Immunostimulatory oligonucleotides

Abstract

Compositions and methods are provided for stimulating toll-like receptor 9 (TLR9). More particularly, immunostimulatory oligonucleotides, methods for enhancing the immunostimulatory properties of oligonucleotides, and methods for eliciting an immune response are disclosed herein.

Description

[0001] REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the priority and benefit of European Patent Application Nos. EP17207740.6, EP17207746.3, and EP17207750.5, each filed on December 15, 2017, the disclosures of which are incorporated herein by reference in their entirety.

[0003] SEQUENCE LISTING

[0004] This application contains a sequence listing that has been electronically submitted in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on November 27, 2018, is named 103976.000119 SEQ LISTING_ST25.txt and is 2,233 bytes in size. FIELD OF THE INVENTION

[0005] Compositions and methods for stimulating toll-like receptor 9 (TLR9) are provided. More particularly, immunostimulatory oligonucleotides, methods of enhancing the immunostimulatory properties of oligonucleotides, and methods of eliciting an immune response are disclosed herein. BACKGROUND OF THE INVENTION

[0007] Antibiotic resistance is a global problem that negatively impacts many industries. Methicillin-resistant Staphylococcus aureus ( Staphylococcus aureus )(MRSA) and other "super bugs" are causing havoc in hospitals and doctor's offices, making a visit to a health center potentially lethal. The agricultural industry also faces similar problems. Due to limited space and non-sterile environments, entire herds are at risk of pathogenic infections. For example, a sick cow in close proximity to its herd can exponentially increase the morbidity and mortality rates. Despite the risk of infection, antibiotic treatment is becoming less popular due to increased costs and consumer demands for meat and dairy products that have not been exposed to antibiotics. Moreover, producers who do use antibiotic therapies understand that even broad-spectrum antibiotics are not completely effective against every pathogen that a herd may come into contact with.

[0008] Accordingly, there is a need for non-antibiotic-based therapies for treating or preventing infections in animals. The disclosed compositions and methods are targeted at these and other important needs. SUMMARY OF THE INVENTION

[0010] Disclosed herein are immunostimulatory oligonucleotides comprising at least one CpG motif and a 3'-cholesterol moiety.

[0011] Also provided herein are immunostimulatory compositions comprising the immunostimulatory oligonucleotides.

[0012] Also disclosed are methods for enhancing the immunogenicity of TLR9 ligands, including attaching a cholesterol moiety to the 3'-end of a TLR9 ligand via a linker, wherein the TLR9 ligand is an oligonucleotide having at least one CpG motif.

[0013] Also provided are methods for eliciting a TLR9-mediated immune response in a subject, including administering to the subject any one of the immunostimulatory oligonucleotides or immunostimulatory compositions described herein. Sequence Listing <110> Elanco Animal Health Incorporated <120> Immunostimulatory Oligonucleotides <130> BHC168026-FC <150> EP17207740.6 <151> 2017-12-15 <150> EP17207746.3 <151> 2017-12-15 <150> EP17207750.5 <151> 2017-12-15 <160> 9 <170> PatentIn version 3.5 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> Phosphorothioate (PTO) Bond <223> Synthetic Oligonucleotide 2006-PTO <400> 1 tcgtcgtttt gtcgttttgt cgtt 24 <210> 2 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide 2006-3dT4G5T4 <400> 2 tcgtcgtttt gtcgttttgt cgttttttgg gggtttt 37 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide 2006-T4-PDE <400> 3 tcgtcgtttt gtcgttttgt cgtttttt 28 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide 2007-PDE-T4 <400> 4 tcgtcgttgt cgttttgtcg tttttt 26 <210> 5 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide 2007-T4G5T4 <400> 5 tcgtcgttgt cgttttgtcg ttttttgggg gtttt 35 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide TCG8-T4 <400> 6 tcgtcgtcgt cgtcgtcgtc gtcgtttt 28 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide 2006-G5 <400> 7 tcgtcgtttt gtcgttttgt cgttggggg 29 <210> 8 <211> 37 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide TCG8-T4G5T4 <400> 8 tcgtcgtcgt cgtcgtcgtc gtcgttttgg gggtttt 37 <210> 9 <211> 13 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide PDE-2006 <400> 9 ttttgggggt ttt 13 Brief description of the drawings

[0015] When read in conjunction with the accompanying Figure 1 drawings, the foregoing summary and the following detailed description will be further understood. For purposes of illustrating the disclosed compositions and methods, exemplary embodiments of the compositions and methods are shown in the drawings; however, the compositions and methods are not limited to the specific embodiments disclosed. In the drawings:

[0016] Figure 1 The chemical structure of a cholesterol moiety attached to a hexanediol linker is described.

[0017] Figure 2A And 2B The TLR9-stimulating activities of oligonucleotide PTO-2006, oligonucleotide PDE-2006 with the 3’ TTTTGGGGGTTTT (SEQ ID NO: 9) sequence (“2006-3dT4G5T4”), and oligonucleotide 2006-3dT4G5T4 with a 3'-cholesterol moiety attached via a hexanediol linker as shown (“2006-3dT4G5T43C”) in HEKBlue-hTLR9 cells are compared. Figure 1

[0018] Figure 3 Figure 1 The oligonucleotide PDE-2006 with a 3'TTTT sequence (“2006-T4-PDE”) and the one with Figure 1The ability of oligonucleotide 2006-T4-PDE with a hexanediol linker attached to the cholesterol moiety at the 3'-end of the oligonucleotide ("3Chol-2006-T4-PDE") to elicit a TLR9-mediated immune response in HEKBlue-hTLR9 cells.

[0019] Figure 4A and 4B compared the TLR9-stimulatory profiles of oligonucleotide 2006-3dT4G5T4 and oligonucleotide 2006-3dT4G5T4C.

[0020] Figure 5 compared the TLR9-stimulatory profiles of oligonucleotide 2006-3dT4G5T4 and oligonucleotide 2006-3dT4G5T4C.

