Immunostimulatory oligonucleotides

Abstract

Compositions and methods for stimulating Toll-like receptor 9 (TLR9) are provided. More particularly, immunostimulatory oligonucleotides, methods for enhancing the immunostimulatory properties of oligonucleotides, and methods for inducing an immune response are disclosed. [Selection diagram] None

Description

[Technical Field]

[0001] This application claims priority to and the benefit of European Patent Applications EP17207740.6, EP17207746.3, and EP17207750.5, each filed on December 15, 2017, the disclosures of which are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on November 27, 2018, is named 103976.000119SEQLISTING_ST25.txt and is 2,233 bytes in size.

[0003] Compositions and methods for stimulating Toll-like receptor 9 (TLR9) are provided. More particularly, immunostimulatory oligonucleotides, methods for enhancing the immunostimulatory properties of oligonucleotides, and methods for inducing an immune response are disclosed herein. [Background technology]

[0004] Antibiotic resistance is a global problem adversely affecting many industries. Methicillin-resistant Staphylococcus aureus (MRSA) and other "superbugs" wreak havoc in hospitals and doctor's offices, turning health center visits into fatalities. The agricultural industry faces similar challenges. Due to limited space and non-sterile environments, entire herds are at risk of pathogenic infection. For example, the presence of a single sick cow in close proximity to the rest of the herd can exponentially increase morbidity and mortality. Despite the risk of infection, antibiotic treatment is becoming more unfavorable due to increasing costs and consumer demand for antibiotic-free meat and dairy products. And those producers who use antibiotic therapy understand that even broad-spectrum antibiotics are not completely effective against all pathogens that may come into contact with the herd.

[0005] Thus, there is a need for non-antibiotic-based therapies for treating or preventing infections in animals. The disclosed compositions and methods address these and other important needs.

[0006] Summary of the Invention Disclosed herein are immunostimulatory oligonucleotides that contain at least one CpG motif and a 3' cholesteryl moiety.

[0007] Also provided herein are immunostimulatory compositions comprising immunostimulatory oligonucleotides.

[0008] Also disclosed is a method for enhancing the immunogenicity of a TLR9 ligand, comprising attaching a cholesteryl 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.

[0009] Also provided are methods for inducing a TLR9-mediated immune response in a subject comprising administering to the subject any one of the immunostimulatory oligonucleotides or immunostimulatory compositions described herein.

