Adjuvant composition, pharmaceutical composition comprising the same, and method for preventing or treating disease caused by viral infection, bacterial infection, protozoal infection or cancer

Abstract

This invention discloses an adjuvant composition comprising: a saponin conjugate as shown in formula (I) or a pharmaceutically acceptable salt or hydrate thereof; and a Toll-like receptor (TLR) agonist, wherein formula (I) is as follows:

Description

[Technical Field]

[0001] This invention relates to a vaccine adjuvant, particularly an adjuvant composition, a pharmaceutical composition containing the adjuvant, and its use in the preparation of a medicament for the prevention or treatment of diseases caused by viral infections, bacterial infections, protozoan infections, or cancer. [Previous Technology]

[0002] Vaccination is one of the most effective and widely used strategies for preventing and combating infectious and certain non-infectious diseases. Challenges in vaccine development include the need to elicit humoral and cellular immunity, including pathogen-specific T-cell responses, both essential factors for effective vaccines against various diseases such as HIV, malaria, tuberculosis, and cancer. Adjuvants, which enhance immune responses to antigens and improve vaccine efficacy, are an important component of many vaccines. However, few adjuvants are sufficiently safe and clinically available to induce cellular immunity.

[0003] Saponin adjuvants have demonstrated excellent immunostimulatory properties and are renowned for their ability to induce strong humoral and cellular immune responses. Saponins used in vaccine adjuvants include saponins (Quillaja saponaria Molina), triterpenoid glycosides isolated from the Chilean soap bark tree. Four major triterpenoid glycosides have been isolated from the saponin extract and identified as QS-7, QS-17, QS-18, and QS-21 (saponin fractions-7, 17, 18, and 21). These saponins all share the same triterpenoid backbone quillaic acid, flanked by branched trisaccharides β-D-Gal-(1→2)-[β-D-Xyl-(1→3)]-β-D-GlcA at the 3-O positions. QS-21 also contains a fucose-linked 4-O-acyl stereochemically rich fatty acyl chain 1, consisting of a linear tetrasaccharide moiety β-D-Apif / Xylp-(1→3)-β-D-Xyl-(1→4)-α-L-Rha-(1→2)-β-D-Fuc at position 28-O and a fucose-linked 4-O-acyl stereochemically rich fatty acyl chain 1, wherein the ratio of apiose to xylose substituted moieties is 65:35.

[0004] QS-7, QS-17, QS-18 and QS-21 are water-soluble saponins, among which QS-21 has particularly good solubility and has been reported to bind with cholesterol. Compound Acyl chain R1 R2 R3 R4 QS-7 Ac β-D-Apif β-D-Glcp N / A α-L-Rhap QS-17 1 β-D-Apif β-D-Glcp α-L-Rhap H QS-18 1 β-D-Apif β-D-Glcp H H QS-21 api 1 β-D-Apif H H H QS-21 xyl β-D-Xylp Apif: apiofuranose, Xylp: xylopyranose, Glucp: glucopyranose, Rhap: rhamnopyranose

[0005] The excellent adjuvant activity of QS-21 has led to its use in hundreds of recent and ongoing vaccine clinical trials across numerous fields, including SARS-CoV-2, malaria, shingles, Alzheimer's disease, HIV-1, respiratory syncytial virus (RSV), melanoma, breast cancer, small cell lung cancer, and prostate cancer. In these studies, natural saponins are formulated into liposomes or micelles to reduce their toxicity. The most advanced formulation is AS01. GSK's shingles vaccine (Shingrix®) uses AS01 as an adjuvant. AS01 is formed by combining GSK's TLR4 agonist monophosphoryl lipid A (MPL) (purified from Salmonella minnesota) with QS-21 and forming the formulation as DOPC / cholesterol liposomes. MPL is formulated in DOPC / cholesterol liposomes, while QS-21 is prepared in an aqueous formulation and mixed with liposome MPL, which may result in at least a portion of QS-21 being encapsulated in the liposomes. As disclosed in U.S. Patent No. 7,939,084, in 2023, the Arexvy vaccine (an adjuvanted respiratory tract fusion virus vaccine) was approved for the prevention of lower respiratory tract disease (LRTD) caused by RSV in people aged 60 years and older. This was the world's first approved RSV vaccine for the elderly, and it also included the AS01 adjuvant, albeit at a lower dose. The clinical report of this vaccine was published in the Journal of Infectious Diseases, 2024 Jul 15;230(1): e102-e110. While the combination of these two natural products clearly produces a highly immunogenic and effective adjuvant, it faces challenges in production, large-scale manufacturing, and reactivity (particularly injection site pain). For healthy (non-cancer) adults and children, the highest practically tolerated dose is 25-50 micrograms, which is an immunologically suboptimal dose. Therefore, the clinical success of a vaccine still hinges on identifying and acquiring novel, more tolerable, and potent adjuvants.

[0006] AFLQ is also a QS-21-based adjuvant system, which includes monophospholipid A, as disclosed in U.S. Patent No. 10,434,167.

[0007] Another adjuvant system based on QS-21, AS15, which contains CpG 7909 (a TLR9 agonist), is currently in the clinical trial stage. The relevant research has been published in the Journal of Applied Toxicology, 36(2), 238-256.

[0008] Another QS-21-based adjuvant system, such as TQL-1055, in combination with a TLR agonist, is disclosed in U.S. Patent No. 11,324,821B2. This system was also published in the journal Human Vaccines & Immunotherapeutics on January 8, 2024; 20(1):2302070.

[0009] The widespread use of QS adjuvants is limited by their dose-limiting toxicity, insufficient stability, unclear molecular mechanism of action, and procurement difficulties, as revealed in this journal (Expert Rev Vaccines. 2011 Apr;10(4):463–470). The availability of QS-21 relies on bark extracts, which have low yields, thus facing the problem of unsustainable supply / procurement. While the isolation of natural products can be scaled up, variations in the source, location, or growth process of starting materials, or changes in the location of natural product isolation, can lead to unacceptable variations in the final product. This results in the inability to produce adjuvant components at multiple locations or overcome the shortage of starting material suppliers, thus creating challenges to production and scalability. The synthetic saponin (Synsap) disclosed in this invention provides a sustainable saponin adjuvant option and offers several advantages, such as well-defined structure, high purity, and scalability. More importantly, synthetic saponins have a clearer structure-activity relationship compared to natural saponins with multiple pharmacological / toxicological activities. Pure synthetic saponins can produce a more selective mechanism of action, making the occurrence of side effects more controllable. When adjuvants with different mechanisms of immune enhancement are combined, there is a greater opportunity to design adjuvant systems that can provide a wide range of vaccine applications, especially in terms of their efficacy or antigen activation across different age groups, thus leading to a more favorable range of applications. [Summary of the Invention]

[0010] This invention provides an adjuvant composition comprising a synthetic saponin (Synsap) of formula (I) or a pharmaceutically acceptable salt or solvate thereof, and a Toll-like receptor (TLR) agonist to produce a beneficial and / or synergistic effect. This invention also provides pharmaceutical compositions comprising these adjuvant compositions, and the use of the pharmaceutical composition in the preparation of a medicament for the prevention or treatment of diseases caused by viral infections, bacterial infections, protozoan infections, or cancer.

