sequences within the known protein that are responsible for binding to the target TLR. For example, libraries of phage displaying overlapping peptides (e.g., between 5 and 20 amino acids) spanning the entire region of Measles Virus hemagglutinin (HA, a TLR2 ligand), respiratory syncytial virus fusion protein (RSV F, a TLR4 ligand), or E. coli flagellin (fliC, a TLR5 ligand) may be constructed. For example, to construct a BPL, synthetic oligonucleotides covering the entire coding region of the polypeptide of interest are converted to double-stranded molecules, digested with EcoRΪ and Hindill restriction enzymes, and ligated into the T7SELECT bacteriophage vector (Novagen). The ligation reactions are packaged in vitro and amplified by either the plate or liquid culture method (according to manufacturer's instructions). The amplified phage are titered (according to manufacturer's instructions) to evaluate the total number of independent clones present in the library (i.e., the complexity of the library). In preferred embodiments the complexity of a BPL is at least 10². In particularly preferred embodiments the complexity of a BPL is at least 10³.

Another type of phage display library is a random peptide library (RPL). For example, libraries of phage displaying random peptides of from 5 to 30 amino acids in length are constructed essentially as described above for biased peptide libraries, but utilizing oligonucleotides of defined length and random sequences. Such RPLs may used to identify polypeptide ligands of TLRs. In preferred embodiments the complexity of a RPL is at least 10⁷. In particularly preferred embodiments the complexity of a RPL is at least 10⁹. It is preferred that RPLs be constructed with only 32 codons (e.g. in the form NNK or NNS where N=A/T/G/C; K=G/T; S=G/C), thus reducing the redundancy inherent in the genetic code from a maximum codon number of 64 to 32 by eliminating redundant codons. Thus, for example, a 6-amino acid residue library displaying all possible hexapeptides requires 32⁶ (~10⁹) unique clones. Another type of phage display library is a biased, random peptide library. In such libraries a known polypeptide TLR ligand is subjected to structure- function analysis by random mutation of the various positions of the polypeptide (i.e., different amino acid positions are coordinately or independently randomized). Such a library may be used to identify the critical amino acid residues for TLR binding within a known polypeptide TLR ligand and/or to identify sequence variants of known polypeptide TLR ligands that exhibit altered TLR binding specificity and/or activity. For example, as discussed above the pentapeptide ALTTE is a known polypeptide TLR2 ligand. A biased, random peptide library may be constructed representing each of the sequences XLTTE (SEQ ID NO: 5), AXTTE (SEQ ID NO: 6), ALXTE (SEQ ID NO: 7), ALTXE (SEQ ID NO: 8) and ALTTX (SEQ ID NO: 9), and/or XXTTE₃ AXXTE, ALXXE, ALTXX, etc (wherein X = any amino acid). Such a library may be constructed essentially as described above for biased peptide libraries, utilizing oligonucleotides of 15 nucleotides in length and the appropriate sequences. Another type of phage display library is based on a cDNA library. For example, libraries of phage displaying bacterial-derived polypeptides may be constructed as described above for biased peptide libraries using cDNA derived from a microbial, e.g., bacterial source of choice. Such cDNA libraries may be used to identify polypeptide TLR ligands from particular pathogenic or non-pathogenic microbes. In order to obtain bacterial cDNA, bacterial mRNA is isolated and reversed-transcribed into cDNA. For example, a PCR-ready single-stranded cDNA library made from total RNA of E. coli strain C600 is commercially available (Qbiogene). . Another type of phage display library is a constrained, cyclic peptide library. In such libraries, each peptide insert (e.g. a random peptide of from 5 to 30 amino acids in length) is flanked by cysteine residues (e.g., the peptide insert is of the sequence Cys-N_x-Cys). These cysteine residues form a disulfide bond, forcing the peptide insert into a loop or cyclic structure. This cyclization restricts conformational freedom, stabilizing the functional presentation of the peptide insert and potentially improving binding affinity of the peptide insert for target sites due to a reduction in entropy.

A variety of pre-made phage display libraries, including random peptide libraries and human and mouse cDNA libraries, are commercially available, for example, from Novagen, New England Biolabs, and Spring Bioscience.

Methods for the amplification and isolation of phage (e.g., of phage display libraries) are well known in the art. See, for example, Barbas et at, eds. Phage Display: A Laboratory Manual (CSHL Press: 2001).

Phage portions

In step iii) of the method to identify a phage population enriched for specific binding to a TLR, the test phage retained in step ii) are divided into a least a first phage portion and a second phage portion. In preferred embodiments, these retained test phage are divided into a first phage portion and a second phage portion.

In preferred embodiments, the number of test phage in the first phage portion is approximately equal to the number of phage in the second phage portion. However, embodiments wherein the number of test phage in the first phage portion is not approximately equal to the number of phage in the second phage portion are also contemplated. In such embodiments, the measured numbers of retained test phage of the first and second phage portions [where step f) is performed] and/or the measured numbers of test phage in the amplified first and second phage portion [where step h) is performed] may be normalized to account for the magnitude of difference between the number of test phage in the first and second phage portions, prior to performing step v). We note that it is not necessary to calculate the total number of test phage in the first and second phage portions to appropriately normalize the measured numbers of test phage. For example, where the test phage retained in step ii) are provided as a liquid suspension of phage, the first and second phage portions may an aliquot of 9/10 and 1/10 of the total volume of the liquid suspension, respectively. In this case, the phage numbers measured for the first phage portion may be divided by nine, or the phage numbers measured for the second phage portion may be multiplied by nine, prior to performing step v), in order to account for the difference in number of test phage in the first and second phage portions.

By "approximately equal" is meant that the number of phage in the first phage portion and the number of phage in the second phage portion differ by less than a factor often. In preferred embodiments, the number of phage in the first phage portion and the number of phage in the second phage portion are "approximately equal" in that they differ by less than a factor of 5. In particularly preferred embodiments, the number of phage in the first phage portion and the number of phage in the second phage portion are "approximately equal" in that they differ by less than a factor of 2.

We note that it is not necessary to calculate the total number of test phage retained in step ii) in order to generate first and second phage portions containing approximately equal numbers of test phage. For example, where the test phage retained in step ii) are provided as a liquid suspension of phage, first and second phage portions containing approximately equal numbers of test phage may be generated by providing two aliquots of liquid suspension having the same volume. In particularly preferred embodiments the test phage retained in step ii) are divided into a first phage portion and a second phage portion, and the number of test phage in the first phage portion is approximately equal to the number of phage in the second phage portion (/. e. , the first and second phage portions each contain about half of the total number of retained test phage). For example, where the test phage retained in step ii) are provided as a liquid suspension of phage, first and second phage portions containing approximately equal numbers of test phage may be generated by providing two aliquots of liquid suspension, where each aliquot is one half of the total volume of the liquid suspension.

The number of phage in a phage portion may be determined [e.g., as recited in steps f) and h) of step iv)] by any of the techniques well known in the art. For example, the number of phage in a phage portion may be determined using a plaque formation assay, wherein the number of phage is expressed as plaque forming units (pfu). Determining the number of phage in a sample is commonly referred to as "titering" the phage, while the number of phage so determined is commonly referred to as the "phage titer".

Characterization of the polypeptide encoded by a nucleic acid insert The polypeptide encoded by a nucleic acid insert of a phage may be characterized by any of the methods well established in the art, including, but not limited to, nucleic acid sequencing of the nucleic acid insert, deduction of the polypeptide sequence from the nucleic acid sequence of the insert, direct determination of polypeptide sequence, and analysis of the biological activity of the encoded polypeptide.

For example, nucleic acid inserts of individual T7Select phage may amplified in PCR using the commercially available primers T7SelectUP (5' - GGA GCT GTC GTA TTC CAG TC-3'; SEQ ID NO: 10; Novagen, catalog # 70005) and T7SelectDOWN (5'-AAC CCC TCA AGA CCC GTT TA-3'; SEQ ID NO: 11; Novagen, catalog # 70006). The PCR product DNA may purified using the QlAquick 96 PCR Purification Kit (Qiagen) and subjected to DNA sequencing using T7SelectUP and T7SelectDOWN primers. The amino acid sequence of the encoded polypeptide may then be deduced from the nucleic acid sequence based upon the known genetic code. The polypeptide encoded by the nucleic acid insert of a phage need not be isolated from the phage in order to characterize the polypeptide. For example, the polypeptide encoded by the nucleic acid insert of a phage may be characterized by measuring the ability of the phage to modulate TLR signaling.

In other embodiments, the polypeptide encoded by the nucleic acid insert of a phage is characterized where the polypeptide is free from contamination by phage components.

For example, the polypeptide encoded by a nucleic acid insert may be generated by coupled in vitro transcription and translation. Kits for in vitro transcription and translation are available from a wide variety of commercial sources including Promega, Ambion, Roche Applied Science, Novagen, Invitrogen, PanVera, and Qiagen. For example, kits for in vitro translation using reticulocyte or wheat germ lysates are commercially available from Ambion. For example, using the rabbit reticulocyte lysate system, reticulocyte lysate is programmed with the PCR DNA using TNT T7 Quick for PCR DNA kit (Promega), which couples transcription to translation. To initiate a TNT reaction, the DNA template is incubated at 3O⁰C for 60-90 min in the presence of rabbit reticulocyte lysate, RNA polymerase, amino acid mixture and RNAsin ribonuclease inhibitor. Direct peptide sequencing may be performed, e.g., on the in vitro transcribed and translated polypeptide to determine the amino acid sequence.

An in vitro transcribed and translated polypeptide may be further characterized, e.g., its activity to modulate TLR signaling may be measured.

The ability of the polypeptide encoded by the nucleic acid insert of a phage may be assessed using a variety of assay systems well known in the art.

In one embodiment, the ability of a polypeptide modulate TLR signaling is measured in a dendritic cell (DC) activation assay. For this assay murine or human dendritic cell cultures are obtained. Murine DCs may be generated in vitro as previously described (Lutz et al J lmmun Meth. 1999;223:77-92). In brief, bone marrow cells from 6-8 week old C57BL/6 mice are isolated and cultured for 6 days in medium supplemented with 100 LVmI GMCSF, replenishing half the medium every two days. On day 6, nonadherant cells are harvested and resuspended in medium without GMSCF and used in the DC activation assay. Human DCs may obtained commercially (Cambrex, Walkersville, MD) or generated in vitro from peripheral blood obtained from healthy donors as previously described (Sallusto & Lanzavecchia. J Exp Med 1994; 179:1109-1118). In brief, peripheral blood mononuclear cells (PBMC) are isolated by Ficoll gradient centrifugation. Cells from the 42.5-50% interface are harvested and further purified following magnetic bead depletion of B- and T-cells using antibodies to CD 19 and CD2, respectively. The resulting DC enriched suspension is cultured for 6 days in medium supplemented with 100 U/ml GMCSF and 1000 U/ml IL-4. On day 6, nonadherant cells are harvested and resuspended in medium without cytokines and used in the DC activation assay. For the dendritic cell assay, a polypeptide TLR ligand is added to DC cells in culture and the cultures are incubated for 16 hours. Supernatants are harvested, and cytokine (e.g., IFNγ, TNFα, IL- 12, IL-10 and/or IL-6) concentrations are determined, e.g., by sandwich enzyme-linked immunosorbent assay (ELISA) using matched antibody pairs from BD Pharmingen or R&D Systems, following the manufacturer's instructions. Cells are harvested, and costimulatory molecule expression (e.g., B7-2) is determined by flow cytometry using antibodies from BD Pharrningen or Southern Biotechnology Associates following the manufacturer's instructions; analysis is performed on a Becton Dickinson FACScan running Cellquest software. Functional polypeptide TLR ligands stimulate cytokine and/or co-stimulatory molecule expression in the DC assay.

In another embodiment, the ability of a polypeptide to modulate an NF-κB~reporter gene in a TLR-dependent manner is assessed. As discussed above, one of the shared pathways of TLR signaling results in the activation of the transcription factor NF-κB. Therefore, expression of an NF-κB-dependent reporter gene can serve as an indicator of active TLR signaling. In such an assay, the ability of a polypeptide TLR ligand to modulate expression of an NF-κB-dependent reporter gene in a TLR¹⁰ cell versus in a TLR^hl cell is compared. A polypeptide TLR ligand will induce higher NF-κB-dependent reporter gene expression in a TLR^hl cell than in a TLR¹⁰ cell. For example, HEK293 do not express detectable levels of endogenous TLR2. HEK293 cells harboring an NF-κB-dependent luciferase reporter gene, and ectopically expressing human or mouse TLR2 are available from Invivogen (Catalogue numbers 293-htlr2 and 293-mtlr2, respectively). For such assays, HEK293-TLR2 cells are grown in standard Dulbecco's Modified Eagle Medium (DMEM) medium with 10% Fetal Bovine Serum (FBS) supplemented with blasticidin (10 μg/ml) and then exposed to peptide ligands. Luciferase activity is then quantitated using commercial reagents.

In another embodiment, the ability of a polypeptide to induce interleukin-8 (IL-8) expression in a TLR-dependent manner is assessed. In such an assay, the ability of a polypeptide TLR ligand to induce IL-8 expression in a TLR¹⁰ cell versus in a TLR¹" cell is compared. A polypeptide TLR ligand will significantly induce IL-8 expression to a greater extent expression in a TLR^hl cell than in a TLR¹⁰ cell. For example, HEK293 do not express detectable levels of endogenous TLR2. HEK293 cells ectopically expressing human or mouse TLR2 are available from Invivogen (Catalogue numbers 293-htlr2 and 293-mtlr2, respectively). For such assays, HEK293-TLR2 cells are grown in standard Dulbecco's Modified Eagle Medium (DMEM) medium with 10% Fetal Bovine Serum (FBS) supplemented with blasticidin (10 μg/ml), and then exposed to a polypeptide TLR2 ligand. IL-8 expression may then be quantitated by standard methods well known in the art, including Northern Blotting to detect 1L-8 mRNA, immunostaining of a Western Blot to detect IL-8 protein, fluorescence activated cell sorter (FACS) analysis using an anti-IL-8 antibody, or sandwich enzyme linked immunosorbent assay (ELISA) using matched antibody pairs specific for IL-8.

Novel polypeptide ligands for TLRs

The invention also relates to polypeptide ligands for TLRs identified using the methods of the invention. The novel polypeptide ligands modulate TLR signaling and thereby regulate the Innate Immune Response.

The polypeptide TLR ligands of the invention may be prepared by any of the techniques well known in the art, including translation from coding sequences and in vitro chemical synthesis.

Translation from coding sequences

In one embodiment, the polypeptide TLR ligands of the invention may be prepared by translation of a nucleic acid sequence encoding the polypeptide TLR ligand. Such nucleic acids may be obtained by any of the synthetic or recombinant DNA methods well known in the art. See, for example, Glover, DNA Cloning: A Practical Approach. 2^nd Edition. Volumes I-IV. (Oxford University Press, 1999); Oligonucleotide Synthesis (Gait ed.:1984); Transcription And Translation (Hames & Higgins, eds.:1984); Perbal. A Practical Guide To Molecular Cloning (1984); Ausubel el al, eds. Current Protocols in Molecular Biology, (John Wiley Sc Sons, Inc.: 1994); PCR Primer: A Laboratory Manual, Second Edition. Dieffenbach and Dveksler, eds. (Cold Spring Harbor Laboratory Press: 2003); and Sambrook et al Molecular Cloning: A Laboratory Manual Third Edition (Cold Spring Harbor Laboratory Press: 2001). For example, nucleic acids encoding a polypeptide TLR ligand (e.g., synthetic oligo and polynucleotides) can easily be synthesized by chemical techniques, for example, the phosphotriester method (Matteucci et al. J. Am. Chem. Soc. 1981;103:3185-3191) or using automated synthesis methods.

Translation of the polypeptide TLR ligands of the invention may be achieved in vitro (e.g. via in vitro translation of a linear nucleic acid encoding the polypeptide TLR ligand) or in vivo (e.g. by recombinant expression of an expression construct encoding the polypeptide TLR ligand). Techniques for in vitro and in vivo expression of peptides from a coding sequence are well known in the art. See, for example, Glover, DNA Cloning: A Practical Approach. 2^nd Edition. Volumes J-IV. (Oxford University Press, 1999); Oligonucleotide Synthesis (Gait ed.:1984); Transcription And Translation (Hames & Higgins, eds.:1984); Higgins and Hames, Protein Expression: A Practical Approach. (Oxford University Press, 1999); Animal Cell Culture (Freshney, ed.:1986); Perbal, A Practical Guide To Molecular Cloning (1984); Ausubel et al, eds. Current Protocols in Molecular Biology \ (John Wiley & Sons, Inc.: 1994); and Sambrook et al. Molecular Cloning: A Laboratory Manual, Third Edition (Cold Spring Harbor Laboratory Press: 2001).

In one embodiment, the polypeptide TLR ligands of the invention are prepared by in vitro translation of a nucleic acid encoding the polypeptide TLR ligand. A number of cell-free translation systems have been developed for the translation of isolated mRNA, including rabbit reticulocyte lysate, wheat germ extract, and E. coli S30 extract systems (Jackson and Hunt. Meth Em 1983;96:50-74; Ambion Technical Bulletin #187; and Hurst. Promega Notes 1996;58:8). Kits for in vitro transcription and translation are available from a wide variety of commercial sources including Promega, Ambion, Roche Applied Science, Novagen, Invitrogen, PanVera, and Qiagen. For example, kits for in vitro translation using reticulocyte or wheat germ lysates are commercially available from Ambion. For example, using the rabbit reticulocyte lysate system, reticulocyte lysate is programmed with the PCR DNA using TNT T7 Quick for PCR DNA kit (Promega), which couples transcription to translation. To initiate a TNT reaction, the DNA template is incubated at 3O⁰C for 60-90 min in the presence of rabbit reticulocyte lysate, RNA polymerase, amino acid mixture and RNAsin ribonuclease inhibitor.

In another embodiment, the polypeptide TLR ligands are translated from an expression construct. For a discussion of expression constructs and expression in host cells, see section TLR¹⁰ cells and TLR^hl cells, above.

In vitro chemical synthesis

The polypeptide TLR ligands of the invention may be prepared via in vitro chemical synthesis by classical methods known in the art. These standard methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, and classical solution synthesis. See, e.g., Menϊfield. J Am. Chem. Soc. 1963;85:2149.

A preferred method for polypeptide synthesis is solid phase synthesis. Solid phase polypeptide synthesis procedures are well-known in the art. See, e.g., Stewart Solid Phase Peptide Syntheses (Freeman and Co.: San Francisco: 1969); 2002/2003 General Catalog from Novabiochem Corp, San Diego, USA; and Goodman Synthesis of Peptides and Peptidomimetics (Houben-Weyl, Stuttgart:2002). In solid phase synthesis, synthesis is typically commenced from the C-terminal end of the polypeptide using an α-amino protected resin. A suitable starting material can be prepared, for instance, by attaching the required α-amino acid to a chloromethylated resin, a hydroxymethyl resin, a polystyrene resin, a benzhydrylamine resin, or the like. One such chloromethylated resin is sold under the trade name BIO-BEADS SX- 1 by Bio Rad Laboratories (Richmond, CA). The preparation of the hydroxymethyl resin has been described (Bodonszky el al. Chem. Ind. London 1966;38:1597). The benzhydrylamine (BHA) resin has been described (Pietta and Marshall. Chem. Commun. 1970; 650), and the hydrochloride form is commercially available from Beckman Instruments, Inc. (Palo Alto, CA). For example, an α-amino protected amino acid may be coupled to a chloromethylated resin with the aid of a cesium bicarbonate catalyst (Gisin. HeIv. Chim. Acta 1973:56:1467). After initial coupling, the α-amino protecting group is removed, for example, using trifluoroacetic acid (TFA) or hydrochloric acid (HCl) solutions in organic solvents at room temperature. Thereafter, α-amino protected amino acids are successively coupled to a growing support-bound polypeptide chain. The α-amino protecting groups are those known to be useful in the art of stepwise synthesis of polypeptides, including: acyl-type protecting groups (e.g., formyl, trifluoroacetyl, acetyl), aromatic urethane-type protecting groups [e.g., benzyloxycarboyl (Cbz) and substituted Cbz], aliphatic urethane protecting groups [e.g., t-butyloxycarbonyl (Boc), isopropyloxycarbonyl, cyclohexyloxycarbonyl], and alkyl type protecting groups (e.g., benzyl, triphenylmethyl), fluorenylmethyl oxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), and l-(4,4-dimethyl~2,6-dioxocyclohex-l-ylidene)ethyl (Dde). The side chain protecting groups (typically ethers, esters, trityl, PMC, and the like) remain intact during coupling and are not split off during the deprotection of the amino-terminus protecting group or during coupling. The side chain protecting group must be removable upon the completion of the synthesis of the final polypeptide and under reaction conditions that will not alter the target polypeptide. The side chain protecting groups for Tyr include tetrahydropyranyl, tert- butyl, trityl, benzyl, Cbz, Z-Br-Cbz, and 2,5-dichlorobenzyl. The side chain protecting groups for Asp include benzyl, 2,6-dichlorobenzyl, methyl, ethyl, and cyclohexyl. The side chain protecting groups for Thr and Ser include acetyl, benzoyl, trityl, tetrahydropyranyl, benzyl, 2,6-dichlorobenzyl, and Cbz. The side chain protecting groups for Arg include nitro, Tosyl (Tos), Cbz, adamantyloxycarbonyl mesitoylsulfonyl (Mts), 2,2,4,6,7-pentamethyldihydrobenzofurane-5-sulfonyl (Pbf), 4- mthoxy-2,3,6-trimethyl-benzenesulfonyl (Mtr), or Boc. The side chain protecting groups for Lys include Cbz, 2-chlorobenzyloxycarbonyl (2-Cl-Cbz), 2- bromobenzyloxycarbonyl (2-Br-Cbz), Tos, or Boc.