[0021] Figure 6 described the chemical structure of the cholesterol moiety attached to a hexaglycol linker.

[0022] Figure 7 compared oligonucleotide 2006-T4G5T4 with and without a Figure 6 cholesterol-linker moiety attached to its 3'-end, oligonucleotide 2006-T4G5T4 ("2006-T4G5T4-3Chol" or "2006-T4G5T4-3C"), for its TLR9-stimulatory ability.

[0023] Figure 8A and 8B compared the immunogenicity of oligonucleotides with and without 3'-cholesterol modification. Figure 8A compared the ability of oligonucleotide 2007-PDE-T4 oligonucleotide to elicit a TLR9-mediated immune response in HEKBlue-hTLR9 cells with that of oligonucleotide 2007-PDE-T4 oligonucleotide with a Figure 1 cholesterol moiety attached via a hexanediol linker ("2007-PDE-T4-3Ch"), and Figure 8B compared oligonucleotide 2007-PDE-T4 with a 3’ GGGGGTTTT sequence ("2007-T4G5T4") and oligonucleotide 2007-T4G5T4 with a Figure 1 cholesterol moiety attached via a hexanediol linker as shown in

[0024] Figure 9A and 9B compared the immunogenicity of oligonucleotides with and without 3'-cholesterol modification. More specifically, Figure 9AIllustrated the immunogenicity of 2006-PTO, 2006-3dT4G5T4, and 2006-3dT4G5T4C oligonucleotides in Ramos-Blue cells via TLR9-mediated immune responses, and Figure 9B illustrated the results in a narrower concentration range of Figure 9A as described in

[0025] Figure 10A and 10B compared the ability of oligonucleotides with or without a 3'-cholesterol moiety attached via a hexanediol linker to elicit TLR9-mediated immune responses in Ramos-Blue cells. More specifically, Figure 10A illustrated the relative ability of oligonucleotides 2006-3dT4G5T4 and 2006-3dT4G5T4C to elicit TLR9-mediated immune responses. Figure 10B illustrated the results in a narrower concentration range of Figure 10A as described in

[0026] Figure 11 compared the immunogenicity in Ramos-Blue of oligonucleotide 2006-T4-PDE and oligonucleotide 2006-T4-PDE with a Figure 1 3'-cholesterol moiety attached via a hexanediol linker as shown (''3Chol-2006-T4-PD'').

[0027] Figure 12 compared the ability of oligonucleotides 2006-3dT4G5T4 and oligonucleotide 2006-3dT4G5T4-3Chol to elicit TLR9-mediated immune responses in Ramos-Blue cells.

[0028] Figure 13A 、 13B and 13C compared the ability of 2007-PDE-T4, 2007-T4G5T4, and TCG8-T4 oligonucleotides with or without a cholesterol moiety attached to the 3'-end of the oligonucleotide to stimulate TLR9 in Ramos-Blue cells. Figure 13A Illustrated the different immunogenicity of oligonucleotides 2007-PDE-T4 and 2007-PDE-T4-Ch3. Figure 13B Illustrated the different immunogenicity of oligonucleotides 2007-T4G5T4 and 2007-T4G5T4-3Ch. Figure 13C Illustrated the different immunogenicity of oligonucleotides TCG8-T4 and TCG8-T4-Ch3.

[0029] Figure 14A 、 14B, 14C and 14D compared the stimulatory activities of several oligonucleotides and cholesterol-modified oligonucleotides on murine TLR9 (“mTLR9”) in HEKBlue-mTLR9 cells. Figure 14A The ability of the unmodified 2007-PDE-T4 oligonucleotide and the oligonucleotide 2007-PDE-T4-3Ch to elicit TLR9-mediated responses in HEKBlue-mTLR9 cells was compared. Figure 14B The ability of the unmodified 2007-T4G5T4 oligonucleotide and the oligonucleotide 2007-T4G5T4-3Ch to elicit TLR9-mediated responses in HEKBlue-mTLR9 cells was compared. Figure 14C The ability of the oligonucleotide TCG8-T4 and the oligonucleotide TCG8-T4-3Ch to elicit TLR9-mediated responses in HEKBlue-mTLR9 cells was compared; and Figure 14D The TCG8-T4G5T4 oligonucleotide and the oligonucleotide with Figure 1 a 3'-cholesterol moiety attached via a hexanediol linker as shown (“TCG8-T4G5T4-3Ch”) were compared for their ability to elicit TLR9-mediated responses in HEKBlue-mTLR9 cells.

[0030] Figure 15 The chemical structure of the cholesterol moiety attached to the hexaethylene glycol linker was described.

[0031] Figure 16A , 16B , 16C and 16D described the effect of modifying the 3' or 5' end of an oligonucleotide with a cholesterol moiety on the ability of the oligonucleotide to elicit TLR9-mediated immune responses. Figure 16A Illustratively described was the ability of the oligonucleotides 2006-PDE-T4, 2006-PDE-T4-Chol, and the oligonucleotide 2006-PDE-T4 with a 5'-cholesterol moiety attached via a hexaethylene glycol linker as shown in Figure 15 (“2006-PDE-T4-5Chol”) to stimulate TLR9 in HEKBlue-hTLR9 cells. Figure 16B Illustratively described were the oligonucleotides 2006-PTO, the oligonucleotide 2006-PDE with a GGGGG 3'-terminal sequence (“2006-G5”), the oligonucleotide 2006-G5 with a 3'-cholesterol moiety attached via a hexaethylene glycol linker as shown in Figure 6 (“2006-G5-3Chol”), and the oligonucleotide with Figure 15The ability of the oligonucleotide 2006-G5 with a 5'-cholesterol moiety attached via a hexaethylene glycol linker (“2006-G5-5Chol”) shown in [Figure] to stimulate TLR9 in HEKBlue-hTLR9 cells. Figure 16C Illustrated are the oligonucleotides 2006-PTO, 2006-T4G5T4, 2006-T4G5T4-3Chol, and having Figure 15 The ability of the oligonucleotide 2006-T4G5T4 with a 5'-cholesterol moiety attached via a hexaethylene glycol linker (“2006-T4G5T4-5Chol”) shown in [Figure] to stimulate a TLR9-mediated immune response in HEKBlue-hTLR9 cells. Figure 16D Illustrated are the oligonucleotides TCG8-T4G5T4, having Figure 6 The ability of the oligonucleotide TCG8-T4G5T4 with a 3'-cholesterol moiety attached via a hexaethylene glycol linker (“TCG8-T4G5T4-3Chol”) and the oligonucleotide TCG8-T4G5T4 with a 5'-cholesterol moiety attached via a hexaethylene glycol linker (“TCG8-T4G5T4-5Chol”) shown in [Figure] to stimulate a TLR9-mediated immune response in HEKBlue-hTLR9 cells. Figure 15 The ability of the oligonucleotide TCG8-T4G5T4 with a 5'-cholesterol moiety attached via a hexaethylene glycol linker (“TCG8-T4G5T4-5Chol”) shown in [Figure] to stimulate a TLR9-mediated immune response in HEKBlue-hTLR9 cells.