[0011] In one aspect, the present invention provides an adjuvant composition comprising: (i) a saponin conjugate or a pharmaceutically acceptable salt or solvate thereof as shown in formula (I): (I), wherein is a single or double bond; W is Me, -CHO, -CH2ORx or -C(O)Ry; wherein Rx is independently hydrogen or an oxygen protecting group selected from the group consisting of alkyl ethers, allyl ethers, benzyl ethers, silyl ethers, acetals, ketals, esters, carbamates and carbonates, and Ry is selected from the group consisting of alkyl, allyl, benzyl, silyl, alkoxy and alkyl carboxylates; V is H or -OH; Y is CH2, -O-, -S-, -NR- or -NH-; Q is CH2, C=O, C=N-OH or C=N-OMe; X Is CH2, —O—, —NH—, —NH—(C=O)—, —(C=O)—NH—, —S—, —(C=O)—O— or —O—(C=O)—; R is a portion selected from the group consisting of aryl, arylaliphatic, cycloaliphatic, heteroarylaliphatic, alkoxy-aliphatic, and aryloxy-aliphatic, or a portion selected from the group consisting of: unsubstituted 5-10 arylaliphatic, substituted 5-10 arylaliphatic, wherein the substituted 5-10 arylaliphatic is substituted by at least one group selected from the group consisting of: alkyl, alkoxy, aryloxy, halogen, haloalkyl, hydroxyl, saturated heterocyclic group having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, sulfur, and combinations thereof; and 4-10 heteroarylaliphatic having 1-4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, sulfur, and combinations thereof, or having the following structures: , , or, wherein Rz is alkyl, Xa is NH or S, and n = 1-20; R1 is independently hydrogen, an oxygen-protecting group selected from the group consisting of alkyl ethers, allyl ethers, benzyl ethers, silyl ethers, acetals, ketals, esters, carbamates, and carbonates, or a carbohydrate having a monosaccharide structure; and Z is hydrogen, or a straight-chain or branched oligosaccharide selectively substituted with at least one group selected from the group consisting of amines, amides, amides, aralkyls, aryls, heteroaryls, aliphatic, heteroaliphatic, cycloaliphatic, and heterocyclic rings; and (ii) a Toll-like receptor (TLR) agonist.

[0012] According to an embodiment of the present invention, Z is a linear tetrasaccharide or trisaccharide, and the first sugar residue is directly linked to Y.

[0013] According to an embodiment of the present invention, Q is C=O and X is —NH—.

[0014] According to an embodiment of the present invention, W is -CHO and V is -OH.

[0015] According to an embodiment of the present invention, R1 is -H.

[0016] According to an embodiment of the present invention, R is selected from the group consisting of: , wherein Rz is an alkyl group.

[0017] According to an embodiment of the present invention, the saponin conjugate is selected from the group consisting of: (i) compounds represented by formula I-1: (I-1), wherein G1 is 10-((2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-((8-(4-methoxyphenyl)octyl)aminomethyl)tetrahydro-2H-pyran-2-yl), 10-((2R,3R,4S,5S,6S)-6-((8-(4-fluorophenyl)octyl)aminomethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl), 10-((2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-((8-(4-phenoxyphenyl)octyl)aminomethyl)tetrahydro-2H-pyran-2-yl), 10-((2R,3R,4S,5S,6S)-6-((8-(4-(4-fluorophenoxy)phenyl)octyl)aminomethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl), 10-((2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-((8-(4-morpholinophenyl)octyl)aminomethyl)tetrahydro-2H-pyran-2-yl) or 10-((2R,3R,4S,5S,6S)-6-((8-(furan-2-yl)octyl)aminomethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl); (ii) Compounds represented by formula I-2: (I-2). Where G2 is 10-((2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-((8-(4-methoxyphenyl)octyl)aminomethyl)tetrahydro-2H-pyran-2-yl), 10-((2R,3R,4S,5S,6S)-6-((8-(4-fluorophenyl)octyl)aminomethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl), 1 0-((2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-((8-(4-phenoxyphenyl)octyl)aminomethyl)tetrahydro-2H-pyran-2-yl) or 10-((2R,3R,4S,5S,6S)-6-((8-(4-(4-fluorophenoxyphenyl)octyl)aminomethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl);(iii) Compounds represented by formula I-3: (I-3), wherein G3 is 10-((2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-((8-(4-methoxyphenyl)octyl)aminomethyl)tetrahydro-2H-pyran-2-yl), 10-((2R,3R,4S,5S,6S)-6-((8-(4-fluorophenyl)octyl)aminomethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl), 1 0-((2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-((8-(4-phenoxyphenyl)octyl)aminomethyl)tetrahydro-2H-pyran-2-yl) or 10-((2R,3R,4S,5S,6S)-6-((8-(4-(4-fluorophenoxyphenyl)octyl)aminomethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl); (iv) compounds represented by formula I-4: (I-4). Where G4 is 10-((2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-((8-(4-methoxyphenyl)octyl)aminomethyl)tetrahydro-2H-pyran-2-yl), 10-((2R,3R,4S,5S,6S)-6-((8-(4-fluorophenyl)octyl)aminomethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl), 10-(2R,3R,4 ... (5S,6S)-3,4,5-trihydroxy-6-((8-(4-phenoxyphenyl)octyl)aminomethyl)tetrahydro-2H-pyran-2-yl) or 10-((2R,3R,4S,5S,6S)-6-((8-(4-(4-fluorophenoxyphenyl)octyl)aminomethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl); and (v) compounds represented by formula I-5: (I-5), wherein Rg is 4-methoxyphenyloctyl, 4-fluorophenyloctyl, 4-phenoxyphenyloctyl or (4-fluorophenoxy)phenyl)octyl.

[0018] According to an embodiment of the present invention, the Toll-like receptor (TLR) agonist is derived from the group consisting of TLR2 agonists, TLR3 agonists, TLR4 agonists, TLR7 agonists, TLR8 agonists and TLR9 agonists.

[0019] According to an embodiment of the present invention, the Toll-like receptor (TLR) agonist is a TLR4 agonist.

[0020] According to an embodiment of the present invention, the TLR4 agonist is selected from the group consisting of PHAD, MPLA, 3D-PHAD, 3D-6A-PHAD, EcML, CRX-527, GSK1795091, E6020, GLA, SLA and combinations thereof.

[0021] According to an embodiment of the present invention, the adjuvant composition further comprises a liposome-forming compound or an emulsion-containing compound.

[0022] According to an embodiment of the present invention, the liposome-forming compound is selected from the group consisting of phospholipids, cholesterol, and combinations thereof, such as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), dioleoyl phosphatidylglycerol (DOPG), dimyristoyl phosphatidylglycerol (DMPG), 1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP), cholesterol, or combinations thereof.

[0023] According to an embodiment of the present invention, the emulsion-containing compound is selected from the group consisting of polysorbate, α-tocopherol, Span 85, glycerol, poloxamer 188 and carboxymethyl cellulose.

[0024] According to an embodiment of the present invention, the adjuvant composition is in the form of an aqueous solution, an oil / water emulsion, or encapsulated in liposomes.

[0025] According to an embodiment of the present invention, the particle size of the adjuvant composition is less than 250 nanometers (nm).

[0026] According to an embodiment of the present invention, the adjuvant composition further comprises squalene.

[0027] On the other hand, the present invention also provides a vaccine composition comprising: an antigen and an adjuvant composition as described above, and optionally a pharmaceutically acceptable excipient.

