Compositions for inducing an immune response

Abstract

The present invention provides a composition substantially free of antigen for inducing an innate immune response in birds or mammals, the composition comprising a saponin, a sterol, a quaternary amine, and a polyacrylic acid polymer, administered to a bird or a mammal. Also provided are methods of using the composition.

Description

[0001] This application is a divisional application of the patent application No. 201480072153.5, with the invention title of "Composition for Inducing an Immune Response", and the application date of November 21, 2014. Field of the Invention

[0002] The present invention relates to methods of using immunomodulators to induce a non-specific response to infection. Background of the Invention

[0003] When the body exhibits an immune response to a particular infectious or immune challenge, the distinction between the innate immune response (innate immunity) and the acquired immune response (antigen-specific adaptive immunity) also becomes apparent.

[0004] The innate immune system is a highly efficient and evolutionarily advanced general defense system. The levels of elements in innate immunity are always low, but are very rapidly activated when stimulated. The stimulation can include the interaction of bacterial signaling molecules with pattern recognition receptors on the surface of body cells or other disease mechanisms. Every day, animals are exposed to potential pathogenic microorganisms through food and water, air, and surfaces they contact. The innate immune system serves to prevent these potential pathogens from causing disease. The innate immune system differs from what is known as adaptive immunity, which includes antibodies and antigen-specific B- and T-lymphocytes, in that the former is always present, acts immediately, and is relatively non-specific for any given pathogen. The adaptive immune system requires the expansion of specific recognition elements and thus takes days to weeks to mount a response. Even after pre-stimulating adaptive immunity through vaccination, it takes three days or more to respond to a pathogen, while the innate immune response is immediate or rapid (within hours). The innate immune response is known to include a variety of effector functions, including phagocytes, complement, etc., but is generally not fully understood.

[0005] Accordingly, there are situations where activation of the innate immune response is highly desirable. For example, in the veterinary field, activation of the innate immune system is particularly useful when an animal has or is about to undergo an environmental change, such as during or immediately after transportation, or in an environment where animals are in close proximity to each other and pathogen spread can be rapid. Summary of the Invention

[0006] The present invention provides methods and compositions for activating the innate immune system. In certain aspects, the present invention provides compositions that are substantially free of antigen and that comprise (or in certain embodiments consist essentially of or consist of) saponins, steroids, quaternary amines, and polyacrylic acid polymers, and optionally at least one glycolipid and immunostimulatory oligonucleotides, for inducing an immune response in mammals or birds. In certain embodiments, the compositions are free of antigen.

[0007] In certain aspects, the present invention provides a method of inducing an immune response in an animal, comprising administering to the animal a composition substantially free of antigen, the composition comprising saponin, steroid, quaternary amine, and polyacrylic acid polymer, wherein the animal is selected from mammals and birds. In some embodiments, the composition is free of antigen.

[0008] In certain aspects, the composition mentioned above further comprises a glycolipid, an immunostimulatory oligonucleotide, or both a glycolipid and an immunostimulatory oligonucleotide.

[0009] In embodiments applicable to the various aspects set forth above, the amount of saponin is from about 1 μg to about 5,000 μg per dose (preferably from about 10 μg to about 50 μg per dose), the amount of steroid is from about 1 μg to about 5,000 μg per dose (preferably from about 10 μg to about 50 μg per dose), the amount of quaternary amine compound is from about 1 μg to about 5,000 μg per dose (preferably from about 1 μg to about 30 μg) per dose, and the amount of polyacrylic acid polymer is from 0.0001% v / v to about 75% v / v (preferably from about 0.01% v / v to about 0.1% v / v per dose). If a glycolipid is present, its amount is generally from about 0.01 mg to about 10 mg per dose (preferably from about 1 mg to about 2 mg per dose). If an immunostimulatory oligonucleotide is present, its amount is generally from 20 μg to about 500 μg per ml (preferably from about 100 μg per ml to about 200 μg per ml).

[0010] The compositions of the present invention can be used to trigger a protective immune response against attacks by a variety of organisms such as viruses, Gram-positive and Gram-negative bacteria. In certain embodiments, the compositions of the present invention are used to trigger a protective immune response against Gram-negative bacteria such as Bordetella bronchiseptica. Brief Description of the Drawings

[0011] Figure 1 Shows the effect of QCDCRT adjuvant on IFNα, MX-1, and OAS mRNA levels.

[0012] Figure 2 Illustrates the effect of QCDCRT adjuvant on virus shedding in a BHV-1 challenge model.

[0013] Detailed Description of Selected Embodiments

[0014] To assist those of ordinary skill in the art in understanding the present invention, the following non-limiting definitions are provided.

[0015] When used in conjunction with a measurable numerical variable, "about" or "approximately" refers to the specified value of the variable and all variable values within the experimental error range of the specified value (e.g., within the 95% confidence interval of the mean) or values within 10% of the specified value, whichever is the larger range.

[0016] "Alkyl" refers to straight-chain and branched saturated hydrocarbon moieties.