[0010] The summary and the following detailed description will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the disclosed compositions and methods, there are shown in the drawings exemplary embodiments of the compositions and methods. However, the compositions and methods are not limited to the particular embodiments disclosed. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows the chemical structure of a cholesteryl moiety attached to a hexanediol linker. [Figure 2]Figures 2A and 2B compare the TLR9-stimulating activity in HEKBlue-hTLR9 cells of oligonucleotide PTO-2006, oligonucleotide PDE-2006 (2006-3dT4G5T4) having the 3'TTTTGGGGGTTTT (SEQ ID NO: 9) sequence, and oligonucleotide 2006-3dT4G5T4 (2006-3dT4G5T43C) having a 3' cholesteryl moiety attached via a hexanediol linker as shown in Figure 1. [Figure 3] Figure 3 shows the ability of the oligonucleotide PDE-2006 with a 3'TTTT sequence (2006-T4-PDE) and the oligonucleotide 2006-T4-PDE with a cholesteryl moiety attached to the 3' end of the oligonucleotide via the hexanediol linker of Figure 1 (3Chol-2006-T4-PDE) to induce a TLR9-mediated immune response in HEKBlue-hTLR9 cells. [Figure 4] Figures 4A and 4B compare the TLR9 stimulatory properties of oligonucleotide 2006-3dT4G5T4 and oligonucleotide 2006-3dT4G5T4C. [Figure 5] FIG. 5 compares the TLR9 stimulatory properties of oligonucleotide 2006-3dT4G5T4 and oligonucleotide 2006-3dT4G5T4C. [Figure 6] FIG. 6 shows the chemical structure of a cholesteryl moiety attached to a hexaethylene glycol linker. [Figure 7] FIG. 7 compares the TLR9 stimulatory ability of oligonucleotide 2006-T4G5T4 with that of oligonucleotide 2006-T4G5T4 having the cholesteryl-linker moiety of FIG. 6 at its 3′ end (“2006-T4G5T4-3Chol” or “2006-T4G5T4-3C”). [Figure 8]Figures 8A and 8B compare the immunogenicity of oligonucleotides with and without a 3' cholesteryl modification. Figure 8A compares the ability of the 2007-PDE-T4 oligonucleotide to induce a TLR9-mediated immune response in HEKBlue-hTLR9 cells with the ability of the 2007-PDE-T4 oligonucleotide (2007-PDE-T4-3Ch), an oligonucleotide with a cholesteryl moiety attached via a hexanediol linker shown in Figure 1, and Figure 8B compares the ability of the 2007-PDE-T4 oligonucleotide (2007-T4G5T4) with a 3' GGGGGTTTT sequence and the 2007-T4G5T4 oligonucleotide (2007-T4G5T4-3Chol) with a cholesteryl moiety attached via a hexanediol linker shown in Figure 1, in HEKBlue-hTLR9 cells. [Figure 9] Figures 9A and 9B compare the immunogenicity of oligonucleotides with and without 3' cholesteryl modifications. More specifically, Figure 9A shows the immunogenicity of the 2006-PTO, 2006-3dT4G5T4, and 2006-3dT4G5T4C oligonucleotides via TLR9-mediated immune responses in Ramos-Blue cells, while Figure 9B shows the results shown in Figure 9A over a narrower concentration range. [Figure 10] Figures 10A and 10B compare the ability of oligonucleotides with and without a 3' cholesteryl moiety attached via a hexanediol linker to induce TLR9-mediated immune responses in Ramos-Blue cells. More specifically, Figure 10A shows the relative ability of oligonucleotides 2006-3dT4G5T4 and 2006-3dT4G5T4C to induce TLR9-mediated immune responses. Figure 10B shows the results of Figure 10A over a narrower concentration range. [Figure 11] Figure 11 compares the immunogenicity in Ramos-Blue of oligonucleotide 2006-T4-PDE with oligonucleotide 2006-T4-PDE having a 3' cholesteryl moiety attached via a hexanediol linker (3Chol-2006-T4-PD) shown in Figure 1. [Figure 12]FIG. 12 compares the ability of oligonucleotide 2006-3dT4G5T4 and oligonucleotide 2006-3dT4G5T4-3Chol to induce a TLR9-mediated immune response in Ramos-Blue cells. [Figure 13] Figures 13A, 13B, and 13C compare the ability of the 2007-PDE-T4, 2007-T4G5T4, and TCG8-T4 oligonucleotides, with and without a cholesteryl moiety attached to the 3' end of the oligonucleotide, to stimulate TLR9 in Ramos-Blue cells. Figure 13A shows the differential immunogenicity of oligonucleotides 2007-PDE-T4 and 2007-PDE-T4-Ch3. Figure 13B shows the differential immunogenicity of oligonucleotides 2007-T4G5T4 and 2007-T4G5T4-3Ch. Figure 13C shows the differential immunogenicity of oligonucleotides TCG8-T4 and TCG8-T4-Ch3. [Figure 14] Figures 14A, 14B, 14C, and 14D compare the stimulatory activity of several oligonucleotides and cholesteryl-modified oligonucleotides on mouse TLR9 (mTLR9) in HEKBlue-mTLR9 cells. Figure 14A compares the ability of the unmodified 2007-PDE-T4 oligonucleotide and the oligonucleotide 2007-PDE-T4-3Ch to induce a TLR9-mediated response in HEKBlue-mTLR9 cells. Figure 14B compares the ability of the unmodified 2007-T4G5T4 oligonucleotide and the oligonucleotide 2007-T4G5T4-3Ch to induce a TLR9-mediated response in HEKBlue-mTLR9 cells. Figure 14C compares the ability of oligonucleotides TCG8-T4 and TCG8-T4-3Ch to induce TLR9-mediated responses in HEKBlue-mTLR9 cells; and Figure 14D compares the ability of the TCG8-T4G5T4 oligonucleotide "TCG8-T4G5T4-3Ch" and the oligonucleotide TCG8-T4G5T4 having a 3' cholesteryl moiety attached via a hexanediol linker shown in Figure 1 to induce TLR9-mediated responses in HEKBlue-mTLR9 cells. [Figure 15]FIG. 15 shows the chemical structure of a cholesteryl moiety attached to a hexaethylene glycol linker. [Figure 16]Figures 16A, 16B, 16C, and 16D show the effect of modifying either the 3' or 5' end of an oligonucleotide with a cholesteryl moiety on the ability of the oligonucleotide to elicit a TLR9-mediated immune response. Figure 16A graphically depicts the ability of oligonucleotides 2006-PDE-T4, 2006-PDE-T4-Chol, and oligonucleotide 2006-PDE-T4 with a 5' cholesteryl moiety attached via a hexaethylene glycol linker (2006-PDE-T4-5Chol) shown in Figure 15 to stimulate TLR9 in HEKBlue-hTLR9 cells. Figure 16B graphically depicts the ability of oligonucleotide 2006-PTO, oligonucleotide 2006-PDE (2006-G5) having a GGGGG 3' end sequence, oligonucleotide 2006-G5 (2006-G5-3Chol) having a 3' cholesteryl moiety attached via a hexaethylene glycol linker shown in Figure 6, and oligonucleotide 2006-G5 (2006-G5-5Chol) having a 5' cholesteryl moiety attached via a hexaethylene glycol linker shown in Figure 15, to stimulate TLR9 in HEKBlue-hTLR9 cells. Figure 16C graphically depicts the ability of oligonucleotide 2006-PTO (2006-T4G5T4-5Chol), 2006-T4G5T4, 2006-T4G5T4-3Chol, and oligonucleotide 2006-T4G5T4, having a 5' cholesteryl moiety attached via a hexaethylene glycol linker as shown in Figure 15, to stimulate a TLR9-mediated immune response in HEKBlue-hTLR9 cells. Figure 16D graphically depicts the ability of oligonucleotide TCG8-T4G5T4, oligonucleotide TCG8-T4G5T4 having a 3' cholesteryl moiety attached via a hexaethylene glycol linker (TCG8-T4G5T4-3Chol) shown in Figure 6, and oligonucleotide TCG8-T4G5T4 having a 5' cholesteryl moiety attached via a hexaethylene glycol linker (TCG8-T4G5T4-5Chol) shown in Figure 15, to stimulate a TLR9-mediated immune response in HEKBlue-hTLR9 cells. [Figure 17]Figures 17A, 17B, 17C, and 17D show the effect of modifying the 3' or 5' end of an oligonucleotide with a cholesteryl moiety. Figure 17A graphically depicts the ability of oligonucleotides 2006-PDE-T4, 2006-PDE-T4-Chol, and 2006-PDE-T4-5Chol to stimulate TLR9 in Ramos-Blue cells. Figure 17B graphically depicts the ability of oligonucleotides 2006-PTO, 2006-G5, 2006-G5-3Chol, and 2006-G5-5Chol to stimulate TLR9 in Ramos-Blue cells. Figure 17C graphically depicts the ability of oligonucleotides 2006-PTO, 2006-T4G5T4, 2006-T4G5T4-3Chol, and 2006-T4G5T4-5Chol to stimulate TLR9-mediated immune responses in Ramos-Blue cells. Figure 17D graphically depicts the ability of oligonucleotides TCG8-T4G5T4, TCG8-T4G5T4-3Chol, and TCG8-T4G5T4-5Chol to stimulate TLR9-mediated immune responses in Ramos-Blue cells. DETAILED DESCRIPTION OF THE INVENTION