[0032] Figure 17A , 17B , 17C and 17D describe the effect of modifying the 3' or 5' end of an oligonucleotide with a cholesterol moiety. Figure 17A Illustrated is the ability of the oligonucleotides 2006-PDE-T4, 2006-PDE-T4-Chol, and 2006-PDE-T4-5Chol to stimulate TLR9 in Ramos-Blue cells. Figure 17B Illustrated is the ability of the oligonucleotides 2006-PTO, 2006-G5, 2006-G5-3Chol, and 2006-G5-5Chol to stimulate TLR9 in Ramos-Blue cells. Figure 17C Illustrated is the ability of the oligonucleotides 2006-PTO, 2006-T4G5T4, 2006-T4G5T4-3Chol, and the oligonucleotide 2006-T4G5T4-5Chol to stimulate a TLR9-mediated immune response in Ramos-Blue cells. Figure 17D Illustrated is the ability of the oligonucleotides TCG8-T4G5T4, TCG8-T4G5T4-3Chol, and TCG8-T4G5T4-5Chol to stimulate a TLR9-mediated immune response in Ramos-Blue cells.

[0033] Exemplary Embodiments Detailed

[0034] The disclosed compositions and methods can be more readily understood by reference to the detailed description, which is to be understood in conjunction with the accompanying drawings, which form a part of the present disclosure. It is to be understood that the disclosed compositions and methods are not limited to the specific compositions and methods described and / or illustrated herein, and the terms used herein are for the purpose of describing particular embodiments by way of example only and are not intended to limit the claimed compositions and methods.

[0035] Unless otherwise specifically noted, any description of possible mechanisms of action or modes or reasons for improvement is meant to be illustrative only, and the disclosed compositions and methods are not bound by the correctness or incorrectness of any such proposed mechanism of action or mode or reason for improvement.

[0036] Throughout this document, the description relates to compositions and methods of using such compositions. Where the disclosure describes or claims a feature or embodiment related to a composition, such feature or embodiment equally applies to the method of using such composition. Similarly, where the disclosure describes or claims a feature or embodiment related to a method of using a composition, such feature or embodiment equally applies to such composition.

[0037] When expressing a range of values, another embodiment includes from one specific value and / or to another specific value. Further, reference to a value stated in a range includes each value within that range. All ranges are inclusive and combinable. When a value is expressed as an approximation by use of the antecedent "about", it will be understood that the specific value forms another embodiment. Unless the context clearly indicates otherwise, reference to a specific numerical value includes at least that specific value.

[0038] It is to be understood that, for clarity, certain features of the disclosed compositions and methods described herein in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, the various features of the disclosed compositions and methods described in the context of a single embodiment may also be provided separately or in any sub-combination.

[0039] As used herein, the singular forms "a", "an" and "the" include the plural.

[0040] As used herein, "CpG motif" refers to a cytosine-guanine dinucleotide sequence. The immunostimulatory nucleic acids described herein contain one or more CpG motifs that, when unmethylated, can interact with toll-like receptor proteins (TLRs) and elicit an immune response.

[0041] As used herein, the term "subject" is used to denote any animal, particularly a mammal, and any type of avian, mammalian, or aquatic species can be treated using the disclosed methods.

[0042] Throughout the specification and claims, various terms are used that refer to aspects of the specification. Unless otherwise indicated, such terms will be given their ordinary meaning in the art. Other specifically defined terms will be interpreted in a manner consistent with the definitions provided herein.

[0043] Disclosed herein are immunostimulatory oligonucleotides comprising at least one CpG motif and a 3'-terminal cholesterol moiety. It has previously been shown that CpG motifs in oligodeoxynucleotides (ODNs) can elicit an immune response in mammals. In some cases, CpG motifs are recognized by toll-like receptors (TLRs). Examples of CpG-recognizing TLRs include, but are not limited to, mammalian homologs of TLR9. Thus, in some aspects of the present disclosure, the CpG-recognizing TLR is a mouse, human, bovine, porcine, equine, or ovine TLR9 homolog. The immunogenicity of ODNs may not be sufficient to elicit an immune response capable of preventing infection in a susceptible population or affected individual. As demonstrated herein, the immunostimulatory properties of ODNs can be enhanced by modifying the oligonucleotide, particularly by adding a thymine run, guanine run, and / or cholesterol moiety at the 3'-end of the ODN.

[0044] The immunostimulatory oligonucleotides of the present disclosure comprise at least one CpG motif. In some embodiments, the immunostimulatory oligonucleotide comprises from 1 to 10 CpG motifs. In other embodiments, the immunostimulatory oligonucleotide may comprise even 20 CpG motifs. Thus, in some embodiments, the immunostimulatory oligonucleotides of the present disclosure comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 CpG motifs. In other embodiments, the immunostimulatory oligonucleotide comprises from 11 to 15 CpG motifs or even from 15 to 20 CpG motifs.