[0017] "Amine" refers to a chemical compound derived from ammonium in which one or more hydrogen atoms have been replaced by hydrocarbon groups. "Quaternary amine" refers to a compound based on ammonium having four hydrocarbon groups.

[0018] "Antigen" refers to any substance that stimulates a specific immune response. The term includes killed, inactivated, attenuated, or modified live bacteria, viruses, or parasites. The term antigen also includes polynucleotides, polypeptides, recombinant proteins, synthetic peptides, protein extracts, cells (including tumor cells), tissues, polysaccharides, or lipids, or fragments thereof, alone or in any combination. The term antigen also includes antibodies, such as anti-idiotypic antibodies or fragments thereof, and may refer to synthetic peptide mimetics that can mimic an antigen or antigenic determinant (epitope).

[0019] As applied to an adjuvant formulation, "substantially consists of" means that the formulation does not contain additional adjuvants or immunomodulatory reagents not mentioned in an amount such that the reagent exerts a measurable adjuvant or immunomodulatory effect.

[0020] "Dose" refers to the unit of a composition administered to a subject and can be a unit of mass or volume.

[0021] "Immunostimulatory molecule" refers to a molecule that elicits an immune response.

[0022] "Parenteral administration" refers to the introduction of a substance, such as a vaccine, into a subject's body through or via a route that does not include the digestive tract.

[0023] "Pharmaceutically acceptable" refers to a substance that is within the scope of good medical evaluation, suitable for contact with a subject's tissue without causing excessive toxicity, radiation, allergic response, etc., having a reasonable benefit-risk ratio, and being effective for its intended use.

[0024] "Saponin" refers to a class of surface-active glycosides of plant origin, consisting of a hydrophilic region (usually several sugar chains) attached to a hydrophobic region of a steroid or triterpene structure.

[0025] "Steroid" refers to any type of organic compound belonging to the biochemical lipid class that contains a four-fused ring system, consisting of three fused cyclohexane (six-carbon) rings plus a fourth cyclopentane (five-carbon) ring. Steroids are generally highly soluble in organic solvents and slightly soluble in water.

[0026] "Sterol" refers to compounds that are biologically produced from terpene precursors in animals. It contains a steroid ring structure with a hydroxyl (OH) group, usually attached to carbon-3. The hydrocarbon chain length of the fatty acid substituent is variable, usually 16 to 20 carbon atoms, and can be saturated or unsaturated. Sterols generally contain one or more double bonds in the ring structure and also contain various substituents attached to the ring. Sterols and their fatty acid esters are substantially insoluble in water.

[0027] "Substantially antigen-free composition" refers to a composition in which the amount of antigen is not sufficient to produce a protective specific immune response or to prevent the successful reproduction and / or elimination (e.g., viral shedding) of the pathogen targeted by the specific immune response generated by the antigen. In some embodiments, the amount of antigen in the substantially antigen-free composition cannot be detected by serological assays such as ELISA.

[0028] "Triterpene" refers to a large and diverse class of naturally occurring organic molecules derived from six five-carbon isoprene (2-methyl-1,3-butadiene) units. Most triterpenes are polycyclic structures that differ from each other in terms of functional groups and their basic carbon skeletons.

[0029] As described herein, the inventors have surprisingly found that compositions comprising saponins, sterols, quaternary amine compounds, polyacrylic acid polymers and optionally one or both of glycolipids and / or immunostimulatory oligonucleotides are capable of triggering a protective immune response in warm-blooded animals (mammals and birds) in the absence of antigen.

[0030] Although warm-blooded animals, namely mammals and birds, are capable of generating innate immune responses and slow-acting adaptive immune responses, they mainly rely on highly evolved adaptive immune mechanisms. Invertebrates, in contrast, lack adaptive immune mechanisms and thus rely entirely on innate immune response mechanisms. Cold-blooded vertebrates such as fish include both innate and adaptive immune response mechanisms. However, compared to warm-blooded vertebrates, the adaptive immune response of cold-blooded vertebrates is relatively underdeveloped, while the innate immune response of cold-blooded vertebrates is relatively more developed. Thus, cold-blooded vertebrates generally rely more on innate immune mechanisms than on adaptive immune mechanisms compared to warm-blooded vertebrates. Because of the differences in the relative development and dependence on the innate immune system between invertebrates and cold-blooded vertebrates compared to warm-blooded animals, the results and findings regarding the innate immune systems of invertebrates and cold-blooded vertebrates cannot be expected to be applicable to the innate immune system of warm-blooded vertebrates. Specifically, the discovery that a compound or composition can stimulate the innate immune system of invertebrates or cold-blooded vertebrates cannot reasonably predict that the compound or composition can be used to stimulate the innate immune system of warm-blooded mammals.

[0031] The innate immune system is a rapidly deployed, highly effective general defense system that provides protection against pathogenic agents. Accordingly, there is a need to identify compounds and compositions that stimulate or enhance the innate immune response system of warm-blooded vertebrates. As described herein, the inventors have surprisingly discovered that compositions comprising one or both of saponins, sterols, quaternary amine compounds, polyacrylic acid polymers, and optionally glycolipids and / or immunostimulatory oligonucleotides (referred to herein as QCDC, QCDCR, QCDCT, and QCDCRT, respectively) effectively activate the innate immune response system of warm-blooded vertebrates and can be used as immunomodulators, i.e., to improve the immune response to challenge in warm-blooded animals (e.g., mammals and birds) independent of antigen. It is surprising and unexpected to find that the QCDC, QCDCR, QCDCT, and QCDCRT compositions described herein can activate the less flexible innate immune system in warm-blooded animals sufficiently effectively.