After removal of the α-amino protecting group, the remaining protected amino acids are coupled stepwise in the desired order. Each protected amino acid is generally reacted in about a 3 -fold excess using an appropriate carboxyl group activator such as 2-(lH-benzotriazol-l-yl)-l,l,3,3 tetramethyluronium hexafluorophosphate (HBTU) or dicyclohexylcarbodimide (DCC) in solution, for example, in methylene chloride (CH₂CIa), N-methyl pyrrolidone, dimethyl fomiamide (DMF), or mixtures thereof.

After the desired amino acid sequence has been completed, the desired polypeptide is decoupled from the resin support by treatment with a reagent, such as trifluoroacetic acid (TFA) or hydrogen fluoride (HF), which not only cleaves the polypeptide from the resin, but also cleaves all remaining side chain protecting groups. When a chloromethylated resin is used, hydrogen fluoride treatment results in the formation of the free peptide acids. When the benzhydrylamine resin is used, hydrogen fluoride treatment results directly in the free peptide amide. Alternatively, when the chloromethylated resin is employed, the side chain protected polypeptide can be decoupled by treatment of the polypeptide resin with ammonia to give the desired side chain protected amide or with an alkylamine to give a side chain protected alkylamide or dialkylamide. Side chain protection is then removed in the usual fashion by treatment with hydrogen fluoride to give the free amides, alkylamides, or dialkylamides. In preparing esters, the resins used to prepare the peptide acids are employed, and the side chain protected polypeptide is cleaved with base and the appropriate alcohol (e.g., methanol). Side chain protecting groups are then removed in the usual fashion by treatment with hydrogen fluoride to obtain the desired ester.

These procedures can also be used to synthesize polypeptides in which amino acids other than the 20 naturally occurring, genetically encoded amino acids are substituted at one, two, or more positions of any of the compounds of the invention. Synthetic amino acids that can be substituted into the polypeptides of the present invention include, but are not limited to, N-methyl, L-hydroxypropyl, L~3, 4- dihydroxyphenylalanyl, δ amino acids such as L- δ-hydroxylysyl and D- δ- methylalanyl, L-α-methylalanyl, β amino acids, and isoquinolyl. D-amino acids and non-naturally occurring synthetic amino acids can also be incorporated into the polypeptides of the present invention.

Polypeptide modifications

One can also modify the amino and/or carboxy termini of the polypeptide TLR ligands of the invention. Amino terminus modifications include methylation (e.g., -NHCH₃ or ~N(CH₃)₂), acetylation (e.g., with acetic acid or a halogenated derivative thereof such as α-chloroacetic acid, α-bromoacetic acid, or α- iodoacetic acid), adding a benzyloxycarbonyl (Cbz) group, or blocking the amino terminus with any blocking group containing a carboxylate functionality defined by RCOO-- or sulfonyl functionality defined by R-SO₂-, where R is selected from alkyl, aryl, heteroaryl, alkyl aryl, and the like, and similar groups. One can also incorporate a desamino acid at the N-terminus (so that there is no N-terminal amino group) to decrease susceptibility to proteases or to restrict the conformation of the polypeptide compound. For example, the N-terminus may be acetylated to yield N- acetylglycine. Carboxy terminus modifications include replacing the free acid with a carboxamide group or forming a cyclic lactam at the carboxy terminus to introduce structural constraints. One can also cyclize the polypeptides of the invention, or incorporate a desamino or descarboxy residue at the termini of the polypeptide, so that there is no terminal amino or carboxyl group, to decrease susceptibility to proteases or to restrict the conformation of the polypeptide. C-terminal functional groups of the compounds of the present invention include amide, amide lower alkyl, amide di (lower alkyl), lower alkoxy, hydroxy, and carboxy, and the lower ester derivatives thereof, and the pharmaceutically acceptable salts thereof.

One can replace the naturally occurring side chains of the 20 genetically encoded amino acids (or the stereoisomeric D amino acids) with other side chains, for instance with groups such as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7- membered alkyl, amide, amide lower alkyl, amide diøower alkyl), lower alkoxy, hydroxy, carboxy and the lower ester derivatives thereof, and with A-, 5-, 6-, to 7- membered heterocyclic. In particular, proline analogues in which the ring size of the proline residue is changed from 5 members to 4, 6, or 7 members can be employed. Cyclic groups can be saturated or unsaturated, and if unsaturated, can be aromatic or non-aromatic. Heterocyclic groups preferably contain one or more nitrogen, oxygen, and/or sulfur heteroatoms. Examples of such groups include the furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g. morpholino), oxazolyl, piperazinyl (e.g., 1 -piperazinyl), piperidyl (e.g., 1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (e.g., 1 -pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g., thiomorpholino), and triazolyl. These heterocyclic groups can be substituted or unsubstituted. Where a group is substituted, the substituent can be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.

One can also readily modify polypeptides by phosphorylation, and other methods (e.g. , as described in Hruby et al. Biochem J 1990;268 :249~262).

The invention also contemplates partially or wholly non-peptidic analogs of the polypeptide TLR ligands of the invention. For example, the peptide compounds of the invention also serve as structural models for non-peptidic compounds with similar biological activity. Those of skill in the art recognize that a variety of techniques are available for constructing compounds with the same or similar desired biological activity as the lead peptide compound, but with more favorable activity than the lead with respect to solubility, stability, and susceptibility to hydrolysis and proteolysis. See, e.g., Morgan and Gainor, Ann. Rep. Med. Chem. 1989;24:243-252. These techniques include replacing the polypeptide backbone with a backbone composed of phosphonates, amidates, carbamates, sulfonamides, secondary amines, or N-methylamino acids.

In one form, the contemplated analogs of polypeptide TLR ligands are polypeptide-containing molecules that mimic elements of protein secondary structure. See, for example, Johnson et al. "Peptide Turn Mimetics," in Biotechnology and Pharmacy. Pezzuto et al, eds. (Chapman and Hall: 1993). Such molecules are expected to permit molecular interactions similar to the natural molecule. In another form, analogs of polypeptides are commonly used in the pharmaceutical industry as non-polypeptide drugs with properties analogous to those of a subject polypeptide (Fauchere Adv. Drug Res. 1986;15:29-69 ; Veber et al. Trends Neurosci. 19S5;8:392- 396; and Evans et al. J Med. Chem. 1987;30:1229-1239), and are usually developed with the aid of computerized molecular modeling. Generally, analogs of polypeptides are structurally similar to the reference polypeptide, but have one or more peptide linkages optionally replaced by a linkage selected from the group consisting of :- CH₂NH-, -CH₂S-, -CH₂-CH₂-, -CH-CH- (cis and trans), -COCH₂-, - CH(OH)CH₂-_.. - CHaSO-and the like. See, for example, Morley Trends Pharmacol. ScL 1980;l : 463468; Hudson et al. Int J Pept Protein Res. 1979;14:177-185; Spatola et al. Life ScL 1986;38:1243-1249; Hann. J. Chem. Soc. Perkin Trans. 1982;l :307-314; Ahnquist et al J. Med. Chem. 1980;23:1392-1398; Jennings- White et al. Tetrahedron Lett. 1982;23:2533; Holladay e/ _7/. Tetrahedron Lett. 1983;24:4401-4404; and Hruby Life ScL 1982;31 :189-199.

Fully synthetic analogs of the polypeptide TLR ligands of the invention can be constructed by structure-based drug design through replacement of amino acids by organic moieties. See, for example, Hughes Philos. Trans. R. Soc. Lond. 1980;290:387-394; Hodgson Biotechnol. 1991 :9:19-21 and Suckling. ScL Prog. 1991 ;75:323-359.

Methods of modulating TLR signaling The invention also provides methods of modulating TLR signaling comprising contacting a cell, wherein the cell comprises a TLR, with a polypeptide TLR ligand identified using the methods of the invention. As used herein, a cell that comprises a TLR. is any cell that contains a given TLR protein, including cells that endogenously express the TLR; cells that do not endogenously express the TLR but are ectopically expressing the TLR; and cells that endogenously express the TLR and are ectopically expressing additional TLR. In preferred embodiments the cells are mammalian cells. In particularly preferred embodiments, the cells are mouse cell or human cells. The cells may be cells cultured in vitro or cells in vivo.

For a discussion of determination of TLR expression status; known TLR2, 4, and 5 expressing and non-expressing cells; and generation of TLR expressing cells see section TLR¹⁰ cells and TLR^hl cells, above.

For a discussion of TLR signaling and assays to detect modulation of TLR signaling see the section Characterization of the polypeptide encoded by a nucleic acid insert, above

Vaccines comprising the polypeptide TLR ligands of the invention

The invention also provides vaccines comprising at least one polypeptide TLR ligand identified by the method of the invention and at least one antigen. These vaccines combine both signals required for the induction of a potent adaptive immune response: an innate immune system signal (i.e. TLR signaling), and an antigen receptor signal (antigen). These vaccines may be used in methods to generate a potent antigen-specific immune response. In particular, these vaccines may used in situations where signaling through a particular TLR receptor signaling is desired.

It is particularly preferred that in the vaccines of the invention the at least one polypeptide TLR ligand and at least one antigen are covalently linked. As used herein, the term "polypeptide TLR ligand: antigen" refers to a vaccine composition comprising at least one polypeptide TLR ligand and at least one antigen, wherein the at least one polypeptide TLR ligand and the at least one antigen are covalently linked. Without intending to be limited by mechanism, it is thought that covalent linkage ensures that every cell that is exposed to antigen also receives an TLR receptor innate immune signal and vice versa. However, vaccines comprising at least one polypeptide TLR ligand and at least one antigen, in which the at least one polypeptide TLR ligand and the at least one antigen are mixed or associated in a non- covalent fashion, e.g. electrostatic interaction, are also contemplated. Composition of the vaccines of the invention

The novel vaccines of the present invention comprise at least one polypeptide TLR ligand identified by the method of the invention and at least one antigen. The antigens used in the vaccines of the present invention can be any type of antigen, including but not limited to pathogen-related antigens, tumor-related antigens, allergy-related antigens, neural defect-related antigens, cardiovascular disease antigens, rheumatoid arthritis-related antigens, other disease-related antigens, hormones, pregnancy-related antigens, embryonic antigens and/or fetal antigens and the like. The antigen component of the vaccine can be derived from sources that include, but are not limited to, bacteria, viruses, fungi, yeast, protozoa, metazoa, tumors, malignant cells, plants, animals, humans, allergens, hormones and amyloidβ peptide. The antigens may be composed of, e.g., polypeptides, lipoproteins, glycoproteins, mucoproteins, lipids, saccharides, lipopolysaccharides, nucleic acids, and the like.

Specific examples of pathogen-related antigens include, but are not limited to, antigens selected from the group consisting of West Nile Virus (WNV, e.g., envelope protein domain EIII antigen) or other Flaviviridae antigens, Listeria monocytogenes {e.g., LLO or p60 antigens), Influenza A vims (e.g., the M2e antigen), vaccinia virus, avipox virus, turkey influenza virus, bovine leukemia virus, feline leukemia virus, chicken pneumovirosis virus, canine parvovirus, equine influenza, Feline rhinotracheitis virus (FHV), Newcastle Disease Virus (NDV), infectious bronchitis virus; Dengue virus, measles virus, Rubella virus, pseudorabies, Epstein- Barr Virus, Human Immunodeficieny Virus (HIV), Simian Immunodeficiency virus (SIV), Equine Herpes Virus (EffV), Bovine Herpes Virus (BHV), cytomegalovirus (CMV), Hantaan, C. tetani, mumps, Morbillivirus, Herpes Simplex Virus type 1, Heipes Simplex Virus type 2, Human cytomegalovirus, Hepatitis A Virus, Hepatitis B Virus, Hepatitis C Vims, Hepatitis E Virus, Respiratory Syncytial Virus, Human Papilloma Virus, Salmonella, Neisseria, Borrelia, Chlamydia, Bordetella, Plasmodium, Toxoplasma, Cryptococcus, Streptococcus, Staphylococcus, Haemophilus, Diptheria, Pertussis, Escherichia, Candida, Aspergillus, Entamoeba, Giardia, and Trypanosoma. The methods and compositions of the present invention can also be used to produce vaccines directed against tumor-associated antigens such as melanoma-associated antigens, mammary cancer-associated antigens, colorectal cancer-associated antigens, prostate cancer-associated antigens and the like. Specific examples of tumor-related or tissue-specific antigens useful in such vaccines include, but are not limited to, antigens selected from the group consisting of prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), Her-2₅ epidermal growth factor receptor, gpl20, p24, and FRAME, In order for tumors to give rise to proliferating and malignant cells, they must become vascularized. Strategies that prevent tumor vascularization have the potential for being therapeutic. The methods and compositions of the present invention can also be used to produce vaccines directed against tumor vascularization. Examples of target antigens for such vaccines are vascular endothelial growth factors, vascular endothelial growth factor receptors, fibroblast growth factors and fibroblast growth factor receptors and the like. Specific examples of allergy-related antigens useful in the methods and compositions of the present invention include, but are not limited to: allergens derived from pollen, such as those derived from trees such as Japanese cedar (Crypt omeria, Crypt omeriajaponicά), grasses (Gramineae), such as orchard-grass (e.g. Dactylis glomerata), weeds such as ragweed (e.g. Ambrosia artemisiifolia); specific examples of pollen allergens including the Japanese cedar pollen allergens Cry j 1 and Cry j 2, and the ragweed allergens Amb a I.I, Amb a 1.2, Amb a 1.3, Amb a 1.4, Amb a II etc.; allergens derived from fungi (e.g. Aspergillus, Candida, Alternaria_^ etc.); allergens derived from mites (e.g. allergens from Dermatophagoides pteronyssinus, Dermatophagoides farinae etc.; specific examples of mite allergens including Der p I, Der p II, Der p III, Der p VII, Der f I, Der f II, Der f III, Der f VII etc.); house dust; allergens derived from animal skin debris, feces and hair (for example, the feline allergen FeI d I); allergens derived from insects (such as scaly hair or scale of moths, butterflies, Chironomidae etc., poisons of the Vespidae, such as Vespa mandarinia); food allergens (eggs, milk, meat, seafood, beans, cereals, fruits, nuts and vegetables etc.); allergens derived from parasites (such as roundworm and nematodes, for example, Anisakis); and protein or peptide based drugs (such as insulin). Many of these allergens are commercially available. Also contemplated in this invention are vaccines directed against antigens that are associated with diseases other than cancer, allergy and asthma. As one example of many, and not by limitation, an extracellular accumulation of a protein cleavage product of β-amyloid precursor protein, called "amyloid-β peptide", is associated with the pathogenesis of Alzheimer's disease. (Janus et al. Nature 2000, 408:979-982 and Morgan et al. Nature 2000, 408:982-985). Thus, the vaccines of the present invention can comprise an amyloid-β polypeptide.

The vaccines of the invention may additionally comprise carrier molecules such as polypeptides {e.g., keyhole limpet hemocyanin (KLH)), liposomes, insoluble salts of aluminum (e.g. aluminum phosphate or aluminum hydroxide), polynucleotides, polyelectrolytes, and water soluble earners (e.g. muramyl dipeptides). A polypeptide TLR ligand and/or antigen can, for example, be covalently linked to a carrier molecule using standard methods. See, for example, Hancock et al. "Synthesis of Peptides for Use as Immunogens," in Methods in Molecular Biology: Immunochemical Protocols, Manson (ed.), pages 23-32 (Humana Press: 1992).

Chemical conjugates

In one embodiment, the vaccines of the invention comprise at least one polypeptide TLR ligand identified by the method of the invention chemically conjugated to at least one antigen. Methods for the chemical conjugation of polypeptides, carbohydrates, and/and lipids are well known in the art. See, for example, Hermanson. Bioconjugate Techniques (Academic Press; 1992); Aslam and Dent, eds. Bioconjugalion: Protein coupling Techniques for the Biomedical Sciences (MacMillan: 1998); and Wong Chemistry of Protein Conjugation and Cross-linking (CRC Press: 1991). For example, in the case of carbohydrate or lipid antigens, functional amino and sulfhydryl groups may be incorporated therein by conventional chemistry. For instance, primary amino groups may be incorporated by reaction with ethylencdiamine in the presence of sodium cyanoborohydride and sulfhydryls may be introduced by reaction of cysteamin dihydrochloride followed by reduction with a standard disulfide reducing agent.

Heterobifunctional crosslinkers, such as sulfosuccinimidyl (4- iodoacetyl) aminobenzoate, which link the epsilon amino group on the D-lysine residues of copolymers of D-lysine and D-glutamate to a sulfhydryl side chain from an amino terminal cysteine residue on the peptide to be coupled, may be used to increase the ratio of polypeptide TLR ligand to antigen in the conjugate,

Polypeptide TLR ligands and polypeptide antigens will contain amino acid side chains such as amino, carbonyl, hydroxyl, or sulfhydryl groups or aromatic rings that can serve as sites for linking the polypeptide TLR ligands and polypeptide antigens to each other, or for linking the polypeptide TLR ligands to an non- polypeptide antigen. Residues that have such functional groups may be added to either the polypeptide TLR ligands or polypeptide antigens. Such residues may be incorporated by solid phase synthesis techniques or recombinant techniques, both of which are well known in the art.

Polypeptide TLR ligands and polypeptide antigens may be chemically conjugated using conventional crosslinking agents such as carbodiimides. Examples of carbodiimides are l-cyclohexyl-3-(2-moipholinyl-(4~ethyl) carbodiimide (CMC), l-ethyl-3-(3-dimethyaminopropyl) carbodiimide (EDC), and l-ethyl-3-(4~azonia~44~dimethylpentyl) carbodiimide.

Examples of other suitable crosslinking agents are cyanogen bromide, glutaraldehyde and succinic anhydride. In general, any of a number of homobifunctional agents including a homobifunctional aldehyde, a hornobifunctional epoxide, a homobifunctional imidoester, a homobifunctional N-hydroxysuccinimide ester, a homobifunctional maleimide, a homobifunctional alkyl halide, a homobifunctional pyridyl disulfide, a homobifunctional aryl halide, a homobifunctional hydrazide, a homobifunctional diazonium derivative and a homobifunctional photoreactive compound may be used. Also included are heterobifunctional compounds, for example, compounds having an amine-reactive and a sulfhydryl-reactive group, compounds with an amine-reactive and a photoreactive group and compounds with a carbonyl-reactive and a sulfhydryl-reactive group.