[0045] Oligonucleotides comprising phosphodiester and / or phosphorothioate bonds between the nucleotides are contemplated herein. In some aspects, the oligonucleotides of the present disclosure comprise phosphodiester bonds between the nucleotides of the oligonucleotide. In other aspects, the oligonucleotide comprises phosphorothioate bonds between the nucleotides of the oligonucleotide. Other bonds are also contemplated herein. For example, the oligonucleotides of the present disclosure may comprise other bonds, including but not limited to, phosphoacetate, methylphosphonate, and phosphonocarboxylate bonds. Some bonds may offer desired advantages over other bonds, such as production cost, ease of production, and / or quality, as well as enhanced immunostimulatory effects.

[0046] In some aspects of the present disclosure, the immunogenicity of oligonucleotides attributable to CpG motifs can be further enhanced by non-CpG sequences. As shown in the examples, adding a thymine extension at the 3' end of the oligonucleotide can improve the ability of the oligonucleotide to elicit a TLR9-mediated immune response. To this end, in some embodiments of the present disclosure, the 3' end sequence of the immunostimulatory oligonucleotide contains a number of thymine nucleotides as the 3' end sequence. In some aspects, the number of thymine nucleotides contains consecutive thymine nucleotides. In some aspects, the number of thymine nucleotides contains four to six consecutive thymine nucleotides. For example, in some embodiments of the present disclosure, the 3' end sequence contains SEQ ID NO: 9. In some embodiments, the oligonucleotide contains SEQ ID NO: 2, 3, 4, 5, 6, or 8. And in some aspects, the 3' end sequence of the oligonucleotide sequence is TTTT.

[0047] Other sequence modifications at the 3' end of the immunostimulatory oligonucleotide may also contribute to enhancing immunogenicity. For example, in some embodiments of the present disclosure, the immunostimulatory oligonucleotide contains a number of guanine nucleotides at or near the 3' end sequence. In some aspects, the 3' end sequence of the immunostimulatory oligonucleotide contains a number of guanine nucleotides. In some aspects, the number of guanine nucleotides contains consecutive guanine nucleotides, such as an oligonucleotide having a 3' end sequence of GGGGG according to the present disclosure. In some aspects, the oligonucleotide contains SEQ ID NO: 7.

[0048] Increasing the immunogenicity of immunostimulatory oligonucleotides is not limited to modifications at the 3' end of the oligonucleotide. Internal sequences can also be modified, for example, to increase the number of CpG motifs. In some aspects, oligonucleotides can be synthesized that contain additional CpG motifs between the 5' and 3' ends of the oligonucleotide. In some aspects, the immunostimulatory oligonucleotide contains the sequence (TCG) n , where n is between 3 and 10. Thus, in some aspects of the present disclosure, the oligonucleotide contains the sequence (TCG) n , where n is 3, 4, 5, 6, 7, 8, 9, or 10.

[0049] In some embodiments of the present disclosure, an immunostimulatory oligonucleotide may include a lipid moiety at the 3'-end to enhance the immunogenic properties of the oligonucleotide. Thus, in some embodiments, a cholesterol moiety is covalently attached via a linker to the 3'-terminal nucleotide of the immunostimulatory oligonucleotide. The cholesterol moiety may increase the immunogenicity of the oligonucleotide by preventing degradation, increasing solubility, creating ligand multivalency by forming higher-order structures (e.g., micelles), increasing the stability of the oligonucleotide in a pharmaceutical composition, or any combination thereof. A linker having at least two moieties capable of forming covalent bonds may bond to the cholesterol moiety and the oligonucleotide. For example, in some embodiments, the linker interacts with the hydroxyl group of the cholesterol moiety to form a covalent bond and interacts with the 3'-terminal nucleotide of the oligonucleotide. In some aspects, the cholesterol moiety is covalently bound to the linker to form a cholesterol-linker moiety. In some aspects, the linker is first attached to the cholesterol moiety, and then the resulting cholesterol-linker is attached to the oligonucleotide. In other aspects, the linker is first attached to the oligonucleotide, and then attached to the cholesterol moiety. In some aspects, the cholesterol-linker is commercially available.

[0050] In addition to having moieties that can bind to the oligonucleotide and the cholesterol moiety, some embodiments of the linker also include a carbon chain, and in some aspects, the carbon chain includes 3 to 12 carbon atoms. For example, a diol can be used as the linker between the cholesterol moiety and the oligonucleotide because the terminal hydroxyl groups can covalently bond to the hydroxyl groups of the oligonucleotide and the cholesterol moiety. In some aspects, the linker includes hexanediol. In some aspects, the cholesterol-linker moiety has the Figure 1 chemical structure described therein. Other embodiments provide linkers that include repeating chemical units. In some aspects, the chemical unit repeats two to twelve times. In some aspects, the repeating chemical unit includes ethylene glycol, and when the ethylene glycol chemical unit repeats six times, the linker includes hexaglycol. A linker that includes hexaglycol can have the Figure 6 chemical structure described therein.

[0051] In some cases, it will be desirable to deliver the oligonucleotides described herein to a subject in need thereof. The oligonucleotides can be delivered as an immunostimulatory composition. Immunostimulatory compositions comprising any of the oligonucleotides disclosed herein are provided. In some aspects, these immunostimulatory compositions comprise an oligonucleotide and other components that affect the immunogenicity, efficacy, and efficiency of the composition. In some embodiments of the present disclosure, the immunostimulatory composition may further comprise a vaccine for the prevention or treatment of an infectious disease, a vector for delivering the oligonucleotide to the subject, a pharmaceutical carrier, or any combination thereof. For example, in some aspects, the oligonucleotide is packaged in a viral vector, which allows for targeted delivery of the oligonucleotide. In some aspects, the oligonucleotide can be added to a cationic liposome delivery vehicle to enhance the ability of the oligonucleotide to cross lipid cell membranes and / or the membranes of organelles containing TLR9.