[0032] Accordingly, in certain embodiments, the present invention provides a composition for improving the immune response of a mammal or bird, the composition comprising a sterol, a saponin, a quaternary amine, a polyacrylic acid polymer, e.g., This four-component composition is referred to as QCDC. The QCDC composition may also contain a glycolipid (R), an immunostimulatory oligonucleotide (T), or both.

[0033] Sterols share a common chemical core, which is a steroid ring structure having a hydroxyl (OH) group, typically attached to carbon-3. The hydrocarbon chains of the fatty acid substituents are typically numbered from 16 to 20 carbon atoms and may be saturated or unsaturated. These sterols commonly contain one or more double bonds in the ring structure and contain a variety of substituents attached to the ring. Sterols and their corresponding fatty acid esters are substantially insoluble in water. Given these chemical similarities, these sterols sharing this chemical core are likely to have similar properties when used in the vaccine compositions of the present invention. Sterols are well known in the art and are commercially available. For example, cholesterol is disclosed in the 12th edition of the Merck Index, p369. Suitable sterols include, but are not limited to, β-sitosterol, stigmasterol, ergosterol, ergocalciferol, and cholesterol.

[0034] Sterols are typically used in an amount of from about 1 μg to about 5,000 μg per dose. They are also used in an amount of from about 1 μg to about 4,000 μg per dose, from about 1 μg to about 3,000 μg per dose, from about 1 μg to about 2,000 μg per dose, from about 1 μg to about 1,000 μg per dose. They are also used in an amount of from about 5 μg to about 750 μg per dose, from about 5 μg to about 500 μg per dose, from about 5 μg to about 200 μg per dose, from about 5 μg to about 100 μg per dose, from about 15 μg to about 100 μg per dose, from about 15 μg to about 30 μg per dose.

[0035] Suitable saponins include triterpenoid saponins. These triterpenoids are a class of surface-active glycosides of plant origin, sharing a common chemical core consisting of a hydrophilic region (usually several sugar chains) bound to a hydrophobic region of a steroid or triterpene structure. Due to these similarities, saponins sharing this chemical core are likely to have similar adjuvant properties. Triterpenoids suitable for use in adjuvant compositions can be from many sources and can be of plant origin or synthetic equivalents, including but not limited to, Quillaja saponaria, tomatine, ginseng extract, mushrooms, and alkaloid glycosides structurally similar to steroid saponins. Thus, triterpenoids suitable for use in adjuvant compositions include saponins, squalene, and lanosterol. In another set of embodiments, the saponin can be, for example, Quil A or another purified or partially purified saponin preparation, which can be obtained commercially. Thus, saponin extracts can be used in the form of, for example, a mixture of QS-7, QS-17, QS-18, and QS-21 or as purified individual components. In one embodiment, Quil A is at least 85% pure. In certain embodiments, the Quil A is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure.

[0036] In the compositions of the present invention, saponins are present in an amount of from about 1 μg to about 5,000 μg per dose. They are also used in amounts of from about 1 μg to about 4,000 μg per dose, from about 1 μg to about 3,000 μg per dose, from about 1 μg to about 2,000 μg per dose, and from about 1 μg to about 1,000 μg per dose. They are also used in amounts of from about 5 μg to about 750 μg per dose, 5 μg to about 500 μg per dose, from about 5 μg to about 200 μg per dose, from about 5 μg to about 100 μg per dose, from about 15 μg to about 100 μg per dose, and from about 15 μg to about 30 μg per dose.

[0037] Quaternary amine compounds are ammonium-based compounds having four hydrocarbon groups. Such hydrocarbon groups are typically restricted to alkyl or aryl groups. In certain embodiments, the quaternary amine compound consists of four alkyl chains, two of which are C 10 -C 20 alkyls, and the remaining two are C1-C4 alkyls. In one set of embodiments, the quaternary amine is didodecyldimethylamine (DDA). In certain embodiments, avridine can be used.

[0038] In the compositions of the present invention, the quaternary amines are present in an amount of from about 1 μg to about 5,000 μg per dose. They are also used in amounts of from about 1 μg to about 4,000 μg per dose, from about 1 μg to about 3,000 μg per dose, from about 1 μg to about 2,000 μg per dose, and from about 1 μg to about 1,000 μg per dose. They are also used in amounts of from about 5 μg to about 750 μg per dose, 5 μg to about 500 μg per dose, from about 5 μg to about 200 μg per dose, from about 5 μg to about 100 μg per dose, from about 15 μg to about 100 μg per dose, and from about 15 μg to about 30 μg per dose. As a specific example, a composition containing DDA adjuvant can be prepared by simply mixing an antigen solution with freshly prepared DDA solution.