[0012] The disclosed compositions and methods may be more readily understood by reference to the following detailed description taken in conjunction with the accompanying drawings, which form a part of this 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 that the terminology used herein is for the purpose of describing particular embodiments by way of example only, and is not intended to limit the compositions and methods described in the claims.

[0013] Unless otherwise specified, any explanation of possible mechanisms or modes of action or reasons for improvement is intended to be exemplary only, and the disclosed compositions and methods are not constrained by the correctness or incorrectness of such suggested mechanisms or modes of action or reasons for improvement.

[0014] Throughout this text, the description refers to compositions and methods of using the compositions. Where the disclosure describes or claims features or embodiments relating to a composition, such features or embodiments are equally applicable to methods of using the composition. Similarly, where the disclosure describes or claims features or embodiments relating to methods of using a composition, such features or embodiments are equally applicable to the composition.

[0015] When a range of values ​​is expressed, another embodiment includes from the one particular value and / or to the other particular value. Moreover, reference to values ​​stated in a range includes each value within that range. All ranges are inclusive and inclusive. When values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. Reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise.

[0016] It is to be understood that certain features of the disclosed compositions and methods that are described herein in the context of separate embodiments can also be provided in combination in a single embodiment. Conversely, various features of the disclosed compositions and methods, while briefly described in the context of a single embodiment, may also be provided separately or in any subcombination.

[0017] As used herein, the singular forms "a," "an," and "the" include the plural forms.

[0018] As used herein, "CpG motif" refers to a cytosine-guanine dinucleotide sequence. The immunostimulatory nucleic acids described herein contain one or more CpG motifs, which, when unmethylated, can interact with Toll-like receptor proteins (TLRs) and elicit an immune response.

[0019] As used herein, the term "subject" is intended to mean any animal, particularly a mammal, and any type of avian, mammalian, or aquatic species can be treated using the disclosed methods.

[0020] Various terms relating to aspects of the specification are used throughout the specification and claims. Such terms have their ordinary meanings in the art unless otherwise indicated. Other specifically defined terms are to be interpreted in a manner consistent with the definitions provided herein.

[0021] Disclosed herein are immunostimulatory oligonucleotides comprising at least one CpG motif and a 3'-terminal cholesteryl moiety. CpG motifs in oligodeoxynucleotides (ODNs) have previously been shown to elicit immune responses in mammals. In some instances, the CpG motif is recognized by a Toll-like receptor (TLR). 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 avoiding infection in susceptible populations or infected individuals. As demonstrated herein, the immunostimulatory properties of ODNs can be enhanced by modifying the oligonucleotide, particularly by adding a thymine run, a guanine run, and / or a cholesteryl moiety to the 3'-end of the ODN.

[0022] The immunostimulatory oligonucleotides of the present disclosure contain at least one CpG motif. In some embodiments, the immunostimulatory oligonucleotides contain between 1 and 10 CpG motifs. In other embodiments, the immunostimulatory oligonucleotides may contain even 20 CpG motifs. Thus, in some aspects, the immunostimulatory oligonucleotides of the present disclosure contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 CpG motifs. In other embodiments, the immunostimulatory oligonucleotides contain between 11 and 15 CpG motifs, or even between 15 and 20 CpG motifs.

[0023] Oligonucleotides containing phosphodiester and / or phosphorothioate linkages between nucleotides are contemplated herein. In some aspects, oligonucleotides of the present disclosure contain phosphodiester linkages between the nucleotides of the oligonucleotide. In other aspects, oligonucleotides contain phosphorothioate linkages between the nucleotides of the oligonucleotide. Other linkages are also contemplated herein. For example, oligonucleotides of the present disclosure may contain other linkages, including, but not limited to, phosphoacetate, methylphosphonate, and phosphonocarboxylate linkages. Some linkages may offer desirable advantages over other linkages, such as production cost, ease and / or quality of production, and enhanced immunostimulatory effects.