Specific examples of such homobifunctional crosslinking agents include the bifunctional N-hydroxysuccinimide esters dithiobis (succinimidylpropionate), disuccinimidyl suberate, and disuccinimidyl tartarate; the bifunctional imidoesters dimethyl adipimidate, dimethyl pimelimidate, and dimethyl suberimidate; the bifunctional sulfhydryl-reactive crosslinkers l,4-di-[3'-(2'-pyridyldithio) propion-amidojbutane, bismaleimidohexane, and bis-N-maleimido-1, 8-octane; the bifunctional aryl lialides 1 ,5-difluoro-2,4-dinitrobenzene and 4,4'-difluoro-3 ,3 '-dinitrophenylsulfone; bifunctional photoreactive agents such as bis- [b-(4-azidosalicylamide)ethyl] disulfide; the bifunctional aldehydes formaldehyde, malondialdehyde, succinaldehyde, glutaraldehyde, and adiphaldehyde; a bifunctional epoxied such as 1 ,4-butaneodiol diglycidyl ether; the bifunctional hydrazides adipic acid dihydrazide, carbohydrazide, and succinic acid dihydrazide; the bifunctional diazoniums o-tolidine, diazotized and bis~diazotized benzidine; the bifunctional alkylhalides NlN '- ethylene-bis(iodoacetamide), NlN'- hexamethylene~bis(iodoacetamide)₃ NlN'- undecamethylene-bis(iodoacetamide), as well as benzylhalides and halomustards, such as ala^'- diiodo-p-xylene sulfonic acid and tri(2-chloroethyl)amine, respectively.

Examples of other common heterobifunctional crosslinking agents that may be used to effect the conjugation of proteins to peptides include, but are not limited to, SMCC (succinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate), MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester), SIAB

(N~succinimidyl(4-iodacteyl) aminobenzoate), SMPB

(succinimidyl-4-(p-maleimidoρhenyl)butyrate), GMBS (N-(y- maleimidobutyryloxy)succinimide ester), MPHB (4-(4-N-maleimidopohenyl) butyric acid hydrazide), M2C2H (4-(N-maleimidomethyl) cyclohexane-l-carboxyl-hydrazide), SMPT (succinimidyloxycarbonyl-a- methyl-a- (2-pyridyldithio)toluene), and SPDP (N-succinimidyl 3-(2-pyridyldithio) propionate). Crosslinking may be accomplished by coupling a carbonyl group to an amine group or to a hydrazide group by reductive animation.

In one embodiment, at least one polypeptide TLR ligand and at least one antigen are linked through polymers, such as PEG, poly-D-lysine, polyvinyl alcohol, polyvinylpyrollidone, immunoglobulins, and copolymers of D-lysine and D- glutamic acid. Conjugation of a polypeptide TLR ligand and an antigen to a polymer linker may be achieved in any number of ways, typically involving one or more crosslinking agents and functional groups on the polypeptide TLR ligand and the antigen. The polymer may also be derivatized to contain functional groups if it does not already possess appropriate functional groups.

Fusion proteins In preferred embodiments, the vaccines of the invention comprise a fusion protein, wherein the fusion protein comprises at least one polypeptide TLR ligand identified by the method of the invention and at least one polypeptide antigen. In one embodiment the polypeptide TLR ligand:antigen fusion protein is obtained by in vitro synthesis of the fusion protein. Such in vitro synthesis may be performed according to any methods well known in the art (see the Section Novel polypeptide ligands for TLRs: In vitro chemical synthesis, above).

In particularly preferred embodiments, the polypeptide TLR ligand:antigen fusion protein is obtained by translation of a nucleic acid sequence encoding the fusion protein. A nucleic acid sequence encoding a polypeptide TLR ligand:antigen fusion protein may be obtained by any of the synthetic or recombinant DNA methods well known in the art. See, for example, Glover, DNA Cloning: A Practical Approach. 2^nd Edition. Volumes I-IV. (Oxford University Press, 1999); Oligonucleotide Synthesis (Gait ed.:1984); Transcription And Translation (Hames & Higgins, eds.:1984); Perbal, A Practical Guide To Molecular Cloning (1984); Ausubel et al., eds. Current Protocols in Molecular Biology, (John Wiley & Sons, Inc.: 1994); PCR Primer; A Laboratory Manual Second Edition. Dieffenbach and Dveksler, eds. (Cold Spring Harbor Laboratory Press: 2003); and Sambrook et al. Molecular Cloning: A Laboratory Manual, Third Edition (Cold Spring Harbor Laboratory Press: 2001 ).

Translation of a nucleic acid sequence encoding a polypeptide TLR ligand:antigen fusion protein may be achieved by any of the in vitro or in vivo methods well known in the art (see the Section Novel polypeptide ligands for TLRs: Translation from coding sequences, above).

Vaccine formulations

Methods of formulating pharmaceutical compositions and vaccines are well-known to those of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18^th Edition, Gennaro, ed, (Mack Publishing Company: 1990)). The vaccines of the invention are administered, e.g., to human or non-human animal subjects, in order to stimulate an immune response specifically against the antigen and preferably to engender immunological memory that leads to mounting of a protective immune response should the subject encounter that antigen at some future time. The vaccines of the invention comprise a polypeptide TLR ligand identified by the method of the invention and at least one antigen, and optionally a pharmaceutically acceptable carrier. As used herein, the phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are "generally regarded as safe", e.g., that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. • Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Other suitable carriers include polypeptides (e.g., keyhole limpet hemocyanin (KLH)), liposomes, insoluble salts of aluminum (e.g. aluminum phosphate or aluminum hydroxide), polynucleotides, polyelectrolytes, and water soluble earners (e.g. muramyl dipeptides). Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.

As discussed above, the vaccines of the invention vaccines combine both signals required for the induction of a potent antigen-specific adaptive immune response: an innate immune system signal (i.e. TLR signaling), and an antigen receptor signal. This combination of signals provides for the induction of a potent immune response without the use of convention adjuvants. Thus, in preferred embodiments, the vaccines of the invention are formulated without conventional adjuvants. However, the invention also contemplates vaccines comprising a polypeptide TLR ligand identified by the method of the invention and at least one antigen, wherein the vaccine additionally comprises an adjuvant. As used herein, the term "adjuvant" refers to a compound or mixture that enhances the immune response to an antigen. An adjuvant can serve as a tissue depot that slowly releases the antigen and also as a lymphoid system activator that non-specifically enhances the immune response (Hood et al., Immunology, Second Ed, 1984, Benjamin/Cummings: Menlo

"0,J"* Park, California, p. 384). Adjuvants include, but are not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, keyhole limpet hemocyanins, and potentially useful human adjuvants such as N-acetyl-muramyl-L-threonyl-D- isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, N- acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(l'-2'-dipalmitoyl-sn-glycero- 3~hydroxyphosphoryloxy)~ethylamine, BCG (bacille Calmette-Gueriή) and Corymb acteήum parvum. Where the vaccine is intended for use in human subjects, the adjuvant should be pharmaceutically acceptable.

Vaccine administration can be oral, parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal (either passively or using iontophoresis or electroporation), transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration or using bioerodible inserts and can be formulated in dosage forms appropriate. for each route of administration. Moreover, the administration may be by continuous infusion or by single or multiple boluses.

The vaccine formulations may include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Hylauronic acid may also be used. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042).

The vaccines may be formulated so as to control the duration of action of the vaccine in a therapeutic application. For examples, controlled release preparations can be prepared through the use of polymers to complex or adsorb the vaccine. For example, biocompatible polymers include matrices of poly(ethylene-co- vinyl acetate) and matrices of a polyanhydride copolymer of a stearic acid dimer and sebacic acid. (Sherwood et al. Bio/Technology 1992; 10: 1446). The rate of release of the vaccine from such a matrix depends upon the molecular weight of the construct, the amount of the construct within the matrix, and the size of dispersed particles. (Saltzman et al. Biophys. J 1989;55: 163; Sherwood et al. Bio/Technology 1992;10: 1446; Ansel et al. Pharmaceutical Dosage Forms and Drug Delivery Systems, 5th Edition (Lea & Febiger 1990); and Remington's Pharmaceutical Sciences, 18th Edition (Mack Publishing Company 1990)). The vaccine can also be conjugated to polyethylene glycol (PEG) to improve stability and extend bioavailability times (e.g., Katre et al.; U.S. Pat. No. 4,766,106).

Contemplated for use herein are oral solid dosage forms, which are described generally in Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton PA 18042) at Chapter 89, which is herein incorporated by reference. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets, pellets, powders, or granules. Also, liposomal or proteinoid encapsulation may be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Patent No. 4,925,673). Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers (e.g., U.S. Patent No. 5,013,556). A description of possible solid dosage forms for the therapeutic is given by Marshall, K. In: Modern Pharmaceutics Edited by G. S. Banker and CT. Rhodes Chapter 10, 1979, herein incorporated by reference. In general, the formulation will include the therapeutic agent and inert ingredients which allow for protection against the stomach environment, and release of the biologically active material in the intestine.

Also contemplated for use herein are liquid dosage forms for oral administration, including pharmaceutically acceptable emulsions, solutions, suspensions, and syrups, which may contain other components including inert diluents; wetting agents, emulsifying and suspending agents; and sweetening, flavoring, coloring, and perfuming agents.

For oral formulations, the location of release may be the stomach, the small intestine (the duodenum, the jejunem, or the ileum), or the large intestine. One skilled in the art has available formulations which will not dissolve in the stomach, yet will release the material in the duodenum or elsewhere in the intestine. Preferably, the release will avoid the deleterious effects of the stomach environment, either by protection of the therapeutic agent or by release of the therapeutic agent beyond the stomach environment, such as in the intestine. To ensure full gastric resistance a coating impermeable to at least pH 5.0 is essential. Examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 5O₅ HPMCP 55, polyvinyl acetate phthalate (PVAP)₅ Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. These coatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which are not intended for protection against the stomach. This can include sugar coatings, or coatings which make the tablet easier to swallow. Capsules may consist of a hard shell (such as gelatin) for delivery of dry therapeutic (/. e. powder), for liquid forms a soft gelatin shell may be used. The shell material of cachets could be thick starch or other edible paper. For pills, lozenges, molded tablets or tablet triturates, moist massing techniques can be used. The formulation of the material for capsule administration could also be as a powder, lightly compressed plugs, or even as tablets. These therapeutics could be prepared by compression. One may dilute or increase the volume of the therapeutic agent with an inert material. These diluents could include carbohydrates, especially mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts may be also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic agent into a solid dosage form. Materials used as disintegrates include but are not limited to starch, including the commercial disintegrant based on starch, Explotab, Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultrarnylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used. The disintegrants may also be insoluble cationic exchange resins. Powdered gums may be used as disintegrants and as binders, and can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants, Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the peptide (or derivative).

An antifrictional agent may be included in the formulation to prevent sticking during the formulation process. Lubricants may be used as a layer between the therapeutic agent and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafiuoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000. Glidants that might improve the flow properties of the therapeutic agent during formulation and to aid rearrangement during compression might be added. The glidants may include starch, talc, pyro genie silica and hydrated silicoaluminate.

To aid dissolution of the therapeutic agent into the aqueous environment a surfactant might be added as a wetting agent. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents might be used and could include benzalkonium chloride or benzethomium chloride. The list of potential nonionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the therapeutic agent either alone or as a mixture in different ratios.

Controlled release oral formulations may be desirable. The therapeutic agent could be incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms, e.g., gums. Slowly degenerating matrices may also be incorporated into the formulation. Some enteric coatings also have a delayed release effect. Another form of a controlled release is by a method based on the Oros therapeutic system (Alza Corp.), i.e. the therapeutic agent is enclosed in a semipermeable membrane which allows water to enter and push agent out through a single small opening due to osmotic effects.

Other coatings may be used for the formulation. These include a variety of sugars which could be applied in a coating pan, The therapeutic agent could also be given in a film coated tablet and the materials used in this instance are divided into 2 groups. The first are the nonenteric materials and include methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose, providone and the polyethylene glycols. The second group consists of the enteric materials that are commonly esters of phthalic acid. A mix of materials might be used to provide the optimum film coating. Film coating may be carried out in a pan coater or in a fluidized bed or by compression coating.

Vaccines according to this invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms may also contain adjuvants, preserving, wetting, emulsifying, and dispersing agents. They can also be manufactured using sterile water, or some other sterile injectable medium, immediately before use.

Regarding the dosage of the vaccines of the present invention, the ordinary skilled practitioner, considering the therapeutic context, age, and general health of the recipient, will be able to ascertain proper dosing. The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment desired. The dosing schedule may vary, depending on the circulation half-life, and the formulation used.

The vaccines of the present invention may be administered in conjunction with one or more additional active ingredients, pharmaceutical compositions, or vaccines.

Methods to stimulate an immune response

The invention also provides methods to stimulate an immune response comprising administering to a subject in need thereof a polypeptide TLR ligand agonist identified by the method of the invention, or a vaccine comprising a TLR ligand agonist identified by the method of the invention. In preferred embodiments the subject is a mammal. In particularly preferred embodiments, the subject is a human. Thus, a polypeptide TLR ligand agonist identified by the method of the invention, or a vaccine comprising a TLR ligand identified by the method of the invention may be administered to subjects, e.g., mammals including humans, in order to stimulate an antigen-specific immune response and preferably to engender immunological memory that leads to mounting of a protective immune response should the subject encounter that antigen at some future time. In one embodiment, the TLR agonist ligands of the invention may be used as a nonspecific immunostimulant. Nonspecific immunostimulation may be desirable in the event of a pandemic or bioterrorist attack, in the treatment of cancer, or in the treatment of immune suppression such as occurs in certain infections (e.g., HIV) or as a result of therapeutic treatment (e.g., certain cytotoxic cancer therapeutics).

Stimulation of an immune response in a subject can be measured by standard tests including, but not limited to, the following: detection of antigen- specific antibody responses, detection of antigen specific T-cell responses, including cytotoxic T-cell responses, direct measurement of peripheral blood lymphocytes; natural killer cell cytotoxicity assays (Provinciali et al J. Immunol. Meth. 1992; 155: 19-24), cell proliferation assays (Vollenweider et al J. Immunol Meth. 1992;149:133-135), immunoassays of immune cells and subsets (Loeffler et al Cytom. 1992; 13: 169-174; and Rivoltini et al. Can. Immunol. Immimother. 1992;34:241-251); and skin tests for cell mediated immunity (Chang et al. Cancer Res. 1993;53: 1043-1050). For an excellent text on methods and analyses for measuring the strength of the immune system, see, for example, Coligan et al., eds. Current Protocols in Immunology, Vol. 1 (Wiley & Sons: 2000).

Methods to inhibit an immune response

The invention also provides methods to inhibit, or antagonize, an immune response comprising administering to a subject in need thereof a polypeptide

TLR antagonist ligand of the invention. In preferred embodiments the subject is a mammal. In particularly preferred embodiments, the subject is a human. Assays for identifying ligands that have activity as TLR antagonists are described in Example 8.

Thus, the polypeptide TLR4 antagonist ligands of the invention may be administered to subjects, e.g., mammals including humans, in order to antagonize TLR and treat an inflammatory disease or disorder. Examples of inflammatory diseases or disorders include, but are not limited to, inflammatory bowel disease, ulcerative colitis, Crohn's disease, leukocyte adhesion deficiency II syndrome, peritonitis, chronic obstructive pulmonary disease, lung inflammation, asthma, septic shock, nephritis, amyloidosis, rheumatoid arthritis, chronic bronchitis, sarcoidosis, scleroderma, lupus, polymyositis, Reiter's syndrome, psoriasis, pelvic inflammatory disease, inflammatory breast disease, orbital inflammatory disease, and autoimmune disorders. The TLR4 antagonist ligands of the invention may also be used to treat or prevent graft versus host disease or transplant rejection in a patient.

In another aspect, the invention provides a method for preventing, in a subject, an inflammatory disease or disorder by administering a polypeptide TLR antagonist ligand of the invention. Subjects at risk for an inflammatory disease or disorder can be identified by, for example, any or a combination of diagnostic or prognostic assays known in the art. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of an inflammatory disease or disorder, such that an inflammatory disease or disorder is prevented or, alternatively, delayed in its progression.

The polypeptide TLR antagonist ligands of the invention may be used alone or in combination with one or more additional anti-inflammatory agents including, but not limited to, non-steroidal anti-inflammatory agents (e.g., NSAIDS), aspirin, corticosteroids, selective COX-2 inhibitors, interleukin-1 antagonists, dihydroorotate synthase inhibitors, p38 MAP kinase inhibitors, TNF-α inhibitors, TNF~α sequestration agents, and methotrexate.

EXAMPLES

The present invention is next described by means of the following examples. However, the use of these and other examples anywhere in the specification is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified form. Likewise, the invention is not limited to any particular preferred embodiments described herein. Indeed, many modifications and variations of the invention may be apparent to those skilled in the art upon reading this specification, and can be made without departing from its spirit and scope. The invention is therefore to be limited only by the terms of the appended claims, along with the full scope of equivalents to which the claims are entitled. In accordance with the present invention there may be employed conventional molecular biology, microbiology, protein expression and purification, antibody, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Glover, DNA Cloning: A Practical Approach. 2^nd Edition. Volumes I-IV. (Oxford University Press, 1999); Oligonucleotide Synthesis (Gait ed.:1984); Nucleic Acid Hybridization (Hames & Higgins eds.:1985); Transcription And Translation (Hames & Higgins, eds.:1984); Higgins and Hames, Protein Expression: A Practical Approach. (Oxford University Press, 1999); Animal Cell Culture (Freshney, ed.:1986); Immobilized Cells And Enzymes (IRL Press: 1986); Perbal, A Practical Guide To Molecular Cloning (1984); Ausubel et al, eds. Current Protocols in Molecular Biology, (John Wiley & Sons, Inc.: 1994); Sambrook et al. Molecular Cloning: A Laboratory Manual Third Edition (Cold Spring Harbor Laboratory Press: 2001); Harlow and Lane. Using Antibodies : A Laboratory Manual (Cold Spring Harbor Laboratory Press: 1999); PCR Primer: A Laboratory Manual Second Edition. Dieffenbach and Dveksler, eds. (Cold Spring Harbor Laboratory Press: 2003); and Hockfield et al. Selected Methods for Antibody and Nucleic Acid Probes (Cold Spring Harbor Laboratory Press: 1993).

EXAMPLE 1: IDENTIFICATION OF CELL LINES FOR USE IN TLR4 LIGAND SCREENS

Materials and Methods

Maintenance of cell lines: HEK293 cells (ATCC Accession # CRL- 1573), HEK293-null cells (Invivogen; cat. # 293-null), and HEK293:hTLR4A/MD2- CD14 cells (Invivogen; cat. #293~htlr4md2cdl4) were maintained in Dulbecco's Modified Eagle Medium (Gibco) with 10% Fetal Bovine Serum (Hyclone) supplemented with lOμg/ml of blasticidin or lOμg/ml of blasticidin and 50μg/ml of hygromycin respectively. Cells were passaged 1 :4 every three days.

TLR4 activity assay with LPS: Cells were plated at a density of 50,000 cells/well in a 96-well tissue culture plate (Falcon) in the growth media described above. Serially diluted concentrations of Ultrapure LPS (Invivogen; cat. # tlrl-pelps), ranging from 50 μg to 50 ng, were added to the cells. Cell supernatants were harvested 16-20 hours later. To detect secreted IL-8, a capture ELISA was performed. First, ELISA plates (Costar; cat. # 9018) were coated with anti-IL-8 capture antibody (Pierce; cat. #M801) and stored at 4⁰C overnight. The following day, the capture antibody solution was removed and BD Assay Diluent (BD Biosciences; cat #555213) was added to each well and the plates were incubated at room temperature for one hour. The plates were then washed twice with PBS-T (IXPBS + 0.05% Tween-20), IL-8 cytokine standard (Pierce; cat #SIL8) and samples (in duplicate) were added to the blocked wells and incubated at room temperature for one hour. The plates were then washed thrice with PBS-T. A biotinylated anti-IL-8 detection antibody (Pierce; cat. #M802B) was then added to each well and incubated for one hour. Plates were then washed with PBS-T and the avidin-horseradish peroxidase conjugate (BD; cat. #554058) was added. After a 30 minute incubation, plates were washed and developed using TMB (Pierce; cat. # 34028). The reaction was stopped by adding 0.25M HCl. Absorbance was read with a FARCyte™ plate reader (Amersham Biosciences) at 450nm.

Results and Discussion

In order to perform appropriately controlled screens for the identification of novel peptide ligands for TLR4, and to perform subsequent bioactivity studies, two cell lines that differ only in their expression of TLR4 and its required accessory molecules MD2 and CD 14 were used.

HEK293 cells do not express TLR4 mRNA transcripts. HEK293 cells engineered to stably express human TLR4 isoform A and human CD 14 and MD2 (HEK293:hTLR4A/MD2-CD14) were obtained from Invivogen (catalog # 293- htlr4md2cdl4). As control, HEK293 cells stably transfected with the empty expression construct (HEK293-null) were obtained from Invivogen (catalog # 293- null). These cells do not express TLR4.