[0028] On the other hand, the present invention also provides a pharmaceutical composition comprising: an antigen and an adjuvant composition as described above.

[0029] According to an embodiment of the present invention, the antigen is selected from one or more of the group consisting of bacteria, viruses, protozoa and tumor-associated antigens.

[0030] On the other hand, the present invention also provides a method for preventing or treating diseases caused by viral infection, bacterial infection, protozoan infection or cancer, comprising the step of administering the pharmaceutical composition as described above to an individual in need.

[0031] On the other hand, the present invention also provides an adjuvant composition that can induce humoral and cellular immune responses.

[0032] In another aspect, the present invention provides a pharmaceutical composition comprising an antigen, an adjuvant composition of the present invention, and optionally a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is a vaccine comprising the antigen and the adjuvant composition of the present invention.

[0033] In another aspect, the present invention provides formulations comprising saponin derivatives, their salt forms, or their solvates. In specific embodiments, the solvates of the formulations may include water, alcohols (including but not limited to methanol, ethanol, butanol, etc.), polyols (including but not limited to glycerol, propylene glycol, polyethylene glycol, etc.), N-methyl-2-pyrrolidone and suitable mixtures thereof, as well as vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.

[0034] In another aspect, the present invention provides formulations of compositions according to the invention in adjuvant systems. In some embodiments, the adjuvant system utilizes a carrier. In some embodiments, the carrier is a particulate carrier, such as metal salt particles, emulsions (e.g., oil-in-water emulsions), polymers, liposomes, or immunostimulating complexes (ISCOM).

[0035] In another aspect, the present invention provides a method for enhancing an immune response to an antigen, comprising administering to a subject an effective amount of a provided vaccine to enhance the subject’s immune response to the antigen.

[0036] In another embodiment, the present invention provides a method for stimulating or enhancing the production of cytokines in a subject, the method particularly comprising administering to the subject any pharmaceutical composition or combination according to the present invention, thereby causing immune cells to secrete cytokines.

[0037] In another aspect, the present invention provides a method for administering a vaccine to a subject, comprising administering the provided vaccine to the subject. In some embodiments, the subject is a human. In some embodiments, the vaccine is administered orally. In other embodiments, the vaccine is administered intramuscularly. In other embodiments, the vaccine is administered subcutaneously. In some embodiments, the antigen administered to the subject may be a cancer, bacterial, viral, protozoan, or autoantigen.

[0038] In another aspect, the present invention provides a kit comprising the pharmaceutical composition or adjuvant composition of the present invention. In some embodiments, the kit includes prescription information. In some embodiments, such a kit includes a combination of the adjuvant composition of the present invention with other immunotherapeutic agents (e.g., vaccines, antibodies). These agents may be packaged individually or together. The kit may also optionally include prescription instructions for the drugs. In some embodiments, the kit includes multiple doses of each agent. The kit may include sufficient quantities of each component to treat a subject for one week, two weeks, three weeks, four weeks, or several months.

[0039] In some embodiments, the kit includes one cycle of immunotherapy. In some embodiments, the kit includes a sufficient amount of pharmaceutical composition to induce long-term immunity against the antigen in the subject.

Implementation Method

[0041] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although similar or equivalent methods and materials to those described herein may be used in practicing or testing embodiments of the invention, exemplary methods and / or materials are described below. In case of any conflict, the patent specification (including definitions) shall prevail. Furthermore, the materials, methods, and examples are for illustrative purposes only and are not intended to be necessarily limiting.

[0042] An adjuvant composition according to an embodiment of the present invention comprises:

[0043] (i) A saponin conjugate or its pharmaceutically acceptable salt or solvate as shown in formula (I): (I), wherein is a single or double bond; W is Me, —CHO, —CH2ORx or —C(O)Ry; wherein Rx is independently hydrogen or an oxygen protecting group selected from the group consisting of alkyl ethers, allyl ethers, benzyl ethers, silyl ethers, acetals, ketals, esters, carbamates and carbonates, and Ry is selected from the group consisting of alkyl, allyl, benzyl, silyl, alkoxy and alkyl carboxylates; V is H or —OH; Y is CH2, —O—, —S—, —NR— or —NH—; Q is CH2, C=O, C=N—OH or C=N—OMe; X is CH2, —O—, —NH—, —NH—(C=O)—, —(C=O)—NH—, —S—, —(C=O)—O— Or —O—(C=O)—; R is a portion selected from the group consisting of aryl, arylaliphatic, cycloaliphatic, heteroarylaliphatic, alkoxy-aliphatic and aryloxy-aliphatic, or a portion selected from the group consisting of: unsubstituted 5-10 arylaliphatic, substituted 5-10 arylaliphatic, wherein the substituted 5-10 arylaliphatic is substituted by at least one group selected from the group consisting of: alkyl, alkoxy, aryloxy, halogen, haloalkyl, hydroxyl, saturated heterocyclic group having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, sulfur and combinations thereof; and 4-10 heteroarylaliphatic having 1-4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, sulfur and combinations thereof, or having the following structures: , , or, wherein Rz is alkyl, Xa is NH or S, n=1-20; R1 is independently hydrogen, an oxygen-protecting group selected from the group consisting of alkyl ethers, allyl ethers, benzyl ethers, silyl ethers, acetals, ketals, esters, carbamates, and carbonates, or a carbohydrate having a monosaccharide structure; and Z is hydrogen, or a straight-chain or branched oligosaccharide selectively substituted with at least one group selected from the group consisting of amines, amides, amides, aralkyls, aryls, heteroaryls, aliphatic, heteroaliphatic, cycloaliphatic, and heterocyclic rings; and

[0044] (ii) Toll-like receptor (TLR) agonists.

[0045] In one embodiment, Z is a linear tetrasaccharide or trisaccharide, and the first sugar residue is directly linked to Y.

[0046] In one embodiment, Q is C=O and X is —NH—.

[0047] In one embodiment, W is -CHO and V is -OH.

[0048] In one embodiment, R1 is -H.

[0049] In one embodiment, R is selected from the group consisting of: , where Rz is an alkyl group.

[0050] In one embodiment, the saponin conjugate is selected from the group consisting of:

[0051] (i) The compound represented by formula I-1: (I-1),

[0052] wherein G1 is 10-((2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-((8-(4-methoxyphenyl)octyl)aminomethyl)tetrahydro-2H-pyran-2-yl), 10-((2R,3R,4S,5S,6S)-6-((8-(4-fluorophenyl)octyl)aminomethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl), 10-((2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-((8-(4-phenoxyphenyl)octyl)aminomethyl)tetrahydro-2H-pyran-2-yl), ), 10-((2R,3R,4S,5S,6S)-6-((8-(4-(4-fluorophenoxy)phenyl)octyl)aminomethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl), 10-((2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-((8-(4-morpholinophenyl)octyl)aminomethyl)tetrahydro-2H-pyran-2-yl) or 10-((2R,3R,4S,5S,6S)-6-((8-(furan-2-yl)octyl)aminomethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl);