[0052] Infectious diseases that can be treated or prevented by administering the immunostimulatory oligonucleotides or immunostimulatory compositions described herein include, but are not limited to, viral, bacterial, fungal, worm, or other parasite infections. It is contemplated that administration of the immunostimulatory oligonucleotides or compositions of the present disclosure results in the generation of an immune response that is detrimental to the environment of the invading pathogen. Thus, the invading pathogen may not be able to establish an infection that results in a negatively altered health condition in the host organism. Administration of the immunostimulatory oligonucleotides and / or compositions can provide a non-antigen-specific immune response that enhances or acts in parallel with the antigen-specific immune response against the invading pathogen.

[0053] In some aspects, the immunostimulatory composition comprising an oligonucleotide can further comprise a vaccine for the prevention or treatment of an infectious disease. For reasons of efficiency, a combination of an oligonucleotide and a vaccine can be made because separately delivering multiple drugs increases the cost of treatment. The oligonucleotide and the vaccine can also be delivered as a single immunostimulatory composition to elicit a non-antigen-specific immune response against any current infection and to initiate the development of an antigen-specific immune response.

[0054] The present invention also contemplates immunostimulatory compositions comprising the oligonucleotides described herein and a pharmaceutically acceptable carrier. In some aspects, the pharmaceutically acceptable carrier is any pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier renders the composition suitable for administration by a route selected from: intravenous, intramuscular, intramammary, intradermal, intraperitoneal, subcutaneous, by spray, by aerosol, in ovo, mucosal, transdermal, by immersion, oral, intraocular, intratracheal, intranasal, pulmonary, rectal, or other means known to those skilled in the art. One or more pharmaceutically acceptable carriers may be a diluent, adjuvant, excipient, or vehicle administered with the immunostimulatory composition. Such carriers may be 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. For example, 0.4% saline and 0.3% glycine may be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional well-known sterilization techniques, such as filtration. The composition may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, stabilizers, thickening agents, lubricants, and coloring agents, among others. The concentration of the molecules of the present invention in such pharmaceutical formulations may vary widely, i.e., from less than about 0.5% by weight, usually to at least about 1% to as high as 15 or 20%, and will be selected primarily based on the required dose, fluid volume, viscosity, etc., according to the particular mode of administration chosen. Suitable carriers and formulations, including other human proteins such as human serum albumin, are described, for example, in Remington: The Science and Practice of Pharmacy, 21st Edition, Troy, D.B., ed., Lippincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing pp. 691-1092 (see particularly pp. 958-989).

[0055] In some embodiments, the oligonucleotide and the carrier are coupled, such as chemically coupled. As used to describe the relationship between the oligonucleotide and the carrier, "coupled" refers to the physical association of the oligonucleotide and the carrier. The oligonucleotide and the carrier may be considered to be coupled when they bind to each other, interact with each other, or combine, or otherwise associate with each other.

[0056] In some embodiments, the immunostimulatory composition described herein further comprises a hapten. In some aspects, the immunostimulatory oligonucleotide is linked to the hapten. The hapten may elicit an immune response against a specific microorganism (e.g., Escherichia coli ( E. coli ), or Salmonella spp. ( Salmonella), while immunostimulatory oligonucleotides elicit a non-specific immune response mediated by the interaction of TLR9 with the oligonucleotide. These and other infectious microorganisms are of particular importance to large agricultural producers such as cattle, sheep, and swine producers.

[0057] Methods for enhancing the immunogenicity of TLR9 ligands are also provided, including attaching a cholesterol moiety to the ligand, where the ligand is an immunostimulatory oligonucleotide having at least one CpG motif, and where the cholesterol moiety is attached via a linker to the 3'-terminal nucleotide of the oligonucleotide.

[0058] Other methods disclosed herein provide for eliciting a TLR9-mediated immune response in a subject in need thereof, including administering to the subject an oligonucleotide having a plurality of CpG motifs and a cholesterol-linker moiety attached to the 3'-terminal nucleotide of the oligonucleotide. In some aspects of the methods for eliciting a TLR9-mediated immune response, the oligonucleotide is administered as an immunostimulatory composition.

[0059] In some embodiments of the present disclosure, the subject to which the immunostimulatory oligonucleotide or immunostimulatory composition is administered is an animal. In some aspects, the animal is at an elevated risk of being infected by a pathogen and especially a pathogen having a CpG-based pathogen-associated molecular pattern (PAMP). When the immunostimulatory oligonucleotide and / or immunostimulatory composition is administered to such an animal, the TLR9-mediated immune response will help prevent pathogen infection or alleviate the symptoms caused by the pathogen. Those skilled in the art will understand that the immunostimulatory oligonucleotides of the present invention need not be specific for a particular pathogen, but rather stimulate a non-antigen-specific immune response. The oligonucleotides also need not be specific for a particular animal. Thus, in some aspects of the present disclosure, the subject is a mammal. In some aspects, the subject is a herd or farm animal such as a pig, cow, horse, or sheep. Administration to herd animals may help prevent the spread of infection to a large animal population under crowded conditions such as pens and / or sharing a common feed or water source. The oligonucleotides of the present disclosure offer distinct advantages over traditional forms of infection prevention treatment, as the use of antibiotics is becoming more unpopular, especially in the presence of bacterial resistance to antibiotic treatment.

[0060] In some embodiments, the subject can be a human. As with herd animals, resistance to antibiotics used by humans is becoming common in bacteria, and treatment options for resistant infections are limited. The oligonucleotides and methods of the present disclosure provide a much-needed solution for so-called "superbugs" such as methicillin-resistant Staphylococcus aureus.

[0061] The subject to which the immunostimulatory oligonucleotide or composition is administered is also expected to be a mouse, rat, hamster, gerbil, or other rodent in this article. The subject can also be non-mammalian. For example, in some aspects, the subject is an aquatic species. Examples

[0062] The following examples are provided to further describe some of the embodiments disclosed herein. The examples are illustrative and not limiting of the disclosed embodiments.