The ability of these TLR4 expressing and non-expressing cells to respond to a TLR4 ligand was assessed by quantitating IL-8 secretion of each cell type following exposure to the TLR4 ligand LPS. As expected HEK293-null cells (Invivogen; cat. # 293 -null) do not respond to LPS (see Figure 3). In contrast to HEK293-null cells, the HEK293:hTLR4A/MD2-CD14 cells are responsive to LPS stimulation (see Figure 3).

Similar results were obtained is assays using HEK293 cells (ATCC Accession # CRL-1573) and HEK293:hTLR4A/MD2-CD14 cells (Invivogen; cat, #293-htlr4md2cdl4).

Thus we have identified suitable TLR4 expressing and non-expressing cells for use in TLR4 screening and bioactivity studies.

EXAMPLE 2: GENERATION OF RANDOM PEPTIDE PHAGE DISPLAY LIBRARIES

Materials and Methods

Construction of constrained cyclic peptide libraries: Two constrained cyclic peptide phage display libraries whose variable regions possess the following amino acid structure: C-X₇-C (cyclic 7-mer) and C-Xi o-C (cyclic 10~mer), where C is a cysteine and X is any residue, were created. For each library, the variable region was generated using an extension reaction.

Random oligonucleotides were ordered PAGE purified from The Midland Certified Reagent Company. An EcoRI restriction enzyme site on the 5" end and a HindIII site on the 3' end were included for cloning purposes. In addition, the 3' end contained additional flanking nucleotides creating a "handle".

For the cyclic 10-mer inserts the random oligonucleotide was 5'-CAT GCC CGG AAT T CC TGC NNK NNK NNK NNK NNK NNK NNK NNK NNK NNK TGC GGA GGA GGA T AA AAG CTT TCG AGA C-3' (SEQ ID NO: 12).

For the cyclic 7-mer inserts the random oligonucleotide was 5'-CAT GCC CGG AAT TCC TGC NNK NNK NNK NNK NNK NNK NNK TGC GGA GGA GGA TAA AAG CTT TCG AGA C-3' (SEQ ID NO: 13).

For both oligonucleotides the 5' EcoRI and 3' HindIII sites are indicated by underlining and the variable region of the insert and nucleotides encoding the flanking cysteine residues are in bold. Amino acids in the variable region are encoded by NNK₃ where N=A/T/G/C and K=G/T. This nucleotide configuration reduces the number of possible codons from 64 to 32 while preserving the relative representation of each amino acid. In addition, the NNK configuration reduces the number of possible stop codons from three to one.

A universal oligonucleotide, 5'-GTC TCG AAA GCT TTT ATC CTC C'3' (SEQ ID NO: 14) containing a HindIII site (underlined) was ordered PAGE purified from The Midland Certified Reagent Company. This universal oligonucleotide was annealed to the 3 ' "handle" serving as a primer for the extension reaction. The annealing reaction was performed as follows: 5 μg of random oligonucleotide were mixed with 3 molar equivalents of the universal primer in dH₂θ with 10OmM NaCl. The mixture was heated to 95⁰C for two minutes in a heat block. After that time, the heat block was turned off and allowed to cool to room temperature.

The annealed oligonucleotides were then added to an extension reaction mediated by the Klenow fragment of DNA polymerase I (New England Biolabs). The extension reaction was performed at 37⁰C for 10 minutes, followed by an incubation at 65⁰C for 15 minutes to inactivate the Klenow. The extended duplex was digested with 50U of both EcoRI (New England Biolabs) and HindIIl (New England Biolabs) for 2 hours at 37⁰C. The digested products were separated by polyacrylamide gel electrophoresis, the bands of the correct size were excised from the gel, placed in 500μl of elution buffer (1OmM magnesium acetate, 0.1%SDS, 50OmM ammonium acetate) and incubated overnight, with shaking, at 37⁰C. The following day the eluted DNA was purified by phenolxhloroform extraction followed by a standard ethanol precipitation.

The purified insert was ligated into T7 Select Vector arms (Novagen; cat. # 70548), using 0.6 Weiss Units of T4 DNA ligase (New England Biolabs). The entire ligation reaction was added to T7 Packaging Extract as per manufacturer's protocol (Novagen; cat. #70014). Using the bacterial strain 5615 (Novagen), the titer of the initial library was determined by a phage plaque assay (Novagen; T7Select System). Both the 7-mer and 10-mer cyclic peptide libraries have 5x10⁸ individual clones which approaches the upper achievable limit of the phage display system. Sequencing of phage inserts: The inserts of 96 randomly selected phage isolates from both the 7mer and lOmer libraries were PCR amplified and sequenced. 300μl lysates of each isolate were generated, lμl of each lysate was put into a 50μl PCR reaction containing High Fidelity SuperMix (Invitrogen) and T7Up and T7Down primers (Novagen). lOμl of each PCR reaction was mixed with 9pmol of the T7Down primer and sequenced (McLab Sequencing).

Results and Discussion

"Constrained" peptide libraries were constructed by inserting a flanking cysteine residue at both the N and C terminus of the random peptide sequence (Cys-N_(X)-Cys). The two cysteines form a disulfide bond that forces the random sequence into a loop or cyclic structure. This cyclization restricts conformational freedom, stabilizing the functional presentation of the peptide and potentially improving the binding affinity for target sites due to a reduction in entropy. Two cyclic libraries (7-mer and 10-mer random peptide libraries) were generated using an extension strategy as described in Figure 4. The peptide insert was PCR amplified and sequenced from 96 phage clones from the 7-mer and the 10- mer random peptide libraries. Sequence analysis confirmed that peptides of the specified length and flanked by cysteines had been successfully cloned into the phage vector.

A public database, RELIC (Receptor LIgand Contacts; http://relic.bio.anl.gov/), was specifically designed for the analysis of phage display data. The programs available through RELIC will assist in identifying consensus sequences and motifs that are enriched after selection. Additionally, since unselected libraries typically contain a degree of bias in amino acid representation and distribution, the initial bias of our libraries must be analyzed. To this end, 96 independent phage clones from both the unselected lOmer and 7mer libraries were sequenced and the amino acid representation and diversity within the libraries was determined using RELIC. In the lOmer library, glycine, arginine, and valine are over- represented while residues such as proline, isoleucine, and lysine are under- represented. Similar to the lOmer library, proline, isoleucine and lysine are also under-represented in the 7mer library, perhaps indicating that these residues are refractory to an enforced cyclic structure. This analysis highlights amino acid bias inherent to the libraries and, hence, will provide critical assistance in determining if motifs identified by phage display screens reflect specificity in target binding or initial library bias. EXAMPLE 3: SCREENING ASSAY FOR POLYPEPTIDE TLR LIGANDS

Materials and Methods

Screening of phage display libraries for polypeptide TLR Hgands: Phage display libraries were screened to identify polypeptide TLR Hgands according to the following procedure (see Figure 5).

A phage display library was subjected to a first phase of screening in order to reduce non-specific binding (i.e., binding not mediated by the TLR of interest). In this first phase of screening, the phage display library was incubated on a cell suspension of in vitro cultured cells that express minimal amounts of the TLR of interest (TLR¹⁰). Phage that did not bind to the TLR¹⁰ cells were retained by collecting the cell culture supernatant containing the unbound phage. This process was repeated once (for a total of two screening cycles) to yield a phage population having reduced non-specific binding. This phage population having reduced non-specific binding as retained at the end of the first phase of screening was then divided into a first phage portion and a second phage portion by dividing the supernatant containing unbound phage into equal halves (by volume).

The first phage portion and the second phage portion were then subjected to a second phase of screening in order to produce a phage population enriched for specific binding to the target TLR. In this second phase of screening, the first phage portion was incubated on a cell suspension of in vitro cultured cells that express the relevant TLR (TLR¹") in order to capture phage with binding specificity for the target TLR, and the second phage portion was incubated on in vitro cultured cells that express minimal amounts of the TLR of interest (TLR¹⁰) in order to capture phage with non-specific binding. The phage of the first phage portion that bound to the TLR¹¹¹ cells and the phage of the second phage portion that bound to the TLR¹⁰ cells were each simultaneously retained and amplified (e.g., by direct liquid amplification in E.coli, strain 5615). The amplified phage of the first phage portion and the amplified phage of the second phage portion were each titered to determine the number of phage in each amplified portion. The amplified phage portions were then used for a subsequent round of screening following the same steps. This

-6S- screening process was repeated three times (for a total of four screening cycles in the second phase of screening).

For each cycle of the second phase of screening, the number of retained phage of the first phage portion and the number of retained phage of the second phage portion were plotted on a line graph to provide a round-by-round comparison of the number of phage recovered. The number of retained phage of the second phage portion provides a measurement of the number of phage having nonspecific binding recovered in the screening assay. By comparing the number for phage having non-specific binding to the number of retained phage of the first phage portion, one can ascertain the approximate proportion of the phage of the first phage portion that represent phage having TLR-specific binding. Otherwise stated, where the number of retained phage of the first phage portion is greater than the number of retained phage of the second phage portion, the subsequently amplified first phage portion represents a phage population enriched for specific binding to a TLR. Individual phage clones from the phage population enriched for specific binding to a TLR were isolated via plaque formation in E. coli. These individual phage clones were then further characterized to identify polypeptide ligands for a TLR.

Results and Discussion Phage display libraries were enriched for those phage that display peptides that specifically mediate TLR-binding by a combined negative screening plus positive screening method as outlined in Figure 5, This method combined a first phase of negative screening with a second phase of positive screening to yield a phage population enriched for specific binding to TLR. Due to the large collection of membrane bound proteins, whole cell screening of phage display libraries is associated with a high degree of non-specific background. In order to control for this background, the supernatant containing unbound phage obtained following the first phase of negative screening is divided and incubated in parallel on both TLR^hl and TLR¹⁰ cells. If phage enrichment observed on the TLR^hl cells is greater than that observed on TLR¹⁰ cells, this indicates TLR- specificity. Individual phage clones from the phage population enriched for specific binding to a TLR were isolated via plaque formation in E. coli. These individual clones were then further characterized to identify polypeptide ligands for a TLR.

EXAMPLE 4: SCREENING ASSAY TO IDENTIFY A PHAGE POPULATION ENRICHED FOR SPECIFIC BINDING TO TLR4

Materials and Methods

Generation of random peptide phage display libraries: Constrained 7- mer and 10-mer cyclic peptide phage display libraries were generated as described in EXAMPLE 2, above.

Generation of phage displaying an S-Tag polypeptide: The S-tag nucleotide sequence is 5'-ATG AAA GAA ACC GCT GCT GCT AAA TTC GAA CGC CAG CAC ATG GAC AGC CCA-3' (SEQ ID NO: 15). The S-tag amino acid sequence is MKETAAAKFERQHMDSP (SEQ ID NO: 16). Double stranded DNA encoding the S-tag peptide sequence was ligated to the T7Select 10-3 bacteriophage vector (Novagen). The ligation reactions were packaged in vitro and titered using the host E. coli strain BLR5615 that was grown in M9TB (Novagen). The recombinant phage was then amplified. Ligation, packaging, and amplification were performed according to manufacturer's instructions.

Mid-scale phage lysates: To amplify phage libraries for use in a whole cell screening assay, the packaged phage extracts described above were added to 1OmL of 5615 bacteria (Novagen) at OD₆₀₀ 0.6 and placed in a 37⁰C shaking incubator until lysis was observed (approximately 2 hours). The phage lysate was clarified by spinning at 8,000*g for 10 minutes. After the spin, the phage lysate supernatant was retained. The phage titer after liquid amplification was reproducibly 10ⁿ pfu/mL.

The Luria Broth (LB) buffer of the phage lysate supernatant was exchanged with Dulbecco's Modified Eagle Medium (DMEM; Gibco) as follows: First, 5mL of phage lysate supernatant was added to an Amicon Ultra Centrifugal Filter (Millipore; cat. #UFC903024) and spun at 2000*g for 10 minutes. Following the first spin, two washes with DMEM were performed. Finally, the phage lysates were resuspended in 5mL of DMEM. This procedure does not result in a loss of phage titer.

Whole cell screening of phage display libraries to identify a phage population enriched for specific binding to TLR4: 5x10⁶ HEK293 cells (ATCC Accession # CRL-1573) were harvested, pelleted by centrifugation, and resuspended in 500μl of growth media (DMEM+10%FBS). ImL of phage lysate (10-mer library lysate, 7-mer library lysate, or S-Tag phage lysate) in DMEM (total of approximately 10¹⁰ phage) was added to the resuspended cells and the cell and phage mixture was rotated at 4⁰C for 1 hour. The mixture was spun down at low speed for 5 minutes and the supernatant containing unbound phage was transferred to a second (pelleted) aliquot of 5x10⁶ HEK293 cells. The cells were rotated for 1 hour and spun down once more,

At this point the supernatant was collected and split in equal halves between IxIO⁶ HEK293:hTLR4A/MD2-CD14 cells and 1x10⁶ HEK293 cells (ATCC Accession # CRL-1573) resuspended in 500μl of growth media. The cells were rotated at 4⁰C for 1 hour and spun down at low speed. The supernatant was removed and the cells washed with DMEM at 4⁰C three times. After the last wash, the cells were resuspended in 500μl of DMEM. A small aliquot was used to determine phage titer and the rest was amplified and used to repeat the positive screening. In total, four rounds of positive screening were performed. For each round, phage titer was used to monitor enrichment for TLR-specific phage.

Results and Discussion

Constrained, cyclic random peptide (10-mer and 7-mer) phage display libraries were screened for polypeptide TLR4 ligands according to the procedure describe in EXAMPLE 3, above. As a further control for non-specific binding, phage lysate of an S-Tag phage was also screened according to this procedure.

In these screens, approximately 10¹⁰ phage were subjected to two rounds of negative screening in a first phase. Then the supernatant containing unbound phage retained after two rounds of negative screening was divided in half to yield two portions. One portion was subjected to four rounds of positive screening on

TLR4-expressing cells (HEK293:hTLR4A/MD2-CD14 cells). In parallel, the second portion was subjected to four rounds of positive screening on cells that do not express TLR4, HEK293 cells (ATCC Accession # CRL-1573). During each round of positive screening, the phage of each portion were titered as a measure of enrichment.

For the 10-mer phage display library, after four rounds of positive screening on TLR4 expressing cells, phage titers had increased 5000 fold (see Figure

6). In contrast, after four rounds of positive screening on cells not expressing TLR4, the 10-mer phage display library and the S-Tag phage showed an enrichment of only

700 fold.

Thus, the 10-mer phage display library showed enrichment for TLR4- specific phage following four rounds of positive screening on TLR4 expressing cells. Furthermore, the screening method provided a population of phage containing 10-mer random peptide inserts that is enriched for specific binding to TLR4.

Similar results were obtained using the 7-mer phage display library.

EXAMPLE 5: IDENTIFICATION OF POLYPEPTIDE TLR4 LIGANDS: CHARACTERIZATION OF PHAGE ISOLATES BY SEQUENCING OF

PHAGE INSERTS AND BY WHOLE CELL ELISA

Materials and Methods

Sequencing of phage inserts: Individual phage clones from phage populations enriched for specific binding to TLR4 were isolated via plaque formation in E. coll The DNA inserts of individual phage were PCR amplified and sequenced as described in Example 2, above.

Whole cell ELISA: HEK293-null cells (Invivogen; cat, # 293-null) or HEK293:hTLR4A/MD2-CD14 cells (Invivogen; cat. #293-htlr4md2cdl4) cells were grown overnight on poly-D-lysine coated 96-well plates (BD Biosciences) to yield TLR4+ and TLR4- plates, respectively. The following day, an individual phage isolate was added to parallel wells of both the TLR4+ and TLR4- plates. A standard curve, using titrations of phage bound directly to the plate, was also included. After a 1 hour incubation at room temperature, plates were washed with DMEM supplemented with 10OmM HEPES. A monoclonal antibody against the tail fiber of T7 phage (Novagen; cat. #71530) was added to the wells. After a one hour incubation at room temperature, the wells were washed twice with DMEM (10OmM HEPES). A

-79. goat anti-mouse Ig antibody conjugated with HRP was added and incubated for 30 minutes. Finally, the wells were washed five times with the DMEM+HEPES solution and the wells developed with TMB (Pierce; cat. # 34028).

Calculation of TLR4-specific binding: The binding specificity of each phage isolate was determined by: 1) averaging the values of duplicate samples and standard curve values; 2) determining a phage titer for each isolate based on the standard curve; 3) subtracting the phage titer from the negative control S-Tag phage from the phage titers obtained with phage isolates; and 4) dividing the TLR4+ titer by the relevant TLR4- titer. A TLR4+:TLR4- ratio of 1 indicates equal binding on to both cell types by the phage isolate, i.e., a lack of specificity, while a value greater than 1 indicates specificity for TLR4+ cells

Results and Discussion

Randomly picked individual phage clones from phage populations enriched for specific binding to TLR4 were isolated via plaque formation in E. coli, their nucleic acid inserts sequenced, and their binding specificity for TLR4 quantitated using the whole cell ELISA assay.

Because whole cell screens are associated with a high degree of nonspecific background, it is critical to develop an assay that will rapidly confirm the specificity of individual phage clones. A monoclonal antibody against the T7 phage tail fiber has recently become commercially available, making it possible to confirm TLR specific binding by whole cell ELISA. Wells that are positive on the TLR4+ plate but not on the corresponding TLR4- plate contain phage expressing a TLR4 binding peptide and should be investigated further. As a negative control for binding, phage expressing the S-Tag peptide were used. 96 randomly selected phage clones from the enriched phage population of the 10-mer phage display library were isolated, their nucleic acid inserts sequenced to determine the amino acid sequence of the encoded peptide, and their binding specificity quantitated. Of these phage clones, 18 showed specificity for binding to TLR4 (i.e., a TLR4+:TLR4~ binding ratio > 1). The amino acid sequence of the peptide insert and the TLR4+:TLR4~ binding ratio for these phage clones are given in Table 1. Table 1: Peptide insert sequence and TLR4+:TLR4~ binding ratio (BR) for 10-mer phage isolates

* Note that in some cases the peptide insert sequence is shorter than 10 amino acids due to the presence of a stop codon in the encoding nucleic acid insert.

96 randomly selected phage clones from the enriched phage population of the 7-mer phage display library were isolated, their nucleic acid inserts sequenced to determine the amino acid sequence of the encoded peptide, and their binding specificity quantitated. Of these phage clones, 18 showed specificity for binding to TLR4 (i.e., a TLR4+:TLR4- binding ratio > 1). The amino acid sequence of the peptide insert and the TLR4+:TLR4- binding ratio for these phage clones are given in Table 2.

* Note that in some cases the peptide insert sequence is shorter than 7 amino acids due to the presence of a stop codon in the encoding nucleic acid insert. The data presented in Table 1 and Table 2 confirm that the phage population enriched for specific binding to TLR4, as identified by the screening method of the invention, contains individual phage having specificity of binding for TLR4. Furthermore, the peptide inserts of the individual phage having specificity of binding for TLR4 are polypeptide TLR4 ligands. These peptide inserts have been identified as polypeptide TLR4 ligands.

Furthermore, these data confirm that we have established a reliable assay which can be used as a secondary screen to confirm the TLR4 binding specificity of phage isolates ltom phage populations enriched for specific binding to TLR4 by the screening method of the invention.