[0053] (ii) The compound represented by formula I-2: (I-2),

[0054] wherein G2 is 10-((2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-((8-(4-methoxyphenyl)octyl)aminomethyl)tetrahydro-2H-pyran-2-yl) or 10-((2R,3R,4S,5S,6S)-6-((8-(4-fluorophenyl)octyl)aminomethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl) ), 10-((2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-((8-(4-phenoxyphenyl)octyl)aminomethyl)tetrahydro-2H-pyran-2-yl) or 10-((2R,3R,4S,5S,6S)-6-((8-(4-(4-fluorophenoxyphenyl)octyl)aminomethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl);

[0055] (iii)The compound represented by formula I-3: (I-3),

[0056] wherein G3 is 10-((2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-((8-(4-methoxyphenyl)octyl)aminomethyl)tetrahydro-2H-pyran-2-yl) or 10-((2R,3R,4S,5S,6S)-6-((8-(4-fluorophenyl)octyl)aminomethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl) ), 10-((2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-((8-(4-phenoxyphenyl)octyl)aminomethyl)tetrahydro-2H-pyran-2-yl) or 10-((2R,3R,4S,5S,6S)-6-((8-(4-(4-fluorophenoxyphenyl)octyl)aminomethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl);

[0057] (iv) The compound represented by formula I-4: (I-4),

[0058] wherein G4 is 10-((2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-((8-(4-methoxyphenyl)octyl)aminomethyl)tetrahydro-2H-pyran-2-yl) or 10-((2R,3R,4S,5S,6S)-6-((8-(4-fluorophenyl)octyl)aminomethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl) 10-((2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-((8-(4-phenoxyphenyl)octyl)aminomethyl)tetrahydro-2H-pyran-2-yl) or 10-((2R,3R,4S,5S,6S)-6-((8-(4-(4-fluorophenoxyphenyl)octyl)aminomethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl); and

[0059] (v) The compound represented by formula I-5: (I-5),

[0060] wherein Rg is 4-methoxyphenyl octyl, 4-fluorophenyl octyl, 4-phenoxyphenyl octyl or (4-fluorophenoxy)phenyl)octyl.

[0061] In one embodiment, the TLR agonist is selected from the group consisting of TLR2 agonists, TLR3 agonists, TLR4 agonists, TLR7 agonists, TLR8 agonists and TLR9 agonists.

[0062] In one embodiment, the TLR agonist is a TLR4 agonist.

[0063] In one embodiment, the TLR4 agonist is selected from the group consisting of PHAD, MPLA, 3D-PHAD, 3D-6A-PHAD, EcML, CRX-527, GSK1795091, E6020, GLA, SLA and combinations thereof.

[0064] In one embodiment, the adjuvant composition of the present invention further comprises a liposome-forming compound or an emulsion-containing compound.

[0065] In one embodiment, the emulsion-containing compound is selected from compounds in the group consisting of polysorbate, α-tocopherol, Span 85, glycerol, poloxamer 188 and carboxymethyl cellulose.

[0066] The liposome-forming compound is selected from the group consisting of phospholipids, cholesterol, and combinations thereof, such as: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), dioleoyl phosphatidylglycerol (DOPG), dimyristoyl phosphatidylglycerol (DMPG), and 1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP).

[0067] In one embodiment, the adjuvant composition is in the form of an aqueous solution, an oil-in-water emulsion, or encapsulated in liposomes.

[0068] In one embodiment, the adjuvant composition has a particle size of less than 250 nanometers. Preferably, the adjuvant composition has a particle size of 50 to 200 nm.

[0069] In one embodiment, the adjuvant composition further comprises squalene.

[0070] A pharmaceutical composition according to another embodiment of the present invention comprises: an antigen and an adjuvant composition as described above.

[0071] In some embodiments, the pharmaceutical composition is a vaccine.

[0072] In some embodiments, the antigen is selected from one or more of the group consisting of bacteria, viruses, protozoa and tumor-associated antigens.

[0073] According to another embodiment of the present invention, a method for preventing or treating diseases caused by viral infection, bacterial infection, protozoan infection or cancer is provided, comprising the step of administering the pharmaceutical composition as described above to a subject in need.

[0075] Toll-like receptor (TLR) agonists as adjuvants

[0076] TLRs are transmembrane pattern recognition receptors that can detect invading pathogens and initiate innate and adaptive immune responses. Different TLRs are expressed in different subsets of immune and non-immune cell types, including macrophages, dendritic cells, B cells, T cells, fibroblasts, and epithelial cells.

[0077] Human TLR2 is expressed on the cell membrane surface in a heterodimer form, particularly in various immune cell types, including neutrophils, macrophages, monocytes, basophils, T cells, B cells, natural killer (NK) cells, and immature dendritic cells (DCs). TLR2 exists in two distinct conformations: TLR2 / 6 and TLR2 / 1. The involvement of TLR2 / TLR1 agonists (including Pam3CysSerLys4 (Pam3CSK4) and its derivatives) plays a crucial role in enhancing B cell responses and inducing T cell stimulation. This underscores their effectiveness in a range of experimental vaccine models.

[0078] In some embodiments, exemplary TLR2 agonists are described as follows: TLR2 agonists structure Pam3CysSerLys4 CU-T12-9 FSL-1

[0079] TLR3 is located within the endosome and recognizes dsRNA produced during viral replication within infected cells. TLR3 is unique among known TLRs because it signals to activate NF-κB-regulated gene expression. This signaling cascade leads to T-helper 1 (Th1) polarized immune responses, thereby providing protection against viral infection, making TLR3 agonists attractive adjuvant candidates for vaccines against intracellular pathogens. dsRNA mimics poly(I:C) to induce NF-κB activation and, through TLR3 ligation, leads to IFN-β induction.

[0080] In some embodiments, exemplary TLR3 agonists are described as follows: TLR3 agonists structure Poly(I:C) NexaVant Novel dsRNA TLR3 agonist (275 kDa)

[0081] TLR4 agonists are recognized for their innate immunostimulatory capabilities, initiating a cascade of signal transduction events that ultimately activate the adaptive immune system. Common TLR4 agonists include biological monophosphoryl lipid A (MPLA) derivatives, such as EcML, and synthetic MPLA-like structures, such as PHAD and CRX-527. Combination vaccine adjuvants consisting of TLR4 agonists and saponins have the potential to elicit stronger, more targeted, and more balanced immune responses.

[0082] In some embodiments, exemplary TLR4 agonists are described as follows: TLR4 agonists structure MPLA PHAD 3D-PHAD 3D-6A-PHAD EcML CRX-527 GSK1795091 (CRX-601) E6020 GLA SLA 2002

[0083] TLR7 / 8 agonists (e.g., imiquimod and resiquimod) have been approved by the FDA for use as a single agent. Imiquimod is approved for topical administration to treat basal cell carcinoma in immunocompetent adults and has been shown to be effective in treating non-genital warts, molluscum contagiosum, squamous cell carcinoma, and malignant freckles caused by human papillomavirus (HPV). As predicted by TLR7 / 8 agonists, imiquimod stimulates local induction of IFN-α, TNF, IL-6, and IL-12 at the administration site and induces cytotoxic T cell responses.