[0024] In some aspects of the present disclosure, the immunogenicity of oligonucleotides due to CpG motifs can be further enhanced by non-CpG sequences. As shown in the Examples, the addition of a thymine run to the 3' end of an oligonucleotide can improve the ability of the oligonucleotide to elicit a TLR9-mediated immune response. Thus, in some embodiments of the present disclosure, the 3' terminal sequence of an immunostimulatory oligonucleotide comprises a plurality of thymine nucleotides as the 3' terminal sequence. In some aspects, this plurality of thymine nucleotides comprises consecutive thymine nucleotides. In some aspects, the plurality of thymine nucleotides comprises between 4 and 6 consecutive thymine nucleotides. For example, in some embodiments of the present disclosure, the 3' terminal sequence comprises SEQ ID NO: 9. In some embodiments, the oligonucleotide comprises SEQ ID NO: 2, 3, 4, 5, 6, or 8. And in some embodiments, the 3' terminal sequence of the oligonucleotide sequence is TTTT.

[0025] Other sequence modifications to the 3' end of the immunostimulatory oligonucleotide may also contribute to enhanced immunogenicity. For example, in some embodiments of the present disclosure, the immunostimulatory oligonucleotide comprises multiple guanine nucleotides at or near the 3' end sequence. In some aspects, the 3' end sequence of the immunostimulatory oligonucleotide comprises multiple guanine nucleotides. In some aspects, the multiple guanine nucleotides comprise consecutive guanine nucleotides, such as an oligonucleotide according to the present disclosure having a 3' end sequence of GGGGG. In some embodiments, the oligonucleotide comprises SEQ ID NO:7.

[0026] Increasing the immunogenicity of immunostimulatory oligonucleotides is not limited to modifications at the 3' end of the oligonucleotide. For example, internal sequences can also be modified to increase the number of CpG motifs. In some aspects, oligonucleotides can be synthesized to contain additional CpG motifs between the 5' and 3' ends of the oligonucleotide. In some aspects, immunostimulatory oligonucleotides comprise the sequence (TCG)n, where n is between 3 and 10. Thus, in some embodiments of the present disclosure, oligonucleotides comprise the sequence (TCG)n, where n is 3, 4, 5, 6, 7, 8, 9, or 10.

[0027] In some embodiments of the present disclosure, the immunostimulatory oligonucleotide may contain a lipid moiety at the 3'-end to enhance the immunogenic properties of the oligonucleotide. Thus, in some embodiments, a cholesteryl moiety is covalently attached to the 3'-terminal nucleotide of the immunostimulatory oligonucleotide via a linker. The cholesteryl moiety is likely to prevent degradation, increase solubility, generate ligand multivalency by forming higher-order structures (e.g., micelles), increase the stability of the oligonucleotide in pharmaceutical compositions, increase the immunogenicity of the oligonucleotide, or any combination thereof. A linker having at least two moieties capable of forming a covalent bond can be attached to the cholesteryl moiety and the oligonucleotide. For example, in some embodiments, the linker interacts with the hydroxyl group of the cholesteryl moiety to form a covalent bond and interacts with the 3'-terminal nucleotide of the oligonucleotide. In some embodiments, the cholesteryl moiety is covalently attached to the linker to form a cholesteryl-linker moiety. In some embodiments, the linker is first attached to the cholesteryl moiety, and then the resulting cholesteryl-linker is attached to the oligonucleotide. In other embodiments, the linker is first attached to the oligonucleotide and then to the cholesteryl moiety. In some embodiments, the cholesteryl-linker is commercially available.

[0028] In addition to having a moiety capable of binding to the oligonucleotide and the cholesteryl moiety, some embodiments of the linker include a carbon chain, and in some embodiments, the carbon chain includes between 3 and 12 carbon atoms. For example, a diol can be used as a linker between the cholesteryl moiety and the oligonucleotide, since its terminal hydroxyl group can covalently bond with the hydroxyl groups of the oligonucleotide and the cholesteryl moiety. In some embodiments, the linker includes hexanediol. In some embodiments, the cholesteryl-linker moiety has the chemical structure shown in Figure 1. In other embodiments, linkers are provided that include repeating chemical units. The chemical unit, in some aspects, is repeated between 2 and 12 times. In some embodiments, the repeating chemical unit includes ethylene glycol, and when the ethylene glycol chemical unit is repeated 6 times, the linker includes hexaethylene glycol. A linker including hexaethylene glycol can have the chemical structure shown in Figure 6.

[0029] In some situations, it may be desirable to deliver the oligonucleotides described herein to a subject in need thereof. The oligonucleotides can be delivered as immunostimulatory compositions. Immunostimulatory compositions comprising any of the oligonucleotides disclosed herein are provided. In some aspects, these immunostimulatory compositions comprise the oligonucleotide as well as other components that affect the immunogenicity, efficacy, and efficiency of the composition. In some embodiments of the present disclosure, the immunostimulatory composition may include, in addition to the immunostimulatory oligonucleotide, a vaccine for preventing or treating an infectious disease, a vector for delivering the oligonucleotide to a subject, a pharmaceutical carrier, or any combination thereof. For example, in some embodiments, the oligonucleotide is packaged in a viral vector that allows for targeted delivery of the oligonucleotide. In some aspects, the oligonucleotide may be added to a cationic liposome delivery vehicle to enhance the ability of the oligonucleotide to cross lipid cell membranes and / or membranes of cellular organelles containing TLR9.