EXAMPLE 6: IDENTIFICATION OF POLYPEPTIDE TLR4 LIGANDS: CHARACTERIZATION OF PHAGE ISOLATES BY PHAGE CAPTURE

ASSAY

Materials and Methods Generation of phage displaying FHC, a polypeptide TLRS ligand:

The coding region of the E. coli flagellin (/7/^'C) gene (SEQ ID NO: 117) was cloned into the T7SELECT phage display vector (Novagen). Double stranded DNA encoding E. coli fliC was ligated to the T7Select 10-3 bacteriophage vector (Novagen). The ligation reactions were packaged in vitro and titered using the host E. coli strain BLR5615 that was grown in M9TB (Novagen). The recombinant phage was then amplified. Ligation, packaging, and amplification were performed according to manufacturer's instructions. This phage displays the E. coli flagellin (fliC) protein, having the amino acid sequence of SEQ ID NO: 118, on the surface of the phage. Phage capture assay: 96 well plates coated with a monoclonal antibody against the tail fiber of T7 phage (Novagen; cat. # 75131) were blocked with BD Assay Diluent (BD; cat #555213) and then incubated with an individual phage isolate at 37⁰C. Plates were then washed extensively with PBS containing 0.05% Tween and 50μg/mL polymyxin B (Invivogen, cat. #tlrl-pmb) to remove endotoxin. Plates were washed again with tissue culture media (DMEM, 10% FBS) supplemented with 50μg/mL polymyxin B to remove residual detergent. HEK293- null cells (Invivogen; cat. # 293-null) or HEK293:hTLR4A/MD2~CD14 cells (Invivogen; cat. #293-htlr4md2cdl4) cells were added in tissue culture media (containing polymyxin B) to parallel wells and incubated overnight at 37⁰C. The cell culture supernatants were harvested the following day and an ELISA for IL-8 was performed (as described in EXAMPLE 5, above) to assess TLR4-dependent activation of the cells. The detergent wash steps and the inclusion of polymyxin B at each step was essential for reducing the endotoxin in the phage lysates to allow for an observation of peptide-specific signal.

Results and Discussion Some of the randomly picked individual phage clones isolated and sequenced in EXAMPLE 5 that showed TLR4-specific binding (i.e., a TLR4+:TLR4- binding ratio >1) were further characterized. The ability of a phage isolate to function as a TLR4 agonist was quantitated in a phage capture assay. This phage capture assay gives rapid, preliminary insight into whether the peptide insert of a phage isolate is a TLR4 agonist.

In the phage capture assay, the ability of a phage isolate to function as a TLR4 agonist was quantitated based upon the induction of IL-8 secretion (in pg/ml) by cells expressing TLR4 upon exposure to the phage isolate. The tested phage isolates and their agonist activity on cells expressing TLR4, expressed as IL-8 secretion in pg/ml, are given in Table 3 and Table 4.

A measure of IL-8 secretion by cells not expressing TLR4 served as a negative control. Cells not expressing TLR4 were not activated by any of the phage isolates tested (i.e., secreted less than 100 pg/ml of IL-8 in response to each of the phage isolates). A measure of IL-8 secretion (in pg/ml) by cells expressing TLR4 upon exposure to a FIiC phage (FIiC is a TLR5 ligand but not a TLR4 ligand) served as a further negative control. Table 3: 10-mer phage isolates and agonist activity values

Table 4: 7-mer phage isolates and agonist activity values

The results of these assays demonstrate that the 10-mer phage isolate

D2 and the 7-mer phage isolates CS, C9, D9, C2, G6, GlO, A6 and D8 each showed TLR4 agonist activity greater than that of the FIiC negative control phage isolate. In particular, the 10-mer phage isolate and the 7-mer phage isolates C8, C9, and D9 showed TLR4 agonist activity at least 2-fold greater than that of the FIiC negative control. Furthermore, this agonist activity is specific to TLR4, as it is not observed when cells not expressing TLR4 are exposed to the phage isolates. Thus the peptide inserts of the 10-mer phage isolate D2 and the 7-mer phage isolates C8, C9, D9, C2, G6, GlO₅ A6 and D8 are identified as polypeptide TLR4 ligands having TLR4 agonist activity. The results of these assays indicated that that the 10-mer phage isolates

A2 and G4 and the 7-mer phage isolates F9, Fl O. H5, F6, and B8 do not have measurable TLR4 agonist activity. It is possible that these phage isolates act as TLR4 antagonists. Similarly, it is possible that the peptide inserts of the 10-mer phage isolates A2 and G4 and the 7-mer phage isolates F9, FlO, H5, F6, and B8 are polypeptide TLR4 ligands having TLR4 antagonist activity.

EXAMPLE 7: CHARACTERIZATION OF POLYPEPTIDE TLR4 LIGANDS BY ENDOTOXIN-FREE BIOACTIVITY ASSAYS

Materials and Methods

Synthetic polypeptide TLR4 ligands: The following synthetic peptides were synthesized by BaChem:

RNS-CEDMVYRIGVPC-G₄-H₄ (SEQ ID NO: 58)

RNS-SEDMVYRIGVPS-G₄-H₄ (SEQ ID NO: 59)

RNS-CRDIPGARRQAC-G₄-H₄ (SEQ ID NO: 60)

RNS-CEDMVYRIGVPC-G4 (SEQ ID NO: 61)

Results and Discussion

The agonist activity of polypeptide TLR4 ligands may also be measured using endotoxin-free tests. For such tests, endotoxin-free polypeptide

TLR4 ligands are obtained, for example, by cloning and expression of polypeptide TLR4 ligands in an endotoxin-free system such as mammalian cell lines or by in vitro chemical synthesis.

To this end, the following synthetic peptides were synthesized:

RNS-CEDMVYRIGVPC-G₄-H₄ (SEQ ID NO : 58) RNS-SEDMVYRIGVPS-G₄-H₄ (SEQ ID NO: 59) RNS-CRDIPGARRQAC-G₄-H₄ (SEQ ID NO: 60)

RNS-CEDMVYRIGVPC-G4 (SEQ ID NO: 61)

The first of these synthetic peptides contains the 10-mer peptide sequence of clone D2 (EDM V YRIGVP, SEQ ID NO: 19) with two flanking cysteines and the 3 to 4 amino acids present at the amino and carboxy ends (respectively) of the peptide in the context of the phage coat of the D2 phage isolate. This first synthetic peptide also contains a 4-His tag (SEQ ID NO: 62) to allow for ease of detection in the detection in the phage capture assay. The second synthetic peptide contains flanking serine residues in the place of the flanking cysteine residues. The third synthetic peptide contains a cyclic lOmer sequence derived from enriched phage isolate F3 (RDIPGARRQA; SEQ ID NO: 63), which does not exhibit TLR4-specific binding or agonist activity, in place of the D2 10-mer sequence. The fourth synthetic peptide does not contain the His tag.

These synthetic peptides, and other endotoxin-free polypeptide TLR4 ligands, were tested for TLR4 specific binding (as described in EXAMPLE 5, above); TLR4 agonist activity (as described in EXAMPLE 6, above). These synthetic peptides, and other endotoxin-free polypeptide TLR4 ligands, will be tested for TLR4 antagonist activity (as described in EXAMPLE 8, below).

EXAMPLE 8: ASSAYS FOR TLR4 ANTAGONIST ACTIVITY

Materials and Methods Phage capture assay: 96 well plates coated with a monoclonal antibody against the tail fiber of T7 phage (Novagen; cat. # 75131) are blocked with BD Assay Diluent (BD; cat #555213) and then incubated with an individual phage isolate at 37⁰C, At least one well is incubated with a S-Tag phage as a negative control. Plates are then washed extensively with PBS containing 0.05% Tween and 50μg/mL polymyxin B (Invivogen; cat. #tlrl-pmb) to remove endotoxin. Plates are washed again with tissue culture media (DMEM, 10% FBS) supplemented with 50μg/mL polymyxin B to remove residual detergent. HEK293:hTLR4A/MD2-CD14 cells in tissue culture media containing a known TLR4 agonist (such as LPS or the 10-mer D2 phage isolate) are added to each well and incubated overnight at 37⁰C. The cell culture supernatants are harvested the following day and an ELISA for IL- 8 is performed (as described in EXAMPLE 5, above) to assess TLR4-dependent activation of the cells.

NF~κB~dependent luciferase reporter assay: An individual phage isolate peptide is monitored for the ability to antagonize TLR4-dependent activation of an NF-κB-dependent luciferase reporter gene in cell lines expressing TLR4. Cells stably transfected with an NF-κB luciferase reporter construct may constitutively express TLR4, or may be engineered to overexpress TLR4. Cells seeded in a 96-well microplate are exposed to a known TRL4 agonist (such as LPS or the 10-mer D2 phage isolate) plus an individual phage isolate for four to five hours at 37⁰C. The S- Tag phage isolate serves as a negative control. NF-κB-dependent luciferase activity is measured using the Steady-Glo Luciferase Assay System by Promega (E2510), following the manufacturer's instructions. Luminescence is measured on a microplate luminometer (FARCyte, Amersham). Antagonist activity of a phage isolate is expressed as the ICso, i.e., the concentration that yields a response that is 50% of the maximal response obtained with the S-Tag control phage. The EC₅₀ values are normalized to protein concentration as determined in the ELISA described above.

Results and Discussion

To determine if individual phage isolates from phage populations enriched for specific binding to TLR4 act as TLR4 antagonists, competition assays will be performed. In such assays, the ability of the individual phage isolates to inhibit induction of IL-8 secretion by a known TLR4 agonist (such as LPS or the 10- mer D2 isolate) is quantitated using the phage capture assay or an NF-κB-dependent reporter gene assay, In the phage capture assays, those phage isolates that provide for reduced IL-8 secretion (in pg/ml) as compared to the S-Tag phage (negative control) are TLR4 antagonists. In the NF-κB-dependent reporter gene assay, those phage isolates that provide for reduced luciferase activity as compared to the S-Tag phage (negative control) are TLR4 antagonists

The 10-mer phage isolates A2 and G4 and the 7-mer phage isolates F9, FlO₅ H5, F6, and B8 are tested in these assays to quantitate their activity as TLR4 antagonists.

EXAMPLE 9: IN VITRO TRANSCRIPTION AND TRANSLATION OF

POLYPEPTIDE TLR4 LIGANDS

Materials and Methods

Generation of DNA inserts by PCR: Individual T7SELECT phage clones from the phage population enriched for specific binding to TLR4 are isolated via plaque formation in E. coli. The individual T7SELECT phage clones are dispensed in a 96-well plate, which serves as a master plate. Duplicate samples are subjected to PCR using phage specific primers, T7FOR (5'-GAA TTG TAA TAC GAC TCA CTA TAG GGA GGT GAT GAA GAT ACC CCA CC-3'; SEQ ID NO: 64), and T7REV (5'-TAA TAC GAC TCA CTA TAG GGC GAA GTG TAT CAA CAA GCT GG-3'; SEQ ID NO: 65) that flank the phage inserts. The forward primer is about 600 bp away from the insert and is designed to incorporate the T7 promoter upstream of the Kozak sequence (KZ), which is critical for optimal translation of eukaryotic genes, and a 6X HIS~tag (SEQ ID NO: 4) sequence (open circle). The reverse primer includes the myc sequence at the c-terminus of the peptide. Therefore, the PCR product will contain all the signals necessary for optimal transcription and translation (T7 promoter, Kozak sequence and the ATG initiation codon), as well as and sequences encoding an N-terminal 6X HIS tag (SEQ ID NO: 4) and a C-terminal myc tag for capture, detection and quantitation of the translated protein. The PCR products are purified using the QIAquick 96 PCR Purification Kit (Qiagen),

In vitro TNT: Rabbit reticulocyte lysate is programmed with the PCR DNA using TNT T7 Quick for PCR DNA kit (Promega), which couples transcription to translation. To initiate a TNT reaction, the DNA template is incubated at 3O⁰C for 60-90 min in the presence of rabbit reticulocyte lysate, RNA polymerase, amino acid mixture and RNAsin ribonuclease inhibitor. lmmunoanalysis of the in vitro translated protein: Immunoanalysis is used to confirm translation of the polypeptide TLR4 ligand. In these assays, an aliquot of the TNT reaction is analyzed by western blot using antibodies specific for one of the engineered tags, or by ELISA to allow normalization for protein levels across multiple samples. For a sandwich ELISA, 6X HIS-tagged (SEQ ID NO: 4) protein is captured on Ni-NTA microplates and detected with an antibody to one of the heterologous tags (i.e., anti-c-myc).

NF- kB-depen dent lucif erase reporter assay: An aliquot of the in vitro synthesized polypeptide TLR4 ligand is monitored for the ability to activate an NF-κB~dependent luciferase reporter gene in cell lines expressing TLR4. Cells stably transfected with an NF-κB luciferase reporter construct may constitutively express TLR4, or may be engineered to overexpress TLR4. Cells seeded in a 96-well microplate are exposed to test peptide for four to five hours at 37⁰C. NF-κB- dependent luciferase activity is measured using the Steady-Glo Luciferase Assay System by Promega (E2510), following the manufacturer's instructions. Luminescence is measured on a microplate luminometer (FARCyte, Amersham). Agonist activity of a polypeptide TLR4 ligand is expressed as the EC₅₀, i.e., the concentration that yields a response that is 50% of the maximal response obtained with the appropriate control reagent, such as LPS. The EC₅₀ values are normalized to protein concentration as determined in the ELISA described above.

Dendritic cell activation assay: For this assay murine or human dendritic cell cultures are obtained. Murine DCs are generated in vitro as previously described (Lutz et al. J Immurt Meth. 1999;223:77-92). In brief, bone marrow cells from 6-8 week old C57BL/6 mice are isolated and cultured for 6 days in medium supplemented with 100 U/ml GMCSF, replenishing half the medium every two days. On day 6, nonadherant cells are harvested and resuspended in medium without GMSCF and used in the DC activation assay. Human DCs are obtained commercially (Cambrex, Walkersville, MD) or generated in vitro from peripheral blood obtained from healthy donors as previously described (Sallusto & Lanzavecchia. J Exp Med 1994; 179: 1109-1118). In brief, peripheral blood mononuclear cells (PBMC) are isolated by Ficoll gradient centrifugation. Cells from the 42.5-50% interface are harvested and further purified following magnetic bead depletion of B and T cells using antibodies to CD 19 and CD2, respectively. The resulting DC enriched suspension is cultured for 6 days in medium supplemented with 100 U/ml GMCSF and 1000 U/ml IL-4. On day 6, nonadherant cells are harvested and resuspended in medium without cytokines and used in the DC activation assay. An aliquot of the in vitro synthesized polypeptide TLR4 ligand is added to DC culture and the cultures are incubated for 16 hours. Supernatants are harvested, and cytokine (IFNγ, TNFα, IL- 12 p70, IL-10 and IL-6) concentrations are determined by sandwich enzyme-linked immunosorbent assay (ELISA) using matched antibody pairs from BD Pharmingen or R&D Systems, following the manufacturer's instructions. Cells are harvested, and costimulatory molecule expression (e.g., B7-2) is determined by flow cytometry using antibodies from BD Pharmingen or Southern Biotechnology Associates following the manufacturer's instructions; analysis is performed on a Becton Dickinson FACScan running Cellquest software. Results and Discussion

In vitro TNT reactions are used to generate endotoxin-free polypeptide TLR4 ligands. These endotoxin-free polypeptide TLR4 ligands are then assessed for TLR4 agonist activity.

EXAMPLE 10: LIGASE INDEPENDENT CLONING FOR IN VITRO

ANALYSIS OF POLYPEPTIDE TLR4 LIGAND ACTIVITY

Materials and Methods

Ligase independent cloning: Individual T7SELECT phage clones from the phage population enriched for specific binding to TLR4 are subjected to PCR to isolate the nucleotide sequences encoding the TLR4-binding peptides. PCR is performed using the primers T7~LlCf (5'-GAC GAC GAC AAG ATT GAG ACC ACT CAG AAC AAG GCC GCA CTT ACC GAC C-3'; SEQ ID NO: 66) and T7- LICr (5'-GAG GAG AAG CCC GGT CTA TTA CTC GAG TGC GGC CGC AAG- 3'; SEQ ID NO: 67) at 10 pmol each with phage lysate at 1 :20 dilution using the Taq polymerase master mix (Invitrogen) at 1 :2 dilution. PCR cycling conditions are as follows: denaturation at 95°C for 5min; 30 cycles of denaturation step at 95⁰C for30 sec, annealing step at 58⁰C for 30 sec, and extension at 72⁰C for 30sec; and a final extension at 72⁰C for 1 Omin.

These sequences are then cloned into the pET-LIC24 and pMTBip-LIC vectors via ligase independent cloning (LIC). For LIC, an ~800 bp PCR fragment, which includes a portion of the phage coat protein encoding sequence to facilitate expression and purification, is treated with T7 DNA polymerase in the presence of dATP and cloned into the linearized pET-LIC24 vector.

To construct the pET-LIC24 vector, a unique BseRl site is introduced into pET24a (Novagen). In order to introduce the BseRΪ site the 5'-phosphorylated primers pET24a-LICf (5'-TAT GCA TCA TCA CCA TCA CCA TGA TGA CGA CGA CAA GAG CCC GGG CTT CTC CTC AGC-3'; SEQ ID NO: 68) and pET24a- LlC-r (5'-TCA GCT GAG GAG AAG CCC GGG CTC TTG TCG TCG TCA TCA TGG TGA TGG TGA TGA TGC A-3'; SEQ ID NO: 69) are annealed and cloned into Ndel and BpuWOll digested pET24a via cohesive end ligation. The resulting construct is then digested with BseRl and treated with T4 DNA polymerase in the presence of dTTP to generate the pET-LIC24 vector . pMT-Bip-LIC is constructed in the same way as pET-LIC24 by inserting an annealed oligonucleotide into UgIII and MwI digested vector pMTBip/V5-HisA. (Invitrogen). The annealed oligonucleotide is made using the 5'- phosphorylated primers pMTBip-LICf (5'-GAT CTC ATC ATC ACC ATC ACC ATG ATG ACG ACG ACA AGA GCC CGG GCT TCT CCT CAA-3'; SEQ ID NO: 70) and pMTBip-LICr (5'-CGC GTT GAG GAG AAG CCC GGG CTC TTG TCG TCG TCA TCA TGG TGA TGG TGA TGA TGA-3'; SEQ ID NO: 71). Protein expression in E. coli; E. coli strain BLR (DE3) pLysS strain

(Invitrogen) is transformed with pET-LIC plasmid DNA using a commercially available kit (Qiagen). A colony is inoculated into 2-ml LB containing 100 μg/ml carbenicillin, 34 μg/ml chloramphenicol supplemented with 0.5% glucose and grown overnight at 37⁰C with shaking. A fresh 2-ml culture is inoculated with a 1:20 dilution of the overnight culture and grown at 37⁰C for several hours until ODeoo ^~ 0.5-0.8. Protein expression is induced by the addition of IPTG to 1 niM for 3 hours.

Ni-NTA protein purification: E. coli cells transformed with the construct of interest are grown and induced as described above. The cells are harvested by centrifugation (7000 rpm x 7 minutes in a Sorvall RC5C centrifuge) and the pellet re-suspended in lysis Buffer B (100 mM NaH2PO4, 10 mM Tris-HCl, 8 M urea, pH 8 adjusted with NaOH) and 10 mM imidazol. The suspension is freeze- thawed 4 times in a dry ice bath. The cell lysate is centrifuged (40,000 g for one hour in a Beckman Optima L ultracentrifuge) to separate the soluble fraction from inclusion bodies. The supernatant is mixed with 1ml Ni-NTA resin (Qiagen Ni-NTA) that has been equilibrated with buffer B and binding of the proteins is allowed to proceed at 4 ⁰C for 2-3 h a roller. The material is then loaded unto 1 cm-diameter column. The bound material is then washed 2 times with 30ml wash buffer (Buffer B + 2OmM imidazol). The proteins are eluted in two rounds with 3ml elution buffer twice (Buffer B+250mM imidazol). The eluates are combined and the pools are used to perform a serial dialysis starting with 1 L of buffer (Buffer B + 250 mM imidazol:2x PBS in a ratio of 1 :1) with change in buffer every 4-8 h. The final dialysis step is performed with two changes of PBS overnight. The integrity of the proteins is verified by SDS-PAGE and immunoblot. Greater than 95% purity can be achieved. Optionally, to further reduce endotoxin contamination, the protein is chromatographed through Superdex 200 gel filtration in the presence of 1% deoxycholate to separate protein and endotoxin. A second round of Superdex 200 gel filtration in the absence of deoxycholate removes the detergent from the protein sample. Purified protein is concentrated and dialyzed against Ix PBS, 1% glycerol. The protein is aliquoted and stored at -8O⁰C.