[0084] In some embodiments, exemplary TLR7 / 8 agonists are described as follows: TLR7 / 8 agonist structure Resiquimod (R848) Imiquimod (R837) CL264 Gardiquimod CL075 CL097 TL8-506 Telratolimod (3M-052) GSK2245035 AZD8848 PF-4878691

[0085] TLR9 is a pattern recognition receptor primarily located within immune cells, such as dendritic cells, macrophages, natural killer cells, and other antigen-presenting cells (APCs). The TLR9 agonists disclosed in this article can stimulate an inflammatory cascade, promoting the phagocytosis and clearance of microorganisms and cancer cells, while simultaneously triggering an adaptive immune response. Several TLR9 agonists have been approved or are in clinical trials. For example, CpG 1018 is approved for use with hepatitis B virus (HBV) and COVID vaccines, and CpG 7909 is approved for applications such as cancer treatment and HBV vaccine trials.

[0086] In some embodiments, exemplary TLR9 agonists are described as follows: TLR9 agonist sequence ODN 1585 5'-ggGGTCAACGTTGAgggggg-3' (SEQ ID NO: 1) CpG 1018 5′-TGACTGTGAACGTTCGAGATGA-3′ (SEQ ID NO: 2) ODN 1668 5'-TCCATGACGTTTCCTGATGCT-3' (SEQ ID NO: 3) ODN 1826 5′-TCCATGACGTTTCCTGACGTT-3′ (SEQ ID NO: 4) ODN 2395 5′-TCGTCGTTTTCGGCGCGCGCCG-3′ (SEQ ID NO: 5) ODN 2006 (ODN 7909) 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 6) ODN 2007 5'-CGTCGTTGTCGTTTTTGTCGTT-3' (SEQ ID NO: 7) ODN 10101 5'-TCGTCGTTTCGCGCGCGCCG-3' (SEQ ID NO: 8) ODN D-SL01 5'-TCGCGACGTTCGCCCGACGTTCGGTA-3' (SEQ ID NO: 9) ODN D-SL03 5'-TCGCGAACGTTCCGCCGCGTTCGAACGCGG-3' (SEQ ID NO: 10)

[0087] In some embodiments, the Toll-like receptor (TLR) agonist used in the adjuvant composition disclosed herein is selected from the TLR2 agonists, TLR3 agonists, TLR4 agonists, TLR7 / 8 agonists or TLR9 agonists listed in the table above.

[0088] Fish oil as an adjuvant

[0089] Fish oil (including squalene and squalane) is readily available from commercial sources or can be obtained by methods known in the art. Fish contain oils that are easily extracted and utilized. Examples of such oils include cod liver oil, shark liver oil, and whale oil, which can be used as adjuvants in this disclosure. In some embodiments, squalene and squalane (saturated forms of squalene) can be used, with squalene being preferred. Squalene and squalane are commercially available or can be obtained by methods known in the art.

[0090] In some embodiments, squalene or squalane may be used in the form of an oil / water emulsion. The emulsion may contain one or more additional oils. The additional oils may be tocopherols, such as α-, β-, γ-, δ-, ε-, or ξ-tocopherol, with α-tocopherol being preferred. In some embodiments, D-α-tocopherol and / or DL-α-tocopherol may be used, with DL-α-tocopherol being preferred.

[0091] Vaccine adjuvant

[0092] Despite the promising outlook for FDA-approved adjuvants, there remains a need for novel adjuvant systems capable of enhancing vaccine immunogenicity and durability while reducing potential side effects. Furthermore, such novel adjuvant systems exhibit good compatibility with a variety of active ingredients. This disclosure reveals novel adjuvant compositions that achieve a delicate balance between enhancing vaccine efficacy and ensuring safety to meet this need.

[0093] The inventors have discovered that co-formulation with a Toll-like receptor (TLR) agonist can enhance the immunostimulatory activity of the saponin conjugate of Formula I or a pharmaceutically acceptable salt or solvation thereof. Therefore, this disclosure provides a novel adjuvant composition (i.e., a novel adjuvant system) comprising (i) a saponin conjugate of Formula I or a pharmaceutically acceptable salt or solvation thereof, and (ii) a Toll-like receptor (TLR) agonist, which imparts a favorable and / or synergistic effect.

[0094] In some embodiments, the pharmaceutical combinations disclosed herein include a saponin conjugate of formula I or a pharmaceutically acceptable salt or solvate thereof and a Toll-like receptor (TLR) agonist.

[0095] In some embodiments, the weight ratio of the Toll-like receptor (TLR) agonist to the saponin conjugate ranges from about 10:1 to about 1:100.

[0096] In some embodiments, the saponin conjugates disclosed herein, or their pharmaceutically acceptable salts or solvates, can be dissolved by a solubilizer to increase solubility. The solubilizer can be used alone or in combination in appropriate proportions. In this disclosure, suitable solubilizers include, but are not limited to: citric acid, hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium stearyl fumarate, methacrylic acid copolymer LD, methylcellulose, sodium dodecyl sulfate, polyoxyethylene 40 stearate, purified shellac, sodium dehydroacetate, polysorbate 20, polysorbate 80, fumaric acid, sodium DL-hydroacetate, polysorbate 20, polysorbate 80, fumaric acid, DL-malic acid, L-ascorbyl stearate, L-asparagine acid, adipic acid, aminoalkyl methacrylate copolymer E, and propylene glycol alginate. alginate), casein, sodium casein, carboxyvinyl polymer, carboxymethyl ethyl cellulose, agar powder, guar gum, succinic acid, copolyvidone, cellulose acetate phthalate, tartaric acid, dioctylsodium sulfosuccinate, zein, skim milk powder, sorbitan trioleate, lactic acid, aluminum lactate, ascorbyl palmitate, hydroxyethyl methyl cellulose, hydroxypropyl methylcellulose acetate succinate, polyoxyethylene (105) polyoxypropylene (5) glycol, polyoxyethylene hydrogenated castor oil 60, polyoxyethylene 35 castor oil, poly(4-styrene sulfonate) sodium(poly(sodium) 4-styrenesulfonate), polyvinyl acetal diethylamino acetateAcetate), polyvinyl alcohol, maleic acid, methacrylic acid copolymer S, lauromacrogol, sulfuric acid, aluminum sulfate, phosphoric acid, calcium dihydrogen phosphate, sodium dodecylbenzenesulfonate, vinyl pyrrolidone-vinyl acetate copolymer, sodium lauroyl sarcosinate, acetyl tryptophan, sodium methyl sulfate, sodium ethyl sulfate, sodium butyl sulfate, sodium octyl sulfate, sodium decyl sulfate, sodium tetradecyl sulfate, sodium hexadecyl sulfate, and sodium octadecyl sulfate.

[0097] In some embodiments, a saponin conjugate of Formula I or a pharmaceutically acceptable salt or solvate thereof and a Toll-like receptor (TLR) agonist are encapsulated in liposomes. In some embodiments, one or more compounds forming liposomes may be used to prepare liposomes. The compounds that form liposomes may be selected from the group consisting of phospholipids, such as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), dioleoyl phosphatidylglycerol (DOPG), dimyristoyl phosphatidylglycerol (DMPG), 1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP), cholesterol, or combinations thereof.

[0098] In some embodiments, the saponin conjugate of Formula I or a pharmaceutically acceptable salt or solvate thereof is in the form of an oil / water emulsion. In some embodiments, one or more compounds comprising an emulsion may improve the stability and immunostimulatory properties of the adjuvant composition. The emulsion may be selected from compounds selected from the group consisting of polysorbate, α-tocopherol, Span 85, glycerol, poloxamer 188, and carboxymethyl cellulose.