[0030] 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, helminthic, or other parasitic infections. Administration of the immunostimulatory oligonucleotides or compositions of the present disclosure is intended to generate an immune response that creates an environment hostile to invading pathogens. Thus, invading pathogens may not be able to establish sufficient infection to worsen the health of the host organism. Administration of the immunostimulatory oligonucleotides and / or compositions may provide a non-antigen-specific immune response that augments or acts in parallel with the antigen-specific immune response against the invading pathogen.

[0031] In some aspects, the immunostimulatory composition comprising an oligonucleotide may further comprise a vaccine for preventing or treating an infectious disease. The combination of an oligonucleotide and a vaccine may be performed for efficiency reasons, since delivering multiple drugs separately increases the cost of treatment. The oligonucleotide and the vaccine may also be delivered as a single immunostimulatory composition to induce a non-antigen-specific immune response against any current infection, as well as to initiate the development of an antigen-specific immune response.

[0032] 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 is adapted for administration of the composition by a route selected from intravenous, intramuscular, intramammary, intradermal, intraperitoneal, subcutaneous, spray, aerosol, in ovo, mucosal, transdermal, immersion, oral, intraocular, intratracheal, intranasal, pulmonary, rectal, or other means known to those skilled in the art. The pharmaceutically acceptable carrier may be a diluent, adjuvant, excipient, or vehicle in which the immunostimulatory composition is administered. Such vehicles 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, etc. 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 (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, stabilizers, thickeners, lubricants, colorants, and the like. The concentration of the molecules of the invention in such pharmaceutical formulations varies widely, i.e., less than about 0.5%, usually ranging from at least about 1% to 15 or 20% by weight, but will be selected primarily based on the required dosage, fluid volume, viscosity, etc., according to the particular method of administration selected. Suitable vehicles and formulations containing other human proteins, such as human serum albumin, are described, for example, in Remington: See The Science and Practice of Pharmacy, 21st Edition, Troy, DB ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092 (especially pages 958-989).

[0033] In some embodiments, the oligonucleotide and the carrier are, for example, chemically bonded.When used to describe the relationship between the oligonucleotide and the carrier, "linkage" refers to the physical bond between the oligonucleotide and the carrier.When the oligonucleotide and the carrier are bonded to each other, interact with each other, or are bonded or otherwise bonded, they can be considered to be coupled.

[0034] The immunostimulatory compositions described herein further comprise a hapten in some embodiments. In some aspects, an immunostimulatory oligonucleotide is linked to the hapten. Haptens can induce an immune response against specific microorganisms, such as E. coli or Salmonella, while immunostimulatory oligonucleotides induce a non-specific immune response mediated by TLR9 interaction with the oligonucleotide. These and other infectious microorganisms are of particular concern to large agricultural producers, such as cattle, sheep, and swine producers.

[0035] Also provided is a method for enhancing the immunogenicity of a TLR9 ligand comprising attaching a cholesteryl moiety to the ligand, wherein the ligand is an immunostimulatory oligonucleotide having at least one CpG motif, and wherein the cholesteryl moiety is attached to the 3' terminal nucleotide of the oligonucleotide via a linker.

[0036] Another method disclosed herein provides a method for inducing a TLR9-mediated immune response in a subject in need thereof, comprising administering to the subject an oligonucleotide having multiple CpG motifs and a cholesteryl-linker moiety attached to the 3'-terminal nucleotide of the oligonucleotide. In some aspects of the methods for inducing a TLR9-mediated immune response, the oligonucleotide is administered as an immunostimulatory composition.

[0037] In certain 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 increased risk of infection by pathogens, particularly pathogens that have CpG-based pathogen-associated molecular patterns (PAMPs). When the immunostimulatory oligonucleotide and / or immunostimulatory composition is administered to such an animal, the TLR9-mediated immune response helps prevent infection by the pathogen or alleviate symptoms caused by the pathogen. Those skilled in the art will understand that the immunostimulatory oligonucleotides of the present invention need not be specific to a particular pathogen, but rather stimulate a non-antigen-specific immune response. The oligonucleotide also need not be specific to 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, cattle, horse, or sheep. Administration to livestock herds helps prevent the spread of infection to large groups of animals in crowded conditions, such as barns and / or sharing common feed and water sources. The oligonucleotides of the present disclosure offer distinct advantages over traditional forms of preventative treatment of infection in that the use of antibiotics is becoming less desirable, especially with the emergence of bacterial resistance to antibiotic treatment.

[0038] In some embodiments, the subject may be a human. Similar to livestock, resistance to antibiotics consumed by humans is becoming more common in bacteria, and treatment options for resistant infections are limited. The oligonucleotides and methods of the present disclosure provide a much-needed solution to so-called "superbugs," such as methicillin-resistant Staphylococcus aureus.