Protein expression in Drosophila S-2 cells: The pMTBip-LIC vectors are used to direct recombinant peptide expression in Drosophila S-2 cells. Conditioned medium from S~2 cells expressing the recombinant peptide may be directly used in bioassays to confirm the activity of the TLR4-binding peptide. Drosophila S-2 cells and the Drosophila Expression System (DES) complete kit is obtained from Invitrogen (catalog#: K5120-01, K4120-01, K5130-1 and K4130-01). The growth and passaging of the S-2 cells, transfection and harvesting of the conditioned medium are performed according to manufacturer's protocol. In vitro IL-8 assay: HEK293 :Null and HEK293 :hTLR4A/MD2-CD 14 cells (see EXAMPLE 1, above) are seeded in 96-well microplates (50,000 cells/well), and aliquots of either purified recombinant peptide expressed in E. coli or conditioned medium from S-2 cells expressing recombinant peptide are added. As a positive control, cells are incubated with the Ultrapure LPS (Invivogen; cat. #tlrpelps). The microplates are then incubated overnight. The conditioned medium is assayed for the presence of IL-8 in a sandwich ELISA using an anti-human IL-8 matched antibody pair (Pierce, catalog # M801E and # M802B) following the manufacturer's instructions. Optical density is measured using a microplate spectrophotometer (FARCyte, Amersham).

Results and Discussion

Ligase independent cloning is used to generate expression vectors for the expression of polypeptide TLR4 ligands in E. coli and in Drosophila S2. The expressed polypeptide TLR4 ligands are then assessed for TLR4 agonist activity in an IL-8 induction assay. EXAMPLE 11: A POLYPEPTIDE ΥLR4-LIGANO:LISTERIA LLO-p60

ANTIGEN FUSION PROTEIN VACCINE

Materials and Methods

Cloning of polypeptide TLR4 ligands into E, coli: Double stranded DNA encoding the polypeptide TLR4 ligand is ligated upstream of sequences encoding a fusion protein of antigenic MHC class I and II epitopes of L. monocytogenes proteins LLO and p60. The amino acid sequence of the L. monocytogenes LLO-pόO fusion protein is given in SEQ ID NO: 98. These ligated sequences encoding a polypeptide TLR4 liganfr.Listeria LLO-p60 antigen fusion protein are inserted into a plasmid expression vector. The expression construct is engineered by using convenient restriction enzyme sites or by PCR.

For example, sequences encoding the polypeptide TLR4 ligand are inserted upstream of the LLO~p60 encoding sequence in the expression construct T7.LIST (Figure 7), where T7.LIST is assembled as described below. In this case, the expressed fusion protein will contain both a V5 epitope (GKPIPNPLLGLDST; SEQ ID NO: 3) and a 6xHis tag (SEQ ID NO: 4).

Generation of the T7.LIST plasmid: Sequences encoding the Listeria LLO-p60 antigen fusion protein are isolated as follows: First primers LLOF7 (5⁵- CTT AAA GAA TTC CCA ATC GAA AAG AAA CAC GCG GAT G-3\; SEQ ID NO: 72) and LLOR3 (5'-TTC TAC TAA TTC CGA GTT CGC TTT TAC GAG-3'; SEQ ID NO: 73) are used to amplify a 5' portion of the LLO sequences. Next primers LLOF6 (5'-CTC GTA AAA GCG AAC TCG GAA TTA GTA GAA-3'; SEQ ID NO: 74) and P60R7 (5' AGA GGT CTC GAG TGT ATT TGT TTT ATT AGC ATT TGT G-3'; SEQ ID NO: 75) are used to amplify the remaining fused 3' portion LLO sequences and the p60 sequences. These two PCR fragments are then joined by a third PCR using the primers LL0F7 and P60R7. This PCR serves to mutate the LLO sequence spanned by LLOR3 and LL0F6 so as to remove the EcoRI site. This product is then ligated into the pCRT7CT-TOPO cloning vector (Invitrogen) to generate the T7.LIST plasmid. In this vector, expression of the chimeric DNA insert is driven by the strong T7 promoter, and the insert is fused in frame to the V5 epitope (GKPIPNPLLGLDST; SEQ ID NO: 3) and polyhistidine (6x His) (SEQ ID NO: 4) is located at the 3' end of the gene (see Figure 7). Protein expression and immunoblot assay: In general, the following protocol will be used to produce recombinant polypeptide TLR4 \iga.nd:Listen^'a LLO- p60 antigen: fusion protein. E. coli strain BL (DE3) pLysS strain (Invitrogen) is transformed with the desired plasmid DNA using a commercially available kit (Qiagen). A colony is inoculated into 2-ml LB containing 100 μg/ml carbenicillin, 34 μg/ml chloramphenicol supplemented with 0.5% glucose and grown overnight at 37⁰C with shaking. A fresh 2-ml culture is inoculated with a 1 :20 dilution of the overnight culture and grown at 37⁰C for several hours until OD_6O0 - 0.5-0.8. Protein expression is induced by the addition of IPTG to 1 mM for 3 hours. The bacteria are harvested by centrifugation and the pellet is re-suspended in 100 μl of Ix SDS-PAGE sample buffer in the presence of β-mercaptoethanol. The samples are boiled for 5 minutes and 1/10 volume of each sample is loaded onto 10% SDS-PAGE gel and electrophoresed. The samples are transferred to PVDF membrane and probed with α- His antibody (Tetra His, Qiagen) at 1 : 1000 dilution followed by rabbit anti-mouse IgG/ AP conjugate (Pierce) at 1 :25,000. The immunoblot is developed using BCIP/NBT colorimetric assay kit (Promega).

Protein purification: Polypeptide TLR4 ligand:Listeria LLO-p60 antigen fusion proteins are expressed with a 6X Histidine tag (SEQ ID NO: 4) to facilitate purification. E coli cells transformed with the construct of interest are grown and induced as described above. Cells are harvested by centrifugation at 7,000 rpm for 7 minutes at 4⁰C in a Sorvall RC5C centrifuge. The cell pellet is resuspended in Buffer A (6 M guanidine HCl, 100 mM NaH₂PO₄, 10 mM Tris-HCl, pH 8.0). The suspension can be frozen at -8O⁰C if necessary. Cells are disrupted by passing through a microfluidizer at 16,000 psi. The lysate is centrifuged at 30,000 rpm in a Beckman Coulter Optima LE-80K Ultracentrifuge for 1 hour. The supernatant is decanted and applied to Nickel -NTA resin at a ratio of ImI resin/1 L cell culture. The clarified supernatant is incubated with equilibrated resin for 2-4 hours by rotating. The resin is washed with 200 volumes of Buffer A. Non-specific protein binding is eliminated by subsequent washing with 200 volumes of Buffer B (8 M urea, 100 mM NaH₂PO₄, 10 mM Tris-HCl, pH 6.3). An additional 200 volume wash with buffer C (10 mM Tris-HCl, pH 8.0, 60% iso-propanol) reduces endotoxin to acceptable level (< 0.1 EU/μg). Protein is eluted with Buffer D (8 M Urea, 100 mM NaH₂PO₄, 10 mM Tris-HCl, pH 4.5) Protein elution is monitored by SDS-PAGE or Western Blot (anti- His, anti-LLO and anti-p60). Greater than 95% purity can be achieved. Endotoxin level may be further reduced by chromatography through Superdex 200 gel filtration in the presence of 1% deoxycholate to separate protein and endotoxin. A second round of Superdex 200 gel filtration in the absence of deoxycholate removes the detergent from the protein sample. Purified protein is concentrated and dialyzed against Ix PBS₅ 1% glycerol. The protein is aliquoted and stored at -8O⁰C.

Endotoxin assay; Endotoxin levels in recombinant fusion proteins is measured using the QCL-1000 Quantitative Chromogenic LAL test kit (BioWhittaker #50-648U), following the manufacturer's instructions for the microplate method.

Confirmation of TLR4 agonist activity in NF-κB luciferase reporter assays: Purified recombinant polypeptide TLR4 ligand:Listeria LLO-p60 antigen fusion proteins are assayed for TLR4 agonist activity and selectively in the NF-κB- dependent luciferase assay as described above. Immunization: Recombinant polypeptide TLR4 UganάiListeria LLO- p60 antigen fusion protein is suspended in phosphate-buffered saline (PBS), without exogenous adjuvant. BALB/c mice (n = 10-20 per group) are immunized by s.c. injection at the base of the tail or in the hind footpad. Initial dosages to be tested range from 0.5 μg to 100 μg/animal. Positive control animals are immunized with 10³ CFU of live L. monocytogenes, while negative control animals receive mock- immunization with PBS alone.

Sublethal L. monocytogenes challenge: Seven days after immunization, BALB/c mice are infected by i.v. injection of 10³ CFU L. monocytogenes in 0.1 ml of PBS. Spleens and livers are removed 72 hours after infection and homogenized in 5 ml of sterile PBS + 0.05% NP-40. Serial dilutions of the homogenates are plated on BHI agar. Colonies are enumerated after 48 hours of incubation. These experiments are performed a minimum of 3 times utilizing 10-20 animals per group. Mean bacterial burden per spleen or liver is compared between treatment groups by Student's t-Test. Lethal L. monocytogenes challenge: Seven days after immunization,

BALB/c mice are infected i.v. (10⁵ CFU) or p.o. (10⁹ CFU) with L. monocytogenes in 0.1 ml of PBS, and monitored daily until all animals have died or been sacrificed for humane reasons. Experiments are performed 3 times utilizing 10-20 animals per group. Mean survival times of different treatment groups are compared by Student's t-Test.

Induction of antigen-specific T-cell responses: CD8 T-cell responses are monitored at specific time points following vaccination (i.e. day 7, 14, 30,120) by quantitating the number of antigen-specific γ-interferon secreting cells using ELISPOT (R&D Systems). At varying time point post-vaccination, T-cells are isolated from the draining lymph nodes and spleens of immunized animals and cultured in microtiter plates coated with capture antibody specific for the cytokine of interest. Synthetic peptides corresponding to the K^d~restricted epitopes, p602i 7-22₅ and LLO₉₁.9₉ are added to cultures for 16 hours. Plates are washed and incubated with anti-IFNγ detecting antibodies as directed by the manufacturer. Similarly, CD4 responses are quantified by IL-4' ELISPOT following stimulation with the I~A^d restricted CD4 epitopes LLOi 89-200> LLO₂16-227, and P6O300-311- Antigen specific responses are quantified using a dissection microscope with statistical analysis by Student's t-Test. For quantitation of CD8 responses, it is also possible to utilize flow cytometric analysis of T cell populations following staining with recombinant MHC Class I tetramer (Beckman Coulter) loaded with the H-2^d restricted epitopes noted above.

Cytotoxic T-lymphocyte (CTL) responses: At specific time points following vaccination (i.e. day 7, 14, 30,120), induction of antigen-specific CTL activity is measured following in vitro restimulation of lymphoid cells from immune and control animals, using a modification of the protocol described by Bouwer and Hinrichs. Briefly, erythrocyte-depleted spleen cells are cultured with Concanavalin A or peptide-pulsed, mitomycin C-treated syngeneic stimulator cells for 72 hours. Effector lymphoblasts are harvested and adjusted to an appropriate concentration for the effector assay. Effector cells are dispensed into round bottom black microtiter plates. Target cells expressing the appropriate antigen (e.g., cells infected with live L. monocytogenes or pulsed with p60 or LLO epitope peptides) are added to the effector cells to yield a final effector :target ratio of at least 40:1. After a four hour incubation, target cell lysis is determined by measuring the release of LDH using the CytoTox ONE fluorescent kit from Promega, following the manufacturer's instructions.

Antibody responses: Antigen-specific antibody titers are measured by ELISA according to standard protocols (see, e.g., Cote-Sierra el al. Infect Immun 2002;70:240-248). For example, immunoglobulin isotype titers in the preimmune and immune sera are measured by using ELISA (Southern Biotechnology Associates, Inc., Birmingham, Ala.). Briefly, 96- well Nuiic-Immuno plates (Nalge Nunc International, Roskilde, Denmark) are coated with 0.5 μ g of COOHgp63 per well, and after exposure to diluted preimmune or immune sera, bound antibodies are detected with horseradish peroxidase-labeled goat anti-mouse IgGl and IgG2a. ELISA titers are specified as the last dilution of the sample whose absorbance was greater than threefold the preimmune serum value. Alternatively, antigen-specific antibodies of different isotypes can be detected by Western blot analysis of sera against lysates of whole L. monocytogenes, using isotype-specific secondary reagents.

Results and Discussion

L monocytogenes is a highly virulent and prevalent food-borne gram- positive bacillus that causes gastroenteritis in otherwise healthy patients (Wing et al. J Infect Dis 2002; 185 Suppl 1 :S18-S24), and more severe complications in immunocompromised patients, including meningitis, encephalitis, bacteremia and morbidity (Crum. Ciirr Gastroenterol Rep 2002;4:287-296 and Frye et al. CHn Infect Dis 2002;35:943-949). In vivo models have identified roles for both T- and B-cells in response to Z. monocytogenes, with protective immunity attributed primarily to CD8 cytotoxic T cells (CTL) (Kersiek and Pamer. Curr Op Immunol 1999:1 1 :400-405). Studies during the past several years have led to the identification of several immunodominant L. monocytogenes epitopes recognized by CD4 and CD8 T cells. In BALB/c mice, several peptides have been identified including the H-2K^d restricted epitopes LLO91.9₉ and p602i7-225 (Pamer et al Nature 1991;353:852-854 and Pamer. J Immunol 1994; 152:686). The vaccine potential for such peptides is supported by studies demonstrating that the transfer of LLOgi.rø-specific CTL into naϊve hosts conveys protection to a lethal challenge with L. monocytogenes when the bacterial challenge is administered within a week of CTL transfer (Harty. J Exp Med 1992;175:1531-1538). The mouse model of listeriosis (Geginat et al. J Immunol 1998; 160:6046-6055) has provided invaluable insights into the mechanisms of disease and the immunological response to infection with L. monocytogenes. This model allows the investigator to study both short-term and memory responses. This mouse model, with modifications, may be employed to confirm the in vivo efficacy and mechanism of action of polypeptide TLR ligands in fusion protein vaccines.

The polypeptide TLR4 ligands on the invention may be used to generate a fusion protein vaccine for Listeria infection. This vaccine comprises a fusion protein of polypeptide TLR4 ligand and antigenic MHC class I and II epitopes of L. monocytogenes proteins LLO and p60 (Listeria monocytogenes LLO-p60 fusion protein, SEQ ID NO: 98). For such vaccines, sequences encoding a polypeptide TLR4 ligmd'.Listeria LLO~p60 antigen fusion protein are inserted into a plasmid expression vector. The expression construct is then expressed in E. coli and the recombinant fusion protein purified based upon the included His tag.

The purified protein is then used to vaccinate mice. At specific time points following vaccination (i.e. day 7, 14, 30,120), animals are examined for antigen-specific humoral and cellular responses, including serum antibody titers, cytokine expression, CTL frequency and cytotoxicity activity, and antigen-specific proliferative responses. Protection versus Listeria infection is confirmed in the vaccinated animals using sublethal and lethal Listeria challenge assays. The polypeptide TLR4 ligand:Listeria LLO-p60 antigen fusion protein vaccine provides strong antigen-specific humoral and cellular immune responses, and provides protective immunity versus Listeria infection.

EXAMPLE 12: CELL LINES ECTOPICALLY EXPRESSING TLRs

Materials and Methods

Generation of cell lines ectopically expressing TLRs: HEK293 (ATCC Accession # CRL- 1573), which had been stably transfected with an NF-κB reporter gene vector containing tandem copies of the NF-κB consensus sequence upstream of a minimal promoter fused to the firefly luciferase gene (κB-LUC), were cultured at 37⁰C under 5% CO₂ in standard Dulbecco's Modified Eagle Medium (DMEM; e.g., Gibco) medium with 10% Fetal Bovine Serum (FBS; e.g., Hyclone). NIH3T3 cells (ATCC Accession # CRL-1658), which had been stably transfected with an NF-κB reporter gene vector containing tandem copies of the NF-κB consensus sequence upstream of a minimal promoter fused to the firefly luciferase gene (κB-LUC), were cultured at 37⁰C under 5% CO₂ in DMEM (e.g., Gibco) medium with 10% FBS (e.g., Hyclone).

The following pUNO-TLR plasmids were obtained from Invivogen: human TLR2 (catalog # puno-htlr2), mouse TLR5 (catalog # puno-mtlr5), and human TLR5 (catalog # puno-htlr5). The following pDUO-CD14/TLR plasmids were obtained from Invivogen: human CD14 plus human TLR2 (catalog # pduo-hcdl4/tlr2) and human CD14 plus human TLR2 (catalog # pduo-hcdl4/tlr4). The pUNO-TLR and pDUO-CD14/TLR plasmids are optimized for the rapid generation of stable transformants and for high levels of expression.

The pUNOTLR or pDUO-CDl4/TLR plasmids were transfected into HEK293 and/or NIH3T3 cells lines using Lyovec (Invivogen), a cationic lipid-based transfection reagent. Transfected cells were cultured at 37⁰C under 5% CO₂ in DMEM (e.g., Gibco) medium with 10% FBS (e.g., Hyclone)supplemented with blasticidin (10 μg/ml). Stably transfected, individual blasticidin-resistant clones were isolated. The cell lines thereby generated are listed in Table 5.

Table 5: HEK293 and NIH3T3 lines ectopically expressing TLRs and CD14. "293" = HEK293 cells. "3T3" = NIH3T3 cells. h=human. m=mouse.

Analysis of TLR expression in HEK293 and NIH3T3 cells:

Individual blasticidin-resistant clones of transfected HEK293 and NIH3T3 have been isolated and characterized by Western blot analysis or flow cytometric analysis using polyclonal antibodies to the appropriate TLR to select clones which over-express the desired receptor.

To prepare whole cell lysate (WCE) for Western Blot analysis, sub- confluent cultures in 10 mm dishes were washed with PBS at room temperature. The following steps were then performed on ice or at 4° C using fresh, ice-cold buffers. Six hundred microliters of RIPA buffer (Santa Cruz Biotechnology Inc., catalog # sc- 24948; RIPA buffer: IXTBS, 1% NP-40, 0.5% Sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail) was added to the culture plate and the contents gently rocked for 15 minutes at 4° C. The cells were then harvested by scraping with a cell scraper and the scraped lysate was transferred to a microcentrifuge tube. The plate was washed once with 0.3 ml of RIPA buffer and combined with first lysate. An aliquot of 10 μl of 10 mg/ml PMSF (Santa Cruz Biotechnology Inc., catalog # sc- 3597) stock was added and the lysate passed through a 21 -gauge needle to shear the DNA. The cell lysate was incubated 30-60 minutes on ice. The cell lysate was microcentrifuged 10,000xg for 10 minutes at 4° C. The lysate supernatant was transferred to a new microfuge tube and the pellet discarded. A 10 μl aliquot of lysate supernatant was loaded onto 10% SDS-PAGE gels and electrophoreses was performed according to standard protocols. The proteins were either stained by Coommassie Blue or transferred from the gels to a nitrocellulose or PVDF membrane using an electroblotting apparatus (BIORAD) according to the manufacturer's protocols. The membrane was then blotted with an anti-TLR antibody (e.g., rabbit anti-hTLR2 polyclonal antibody from Invivogen, catalog # ab-htlr2) and reacted with a secondary antibody (e.g., goat anti-rabbit IgG Fc from Pierce, catalog # 31341).

For flow cytometric analysis, HEK293 cells were removed from culture and resuspended in FACS staining buffer (phosphate buffered saline (PBS) containing 2% bovine serum albumin (BSA) and 0.01% Sodium azide). A total of 10⁵ cells were then stained in a volume of lOOμl of with the biotin labeled monoclonal anti-TLR antibody for 30 minutes at 4⁰C. Cells were then washed 3 times and incubated with streptavidin-FITC conjugated secondary antibody (BD Pharmingen, catalog # 554060). Following incubation at 4⁰C for 30 minutes samples were washed 3x with FACS buffer and then fixed in phosphate buffer containing 3% paraformaldehyde. Samples were then analyzed on a FACScan cytometer (BD Pharmingen) and analyzed using CellQuest software.

Results and Discussion In order to identify and affinity select potent ligands for TLRs from a peptide library displayed on bacteriophage, it is essential to employ cell lines expressing the TLR of choice. HEK293 cells, which endogenously express TLRl, TLR3, TLR5 and TLR6, and NIH3T3 cells, which endogenously express TLRl, TLR4, TLR5 and TLR6, were stably transfected with pUNO-TLR and pDUO- CD14/TLR plasmid constructs from Invivogen. Individual blasticidin-resistant clones were isolated and characterized by Western blot analysis or flow cytometric analysis using polyclonal antibodies to CD 14 and/or the appropriate TLR to select clones which over-express the desired receptor. Using this strategy, we have generated cell lines over-expressing various TLRs and CD 14 as summarized in Table 6.

Table 6: Expression of TLRs and CD14 in HEK293 and NIH3T3 cells. h=human. m=mouse. NT = not tested.