[0099] Although the specific mechanism of action of adjuvants is not fully understood, their involvement with dendritic cells has been reported. In some embodiments, to enhance adjuvant uptake by dendritic cells, the resulting liposomes may have a particle size of less than 250 nm (e.g., 220 nm, 190 nm, 180 nm, 170 nm, 160 nm, 150 nm, 120 nm, 100 nm or smaller). In some embodiments, the lipid composition (including phospholipids, cholesterol, and combinations thereof) is used to encapsulate saponins and Toll-like receptor (TLR) agonists. In some embodiments, the particle size of the liposomes can be analyzed using transmission electron microscopy (TEM) and / or Malvern's Zetasizer Nano.

[0100] Pharmaceutical composition comprising a vaccine composition

[0101] This disclosure also provides pharmaceutical compositions comprising an antigen and an adjuvant composition of the present invention, wherein the adjuvant composition comprises a combination of Synsap or a pharmaceutically acceptable salt or solvation thereof with a Toll-like receptor (TLR) agonist. In some embodiments, the pharmaceutical composition is a vaccine composition. Any animal that may benefit from the compositions of this disclosure is within the scope of objects to be treated or administered the compositions or combinations of this disclosure. In some embodiments, the object is a mammal. In some embodiments, the object is a human.

[0102] In some embodiments, the amount of antigen in the pharmaceutical composition may vary depending on the type of antigen, etc. In some embodiments, each dose may contain 1 to 1000 μg of antigen, for example, 1 μg, 2 μg, 5 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 40 μg, 50 μg, 60 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 800 μg, or 1000 μg. However, this disclosure is not limited thereto.

[0103] In some embodiments, the pharmaceutical compositions disclosed herein may also contain pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical compositions disclosed herein may also contain one or more wetting agents, emulsifiers, and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as colorants, release agents, coating agents, sweeteners and flavorings, preservatives, and antioxidants.

[0104] Vaccines (or their derived antiserum) may be administered for both "preventive" and "therapeutic" purposes. Prophylactic vaccination is intended to prevent or reduce the subsequent manifestations of disease. When used for prevention, the vaccine is administered before any symptoms of disease appear. When used for treatment, the vaccine is administered when or after symptoms indicating that the animal may be infected with a pathogen or has a certain type of cancer are detected. The therapeutic effect of a vaccine is to reduce any actual manifestations of disease. Therefore, vaccines may be administered before or after the actual spread of disease begins.

[0105] Therefore, in one aspect, this disclosure provides a vaccine comprising a combination of one or more bacterial, viral, protozoan, or tumor-associated antigens with an adjuvant of the invention disclosed herein. In some embodiments, the vaccine comprises a combination of a single bacterial, protozoan, viral, or tumor-associated antigen with an adjuvant of the invention disclosed herein.

[0106] In some embodiments, one or more antigens in the provided vaccine are bacterial antigens. In some embodiments, the bacterial antigen is a bacterial-associated antigen selected from *Bordetella pertussis*, *Bordetella parapertussis*, *Bordetella bronchiseptica*, *Borrelia burgdorferi*, *Borrelia spp.*, *Chlamydia trachomatis*, *Helicobacter pylori*, *Chlamydia pneumoniae*, *Ureaplasma urealyticum*, *Mycoplasma pneumoniae*, *Staphylococcus spp.*, *Staphylococcus aureus*, *Streptococcus pyogenes*, and *Streptococcus*. Streptococcus pneumoniae (spp.), Streptococcus pneumoniae, Streptococcus viridans, Enterococcus faecalis, Neisseria meningitidis, Neisseria gonorrhoeae, Bacillus anthracis, Salmonella spp., Salmonella typhi, Vibrio cholera, Pasteurella pestis, Campylobacter spp., Campylobacter jejuni, Clostridium spp., Clostridium difficile, Corynebacterium diphtheriae, Mycobacterium spp.This group comprises Mycobacterium tuberculosis, Pseudomonas aeruginosa, *Treponema* spp., *Leptospria* spp., *Haemophilus ducreyi*, *Haemophilus influenzae*, *Escherichia coli*, *Shigella* spp., *Ehrlichia* spp., *Rickettsia* spp., and combinations thereof.

[0107] In some embodiments, one or more antigens in the provided vaccine are virus-associated antigens. In some embodiments, the virus-associated antigen is a virus-related antigen selected from influenza virus, parainfluenza virus, mumps virus, adenovirus, respiratory syncytial virus, Epstein-Barr virus (EBV), rhinovirus, poliovirus, coxsackievirus, echovirus, rubeola virus, rubella virus, varicella-zoster virus, herpes viruses, herpes simplex virus, herpes simplex virus types 1 to 8, parvoviruses, cytomegalovirus, hepatitis virus, human papillomavirus, alphavirus, flavivirus, bunyavirus, and rabies virus. This group comprises viruses, arenaviruses, filoviruses, HIV-1, HIV-2, HTLV-1, HTLV-II, feline leukemia virus (FeLV), bovine leukemia virus (bovine LV), feline immunodeficiency virus (FeIV), canine distemper virus, canine contagious hepatitis virus, feline calicivirus, feline rhinotracheitis virus, transmissible gastroenteritis virus (TGEV), foot-and-mouth disease virus, coronaviruses, dengue virus, flaviviruses, and combinations thereof.

[0108] In some embodiments, one or more antigens in the provided vaccine are tumor-associated antigens. In some embodiments, the tumor-associated antigen is an antigen selected from the group consisting of: killed tumor cells and their lysate; MAGE-1, MAGE-3 and their peptide fragments; human chorionic gonadotropin and its peptide fragments; oncogenetic antigen and its peptide fragments; alpha-fetoprotein and its peptide fragments; pancreatic oncofetal antigen (POA) and its peptide fragments; prostate-specific antigen and its peptide fragments; MUC-1 and its peptide fragments; CA 125, CA 15-3, CA 19-9, CA 549, CA 195 and their peptide fragments; prostate-specific membrane antigen and its peptide fragments; squamous cell carcinoma-associated antigen and its peptide fragments; ovarian cancer antigen and its peptide fragments; pancreatic cancer-associated antigen and its peptide fragments; Her1 / neu and its peptide fragments; gp-100 and its peptide fragments; mutant K-Ras protein and its peptide fragments; mutant p53 and its peptide fragments; truncated epidermal growth factor receptor. receptor), chimeric proteins p210BCR-ABL, STn, Tn, Lewisx, Lewisy, TF, GM1, GM2, GD2, GD3, Gb3, KH-1, Globo-H, SSEA-4; and mixtures thereof.

[0109] As described above, the adjuvant composition of the present invention can be used as an adjuvant in combination with tumor-associated antigens for use in cancer vaccines. In some embodiments, the vaccine can be used to treat or prevent tumors. In some embodiments, the tumor is a benign tumor. In other embodiments, the tumor is a malignant tumor.

[0110] On the other hand, the present invention relates to a method for immunizing a subject using the vaccine composition of the present invention.

[0111] The pharmaceutical compositions of the present invention can be prepared in a variety of forms. For example, they can be formulated as injectables, as liquid solutions, or as suspensions. While aqueous compositions are preferred, they can also be prepared in solid forms suitable for reconstitution into solutions or suspensions in liquid carriers prior to injection (e.g., lyophilized or spray-dried compositions). Typical suspensions are for intramuscular, intradermal, or subcutaneous injection.