[0039] It is also contemplated herein that the subject to which the immunostimulatory oligonucleotide or composition is administered may be a mouse, rat, hamster, gerbil, or other rodent. The subject may also be a non-mammal. For example, in some aspects, the subject is an aquatic species. [Example]

[0040] The following examples are provided to further illustrate some of the embodiments disclosed herein. The examples are intended to illustrate, but not limit, the disclosed embodiments.

[0041] Example 1: 3'-cholesteryl modification of ODN strongly increases TLR9 stimulating activity

[0042] Human TLR9 recombinant overexpression in HEKBlue, 3'-cholesteryl modification of PDE-ODN(I)

[0043] A 3'-cholesteryl modification (see Figure 1 for the chemical structure of the cholesteryl-linker moiety) was applied to a PDE-ODN (Table 1, 2006-3dT4G5T4) that has considerable activity against human TLR9. The modified and unmodified forms were tested in vitro in HEKBlue-hTLR9 cells (Invivogen), a cell line expressing human TLR9.

[0044] TIFF0007770769000001.tif63170

[0045] TIFF0007770769000002.tif42169

[0046] The results suggest that the TLR9 stimulatory activity of 2006-3dT4G5T4 is significantly improved by the 3'-cholesteryl modification in terms of EC50, which is nearly 30-fold lower for 2006-3dT4G5T43C (Table 2, Figures 2A, 2B).

[0047] 3'-Cholesteryl modification of PDE-ODN (II)

[0048] A cholesteryl moiety (see Figure 1 for the chemical structure of the cholesteryl-linker moiety) was attached to the 3'-terminal nucleotide of 2006-T4-PDE (SEQ ID NO: 3, Table 3), which is known to be a weakly activating ligand of human TLR9. Modified and unmodified 2006-T4-PDE oligonucleotides were administered to HEKBlue-hTLR9 cells in vitro to measure the immunostimulatory effect of the 3'-cholesteryl modification.

[0049] TIFF0007770769000003.tif35168

[0050] The results suggest that the human TLR9 stimulating activity of 2006-T4-PDE is significantly improved by 3'-cholesteryl modification (Table 2, Figure 3).

[0051] 3'-Cholesteryl modification of PDE-ODN (III)

[0052] The 3'-cholesteryl modification (see Figure 1 for the chemical structure of the cholesteryl-linker moiety) was applied to PDE-ODNs with significant activity against human TLR9, 2006-3dT4G5T4 (Table 4). The modified and unmodified forms were tested in vitro in HEKBlue-hTLR9 cells.

[0053] TIFF0007770769000004.tif42169

[0054] TIFF0007770769000005.tif29170

[0055] The results suggest that the TLR9 stimulatory activity of 2006-3dT4G5T4 was significantly improved by the 3'-cholesteryl modification in terms of EC50, which was more than three-fold lower for 2006-3dT4G5T43C (Table 5, Figures 4A, 4B).

[0056] 3'-Cholesteryl modification of PDE-ODN (IV)

[0057] The 3'-cholesteryl modification (see Figure 1 for the chemical structure of the cholesteryl-linker moiety) was applied to a PDE-ODN, 2006-3dT4G5T4, which has considerable activity against human TLR9 (Table 8). The modified and unmodified forms were tested in vitro in HEKBlue-hTLR9 cells.

[0058] TIFF0007770769000006.tif34166

[0059] TIFF0007770769000007.tif29168

[0060] The results suggest that the TLR9 stimulatory activity of 2006-3dT4G5T4 is significantly improved by the 3'-cholesteryl modification in terms of EC50, which is over 36-fold lower for 2006-3dT4G5T43C (Table 9, Figure 5).

[0061] 3'-cholesteryl modification of PDE-ODN (V)

[0062] The 3'-cholesteryl modification applied to PDE-ODN (see Figure 6 for the chemical structure of the cholesteryl-linker moiety) has very weak activity against human TLR9, 2006-3dT4G5T4 (Table 10). The modified and unmodified forms were tested in vitro in HEKBlue-hTLR9 cells.

[0063] TIFF0007770769000008.tif40170

[0064] TIFF0007770769000009.tif27170

[0065] The results suggest that the TLR9 stimulatory activity of 2006-3dT4G5T4 is significantly improved by the 3'-cholesteryl modification, from virtually none to an EC50 of 68 nM (Table 11, Figure 7).

[0066] 3'-Cholesteryl modification of PDE-ODN (VI)

[0067] The 3'-cholesteryl modification (see Figure 1 for the chemical structure of the cholesteryl-linker moiety) was applied to PDE-ODNs that showed very low or no activity against human TLR9, 2007-PDE-T4, or 2007-PDE-T4G5T4 (Table 12). The modified and unmodified forms were tested in vitro in HEKBlue-hTLR9 cells.

[0068] TIFF0007770769000010.tif60164

[0069] TIFF0007770769000011.tif47170

[0070] The results suggest that the TLR9 stimulatory activity of both 2007-PDE-T4 and 2007-PDE-T4G5T4 is significantly improved by the 3'-cholesteryl modification (Figures 8A and 8B), with an EC50 of 24.9 nM for 2007-T4G5T4-3Ch (Table 13).