[0063] Example 1: 3'-cholesteryl modification of ODNs results in greatly increased TLR9-stimulating activity

[0064] Human TLR9, recombinant overexpression in HEKBlue

[0065] 3'-cholesteryl modification of PDE-ODNs (I)

[0066] The 3'-cholesteryl modification (for the chemical structure of the cholesteryl-linker moiety, see Figure 1 ) was applied to PDE-ODN (Table 1, 2006-3dT4G5T4) with moderate activity against human TLR9. In vitro, the modified and unmodified forms were tested in the HEKBlue-hTLR9 cell line (Invivogen) expressing human TLR9.

[0067] Table 1: ODN sequences (lowercase font: PTO key)

[0068]

[0069] Table 2: Half-maximal effective concentration (EC 50 ) and maximum signal velocity (V max )

[0070]

[0071] The results suggest that, in terms of EC 50 , the TLR9-stimulating activity of 2006-3dT4G5T4 was significantly improved upon 3'-cholesteryl modification, which was almost 1 / 30 for 2006-3dT4G5T43C (Table 2, Figure 2A , 2B).

[0072] 3'-cholesteryl modification of PDE-ODNs (II)

[0073] The cholesteryl moiety (for the chemical structure of the cholesteryl-linker moiety, see Figure 1)The 3'-terminal nucleotides of 2006-T4-PDE (SEQ ID NO: 3, Table 3) that are attached to the weakly activating ligand known as human TLR9. Modified and unmodified 2006-T4-PDE oligonucleotides were administered in vitro to HEKBlue-hTLR9 cells to determine the immunostimulatory effect of the 3'-cholesteryl modification.

[0074] Table 3: ODN Sequences

[0075]

[0076] The results suggest that the human TLR9-stimulating activity of 2006-T4-PDE is significantly improved upon 3'-cholesteryl modification (Table 2, Figure 3 ).

[0077] 3'-Cholesteryl Modification of PDE-ODNs (III)

[0078] The 3'-cholesteryl modification (for the chemical structure of the cholesteryl-linker moiety, see Figure 1 ) was applied to the PDE-ODN 2006-3dT4G5T4, which has moderate activity against human TLR9 (Table 4). In vitro, the modified and unmodified forms were tested in HEKBlue-hTLR9 cells.

[0079] Table 4: ODN Sequences

[0080]

[0081] Table 5: Half-Maximal Effective Concentration (EC 50 ) and Maximum Signal Velocity (V max )

[0082]

[0083] The results suggest that, with respect to EC 50 , the TLR9-stimulating activity of 2006-3dT4G5T4 is significantly improved upon 3'-cholesteryl modification, which is up to 1 / 3 for 2006-3dT4G5T4 3C (Table 5, Figure 4A , 4B).

[0084] 3'-Cholesteryl Modification of PDE-ODNs (IV)

[0085] The 3'-cholesteryl modification (for the chemical structure of the cholesteryl-linker moiety, see Figure 1 ) was applied to the PDE-ODN 2006-3dT4G5T4, which has moderate activity against human TLR9 (Table 8). In vitro, the modified and unmodified forms were tested in HEKBlue-hTLR9 cells.

[0086] Table 8: ODN Sequences

[0087]

[0088] Table 9: Half Maximal Effective Concentration (EC 50 ) and Maximum Signal Velocity (V max )

[0089]

[0090] The results suggest that, with respect to EC 50 , the TLR9-stimulatory activity of 2006-3dT4G5T4 was significantly improved upon 3'-cholesteryl modification, being up to 1 / 36 that of 2006-3dT4G5T43C (Table 9, Figure 5 ).

[0091] 3'-Cholesteryl Modification of PDE-ODNs (V)

[0092] The 3'-cholesteryl modification (for the chemical structure of the cholesteryl-linker moiety, see Figure 6 ) was applied to the PDE-ODN, 2006-3dT4G5T4, which has very weak activity against human TLR9 (Table 10). In vitro, the modified and unmodified forms were tested in HEKBlue-hTLR9 cells.

[0093] Table 10: ODN Sequences (Lowercase Font: PTO Keys)

[0094]

[0095] Table 11: Half Maximal Effective Concentration (EC 50 ) and Maximum Signal Velocity (V max )

[0096]

[0097] The results suggest that upon 3'-cholesteryl modification, the TLR9-stimulatory activity of 2006-3dT4G5T4 was improved substantially, from virtually zero to an EC 50 of 68 nM (Table 11, Figure 7 ).

[0098] 3'-Cholesteryl Modification of PDE-ODNs (VI)

[0099] The 3'-cholesteryl modification (for the chemical structure of the cholesteryl-linker moiety, see Figure 1)(i) Applied to PDE-ODNs that have very weak or no activity against human TLR9, 2007-PDE-T4 or 2007-PDE-T4G5T4 (Table 12). In vitro, the modified and unmodified forms were tested in HEKBlue-hTLR9 cells.

[0100] Table 12: ODN sequences

[0101]

[0102] Table 13: Calculation of 50% effective concentration (EC 50 -) and maximum signal velocity (V max )

[0103]

[0104] The results suggest that the TLR9-stimulatory activity of both 2007-PDE-T4 and 2007-PDE-T4G5T4 is massively improved upon 3'-cholesteryl modification ( Figure 8A and 8B ), to an EC 50 of 24.9 nM in the case of 2007-T4G5T4-3Ch (Table 13).

[0105] Example 2: Natural expression of human TLR9 in Ramos-Blue cells

[0106] 3'-cholesteryl modification (I)

[0107] The 3'-cholesteryl modification (for the chemical structure of the cholesteryl-linker moiety, see Figure 1 ) was applied to the PDE-ODN 2006-3dT4G5T4, which has moderate activity against human TLR9 (Table 14). In vitro, the modified and unmodified forms were tested in Ramos-Blue cells. The Ramos-Blue B lymphocyte cell line ((Invivogen, San Diego, CA) stably expresses an NF-κB / AP-1-inducible reporter gene, which allows detection of TLR9 signal transduction.