[0112] The present invention provides a method for enhancing the immune response in a subject, comprising administering to the subject a composition or combination as described herein.

[0113] The vaccine-stimulated immune response described herein typically includes an antibody response, preferably a protective antibody response. Immune responses may also include cellular responses. Methods for assessing antibody and cellular immune responses following immunization are well known in the art.

[0114] Definition

[0115] As used herein, unless otherwise indicated, the following definitions shall apply.

[0116] "Stereoisomers" refer to compounds that have different chirality in one or more stereocenters. Stereoisomers include mirror-image isomers and non-mirror-image isomers.

[0117] "Subject" refers to mammals, including humans and non-human mammals.

[0118] As used herein, the terms "aliphatic," "aliphatic group," or "aliphatic moiety" refer to a fully saturated or branched linear (i.e., unbranched) or substituted or unsubstituted hydrocarbon chain containing one or more unsaturated units, or a fully saturated or branched monocyclic or bicyclic hydrocarbon containing one or more unsaturated units, but not an aromatic hydrocarbon (also referred to herein as "carbocyclic," "alicyclic," or "cycloalkyl") having a single connection point with the rest of the molecule. Unless otherwise stated, an aliphatic group comprises an aliphatic carbon atom. In some embodiments, the aliphatic group comprises 1-20 aliphatic carbon atoms. In some embodiments, the aliphatic group comprises 1-12 aliphatic carbon atoms. In some embodiments, the aliphatic group comprises 1-11 aliphatic carbon atoms. In other embodiments, the aliphatic group comprises 1-10 aliphatic carbon atoms. In other embodiments, the aliphatic group comprises 1-9 aliphatic carbon atoms, and in other embodiments, the aliphatic group comprises 1-8 aliphatic carbon atoms. In other embodiments, the aliphatic group comprises 1-7 aliphatic carbon atoms. In other embodiments, the aliphatic group comprises 1-6 aliphatic carbon atoms. In other embodiments, the aliphatic group comprises 1-5 aliphatic carbon atoms. In other embodiments, the aliphatic group comprises 1-4 aliphatic carbon atoms. In other embodiments, the aliphatic group comprises 1-3 aliphatic carbon atoms, while in other embodiments, the aliphatic group comprises 1-2 aliphatic carbon atoms. Suitable aliphatic groups include, but are not limited to, straight-chain or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl, and mixtures or combinations thereof.

[0119] In some embodiments, cycloaliphatic (or "carbocyclic" or "cycloalkyl") refers to a monocyclic CC hydrocarbon that is fully saturated or contains one or more unsaturated units, but is not an aromatic hydrocarbon having a single connection point with the rest of the molecule. Suitable alicyclic groups include, but are not limited to, cycloalkyl, cycloalkenyl, cycloynyl, (cycloalkyl)alkyl, (cycloalkenyl)alkyl, (cycloynyl)alkyl, (cycloalkyl)alkenyl, (cycloalkenyl)alkenyl, (cycloynyl)alkenyl, (cycloalkyl)alkenyl, or (cycloynyl)alkenyl.

[0120] The term "heteroatom" means one or more of oxygen, sulfur, nitrogen, phosphorus or silicon (including any oxidized form of nitrogen, sulfur, phosphorus or silicon; any basic nitrogen in the tetraamined form or a substituted nitrogen in a heterocycle, such as N (as in 3,4-dihydro-2H-pyrrole), NH (as in pyrrolidinyl) or NR" (as in N-substituted pyrrolidinyl)).

[0121] As used herein, the term "unsaturated" refers to a moiety having one or more double bonds.

[0122] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0123] The term "acetylated group" is used alone or as part of a larger moiety and refers to a group formed by removing a hydroxyl group from a carboxylic acid.

[0124] The terms "arylkyl" and "arylaliphatic" are used interchangeably and refer to an aliphatic group in which a hydrogen atom is replaced by an aryl group. Aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl, cinnamyl, and dihydrocinnamyl.

[0125] The term "aryl" is used alone or as part of a larger moiety, such as in "aryl-aliphatic". The term "aryl" refers to a monocyclic or polycyclic, monovalent aromatic group. In some embodiments, the aryl group has 6 to 20 (C6-20), 6 to 15 (C6-15), 6 to 12 (C6-12), or 6 to 10 (C6-10) ring atoms. In some embodiments of the invention, "aryl" refers to an aromatic ring system, including but not limited to benzyl, phenyl, biphenyl, naphthyl, anthracene, etc., which may have one or more substituents. As used herein, groups formed by the fusion of an aromatic ring with zero or one or more non-aromatic rings are also included within the scope of the term "aryl", such as dihydroindenyl, phthalimino, naphthidylimino, phenidyl, or tetrahydronaphthyl, etc. The term "aryl" can be used interchangeably with the term "aryl ring".

[0126] The term “aryloxy-aliphatic” (or “aralkoxy or arylkoxy” or “aryloxyalkyl”) refers to a monocyclic or bicyclic ring system having a total of 5-14 ring members, wherein at least one ring in the system is aromatic and each ring in the system contains 3-7 ring members.

[0127] The term "heteroaryl" used alone or as part of a larger moiety, such as "heteroaryloxy," "heteroaryl-aliphatic," or "heteroarylalkyl," refers to a group with the following structural characteristics: having 4-10 ring atoms, preferably 4, 5, 6, or 9 ring atoms; having a ring array sharing 6, 10, or 14 electrons; and having 1-5 heteroatoms other than carbon atoms. The term "heteroatom" refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any tetraamined form of basic nitrogen. Heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrroleyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, azole, isozolyl, diazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, indolizinyl, purine, naphridinyl, and pteridinyl.

[0128] As used herein, the terms "heteroaryl" and "heteroary-" also include groups formed by the fusion of a heteroaryl ring with one or more aryl, alicyclic, or heterocyclic rings, with the fusion point on the heteroaryl ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, and quinazolinyl. The heteroaryl group can be monocyclic or bicyclic. The term "heteroaryl" is used interchangeably with the terms "heteroaryl ring," "heteroaryl group," or "heteroarylene group," and any of these terms includes optionally substituted rings. The terms "heteroaryl-aliphatic" and "heteroaryl-alkyl" refer to aliphatic compounds that are partially substituted with heteroaryl groups, wherein the aliphatic and heteroaryl groups are optionally substituted independently.

[0129] As used herein, the term "heteroaliphatic" means an aliphatic group in which one or both carbon atoms are independently substituted with one or more of oxygen, sulfur, nitrogen, or phosphorus. Heteroaliphatic groups may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and include "heterocycle", "heterocyclic group", "heterocyclic aliphatic", or "heterocyclic" groups.

[0130] As used herein, the terms "heterocycle," "heterocyclic group," and "heterocyclic" are used interchangeably and refer to a stable 5-7 membered monocyclic or 7-10 membered bicyclic heterocyclic moiety that is saturated or partially unsaturated and has one or more (preferably 1-4) heteroatoms as defined above, in addition to a carbon atom. When referring to the ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen, for example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur, or nitrogen, nitrogen may be N (e.g., in 3,4-dihydro-2H-pyrrolyl), NH (e.g., in pyrrolidinyl), or +NR (e.g., in N-substituted pyrrolidinyl).

[0131] The heterocyclic ring can be attached to its side group on any heteroatom or carbon atom to form a stable structure, and any ring atom can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic residues include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl.