[0071] Example 2: Human TLR9, naturally expressed in Ramos-Blue B cells 3'-Cholesteryl modification (I)

[0072] A 3'-cholesteryl modification (see Figure 1 for the chemical structure of the cholesteryl-linker moiety) was applied to PDE-ODN 2006-3dT4G5T4, which has considerable activity against human TLR9 (Table 14). The modified and unmodified forms were tested in vitro in Ramos-Blue cells. The Ramos-Blue B lymphocyte line (Invivogen, San Diego, CA) stably expresses an NF-κB / AP-1 inducible reporter gene, allowing detection of TLR9 signaling.

[0073] TIFF0007770769000012.tif60170

[0074] TIFF0007770769000013.tif41166

[0075] The results suggest that the TLR9 stimulating activity of 2006-3dT4G5T4 is significantly improved by 3'-cholesteryl modification in terms of EC50, which is more than 8-fold lower for 2006-3dT4G5T43C (Table 15, Figures 9A and 9B). The modified ODN 2006-3dT4G5T43C also exceeds the activity of the "industry standard" ODN 2006-PTO.

[0076] 3'-Cholesteryl modification (II)

[0077] The 3'-cholesteryl modification (see Figure 1 for the chemical structure of the cholesteryl-linker group) was applied to PDE-ODN 2006-3dT4G5T4, which has considerable activity against human TLR9 (Table 16). The modified and unmodified forms were tested in vitro in Ramos-Blue cells.

[0078] TIFF0007770769000014.tif46163

[0079] TIFF0007770769000015.tif35163

[0080] The results suggest that the TLR9 stimulatory activity of 2006-3dT4G5T4 is significantly improved by the 3'-cholesteryl modification in terms of EC50, which is more than 7-fold lower for 2006-3dT4G5T43C (Table 17, Figures 10A, 10B).

[0081] 3'-Cholesteryl modification of PDE-ODN (III)

[0082] The 3'-cholesteryl modification (see Figure 1 for the chemical structure of the cholesteryl-linker moiety) was applied to PDE-ODNs that exhibited reduced activity against human TLR9, 2006-T4-PDE (Table 18). The modified and unmodified forms were tested in vitro in Ramos-Blue cells.

[0083] TIFF0007770769000016.tif34148

[0084] The results suggest that the human TLR9 stimulating activity of 2006-T4-PDE is significantly improved by 3'-cholesteryl modification (Table 18, Figure 11).

[0085] 3'-Cholesteryl modification of PDE-ODN (IV)

[0086] The 3'-cholesteryl modification (see Figure 6 for the chemical structure of the cholesteryl-linker moiety) was applied to PDE-ODNs with very low activity against human TLR9, 2006-3dT4G5T4 (Table 19). The modified and unmodified forms were tested in vitro in Ramos-Blue cells.

[0087] TIFF0007770769000017.tif40170

[0088] TIFF0007770769000018.tif34168

[0089] The results suggest that the human TLR9 stimulatory activity of 2006-3dT4G5T4 on Ramos-Blue cells is significantly improved by the 3'-cholesteryl modification, by a factor of 13 in terms of EC50 (Table 20, Figure 12).

[0090] 3'-cholesteryl modification of PDE-ODN (V)

[0091] The 3'-cholesteryl modification (see Figure 1 for the chemical structure of the cholesteryl-linker moiety) was applied to PDE-ODNs, 2007-PDE-T4 or 2007-PDE-T4G5T4, which showed very low or no activity against human TLR9 (Table 21). The modified and unmodified forms were tested in vitro in Ramos-Blue cells.

[0092] TIFF0007770769000019.tif61168

[0093] TIFF0007770769000020.tif61163

[0094] The results suggest that the human TLR9 stimulating activity of 2006-3dT4G5T4 in Ramos-Blue cells significantly improves the 3'-cholesteryl modification of all ODNs considered in this experiment (2007-PDE-T4, 2007-T4G5T4, TCG8-T4, Table 22, Figures 13A, 13B and 13C). In the case of 2007-T4G5T4-Ch, an improvement in activity of approximately 14 factors was observed in terms of EC50 compared to its unmodified analogue.

[0095] Example 3: Mouse TLR9 recombinant overexpression in HEKBlue 3'-cholesteryl modification of PDE-ODN The 3'-cholesteryl modification (see Figure 1 for the chemical structure of the cholesteryl-linker moiety) was applied to 2007-PDE-T4, 2007-PDE-T4G5T4, and TCG8-T4, which has very low or no activity against human TLR9 (Table 23). The modified and unmodified forms were tested in vitro in HEKBlue-mTLR9 cells (Invivogen).

[0096] TIFF0007770769000021.tif79170

[0097] The results suggest that the mouse TLR9 stimulating activity of all ODNs considered in this experiment (2007-PDE-T4, 2007-T4G5T4, TCG8-T4, Table 23) in HEKBlue-mTLR9 is significantly improved by 3'-cholesteryl modification in three cases (Figures 14A, 14B and 14C), and slightly improved at lower concentrations in the fourth case (Figure 14D).

[0098] Example 4: Systematic study of unmodified, 3'-cholesteryl-modified, and 5'-cholesteryl-modified ODNs in HEKBlue-hTLR9 and Ramos-Blue cells: Structure-activity relationship (SAR) HEKBlue-hTLR9

[0099] 3'-cholesteryl or 5'-cholesteryl modification (see Figures 6 and 15, respectively, cholesteryl-linker moiety chemical structure) was applied to four different ODNs (Table 24). Modified and unmodified forms were tested in vitro in HEKBlue-hTLR9 cells.