The transfected parent HEK293 and NIH3T3 cell lines harbor an NF- κB-dependent luciferase reporter gene. As discussed above, one of the shared pathways of TLR signaling results in the activation of the transcription factor NF-κB. Therefore, in the cell lines generated here, expression of the NF-κB -dependent reporter gene serves as an indicator of TLR signaling.

EXAMPLE 13: PHAGE DISPLAY LIBRARY CONSTRUCTION

Materials and Methods

Construction of biased peptide libraries (BPL): Libraries of phage displaying overlapping peptides (between 5 and 20 amino acids) spanning the entire region of Measles Virus hemagglutinin (HA, a TLR2 ligand), respiratory syncytial virus fusion protein (RSV F, a TLR4 ligand), or E. coH flagellin (fliC, a TLR5 ligand) are constructed. The nucleotide and amino acid sequences of measles HA (GenBank Accession # D28950) are set forth in SEQ ID NO: 119 and SEQ ID NO: 120, respectively. The nucleotide and amino acid sequences of RSV F (GenBank Accession # D00334) are set forth in SEQ ID NO: 121 and SEQ ID NO: 122, respectively. The nucleotide and amino acid sequences of E. coli fliC are set forth in SEQ ID NO: 117 and SEQ ID NO: 118, respectively.

To construct a library, synthetic oligonucleotides covering the entire coding region of the polypeptide of interest (e.g. RSV F) are converted to double- stranded molecules, digested with EcoRl and HindlU restriction enzymes, and ligated into the T7SELECT bacteriophage vector (Novagen). The ligation reactions are packaged in vitro and amplified by either the plate or liquid culture method (according to manufacturer's instructions). The amplified phage are titered (according to manufacturer's instructions) to evaluate the total number of independent clones present in the library. The amplified library will contain approximately 10"- 10³ individual clones.

Construction of random peptide libraries (RPL): Libraries of phage displaying random peptides of from 5 to 30 amino acids in length are constructed essentially as described above for biased peptide libraries, but utilizing oligonucleotides of defined length and random sequences. It is generally recognized that the major constraints of phage display are the bias and diversity (or completeness) of the RPL. To circumvent the former problem, the RPLs are constructed with only 32 codons (e.g. in the form NNK or NNS where N=A/T/G/C; K=G/T; S=G/C), thus reducing the redundancy inherent in the genetic code from a maximum codon number of 64 to 32 by eliminating redundant codons. For example, a 6-amino acid residue library displaying all possible hexapeptides requires 32⁶ (=10⁹) unique clones and is thus considered a complete library. Assuming a practical upper limit of ~10⁹-10¹⁰ clones, RPLs longer than 7 residues accordingly risk being incomplete. This is not a major concern, since a longer residue library may actually increase the effective library diversity and thus is more suitable for isolating new polypeptide TLR ligands. The constructed libraries have a minimum of 10⁹ individual clones.

Construction of cDNA libraries: Libraries of phage displaying bacterial-derived polypeptides are constructed as described above for biased peptide libraries using cDNA derived from a bacterial source of choice. In order to obtain bacterial cDNA, bacterial mRNA is isolated and reversed-transcribed into cDNA. A PCR-ready single-stranded cDNA library made from total RNA of E. coli strain C600 is commercially available (Qbiogene). 10-mer degenerate oligonucleotides are employed as universal primer to synthesize the second strand of the E. coli cDNA. The amplified products are size-selected (ranging from 500 bp to 2 kb), excised and eluted from 1% agarose gel and ligated in the to T7SelectlO-3b vector (Novagen) which can accommodate proteins up to 1200 amino acids in length.

Results and Discussion

A variety of phage display libraries are constructed for use in the screening assay to identify novel polypeptide TLR ligands. Such libraries include: 1) biased peptide libraries, which may be used to identify functional peptide TLR ligands within known polypeptide sequences; 2) random peptide libraries, which may be used to identify functional TLR ligands among randomly generated peptide sequences of between 5 and 30 amino acids in length; and 3) cDNA libraries, which may be used to identify functional TLR ligands from a microorganism of choice, e.g., the bacterium E. coli.

EXAMPLE 14: SCREENING ASSAY FOR PEPTIDE TLR5 LIGANDS

Materials and Methods

Generation of phage displaying a polypeptide TLRS ligand: The coding region of the E. coli flagellin (fliC) gene (SEQ ID NO: 117) was cloned into the T7SELECT phage display vector (Novagen). Double stranded DNA encoding E. coli fliC was ligated to the T7Select 10-3 bacteriophage vector (Novagen). The ligation reactions were packaged in vitro and titered using the host E. coli strain

BLR5615 that was grown in M9TB (Novagen). The recombinant phage was then amplified. Ligation, packaging, and amplification were performed according to manufacturer's instructions.

Generation of phage displaying an S~Tag polypeptide: The S-tag nucleotide sequence is 5'-ATG AAA GAA ACC GCT GCT GCT AAA TTC GAA CGC CAG CAC ATG GAC AGC CCA-3' (SEQ ID NO: 15). The S-tag amino acid sequence is MKETAAAKFERQHMDSP (SEQ ID NO: 16). Double stranded DNA encoding the S-tag peptide sequence was ligated to the T7Select 10-3 bacteriophage vector (Novagen). The ligation reactions were packaged in vitro and titered using the host E. coli strain BLR5615 that was grown in M9TB (Novagen). The recombinant phage was then amplified. Ligation, packaging, and amplification were performed according to manufacturer's instructions. In order to simulate a random peptide library, 10³ fliC phage were mixed with 10¹⁰ S-tag phage (10^"7 dilution).

NF~κB~dependent luciferase reporter assay; Parental 293 cells and 293.hTLR5 cells (see EXAMPLE 12, above) were incubated with an aliquot of fiiC- expressing T7SELECT phage, or S-tag expressing T7SELECT phage, for four to five hours at 37⁰C. As a negative control, cells were incubated with medium alone. NF- κB-dependent luciferase activity was measured using the Steady-Glo Luciferase Assay System by Promega (E2510), following the manufacturer's instructions. Luminescence was measured on a microplate luminometer (FARCyte, Amersham) and expressed as relative luminescence units (RLU) after subtracting the background reading obtained by exposing cells to the DMEM medium alone.

Results and Discussion

To verify the utility of the screening assay to identify TLR-binding polypeptides, we cloned the E. coli flagellin gene (fliC) into the T7SELECT phage display vector, expressed the protein in T7 phage, and examined binding of the recombinant fliC-phage to the cognate receptor, TLR5. The recombinant fliC-phage were incubated on parental HEK293 cells containing an NF-κB-dependent luciferase reporter construct (293) or on TLR5-overexpressing HEK293 cells containing an NF- κB-dependent luciferase reporter construct (293.hTLR5, see EXAMPLE 12. above), and luciferase activity was measured. The data shown in Figure 9 demonstrate that phage displaying fliC on their surface can bind to and activate TLR5. Moreover, the activation of the reporter gene correlates with over-expression of the appropriate TLR (z.e., TLR5).

In order to simulate a random peptide library, 10³ fliC phage are mixed with 10¹⁰ control S-tag phage (10^"7 dilution) and screened by the method described in EXAMPLE 3. For this screen, the TLR¹⁰ cells are parental HEK293 (TLR5^") cells, and the TLR^hi cells are HEK293 cells ectopically expressing human TLR5 (293.hTLR5, see EXAMPLE 12, above). EXAMPLE 15: SCREENING ASSAY FOR PEPTIDE TLR2 LIGANDS

Materials and Methods

Construction of random peptide libraries (RPL): A pentameric random peptide phage display library of T7 SELECT phage was constructed essentially as described in EXAMPLE 13. A pair of phosphorylated oligonucleotides with the sequence NNBNNBNNBNNBNNB (where N-A/G/C/T, B-G/C/T) flanked at the 5' and 3' ends by EcoRI and HindIII sites, respectively, were synthesized. Equimolar amounts of the oligonucleotides were annealed by heating for 5 min at 9O⁰C with gradual cooling to 25⁰C. The double stranded DNA was ligated to T7Select 10-3 bacteriophage vector (Novagen) that had been previously digested with EcoRI and HindIII. The ligation reactions were packaged in vitro and titered using the host E. coli strain BLR5615 that was grown in M9TB (Novagen), generating 2.5 x 10⁷ clones, representing about 75% coverage of the library. The recombinant phage were subjected to several rounds of amplification to generate a total library of 1.35 x 10¹² phage, ensuring representation in excess of 5 x 10⁴ fold for each clone in the library.

Libraries of phage displaying random peptides 10, 15 and 20 amino acids in length were constructed essentially as described for the pentameric random peptide library, except that the phosphorylated oligonucleotides used were 30, 45, and 60 nucleotides in length, respectively.

Sequencing of phage inserts: Individual phage clones from the enriched pool are isolated via plaque formation in E. coli. The DNA inserts of individual phage are amplified in PCR using the commercially available primers T7SelectUP (5' - GGA GCT GTC GTA TTC CAG TC-3'; SEQ ID NO: 10; Novagen, catalog # 70005) and T7SelectDOWN (5'-AAC CCC TCA AGA CCC GTT TA-3'; SEQ ID NO: 11; Novagen, catalog # 70006). The PCR product DNA is purified using the QIAquick 96 PCR Purification Kit (Qiagen) and subjected to DNA sequencing using T7SelectUP and T7SelectDOWN primers. Peptide synthesis: The synthetic monomer of the DPDSG (SEQ ID NO: 76) motif, as well a concatemerized copy (DPDSG)₅ (SEQ ID NO: 77) peptides are manufactured using solid phase synthesis methodologies and FMOC chemistry.

NF~κB-dependent luciferase reporter assay: Parental 293 cells and 293.hTLR2 cells (see EXAMPLE 12, above) are incubated with an aliquot of test peptide four to five hours at 37⁰C. NF-κB-dependent luciferase activity is measured using the Steady-Glo Luciferase Assay System by Promega (E2510), following the manufacturer's instructions. Luminescence is measured on a microplate luminometer

(FARCyte, Amersham) and expressed as relative luminescence units (RLU) after subtracting the background reading obtained by exposing cells to the DMEM medium alone.

Results and Discussion

We constructed a pentameric random peptide phage display library in T7 phage. This phage library is then screened by the method described in EXAMPLE 3. For this screen, the TLR¹⁰ cells are parental HEK293 (TLR2^") cells, and the TLR^hl cells are HEK293 cells ectopically expressing human TLR2 (293.hTLR2, see EXAMPLE 12, above).

Individual phage clones from the phage population enriched for specific binding to TLR2 are isolated via plaque formation in E. coli and sequenced. The biological activity of the TLR2-binding peptides isolated by the screening method is confirmed using isolated peptides in an NF-κB-dependent reporter gene assay. For this assay, a synthetic peptides corresponding to the peptide inserts of individual phage clones from the phage population enriched for specific binding to TLR2 are incubated on parental HEK293 cells containing an NF-κB- dependent luciferase reporter construct (293) and on TLR2-overexpressing HEK293 cells containing an NF-κB-dependent luciferase reporter construct (293.hTLR2, see EXAMPLE 12, above). Luciferase activity is then measured. This assay is used to show that the synthetic peptides activate luciferase reporter gene expression in a TLR2-dependent manner, and thus, are functional TLR2 ligands. We also constructed 10, 15, and 20 amino acid random peptide phage display libraries in T7SELECT phage. These phage display libraries are pooled in equal proportion and then screened by method as described in EXAMPLE 3. For this screen, the TLR¹⁰ cells are parental HEK293 (TLR2^") cells, and the TLR^hl cells are HEK293 cells ectopically expressing human TLR2 and human CD 14 (293.hTLR2.hCD14, see EXAMPLE 12, above).

EXAMPLE 16: SYNTHETIC PEPTIDES THAT ACT AS TLR4 AGONISTS

IN VITRO

Materials and Methods:

Cell Lines: HEK293 cells (Invivogen; cat. # 293 -null) and

HEK293:TLR4 cells (Invivogen; cat. #293-htlr4md2cdl4) were maintained in Dulbecco's Modified Eagle Medium (Gibco) with 10% Fetal Bovine Serum

(Hyclone) supplemented with 10 μg/ml of blasticidin or 10 μg/ml of blasticidin and

50 μg/ml of hygromycin respectively. Cells were passaged 1 :4 every three days.

RAW267.4 cells (ATCC #TIB-71) were maintained in Dulbecco's Modified Eagle Medium (Gibco) with 10% Fetal Bovine Serum (Hyclone). Cells were passaged 1 :8 every three days.

Synthetic Peptides: Synthetic peptides were made by a commercial vendor (BaChem) using solid phase synthesis. Peptides were HPLC purified (purity > 95%). Peptides were resuspended in either phosphate buffer saline (PBS) or a formulation buffer developed in-house termed Fl 2 Ia. The recipe for F121a is as follows: 10 mM histidine, 10% sucrose (w/v), 1.5% (w/v) polysorbate-80, 0.1 rnM EDTA, 0.5% (v/v) ethanol at pH 6.5. Lyophilized peptides are stored at -20C and peptide solutions are made fresh at the start of each experiment.

TLR4 Bioactivity Assay: Cells were plated at a density of 50,000 cells/ well in a 96-well tissue culture plate (Falcon) in growth media described above. Either Ultrapure LPS (Invivogen; cat, # thi-pelps) or synthetic peptides were added to the cells. Cell supernatants were harvested 16-20 hours later. IL-8 was used as a readout for cellular activation when HEK293 cells were used and TNF was used with RAW264.7 cells.

To detect IL-S (HEK cells), a capture ELISA was performed. First, ELISA plates (Costar; cat. # 9018) were coated with anti-IL-8 capture antibody (Pierce; cat. #M801) and stored at 4°C overnight. The following day, the capture antibody solution was removed and BD Assay Diluent (BD; cat #555213) was added to each well and the plates were incubated at room temperature for one hour. The plates were then washed twice with IXPBS + .05% Tween-20 (PBS-T). IL-8 cytokine standard (Pierce; cat #SIL8) and samples (in duplicate) were added to the blocked wells and incubated at room temperature for one hour. The plates were then washed thrice with PBS-T and biotinylated anti-IL-8 detection antibody (Pierce; cat. #M802B) was added to each well and incubated for one hour. Plates were then washed with PBS-T and the avidin-horseradish peroxidase conjugate (BD; cat. #554058) is added. After a 30 minute incubation, plates were washed and developed using TMB (Pierce; cat. # 34028). The reaction was stopped by adding .25M HCl. Absorbance was read with a FARCyte plate reader at 450nm.

To detect TNF (RAW264.7), a capture ELISA was performed. First, ELISA plates were coated with an anti-TNF capture antibody (BD Pharmingen #557516) in coating buffer (.1M Na₂HPO₄ adjusted to pH 6.0 with NaH₂PO₄) and incubated overnight at 4⁰C. The following morning, the capture antibody solution was removed and the blocking solution, BD Assay Diluent, was added to each well and the plates were incubated at room temperature for one hour. The plates were then washed twice with IXPBS + .05% Tween-20 (PBS-T). TNF standard (BD Pharmingen #554589) and samples (in duplicate) were added to the blocked wells and incubated at room temperature for 1 hour. The remaining steps of the ELISA were performed as described for the IL-8 ELISA above.

Results and Discussion:

As shown in Example 5, above, phage isolates have been identified that specifically activate cells expressing human TLR4 (hTLR4), CD 14 and MD2. In this example, it is shown that a subset of the peptides expressed by active phage isolates act as TLR4 agonists when removed from the structural confines of the T7 phage coat protein.

As a test case, the peptide sequence (EDMVYRIGVP (SEQ ID NO: 19)) derived from the phage isolate D2 described in Example 5, above, was first tested. As shown in Table 7, this peptide contains the insert expressed by the D2 phage isolate including the two flanking cysteines. The three and four amino acids present at the amino and carboxy ends, respectively, of the peptide in the context of the phage coat protein were also included in the synthetic version. Additionally, the peptide contains a 4x-His tag (SEQ ID NO: 62) to allow detection in a binding assay if desired. The three remaining synthetic peptides served as controls. The peptide termed D2.No His is identical to peptide D2 except that the His tag has been removed. The peptide D2.Ser Sub contains serine residues in place of the flanking cysteines to test the requirement of the cyclic nature of the peptide for TLR4 activity. Finally, peptide F3 contains a cyclic lOmer sequence that does not exhibit TLR4 specific binding or agonist activity (Figure 11). AU four peptides were synthesized by a commercial vendor and the presence of a disulfide bond between the flanking cysteines (peptides 1, 2, and 4) was confirmed by an Elman's assay.

Table 7. Synthetic peptides tested for TLR4 agonist activity. The listed peptides were synthesized by a commercial vender and used in cell assays.

Peptides were resuspeneded in PBS and equimolar amounts of each peptide were added to HEK293:hTLR4 cells as well as HEK293:Null cells (hTLR4-). Cell supernatants were harvested 24 hours later and tested for the presence of IL-8 by ELISA. As shown in Figure 10, nanomolar quantities of both D2 and D2.No His activated hTLR4-expressing cells, In contrast, an irrelevant lOmer cyclic peptide (F3) and the serine substituted version of D2 failed to stimulate IL-8 production by HEK293:hTLR4 cells. None of the four peptides activated HEK293:Null cells (data not shown). This study indicates that the synthetic D2 peptide acts as a TLR4- specific agonist in vitro. Additionally, this experiment demonstrates that the activity of D2 is dependent upon the two cysteines responsible for the cyclic conformation of the peptide. Without intending to be bound by theory, the difference between the D2 and D2.No His peptides may be attributable to peptide quality as BaChem experienced great difficulty in synthesizing D2.No His. Alternatively, the D2.No His may be inherently more susceptible to degradation by proteases present in the cell supernatant.

To confirm that D2 acts similarly on mouse and human TLR4 and analyze the activity of D2 in a system that more closely mimics endogenous TLR4 expression levels, D2 on the mouse macrophage cell line RAW264.7 which naturally expresses TLR4 was tested. Titrating molar amounts of peptide were added to RAW264.7 cells. Cell supematants were collected 20 hours later and the presence of TNF was measured by ELISA as a measure of TLR-dependent cell activation. As shown in Figure 11, only D2 and D2-No His activated RAW264.7 cells. This result shows that the cysteine constrained peptide sequence EDMVYRIGVP (SEQ ID NO: 19) is able to activate a mouse cell line expressing endogenous levels of TLR4 in vitro. These data mark the first time, to our knowledge, that a synthetic peptide has been shown to activate through TLR4.

In light of the success with D2, the peptide inserts derived from other active phage isolates (Table 8) identified as described in Figure 5, above, were tested.

Table 8. New Peptide Sequences. Six peptides were synthesized by a commercial vendor. The amino acid sequence of each peptide along with the cyclic library from which it originated is listed in the table. Underlined sequences are the unique portion of the peptide.

In contrast to D2, the majority of the new peptides were not soluble in 1 x PBS. Based on the composition of the peptides, formulation buffer, F121a (described above), was used. The resuspended peptides were added in titrating molar amounts to HEK293 cells overexpressing TLR4, MD2, and CD 14 (Figure 12). The peptide F5 (WWSVGLISW (SEQ ID NO:84)) demonstrated the best activity of the group and behaved similarly to D2 peptide resuspended in Fl 2 Ia. To determine if Fl 21 a has unintended effects on the cell line, the activity of LPS in F121a was compared to that of LPS in PBS. As shown in Figure 12B,

F 121 a does not affect the ability of this cell line to respond to a TLR4 ligand. Finally, peptide F5 does not activate HEK293 cells deficient in TLR4 expression.

Therefore, two peptides, D2 and F5, have been identified which reproducibly activate TLR4+ cells in vitro. Analysis of multiple hits will provide sequence data for use in peptide optimization. Along this line, D2 and F5 peptides share a three amino acid motif with conserved substitutions, providing insight into a putative activation motif (Table 9).

Table 9. Sequence Alignment of Active Peptides. The sequences of D2 and F 5 are shown and the shared three amino acid motif is underlined.