[0039] A variety of polyacrylic acid polymers suitable for the uses of the present invention are commercially available. In certain embodiments, the polyacrylic acid polymer comprises polyacrylic acid, which is sold under the trade name commercially. These polymers have an average equivalent weight of 76. They are produced from primary polymer particles having an average diameter of about 0.2 to 6.0 microns. The polymer swells in water up to 1000 times its original volume and 10 times its original diameter and forms a gel when exposed to a pH environment greater than the pKa of the carboxylic acid groups. At a pH above the pKa of the carboxylic acid groups, ionization of the carboxylic acid groups results in repulsion between the negative charges, contributing to the swelling of the polymer.

[0040] In the compositions of the present invention, the polyacrylic acid polymer is present in an amount of from about 0.0001% to about 75% v / v by volume ratio (v / v). In certain embodiments, it is used in amounts of from about 0.001% v / v to about 50% v / v, from about 0.005% v / v to about 25% v / v, from about 0.01% v / v to about 10% v / v, from about 0.05% v / v to about 2% v / v. In another embodiment, it is used in an amount of from about 0.02% v / v to about 0.1% v / v.

[0041] Suitable glycolipids are generally those that activate the Th2 response. Examples of glycolipids include, but are not limited to, compounds covered by Formula I and other compounds generally described in U.S. Publication No. 20070196384 (Ramasamy et al.).

[0042]

[0043] In the structure of Formula I, R 1 is hydrogen, or a saturated alkyl group having up to 20 carbon atoms; X is -CH2-, -O-, or -NH-; R 2 is hydrogen, or a saturated or unsaturated alkyl group having up to 20 carbon atoms; R 3 、R4 and R 5 are independently hydrogen, -SO4 2- , -PO4 2- , -COC 1-10 alkyl; R 6 is L-alanyl, L-α-aminobutyryl, L-arginyl, L-aspartyl, L-asparaginyl, L-cysteinyl, L-glutamyl, L-glycyl, L-histidyl, L-hydroxyprolyl, L-isoleucyl, L-leucyl, L-lysyl, L-methionyl, L-ornithyl, L-phenylalanyl, L-prolyl, L-serinyl, L-threonyl, L-tyrosyl, L-tryptophanyl and L-valyl or their D-isomers.

[0044] In certain embodiments, a suitable glycolipid is N-(2-deoxy-2-L-leucylamido-β-D-glucopyranosyl)-N-octadecyllaurylamide or a salt thereof. In certain embodiments, the salt is acetate.

[0045] In certain embodiments, in the compositions of the present invention, the glycolipid is present in an amount of from about 0.01 mg to about 10 mg per dose. In certain embodiments, they are used in amounts of from about 0.05 mg to about 7.5 mg per dose, from about 0.05 mg to about 1 mg per dose, from about 0.5 mg to about 2.5 mg per dose, and from about 1 mg to about 2 mg per dose.

[0046] In certain embodiments of the present invention, immunostimulatory oligonucleotides can also be used, in combination with QCDC or QCDCR. Generally, immunostimulatory oligonucleotides contain at least one CG motif and are sometimes referred to as CpG nucleotides. CpG oligonucleotides are characterized by the presence of unmethylated CG dinucleotides (CpG motifs) in a specific base sequence. (Hansel TT, Barnes PJ (eds): New Drugs for Asthma, Allergy and COPD. Prog Respir Res. Basel, Karger, 2001, vol 31, pp 229-232, which is incorporated herein by reference). CpG motifs are present in bacterial DNA and confer immunostimulatory properties to bacterial DNA. CpG motifs are generally not found in eukaryotic DNA, where CG dinucleotides are suppressed and are usually methylated if present.

[0047] The adjuvants of the present invention may include so-called P-type immunostimulatory oligonucleotides, including, for example, modified P-type immunostimulatory oligonucleotides. P-type immunostimulatory oligonucleotides are CpG oligonucleotides characterized by the presence of a palindromic sequence, generally 6-20 nucleotides in length. The presence of such a palindrome makes it possible to form multimers or stem-loop structures. The total length of P-type immunostimulatory oligonucleotides is usually between 19 and 100 nucleotides, such as 19-30 nucleotides, 30-40 nucleotides, 40-50 nucleotides, 50-60 nucleotides, 60-70 nucleotides, 70-80 nucleotides, 80-90 nucleotides, 90-100 nucleotides.

[0048] In certain aspects of the present invention, the immunostimulatory oligonucleotide comprises a 5'TLR activation domain and at least two palindromic regions, one palindromic region being a 5' palindrome of at least 6 nucleotides in length and directly or via a spacer linked to a 3' palindromic region of at least 8 nucleotides in length.

[0049] P-type immunostimulatory oligonucleotides can be modified according to techniques known in the art. For example, J modification refers to iodinated nucleotides. E modification refers to ethylated nucleotides. Thus, an E-modified P-type immunostimulatory oligonucleotide is a P-type immunostimulatory oligonucleotide in which at least one nucleotide (preferably the 5' nucleotide) is ethylated. Additional modifications include the attachment of 6-nitrobenzimidazole, O-methylation, modification of proynyl-dU, inosine modification, 2-bromo vinyl attachment (preferably attached to uridine).