[0100] TIFF0007770769000022.tif152170

[0101] TIFF0007770769000023.tif133170

[0102] In this experiment, due to the relatively high background measurements, 0 (zero) ODN values ​​were subtracted from all data points for EC50 and Vmax calculations.

[0103] For all ODNs tested in this experiment, 3'-cholesteryl modification was most beneficial for activity against human TLR9 expressed in HEK blue cells (Table 24, Figures 16A, 16B, 16C, 16D). Where EC50 / Vmax calculations were possible (2006-G5, 2006-T4G5T4, Table 25), the EC50 of unmodified ODNs was low (2.5 and 48-fold, respectively), while 5'-cholesteryl modification was found to reduce activity. The EC50 of 2006-G5-3Chol and 2006-T4G5T4-3Chol was lower than that of the "industry standard" 2006-PTO, making them candidates for immunomodulatory interference.

[0104] Ramos‐Blue

[0105] 3'-cholesteryl or 5'-cholesteryl modifications (see Figures 6 and 15, respectively, chemical structures of the cholesteryl-linker moiety) were applied to four different ODNs (Table 26). Modified and unmodified versions were tested in vitro in Ramos-Blue cells.

[0106] TIFF0007770769000024.tif151170

[0107] TIFF0007770769000025.tif131170

[0108] In this experiment, 0 (zero) ODN values ​​were subtracted from all data points for EC50 and Vmax calculations to maintain consistency with Table 25.

[0109] For all ODNs examined in this experiment, 3'-cholesteryl modification was most beneficial for activity against endogenously present human TLR9 in Ramos-Blue cells (Table 26, Figures 17A-17D). Where EC50 / Vmax calculations were possible (2006-G5, 2006-T4G5T4, Table 25), the EC50 of unmodified ODNs was low (3- and 10-fold, respectively), while 5'-cholesteryl modification resulted in a loss of activity, except for TCG8-T4G5T4, where both derivatizations resulted in improved activity, although 3'-cholesteryl was more active than 5'-cholesteryl (Figure 17D, Table 26). The EC50 of 2006-G5-3Chol and 2006-T4G5T4-3Chol was lower than the EC50 of the "industry standard" 2006-PTO, making them candidates for immunomodulatory interference.

[0110] Those skilled in the art will understand that many changes and modifications can be made to the preferred embodiments of the present invention and that such changes and modifications can be made without departing from the spirit of the present invention. It is, therefore, intended by the appended claims to cover all such equivalent variations as fall within the true spirit and scope of the invention.

[0111] The disclosures of each patent, patent application, and publication cited or described herein are hereby incorporated by reference in their entirety.

Claims

1. 1. An immunostimulatory oligonucleotide comprising a CpG motif and a 3′ cholesteryl moiety, wherein the cholesteryl moiety is covalently attached to the 3' terminal nucleotide of the immunostimulatory oligonucleotide via a linker; and wherein the immunostimulatory oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 2, 3, 4, 5, 6, 7 or 8.

2. The immunostimulatory oligonucleotide of claim 1, wherein the immunostimulatory oligonucleotide contains phosphodiester bonds or phosphorothioate bonds between nucleotides of the immunostimulatory oligonucleotide.

3. The immunostimulatory oligonucleotide of claim 1 , wherein the immunostimulatory oligonucleotide comprises the nucleotide sequence of SEQ ID NO:

7.

4. The immunostimulatory oligonucleotide of any one of claims 1-2, wherein the immunostimulatory oligonucleotide comprises (TCG)n, where n is between 3 and 10.

5. The immunostimulatory oligonucleotide of any one of claims 1 to 4, wherein the linker comprises a carbon chain.

6. The immunostimulatory oligonucleotide of claim 5, comprising a cholesteryl-linker moiety having the structure: 【Chemistry 1】

7. The immunostimulatory oligonucleotide of any one of claims 1 to 5, wherein the linker comprises repeating chemical units.

8. 8. The immunostimulatory oligonucleotide of claim 7, wherein the repeating chemical unit is repeated between 2 and 12 times.

9. 8. The immunostimulatory oligonucleotide of claim 7, wherein the repeating chemical unit is ethylene glycol.

10. The immunostimulatory oligonucleotide of any one of claims 7 to 9, wherein the linker comprises hexaethylene glycol.

11. The immunostimulatory oligonucleotide of any one of claims 7 to 10, wherein the cholesteryl moiety is covalently linked to a linker to form a cholesteryl-linker moiety.

12. The immunostimulatory oligonucleotide of claim 11, comprising a cholesteryl-linker moiety having the structure: 【Chemistry 2】

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

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

15. An immunostimulatory composition described in claim 13 or 14, wherein the composition further comprises a pharmaceutically acceptable carrier.

16. 1. A method of enhancing the immunogenicity of a TLR9 ligand, comprising attaching a cholesteryl 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 oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 2, 3, 4, 5, 6, 7, or 8; wherein the cholesteryl moiety is covalently attached to the linker to form a cholesteryl-linker moiety; wherein the cholesteryl-linker moiety is 【Transformation 3】 or 【Chemistry 4】 Including, method.

Images