[0108] Table 14: ODN sequences (lowercase font: PTO key)

[0109]

[0110] Table 15: Half-maximal effective concentration (EC 50 ) and maximum signal velocity (V max )

[0111]

[0112] The results suggest that, with respect to EC 50 for 3'-cholesteryl modification, the TLR9-stimulatory activity of 2006-3dT4G5T4 was significantly improved, being at most 1 / 8 for 2006-3dT4G5T43C (Table 15, Figure 9A , 9B). Similarly, the modified ODN 2006-3dT4G5T43C exceeded the activity of the "industry standard" ODN 2006-PTO.

[0113] 3'-Cholesteryl modification (II)

[0114] 3'-Cholesteryl modification (see for the chemical structure of the cholesteryl-linker group Figure 1 ) was applied to the PDE-ODN 2006-3dT4G5T4, which has moderate activity against human TLR9 (Table 16). In vitro, the modified and unmodified forms were tested in Ramos-Blue cells.

[0115] Table 16: ODN sequences

[0116]

[0117] Table 17: Half-maximal effective concentration (EC 50 ) and maximum signal velocity (V max )

[0118]

[0119] The results suggest that, with respect to EC 50 for 3'-cholesteryl modification, the TLR9-stimulatory activity of 2006-3dT4G5T4 was significantly improved, being at most 1 / 7 for 2006-3dT4G5T43C (Table 17, Figure 10A , 10B).

[0120] 3'-Cholesteryl modification of PDE-ODNs (III)

[0121] 3'-Cholesteryl modification (see for the chemical structure of the cholesteryl-linker moiety Figure 1 ) was applied to the PDE-ODN 2006-T4-PDE, which has only weak activity against human TLR9 (Table 18). In vitro, the modified and unmodified forms were tested in Ramos-Blue cells.

[0122] Table 18: ODN sequences

[0123]

[0124] The results suggest that, upon 3'-cholesteryl modification, the human TLR9-stimulating activity of 2006-T4-PDE was significantly improved (Table 18, Figure 11 ).

[0125] 3'-Cholesteryl modification of PDE-ODNs (IV)

[0126] The 3'-cholesteryl modification (for the chemical structure of the cholesteryl-linker moiety, see Figure 6 ) was applied to PDE-ODN 2006-3dT4G5T4, which has very weak activity against human TLR9 (Table 19). In vitro, the modified and unmodified forms were tested in Ramos-Blue cells.

[0127] Table 19: ODN sequences (lowercase font: PTO key)

[0128]

[0129] Table 20: Half-maximal effective concentration (EC 50 ) and maximal signal velocity (V max )

[0130]

[0131] The results suggest that, with respect to EC 50 , upon 3'-cholesteryl modification, the human TLR9-stimulating activity of 2006-3dT4G5T4 against Ramos-Blue cells was significantly improved to 13-fold (Table 20, Figure 12 ).

[0132] 3'-Cholesteryl modification of PDE-ODNs (V)

[0133] The 3'-cholesteryl modification (for the chemical structure of the cholesteryl-linker moiety, see Figure 1 ) was applied to PDE-ODNs 2007-PDE-T4 or 2007-PDE-T4G5T4, which have very weak or no activity against human TLR9 (Table 21). In vitro, the modified and unmodified forms were tested in Ramos-Blue cells.

[0134] Table 21: ODN sequences

[0135]

[0136] Table 22: Half-maximal effective concentration (EC 50 ) and maximal signal velocity (V max )

[0137]

[0138] Results suggest that when considering the 3'-cholesteryl modification of all ODNs in this experiment, the human TLR9-stimulatory activity of 2006-3dT4G5T4 in Ramos-Blue was significantly improved (2007-PDE-T4, 2007-T4G5T4, TCG8-T4, Table 22, Figure 13A , 13B and 13C). In the case of 2007-T4G5T4-Ch, a nearly 14-fold increase in activity was noted compared to its unmodified congener with respect to EC 50 .

[0139] Example 3: Recombinant overexpression of murine TLR9 in HEKBlue

[0140] 3'-Cholesteryl modification of PDE-ODNs

[0141] The 3'-cholesteryl modification (see for the chemical structure of the cholesteryl-linker moiety Figure 1 ) was applied to 2007-PDE-T4, 2007-PDE-T4G5T4, and TCG8-T4 which have very weak or no activity against human TLR9 (Table 23). In vitro, the modified and unmodified forms were tested in HEKBlue-mTLR9 cells (Invivogen).

[0142] Table 23: ODN sequences

[0143]

[0144] Results suggest that in all 3 cases upon 3'-cholesteryl modification, the murine TLR9-stimulatory activity of all ODNs considered in this experiment (2007-PDE-T4, 2007-T4G5T4, TCG8-T4, Table 23) in HEKBlue-mTLR9 was significantly improved ( Figure 14A , 14B and 14C), and in a fourth instance to some extent at low concentrations ( Figure 14D ).

[0145] Example 4: Systematic study of unmodified, 3'-cholesteryl-modified, and 5'-cholesteryl-modified ODNs on HEKBlue-hTLR9 and Ramos-Blue cells: Structure-activity relationship (SAR)

[0146] HEKBlue-hTLR9

[0147] The 3'-cholesteryl or 5'-cholesteryl modification (see Figure 6 and 15 respectively for the chemical structure of the cholesteryl-linker moiety) was applied to 4 different ODNs (Table 24). In vitro, the modified and unmodified forms were tested in HEKBlue-hTLR9 cells.

[0148] Table 24: ODN sequences (lowercase font indicates PTO linkages)

[0149]

[0150] Table 25: Half-maximal effective concentration (EC 50 ) and maximal signal velocity (V max )

[0151]

[0152] In this experiment, due to the relatively high background readings, the zero ODN value was subtracted from each data point for EC50 and Vmax calculations.