[0050] P-type immunostimulatory oligonucleotides may also contain modified internucleotide linkages, including but not limited to, phosphodiester bonds and phosphorothioate bonds. The oligonucleotides can be synthesized or obtained from commercial sources.

[0051] Examples of P-type oligonucleotides and modified P-type oligonucleotides are disclosed in PCT application No. WO2008 / 068638, published on June 12, 2008. Suitable non-limiting examples of modified P-type immunostimulatory oligonucleotides are provided below (in SEQ ID NO 1-10, "*" refers to a phosphorothioate bond, "-" refers to a phosphodiester bond; in SEQ ID NO 11-13 all bonds are phosphodiester bonds).

[0052] SEQ ID NO:1

[0053] 5'T*C-G*T*C-G*A*C-G*A*T*C-G*G*C*G*C-G*C*G*C*C*G 3'

[0054] SEQ ID NO:2

[0055] 5'T*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3'

[0056] SEQ ID NO:3

[0057] 5'T*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G*T 3'

[0058] SEQ ID NO:4

[0059] 5'JU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3'

[0060] SEQ ID NO:5

[0061] 5'JU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G*T 3'

[0062] SEQ ID NO:6

[0063] 5'JU*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G*T 3'

[0064] SEQ ID NO:7

[0065] 5'EU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3'

[0066] SEQ ID NO:8

[0067] 5'JU*C-G*T*C*G*A*C*G*A*T*C*G*G**C*G*G*C*C*G*C*C*G*T 3'

[0068] SEQ ID NO:9

[0069] 5'JU*C*G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C*G*T 3'

[0070] SEQ ID NO:10

[0071] 5'T*C-G*T*C-G*A*C-G*A*T*C-G*G*C*G*C-G*C*G*C*C*G 3'

[0072] SEQ ID NO:11 5'-UUGUUGUUGUUGUUGUUGUU-3'

[0073] SEQ ID NO:12 5'-UUAUUAUUAUUAUUAUUAUU-3'

[0074] SEQ ID NO:13 5'-AAACGCUCAGCCAAAGCAG-3'.

[0075] P-type immunostimulatory oligonucleotides are generally used in the adjuvant compositions described herein in an amount of about 20 μg to about 500 μg per ml. They are also used in amounts of about 25 μg to about 400 mg per ml, about 40 μg to about 250 μg per ml, about 50 μg to about 200 μg per ml, about 100 μg to about 200 μg per ml.

[0076] The method of preparing the compositions described herein is described, for example, in U.S. Publication No. 20090324641 (Dominowski et al., published on December 31, 2009). Briefly, the compounds of the composition can be mixed together, and preferably, the polyacrylic acid polymer is added last. The composition can be microfluidized before adding the polyacrylic acid polymer.

[0077] The compositions described herein generally do not require any specific carrier and can be formulated in an aqueous or other pharmaceutically acceptable buffer. The adjuvant compositions can be made in various forms depending on the route of administration, storage requirements, etc. For example, they can be made in the form of a sterile aqueous solution or dispersion, suitable for injection, or made in a lyophilized form using freeze-drying, vacuum drying or spray-drying techniques. The lyophilized composition can be reconstituted in a stable solution, such as saline or HEPES, before use. Thus, the adjuvant composition can be used in solid, semi-solid or liquid dosage forms.

[0078] Phosphate buffered saline (PBS) can be used as the aqueous buffer medium; the pH value of the buffer can be neutral or slightly alkaline or slightly acidic. Thus, the pH is in the range of pH 6 to 8. A pH of about 7.0 to about 7.3 is common. The buffer strength can be between 10 and 50 mM PO4 and between 10 and 150 mM PO4. In one example, 0.063% PBS is used. The pH can be adjusted using NaOH or HCl as needed. Typical concentrations include 1N to 10N HCl and 1N to 10N NaOH.

[0079] The composition can be homogenized or microfluidized. The composition is subjected to a primary blending process, typically passing through one or more homogenizers one or more times. Any commercially available homogenizer can be used for this purpose, e.g., Ross emulsifier (Hauppauge, NY), Gaulin homogenizer (Everett, Mass), or Microfluidics (Newton, Mass).

[0080] In certain embodiments, saponins are added to a suitable buffer. Then, sterols are slowly added to the saponin solution, followed by slow addition of the quaternary amine compound. If glycolipids and / or immunostimulatory oligonucleotides are present, they are also added. The resulting composition is homogenized and then microfluidized. After microfluidization, a polymer is added to the microfluidized composition. Depending on the components used, the order of these steps can be altered to optimize the preparation of the composition.

[0081] In one embodiment, the composition is homogenized at 10,000 rpm for 3 minutes. Microfluidization can be achieved by using a commercial microfluidizer, such as Model 11OY from Microfluidics (Newton, Mass); Model 30CD from Gaulin (Gaulin Inc., Everett, Mass); and Model 8.30H Rainnie Minilab (Miro Atomizer Food and Dairy, Inc., Hudson, Wis). The operating principle of these microfluidizers is to drive a fluid through a small orifice under high pressure so that two fluid streams interact at high speed in an interaction chamber to form a composition with droplets of submicron size. In one embodiment, the formulation is microfluidized by passing through a chamber with a restricted size of 200 microns at 10,000 ± 500 psi.