EXAMPLE 17: ACTIVITY OF SYNTHETIC PEPTIDES ON MOUSE AND

HUMAN PRIMARY CELLS

Materials and Methods

Generation and Activation of Mouse Bone Marrow Derived Dendritic Cells: Bone marrow cells were flushed from the femurs of C3H/HeN (TLR4+) or C3FI/HeJ (TLR4-) mice using a needle and syringe. Cells were washed in RPMI-1640 supplemented with 10% FBS (HyClone). Red blood cells were removed from the suspension using Red Blood Cell Lysis solution (Sigma) as per manufacturer's protocol. The remaining cells were resuspended in Dendritic Cell Growth Media (RPMI-1640 containing FBS and a 1 :100 dilution of mouse GM-CSF) to promote differentiation from stem cells to bone marrow derived dendritic cells (BMDCs). Cells were plated in 24 well plates at a concentration of 7-8x10⁵cells/mL. Cells were cultured for four days, with media being replenished on day 2 and day 4. At the end of four days, the cells have differentiated into BMDCs as indicated by a distinct change in morphology as well as the upregulation of cell surface markers associated with this cell type.

After four days of differentiation, C3H/HeN(TLR4+:TLR2+) BMDC and C3H/He.T(TLR4-:TLR2+) BMDC were stimulated with either Ultrapure LPS (Invivogen; # thi-pelps), Pam3CSK4 (Invivogen, #tlrl-pms), or peptides synthesized by a commercial vendor (BaChem). All ligands were resuspended in Ix PBS and added directly to the cells in the 24-well plate. After 18 hours, cell supernatants were harvested for analysis.

Generation and Activation of Human Dendritic Cells: Human CD14+ monocytes were obtained from a commercial vendor (Cambrex, #2W-400B). Cells were washed with RPMI- 1640 (Gibco) supplemented with 10% FBS. Cells were resuspended in RPMI- 1640 with FBS and 50ng/mL hGM-CSF (Peprotech, #300-03) and lOOng/mL hIL-4 (R&D Systems, #204~IL) at a concentration of 5x10⁵ cells/mL. Cells were plated in 24 well plates and cultured for five days. Media was replenished on day 3 and day 5. On day 6, cells were stimulated with Ultrapure LPS (Invivogen) or peptides resuspended in the formulation buffer F 12 Ia described in Example 1. Cell supernatants were harvested for analysis at 24 hours and 48 hours post-stimulation.

Analysis of Cytokines in the Cell Supernatant: __To measure the production of cytokines by the BMDCs, the Mouse Inflammation Cytokine Bead

Array Kit (#552364, BD Biosciences) was used as directed by the manufacturer. Human DC supernatants were analyzed using the Human Inflammation CBA Kit

(#551811, BD Biosciences).

Results and Discussion

As described in Example 12, a synthetic cyclic peptide (termed D2; amino acid sequence EDMVYRIGVP (SEQ ID NO: 19) was identified that activates both an HEK cell line transfected with human TLR4 and the RAW macrophage cell line (mouse origin) that endogenously expresses TLR4. These studies demonstrated that the D2 peptide was capable of activating both human and mouse TLR4 in the context of immortalized cell lines. Since cell line systems do not reflect physiological levels of TLR expression, the bioactivity of D2 on both mouse and human primary cells was tested.

Mouse primary cells. The effect of D2 on mouse bone marrow derived dendritic cells (BMDC) was monitored as this system has been used extensively to study TLR4 in an endogenous context. Femur bone marrow cells were isolated from C3H/HeN (TLR4+) and C3H/HeJ (TLR null) mice. These cells were cultured in the presence of GM-CSF over four days, ultimately generating immature BMDC as indicated by the upregulation of CDl Ic and moderate levels of MHClI on the cell surface (data now shown). C3H/HeN (TLR4+:TLR2+) BMDC and C3H/HeJ (TLR4- :TLR2+) BMDC were stimulated with 500 ng/niL or 50 ng/mL of LPS (TLR4 ligand, positive control), peptide D2 at 100 μM or 50 μM, peptide F3 (negative control) at 100 μM or 50 μM, and 500 ng/mL or 50 ng/mL of Pam3Cys (TLR2 ligand). All stimulants were resuspended in Ix PBS. After 18 hours, cell supernatants were harvested for analysis of cytokine/chemokine production.

To measure the production of cytokines by the BMDCs, the Cytokine Bead Array (CBA) from BDBiosciences was used. This array measures six cytokines simultaneously, including TNF, 1L-6, IL-10, IL- 12, IFN-γ, and MCP-I. As shown in Figure 13, C3H/HeN BMDC (TLR2+:TLR4+) produced robust levels of TNF, IL-ό, and MCP-I in response to Pam3CsK4 and LPS. The addition of D2 peptide resulted in low, but detectable, levels of these same cytokines. In contrast, the negative control peptide, F3, did not induce a measurable increase in any of the cytokines tested. C3H/HeJ BMDC (TLR2+:TLR4 null) produced high levels of TNF, IL-6 and MCP-I in response to Pam3CSK4 but not LPS or any of the synthetic peptides tested. Together, these data suggest that D2 stimulates mouse BMDC to produce modest levels of inflammatory cytokines in a TLR4-specific manner, indicating that this peptide is capable of activating mouse primary cells as well as cell lines. Human Primary Cells. It is well established that dendritic cells differentiated from human blood monocytes express TLR4 and are responsive to LPS. Purified CD 14+ monocytes were obtained from a commercial vendor (Cambrex) and were differentiated in complete RPMI media supplemented with 50 ng/mL hGM-CSF and 100 ng/mL hIL-4 for six days. On the sixth day, 100 ng/mL or 10 ng/mL of LPS, 50 μM or 10 μM of D2 peptide or F3 (negative control peptide) were added to the cells. In this experiment, the peptides were resuspended in a formulation buffer (F121a) designed to enhance the stability of the peptides. Cell supematants were collected at 24 and 48 hours post-stimulation.

Cell supematants were analyzed for the presence of six inflammatory cytokines (IL-8, TNF, IL-12, IL-6, IL-I β, and IL-10) as measured by CBA. As expected, LPS induced multiple cytokines in the array including IL-8 and TNF (Figure 14), confirming that these cells are responsive to TLR4 ligands. Of particular interest, D2, but not the negative controls, induced the secretion of IL-8 and TNF (Figure 14), showing that this peptide acts as an agonist of key effector cells of the innate immune system.

EXAMPLE 18: IDENTIFICATION OF ADDITIONAL PHAGE ISOLATES

THAT ACTIVATE TLR4+ CELLS

Materials and Methods

Phage Capture Bioassay for TLR4 Agonists: This example describes some modifications to the phage capture bioassay described in Figure 5, and use of the bioassay to identify additional phage isolates with TLR4 agonist activity. First, TLR4- cells were not tested in parallel with TLR4+ cells. Instead, isolates that test positive on TLR4+ cells were later retested on TLR4- cells. This change was made to allow rapid screening of phage isolates. Secondly, while the IL-8 ELISA is still the assay readout, sample OD was used as a measure of cell activity instead of a value generated by a standard curve. This change allows for more rapid analysis of data and the application of additional samples on each plate. Each phage isolates was tested in duplicate. The value of each duplicate was then compared to the average of all isolates tested on a given plate. Isolates that were at least two standard deviations above the plate average, in duplicate, were scored as positive. Phage isolates that scored positive on TLR4+ cells were then tested on the parental HEK:Null (TLR4-) cells to exclude those that non~specifically activate HEK cells. Results and Discussion

Using the phage capture bioassay originally described in Example 3 and in Figure 5, and modified here, additional phage isolates with TLR4 agonist activity have been identified (Table 10). The sequences of the peptide inserts contained within these active isolates are shown in Table 10, Also, Table 10 shows that these isolates do not activate HEK:Null cells, indicating that their observed activity is dependent upon TLR4.

Table 10. Phage isolates with activity on HEK:TLR4 cells. Peptide sequences derived from phage isolates that activate HEK:TLR4 cells are shown. "Isolate OD" refers to the average of the duplicate OD values from each positive isolate. "Avg Control OD +/- S.D." shows the sample average and standard deviation from the plate on which the corresponding phage isolate was tested. Note that each positive isolate has an average value that is at least 2 standard deviations above the plate average.

These active isolates were unable to activate HEK:Null cells (Figure 14), indicating that their activity is dependent upon TLR4.

Table 11. The activity of phage isolates on HEK cells is dependent upon TLR4. The positive phage isolates identified in Table 10 were tested on HEK:NulI cells. "Isolate OD" refers to the average of the duplicate OD values from each isolate. "Avg Control OD +/- S.D." shows the sample average and standard deviation from the plate on which the corresponding phage isolate was tested. Note that each isolate has an average value that is not greater than 2 standard deviations above the plate average, indicating that these isolates do not activate HEKrNuIl cells.

With the exception of VCEVSDSVMA (SEQ ID NO:91), these peptides inserts were ordered in the form of synthetic peptides (Table 12) and their activity on TLR4+ cells will be analyzed.

Table 12. Synthetic peptides as putative TLR4 agonists. Peptides are currently being synthesized by a commercial vendor (BaChem). Underlined sequences denote the unique portion of the peptide. A disulfide bond will be engineered between the two flanking cysteines.

RNS-CVEEYSSSGVSC-GGGGHHHH (SEQ ID NO:92)

RNS-CLTYGGLEALGC-GGGGHHHH (SEQ ID NO:93)

RNS-CVSSAQEVRVPC-GGGGHHHH (SEQ ID NO:94) ^■ RNS-CSRTDVGVLEVC-GGGGHHHH (SEQ ID NO:95)

RNS-CREKVSRGDKGC-GGGGHHHH (SEQ ID NO:96)

RNS-CDWDAVESEYMC-GGGGHHHH (SEQ ID NO:97)

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

It is further to be understood that all values are approximate, and are provided for description.

Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

-I l l-

Claims

WHAT IS CLAIMED IS:

1. A method to identify a phage population enriched for specific binding to a TLR comprising:

5 i) providing a multiplicity of test phage in the form of a phage display library, wherein each test phage comprises a nucleic acid insert encoding a polypeptide; ii) performing a first phase of screening comprising the steps of: a) contacting a TLR¹⁰ cell with the multiplicity of test phage; b) retaining the test phage that do not bind to the TLR¹⁰ cell; and 10 c) optionally, repeating steps a) and b); iii) dividing the test phage retained in step ii) into at least a first phage portion and a second phage portion; iv) performing a second phase of screening comprising the steps of: d) contacting a TLR^hl cell with the first phage portion, and contacting a ] 5 TLR'° cell with the second phage portion, wherein each TLR is the same TLR as in step ii); e) retaining the test phage of the first phage portion that bind to the TLR^hl cell and retaining the test phage of the second phage portion that bind to the TLR¹⁰ cell; 0 f) optionally, determining the number of retained test phage of the first phage portion and determining the number of retained test phage of the second phage portion; g) amplifying the retained test phage of the first phage portion and amplifying the retained test phage of the second phage portion; 5 h) optionally, determining the number of test phage in the amplified first phage portion and determining the number of test phage in the amplified second phage portion; and i) optionally, repeating steps d) through h); wherein step f) is performed at least once or step h) is performed at least once; and 0 v) performing at least one of steps j) or k): wherein step j) comprises comparing the number of retained test phage of the first phage portion determined in step iv) with the number of retained test phage of the second phage portion determined in step iv); and wherein step k) comprises comparing the number of test phage in the amplified first phage portion determined in step iv) with the number of test phage in the amplified second phage portion determined in step iv), wherein: if the number of retained test phage of the first phage portion determined in step iv) is greater that the number of retained test phage of the second phage portion determined in step iv), or if the number of test phage in the amplified first phage portion determined in step iv) is greater than the number of test phage in the amplified second phage portion determined in step iv), the test phage of the first phage portion amplified in step iv) are identified as a phage population enriched for specific binding to a TLR,

2. The method according to claim 1, wherein steps a) and b) are performed at least two times.

3. The method according to claim 1, wherein steps d) through h) are performed at least four times.

4. The method according to claim 1 , wherein the TLR is a mammalian TLR.

5. The method according to claim 1, wherein the TLR is a TLR2, a TLR4, or a TLR5.

6. The method according to claim 1, wherein the TLR¹⁰ cell of step ii) and the TLR¹⁰ cell of step iv) are the same cell type.

7. The method according to claim 6, wherein the TLR¹⁰ cell of step ii), the TLR¹⁰ cell of step iv), and the TLR¹" cell of step iv) are the same cell type.

8. The method according to claim 7, wherein the TLR¹⁰ cell of step ii), the TLR¹⁰ cell of step iv), and the TLR^hi cell of step iv) are each a HEK293 cell.

9. The method according to claim 7, wherein the TLR¹⁰ cell of step ii), the TLR¹⁰ cell of step iv), and the TLR^hl cell of step iv) are each an NIH3T3 cell.

10. The method according to claim 1, wherein the TLR¹⁰ cell of step ii), the TLR¹⁰ cell of step iv), and the TLR^hl cell of step iv) are each a mammalian cell.

11. A method to identify a polypeptide TLR ligand comprising: i) identifying a phage population enriched for specific binding to a TLR according the to method of claim 1 ; and ii) characterizing the polypeptide encoded by the nucleic acid insert of a test phage of the phage population enriched for specific binding to a TLR.

12. The method according to claim 11, wherein characterizing the polypeptide encoded by the nucleic acid insert of a test phage of the phage population enriched for specific binding to a TLR comprises: i) determining the nucleic acid sequence of the nucleic acid insert; and ii) using the nucleic acid sequence from step i) to deduce the amino acid sequence of the polypeptide encoded by the nucleic acid insert.

13. The method according to claim 11 , wherein characterizing the polypeptide encoded by the nucleic acid insert of a test phage of the phage population enriched for specific binding to a TLR comprises: i) translating the nucleic acid insert to generate the polypeptide encoded by the nucleic acid insert; and ii) characterizing said polypeptide.

14. The method according to claim 13, wherein characterizing said polypeptide comprises determining the amino acid sequence of the polypeptide.

15. The method according to claim 13, wherein characterizing said polypeptide comprises measuring the ability of the polypeptide to modulate TLR signaling.

16. The method according to claim 11 , wherein characterizing the polypeptide encoded by the nucleic acid insert of a test phage of the phage population enriched for specific binding to a TLR comprises measuring the ability of the test phage to modulate TLR signaling.

17. A method to identify a phage population enriched for specific binding to a TLR comprising: i) providing a multiplicity of test phage in the form of a phage display library, wherein each test phage comprises a nucleic acid insert encoding a polypeptide; ii) performing a first phase of screening comprising the steps of: a) contacting a TLR¹⁰ cell with the multiplicity of test phage; b) retaining the test phage that do not bind to the TLR¹⁰ cell; and c) repeating steps a) and b) once; iii) dividing the test phage retained in step ii) into a first phage portion and a second phage portion, wherein the number of test phage in the first phage portion is approximately equal to the number of test phage in the second phage portion; iv) performing a second phase of screening comprising the steps of: d) contacting a TLR^hl cell with the first phage portion, and contacting a TLR ° cell with the second phage portion, wherein each TLR is the same TLR as in step ii); e) retaining the test phage of the first phage portion that bind to the TLR^hl cell and retaining the test phage of the second phage portion that bind to the TLR¹⁰ cell; g) amplifying the retained test phage of the first phage portion and amplifying the retained test phage of the second phage portion; h) determining the number of test phage in the amplified first phage portion and determining the number of test phage in the amplified second phage portion; and i) repeating steps d) through g) three times; and v) comparing the number of test phage in the amplified first phage portion determined in step iv) with the number of test phage in the amplified second phage portion determined in step iv), wherein if the number of test phage in the amplified first phage portion determined in step iv) is greater than the number of test phage in the amplified second phage portion determined in step iv) the test phage of the amplified first phage portion of step iv) are identified as a phage population enriched for specific binding to a TLR.

18. The method according to claim 17, wherein the TLR is a mammalian TLR.

19. The method according to claim 17, wherein the TLR is a TLR2, a TLR4, or a TLR5.

20. The method according to claim 17, wherein the TLR ° cell of step ii) and the TLR ° cell of step iv) are the same cell type.

21. The method according to claim 20, wherein the TLR ° cell of step ii), the TLR¹⁰ cell of step iv), and the TLR^hl cell of step iv) are the same cell type.

22. The method according to claim 21, wherein the TLR¹⁰ cell of step ii), the TLR'⁰ cell of step iv), and the TLR^hi cell of step iv) are each a HEK293 cell.

23. The method according to claim 21 , wherein the TLR¹⁰ cell of step ii), the TLR¹⁰ cell of step iv), and the TLR^hi cell of step iv) are each an NIH3T3 cell.

24. The method according to claim 17, wherein the TLR¹⁰ cell of step ii), the TLR'° cell of step iv), and the TLR^hl cell of step iv) are each a mammalian cell.

25. A method to identify a polypeptide TLR ligand comprising: i) identifying a phage population enriched for specific binding to a TLR according the to method of claim 17; and ii) characterizing the polypeptide encoded by the nucleic acid insert of a test phage of the phage population enriched for specific binding to a TLR.

26. The method according to claim 25, wherein characterizing the polypeptide encoded by the nucleic acid insert of a test phage of the phage population enriched for specific binding to a TLR comprises: i) determining the nucleic acid sequence of the nucleic acid insert; and ii) using the nucleic acid sequence from step i) to deduce the amino acid sequence of the polypeptide encoded by the nucleic acid insert.

27. The method according to claim 25, wherein characterizing the polypeptide encoded by the nucleic acid insert of a test phage of the phage population enriched for specific binding to a TLR comprises: i) translating the nucleic acid insert to generate the polypeptide encoded by the nucleic acid insert; and ii) characterizing said polypeptide.

28. The method according to claim 27, wherein characterizing said polypeptide comprises determining the amino acid sequence of the polypeptide.

29. The method according to claim 27, wherein characterizing said polypeptide comprises measuring the ability of the polypeptide to modulate TLR signaling.

30. The method according to claim 25 wherein characterizing the polypeptide encoded by the nucleic acid insert of a test phage of the phage population enriched for specific binding to a TLR comprises measuring the ability of the test phage to modulate TLR signaling.

31. A phage population enriched for specific binding to a TLR identified by the method of any of claims 1 to 10 and 17 to 24.

32. A polypeptide TLR ligand identified by the method of any of claims 11 to 16 and 25 to 30.

33. A method to stimulate an immune response in a subject comprising administering to a subject in need thereof the polypeptide TLR ligand of claim 32.

34. The method of claim 33, wherein the subject is a mammal.

35. A method of modulating TLR signaling in a cell comprising contacting a cell, wherein the cell comprises the TLR, with the polypeptide TLR ligand of claim 32,

36. The method of claim 35, wherein the cell is a mammalian cell.

37. A vaccine comprising: i) at least one polypeptide TLR ligand of claim 32; ii) at least one antigen; and iii) optionally, a pharmaceutically acceptable carrier.

38. The vaccine of claim 37, wherein the at least one polypeptide TLR ligand and the at least one antigen are covalently linked.

39. The vaccine of claim 37, wherein the at least one antigen is a polypeptide, a lipoprotein, a glycoprotein, a mucoprotein, a lipid, a saccharide, a lipopolysaccharide, or a nucleic acid.

40. The vaccine of claim 37, wherein the at least one antigen is a pathogen-related antigen, a tumor-associated antigen, or an allergen-related antigen.

41. The vaccine of claim 406, wherein the pathogen-related antigen is an Influenza antigen, a Listeria monocytogenes antigen, or a West Nile Virus antigen

42. A method to stimulate an immune response in a subject comprising administering to a subject in need thereof the vaccine of claim 37.

43. The method of claim 42, wherein the subject is a mammal.

44. A polypeptide comprising: i) at least one polypeptide TLR ligand identified by the method of any of claims 11 to 16 and 25 to 30; and ii) at least one polypeptide antigen.

45. The polypeptide of claim 44, wherein the at least one polypeptide antigen is a pathogen-related antigen, a tumor-associated antigen, or an allergen-related antigen.

46. The polypeptide of claim 45, wherein the pathogen-related antigen is an Influenza antigen, a Listeria monocytogenes antigen, or a West Nile Virus antigen.

47. A method to stimulate an immune response in a subject comprising administering to a subject in need thereof the polypeptide claim 44.

48. The method of claim 47, wherein the subject is a mammal.

49. A method of modulating TLR signaling in a cell comprising contacting a cell, wherein the cell comprises the TLR, with the polypeptide of claim 44.

50. The method of claim 49, wherein the cell is a mammalian cell.

51. A vaccine comprising the polypeptide of claim 44 and, optionally, a pharmaceutically acceptable carrier.

52. A method to stimulate an immune response in a subject comprising administering to a subject in need thereof the vaccine of claim 51.

53. The method of claim 52, wherein the subject is a mammal.