[0153] For each ODN studied in this experiment, the 3'-cholesteryl modification was most favorable for the activity of human TLR9 expressed in HEKblue cells (Table 24, Figure 16A , 16B, 16C, 16D). In those cases where EC 50 / V max calculations were possible (2006-G5, 2006-T4G5T4, Table 25), the EC 50 of the unmodified ODNs was found to be lower (by factors of 2.5 and 48 respectively), while the 5'-cholesteryl modification led to loss of activity. The EC 50 of 2006-G5-3Chol and 2006-T4G5T4-3Chol was lower than those of the "industry standard" 2006-PTO, thus making them candidates for immunomodulatory intervention.

[0154] Ramos-Blue

[0155] The 3'-cholesteryl or 5'-cholesteryl modification (see Figure 6 and 15 respectively for the chemical structure of the cholesteryl-linker moiety) was applied to 4 different ODNs (Table 26). In vitro, the modified and unmodified forms were tested in Ramos-Blue cells.

[0156] Table 26: ODN sequences (lowercase font: PTO linkages)

[0157] ​​​​​​​​​​​​​​​​​​​​​​

[0158] Table 27: Half-maximal concentration (EC 50 50 max max

[0159]

[0160] In this experiment, to be consistent with Table 25), for EC50 and Vmax calculations, the zero ODN value was subtracted from each data point.

[0161] For each ODN studied in this experiment, the 3'-cholesteryl modification was most favorable for the activity of human TLR9 endogenously present in Ramos-Blue cells (Table 26, Figure 17A - 17D ). In those cases where EC 50 50 max / V 50 max Figure 17D calculations were possible (2006-G5, 2006-T4G5T4, Table 25), it was found that the EC 50 50 of the unmodified ODNs was lower (by a factor of 3 and 10, respectively), while 5'-cholesteryl modification led to loss of activity except for TCG8-T4G5T4, and in the case of TCG8-T4G5T4 both derivatizations led to increased activity, but more so for 3'-cholesteryl than for 5'-cholesteryl (

[0162] The EC 50 50 of 2006-G5-3Chol and 2006-T4G5T4-3Chol was lower than those of the "industry standard" 2006-PTO, making them candidates for immunomodulatory intervention.

[0162] Those skilled in the art will understand that many changes and modifications can be made to the preferred embodiments of the present invention, and such changes and modifications can be made without departing from the spirit of the present invention. Accordingly, it is intended that the appended claims cover all such equivalent variations that fall within the true spirit and scope of the present invention.

[0163] The disclosure of each patent, patent application, and publication cited or described in this document is hereby incorporated by reference in its entirety.

Claims

1. An immunostimulatory oligonucleotide comprising at least one CpG motif and a 3'-cholesterol moiety, wherein the cholesterol moiety is covalently attached via a linker to the 3'-terminal nucleotide of the immunostimulatory oligonucleotide, wherein the linker comprises a cholesterol-linker moiety having the following structure: wherein the immunostimulatory oligonucleotide comprises a 3'-terminal sequence that comprises a number of thymine nucleotides, and wherein the 3'-terminal sequence comprises SEQ ID NO:

9.

2. The immunostimulatory oligonucleotide of claim 1, wherein the immunostimulatory oligonucleotide comprises phosphodiester bonds between the nucleotides of the immunostimulatory oligonucleotide.

3. The immunostimulatory oligonucleotide of claim 1, wherein the immunostimulatory oligonucleotide comprises phosphorothioate bonds between the nucleotides of the immunostimulatory oligonucleotide.

4. The immunostimulatory oligonucleotide of claim 1, wherein the number of thymine nucleotides comprises consecutive thymine nucleotides.

5. The immunostimulatory oligonucleotide of claim 1, wherein the number of thymine nucleotides comprises 4-6 consecutive thymine nucleotides.

6. The immunostimulatory oligonucleotide of claim 1, wherein the immunostimulatory oligonucleotide comprises SEQ ID NO: 2, 3, 4, 5, 6 or 8.

7. The immunostimulatory oligonucleotide of claim 1, wherein the 3'-terminal sequence is TTTT.

8. The immunostimulatory oligonucleotide of claim 1, wherein the 3'-terminal sequence of the immunostimulatory oligonucleotide comprises a number of guanine nucleotides.

9. The immunostimulatory oligonucleotide of claim 8, wherein the number of guanine nucleotides comprises consecutive guanine nucleotides.

10. The immunostimulatory oligonucleotide of claim 8 or 9, wherein the 3'-terminal sequence is GGGGG.

11. The immunostimulatory oligonucleotide of any one of claims 8-9, wherein the immunostimulatory oligonucleotide comprises SEQ ID NO:

7.

12. The immunostimulatory oligonucleotide of any one of claims 1-5, wherein the immunostimulatory oligonucleotide comprises (TCG) n , where n is between 3 and 10.

13. An immunostimulatory composition comprising the immunostimulatory oligonucleotide of any one of claims 1-12.

14. The immunostimulatory composition of claim 13, which further comprises a vaccine for preventing or treating an infectious disease.

15. The immunostimulatory composition of claim 13 or 14, which further comprises a carrier.

16. The immunostimulatory composition of claim 15, wherein the carrier is a viral carrier.

17. The immunostimulatory composition of claim 16, wherein the oligonucleotide is packaged in the viral carrier.

18. The immunostimulatory composition of any one of claims 13-14 and 16-17, which further comprises a pharmaceutically acceptable carrier.

19. The immunostimulatory composition of claim 18, wherein the oligonucleotide and the pharmaceutically acceptable carrier are covalently coupled.

20. The immunostimulatory composition of any one of claims 13-17 and 19, which further comprises a hapten.

21. The immunostimulatory composition of claim 20, wherein the oligonucleotide and the hapten are covalently coupled.

22. A method for enhancing the immunogenicity of a TLR9 ligand, which comprises attaching a cholesterol moiety to the 3'-end of the TLR9 ligand via a linker, wherein the TLR9 ligand is an oligonucleotide having at least one CpG motif, wherein the cholesterol moiety is covalently bound to the linker to form a cholesterol-linker moiety comprising: wherein the immunostimulatory oligonucleotide comprises a 3'-end sequence that comprises a plurality of thymine nucleotides, and wherein the 3'-end sequence comprises SEQ ID NO: 9.

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