[0082] The size of the dose of the composition is generally from about 0.05 mL to about 5 mL, including the endpoints, and depends on the subject and the antigen. For example, for dogs or cats, a dose of about 1 mL is typically used, while for cattle, a dose of about 2 - 5 mL is generally used. However, these adjuvants can also be formulated in microdoses, where a dose of about 100 μL can be used, e.g., for administration to chickens.

[0083] Routes of administration for the adjuvant composition include parenteral, oral, oro-nasal, intranasal, intratracheal, topical, and in ovo. Any suitable device can be used to administer the composition, including syringes, droppers, needleless injection devices, patches, etc. The route and device chosen for use will depend on the components of the composition and the size of the animal.

[0084] Different mammals and birds are suitable for treatment by the methods of the present invention. The animals include, but are not limited to, dogs, cats, horses, sheep, cattle, pigs, geese, chickens, ducks, etc.

[0085] One advantageous aspect of the present invention is that it can be administered to a test mammal or bird when the animal's immune system is weakened, when the animal is in a high-stress environment, or when there is a high likelihood of pathogen exposure. For example, the animal can be treated immediately before, during, or after transportation, or when the animal is in quarantine. Sometimes, suitable antigens are not easily available or are even unknown.

[0086] Activation of the innate immune system is particularly important if a disease is known to have a rapid onset. In addition, the present invention can be used for animals kept in crowded conditions, such as in a chicken farm.

[0087] Diseases can be caused by such a wide range of pathogens as viruses, Gram-positive bacteria, and Gram-negative bacteria.

[0088] Viruses include, but are not limited to, Avian herpesvirus, Bovine herpesviruses, Canine herpesviruses, Equine herpesviruses, Feline viral rhinotracheitis virus, Marek's disease virus, Ovine herpesviruses, Porcine herpesviruses, Pseudorabies virus, Avian paramyxoviruses, Bovine respiratory syncytial virus, Canine distemper virus, Canine parainfluenza virus, canine adenovirus, canine parvovirus, Bovine Parainfluenza virus 3, Ovine parainfluenza 3, Rinderpest virus, Border disease virus, Bovine viral diarrhea virus (BVDV), BVDV type I, BVDV type II, Classical swine fever virus, Avian Leukosis virus, Bovine immunodeficiency virus, Bovine leukemia virus, Bovine tuberculosis, Equine infectious anemia virus, Feline immunodeficiency virus, FeLV (Feline leukemia virus), Newcastle Disease virus, Ovine progressive pneumoniavirus), Ovine pulmonary adenocarcinoma virus, Canine coronavirus (CCV), pantropic CCV, Canine respiratory coronavirus, Bovine coronavirus, Feline Calicivirus, Feline enteric coronavirus, Feline infectious peritonitis virus, Porcine epidemic diarrhea virus, Porcine hemagglutinating encephalomyelitis virus, Porcine parvovirus, Porcine Circovirus (PCV) Type I, PCV Type II, Porcine Reproductive and Respiratory Syndrome (PRRS) Virus, Transmissible gastroenteritis virus, Turkey coronavirus, Bovine ephemeral fever virus, Rabies, Rotovirus, Vesicular stomatitis virus, lentivirus, Avian influenza, Rhinoviruses, Equine influenza virus, Swine influenza virus, Canine influenza virus, Feline influenza virus, Human influenza virus, Eastern Equine encephalitis virus (EEE), Venezuelan equine encephalitisvirus), West Nile virus, Western equine encephalitis virus, human immunodeficiency virus, human papilloma virus, varicella zoster virus, hepatitis B virus, rhinovirus, and measles virus.

[0089] Gram-positive bacteria have a thick peptidoglycan layer on the cell membrane surface. Non-limiting examples of Gram-positive bacteria are Streptococcus and Staphylococcus. Specific examples include, but are not limited to, bacteria from the following genera: Actinomyces, Bacillus, Clostridium, Corynebacterium, Enterococcus, Listeria, Nocardia, Propionibacterium, Rhodococcus, Staphylococcus, Streptococcus, and Viridans. Exemplary species include, but are not limited to, Streptococcus agalactiae, Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus equi, Clostridium tetani, Clostridium botulinum, Clostridium perfringes, Clostridium difficile, Bacillus anthracis, Listeria monocytogenes.

[0090] Compared to Gram-positive bacteria, Gram-negative bacteria lack this peptidoglycan layer. The pathogenicity of Gram-negative bacteria is often associated with certain components of the Gram-negative cell envelope, particularly the lipopolysaccharide layer (also known as the LPS or endotoxin layer). Specific examples include, but are not limited to, bacteria from the following genera: Acinetobacter, Bordetella, Brachyspira, Burkholderia, Brucella, Cardiobacterium, Citrobacter, Coxiella, Enterobacter, Escherichia, Fusobacterium, Haemophilus, Helicobacter, Klebsiella, Legionella, Moxarella, Morganella, Neisseria, Proteus, Pseudomonas, Rickettsia, Salmonella, Serratia, Spirochaeta, Vibrio. Exemplary species include, but are not limited to, Bordetella bronchiseptica, Brucella canis, Brucellssuis, Burkholderia mallei, Klebsiella pneumoniae, Serratia marcescens, and Enterobacter cloacae.

[0091] Parasites include, but are not limited to, Anaplasma, Fasciola hepatica, Coccidia, Eimeria spp, Neospora caninum, Toxoplasma gondii, Giardia, Dirofilaria, Ancylostoma, Trypanosoma spp., Leishmania spp., Trichomonas spp., Cryptosporidium parvum, Babesia, Schistosoma, Taenia, Strongyloides, Ascaris, Trichinella, Sarcocystis, Hammondia, and Isopsora, and combinations thereof. Also included are ectoparasites, including but not limited to ticks, including species of Ixodes, Rhipicephalus, Dermacentor, Amblyomma, Boophilus, Hyalomma, and Haemaphysalis, and combinations thereof.

[0092] Fungal pathogens include Candida, Microsporum, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis, Stachybotrys.

[0093] The following examples are presented as exemplary embodiments and should not be regarded as limiting the scope of the invention. Many variations, modifications, alterations, and other uses and applications of the invention will be apparent to those skilled in the art. Examples

[0094] Example 1: Protection by QCDC against Bordetella bronchiseptica challenge

[0095] Fifty Swiss outbred CF-1 mice were randomly divided into two groups of 15 mice each and one group of 20 mice. Fifteen mice, approximately 18 - 20 g, were inoculated with (It is a lecithin oil emulsion) QAC (Quil A and cholesterol (50 μg / 50 μg per dose), or aqueous QCDC (Quil A / cholesterol / DDA / were inoculated with an adjuvant preparation of 20 μg / 20 μg / 10 μg / 0.05%). Each mouse received two injections via the intraperitoneal route, 0.2 ml each time, with a two-week interval. The 20 mice were designated as non-inoculated controls. Two weeks after the second injection, all three groups of mice were intraperitoneally challenged with Bordetella bronchiseptica strain B133.

[0096] The challenge culture was prepared as follows: Briefly, the challenge organism was cultured on trypticase blood agar plates at 35 - 37 °C for about 24 hours. The plates were washed with peptone saline solution, and the culture was adjusted to a light transmittance of about 75% at 600 nm. Each mouse received approximately 8×10 7 colony-forming units of Bordetella bronchiseptica strain B133 via the intraperitoneal route. The number of surviving mice was confirmed 7 days after the challenge.

[0097] The survival results are provided in Table 1

[0098] Treatment (N) Surviving number Survival % at 7 days post - attack QAC(15) 1 6.7 QCDC(15) 10 66.7 Unvaccinated(20) 4 20.0

[0099] These results indicate that treatment with QCDC without specific antigen is sufficient to generate a protective response against Gram-negative bacteria in the Bordetella bronchiseptica model.

[0100] Example 2: QCDCRT reduces the viral titer of BHV-1 (Bovine herpesvirus 1) in the challenge of neonatal calves.

[0101] The effect of the preparation QCDCRT (described below) on the activation of the innate immune system was evaluated by challenging calves with BHV-1. Animals that had not previously received a vaccine containing BHV moieties were selected (10 animals per group). Eligible test animals were screened, and only those with a BHV serum neutralization titer of less than 1:2 were selected.

[0102] The animals were acclimated to the environment for approximately 5 - 7 days before the challenge. During the acclimation period, the animals were fed an antibiotic-free free-choice total mixed ration. Water was provided ad libitum. Before arrival, the animals received and

[0103] The treatment conditions are summarized in Table 2 below.

[0104] Table 2

[0105]

[0106] On day 0, a 2 ml volume was administered subcutaneously. Twelve hours after inoculation, an intranasal administration was performed for BHV-1 challenge (2 ml per nostril, 4 ml per animal). Before inoculation and at the time of challenge, blood samples and nasal swab samples were collected. In addition, additional nasal swabs were collected daily until day 14, and another blood sample was taken on day 14.

[0107] The amounts of IFN-α, MX-1, and OAS mRNA determined by qPCR. The results obtained are summarized in Figure 1 .

[0108] Twelve hours after treatment, the IFN-α level did not increase significantly. However, the levels of MX-1 and OAS mRNA increased in groups T02, T03, and T04, with T02 and T03 being the most effective. Since MX-1 and OAS are downstream of IFNα, it is possible that the mRNA level of IFNα had increased at an earlier time point.

[0109] The timeline of virus shedding is shown in Figure 2 . The differences between the control group (T01) and the treatment groups (T02 - T04) were observed on days 3 - 5. In group T01 (negative control) on day 4, shedding reached a peak of approximately 4x10 7 units, while in the treatment groups, it was approximately 1 - 1.5×10 7 units. By day 5, the differences between the control group and the treatment groups disappeared, and by day 9, there were no significant differences in the percentage of virus shedding. All groups showed clinical symptoms of the disease.

[0110] The present invention relates to the following embodiments:

[0111] 1. A method for inducing an immune response in a mammal or bird, comprising administering to the mammal or bird a composition substantially free of antigen, which comprises saponin, sterol, quaternary amine, and polyacrylic acid polymer.

[0112] 2. The method of embodiment 1, wherein the saponin is a triterpenoid saponin, the sterol is cholesterol, and the quaternary amine is DDA.

[0113] 3. The method of embodiment 2, wherein the triterpenoid saponin is Quil A or a purified part thereof.

[0114] 4. The method of any one of embodiments 1 - 3, wherein the saponin is present in an amount of 1 μg to about 5,000 μg per dose, the sterol is present in an amount of 1 μg to about 5,000 μg per dose, the quaternary amine is present in an amount of 1 μg to about 5,000 μg per dose, and the polyacrylic acid polymer is present in an amount of 0.0001% volume / volume (v / v) to about 75% v / v.

[0115] 5. The method according to any one of Embodiments 1-4, wherein the composition consists essentially of saponin, sterol, quaternary amine, and polyacrylic acid polymer.

[0116] 6. The method according to any one of Embodiments 1-4, wherein the composition further comprises glycolipid.

[0117] 7. The method according to Embodiment 6, wherein the glycolipid is N-(2-deoxy-2-L-leucylamino-β-D-glucopyranosyl)-N-octadecyllaurylamide or a salt thereof.

[0118] 8. The method according to any one of Embodiments 6 and 7, wherein the composition does not contain immunostimulatory oligonucleotides.

[0119] 9. The method according to any one of Embodiments 6-8, wherein the composition consists essentially of glycolipid, saponin, sterol, quaternary amine, and polyacrylic acid polymer.

[0120] 10. The method according to any one of Embodiments 6-9, wherein the glycolipid is present in an amount of 0.01 mg to about 10 mg per dose.

[0121] 11. The method according to any one of Embodiments 1-4, 6 or 7, wherein the composition further comprises immunostimulatory oligonucleotides.

[0122] 12. The method according to Embodiment 11, wherein the composition consists essentially of the immunostimulatory oligonucleotides, saponin, sterol, quaternary amine, and polyacrylic acid polymer.

[0123] 13. The method according to Embodiment 11, wherein the composition consists essentially of immunostimulatory oligonucleotides, glycolipid, saponin, sterol, quaternary amine, and polyacrylic acid polymer.

[0124] 14. The method according to any one of Embodiments 11-13, wherein the immunostimulatory oligonucleotides are present at a concentration of 20 μg to about 500 μg per ml.

[0125] 15. The method according to any one of Embodiments 1-14, wherein the immune response is an immunoprotective response to an infection.

[0126] 16. The method according to Embodiment 15, wherein the infection is a viral infection.

[0127] 17. The method according to Embodiment 15, wherein the infection is a bacterial infection.

[0128] 18. The method according to Embodiment 17, wherein the bacterial infection is caused by Gram-negative bacteria.

[0129] 19. The method according to Embodiment 18, wherein the Gram-negative bacteria is Bordetella bronchiseptica.

[0130] The method of any one of embodiments 1-10, wherein the composition is free of antigen.

[0131] All cited publications are hereby incorporated by reference in their entirety herein.

[0132] While the foregoing specification teaches the principles of the present invention and provides examples for illustrative purposes, those skilled in the art will understand that various changes in form and detail may be made by reading the present disclosure without departing from the true scope of the invention.

Claims

1. Use of a composition in the preparation of a medicament for inducing an immune response against Bordetella bronchiseptica in a non-human mammal, wherein the composition comprises Quil A, cholesterol, DDA and CARBOPOL®, and the composition does not contain an antigen specific to Bordetella bronchiseptica.

2. The use according to claim 1, wherein Quil A is present in an amount of 1 μg to 5,000 μg per dose, cholesterol is present in an amount of 1 μg to 5,000 μg per dose, DDA is present in an amount of 1 μg to 5,000 μg per dose, and CARBOPOL® is present in an amount of 0.0001% volume / volume (v / v) to 75% v / v.

3. The use according to claim 1 or 2, wherein the composition consists of Quil A, cholesterol, DDA and CARBOPOL®.

4. The use according to claim 1 or 2, wherein the composition further comprises the glycolipid N-(2-deoxy-2-L-leucylamino-β-D-glucopyranosyl)-N-octadecyllaurylamide or a salt thereof.

5. The use according to claim 4, wherein the glycolipid is BayR1005®.

6. The use according to claim 1 or 2, wherein the composition does not contain immunostimulatory oligonucleotides.

7. The use according to claim 1 or 2, wherein the composition consists of the glycolipid BayR1005®, Quil A, cholesterol, DDA and CARBOPOL®.

8. The use according to claim 4, wherein the glycolipid is present in an amount of 0.01 mg to 10 mg per dose.

9. The use according to claim 1 or 2, wherein the composition does not contain an antigen.

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