[0132] The terms “heterocycle,” “heterocyclic group,” “heterocyclic ring,” “heterocyclic moiety,” and “heterocyclic group” are used interchangeably herein and also include groups in which the heterocyclic ring is fused with one or more aryl, heteroaryl, or alicyclic rings, such as indololinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl. The heterocyclic group may be monocyclic or bicyclic.

[0133] The term "heterocyclic aliphatic" refers to an alkyl group substituted with a heterocyclic group, wherein the aliphatic group and the heterocyclic moiety are optionally substituted independently.

[0134] As used herein, the term "partially unsaturated" means that the ring portion includes at least one double or triple bond. The term "partially unsaturated" is intended to cover rings having multiple unsaturated sites, but does not include aryl or heteroaryl portions as defined herein.

[0135] On the other hand, the present invention provides a "medically acceptable" composition comprising a therapeutically effective amount of one or more of the compounds described herein, formulated together with one or more pharmaceutically acceptable carriers (additives) and / or diluents. As detailed, the pharmaceutical compositions of the present invention can be specifically formulated for administration in solid or liquid form, including forms suitable for: oral administration, such as drenching (aqueous or non-aqueous solutions or suspensions), tablets (e.g., tablets targeted for buccal, sublingual and systemic absorption), pills, powders, granules, ointments applied to the tongue; non-enteral administration, such as intramuscular, subcutaneous, intravenous or epidural injection in the form of sterile solutions or suspensions or sustained-release formulations; topical application, such as in the form of creams, ointments or controlled-release sprays or patches applied to the lungs, skin or mouth; intravaginal or rectal, such as pessaries, creams or foams; sublingual; ocular; transdermal; or nasal, pulmonary and other mucosal surfaces.

[0136] The phrase “medically acceptable” as used herein means that a compound, composition, material and / or dosage form is suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions or other problems or complications, within reasonable medical judgment, and has a reasonable benefit / risk ratio.

[0137] As used herein, the phrase "medically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or medium. Such as liquid or solid fillers, excipients, diluents, or solvent encapsulating materials, involve the transport or delivery of a target compound from one part of the body to another. Each carrier must be "acceptable" in the sense of compatibility with other components of the formulation and harmlessness to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars, such as glucose, lactose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; diols, such as propylene glycol; and polyols. Examples include glycerol, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginate; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; pH buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic and compatible substances used in pharmaceutical preparations.

[0138] As used herein, the term "medically acceptable salt" means a salt that, within reasonable medical judgment, is suitable for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., and has a reasonable benefit / risk ratio. Medically acceptable salts are well known in the art. For example, SMBerge et al. detailed medically acceptable salts in the Journal of Pharmaceutical Sciences (J. Pharmaceutical Sciences, 1977, 66, 1-19), which is incorporated herein by reference. Medically acceptable salts of the compounds of the present invention include salts derived from suitable inorganic and organic acids and bases. Examples of medically acceptable non-toxic acid addition salts are salts formed by an amino group with an inorganic acid (e.g., hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or with an organic acid (e.g., acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid), or salts of an amino group formed using other methods in the art (e.g., ion exchange). Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucono-enolate, glyceryl phosphate, gluconate, hemisulfate, heptaate, hexanoate, hydroiodate, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, dodecyl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, papoate, pectate, persulfate, 3-phenylpropionate, phosphate, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc.

[0139] In some cases, the compounds of the present invention can form pharmaceutically acceptable salts with inorganic or organic acids or with inorganic or organic bases. In these cases, the term "pharmaceutically acceptable salt" refers to relatively non-toxic inorganic and organic base addition salts of the compounds of the present invention, or relatively non-toxic inorganic and organic acid addition salts of the compounds of the present invention. These salts can also be prepared in situ during the manufacture of drug delivery carriers or dosage forms, or by reacting purified compounds with a suitable base or acid alone.

[0140] In some cases, the compounds of the present invention or their pharmaceutically acceptable salts may be able to form pharmaceutically acceptable solvates. The term "solvate" refers to a solvation form containing a stoichiometric or non-stoichiometric solvent. Some compounds tend to trap solvent molecules, thereby forming solvates. When the solvent is water, the term "hydrate" is used, and for the avoidance of any doubt, the term "solvate" encompasses the term "hydrate". Embodiments of hydrates include, but are not limited to, monohydrates and dihydrates. Examples of solvates include, but are not limited to, ethanol solvates and acetone solvates.

[0141] Unless otherwise stated, the structures described herein are also intended to include all isomers of the structures (e.g., mirror-image isomers, non-mirror-image isomers, and geometric configurations); for example, R and S configurations of each stereocenter, Z and E double bond isomers, and Z and E configuration isomers. Therefore, single stereochemical isomers, mirror-image isomers, non-mirror-image isomers, and geometric (or configurational) mixtures of the compounds of the present invention are all within the scope of the present invention. Unless otherwise stated, all tautomers of the compounds of the present invention are within the scope of the present invention.

[0142] The provided compounds may contain one or more sugar moieties. Unless otherwise stated, D- and L-configurations and mixtures thereof are within the scope of this invention. Unless otherwise stated, embodiments with C- and S-connections and mixtures thereof are contemplated in this invention.

[0143] Furthermore, unless otherwise stated, the structures described herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the structures of the present invention, including those with hydrogen replaced by deuterium or tritium or carbon replaced by carbon-13 or carbon-14, are within the scope of the present invention. Such compounds can be used, for example, as analytical tools, probes in bioanalysis, or therapeutic agents according to the present invention.

[0144] According to embodiments of the present invention, the phrase "protecting group" as used herein refers to a temporarily modified potentially reactive functional group that is protected from undesirable chemical transformations. Examples of such protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketals. Of course, other suitable protecting groups may be used. Furthermore, Greene and Wuts describe a variety of protecting groups (as above).

[0145] Furthermore, in one embodiment, the present invention provides a method for stimulating, suppressing, inhibiting or regulating an immune response in an individual, the method particularly comprising administering to an individual any one of the compounds of the present invention or any combination thereof.

[0146] Furthermore, in one embodiment, the present invention provides a method for stimulating, suppressing, inhibiting or modulating an immune response in an individual, the method particularly comprising administering a pharmaceutical composition to an individual, said pharmaceutical composition particularly comprising any compound of the present invention or any combination thereof and one or more pharmaceutically acceptable excipients.

[0147] Furthermore, in one embodiment, "pharmaceutical composition" may refer to one or more of the compounds of the present invention, along with suitable excipients and / or carriers, suitable for stimulating, suppressing, inhibiting or regulating an immune response in an individual.

[0148] In one embodiment, "therapeutic effective amount" may refer to an amount that provides a therapeutic effect for a particular condition and administration regimen. In one embodiment, such a composition may be administered by any method known in the art.

[0149] As described herein, the compounds of the present invention may comprise an "optionally substituted" portion. Generally, the term "substituted," whether or not it is preceded by the term "optionally," means that one or more hydrogens of the specified portion are substituted by suitable substituents. Unless otherwise stated, the "optionally substituted" group may have suitable substituents at each substituted position of the group, and when more than one position in any particular structure may be substituted by more than one substituent selected from the particular group, the substituents at each position may be the same or different. The combinations of substituents contemplated in the present invention are preferably those that result in the formation of stable or chemically viable compounds.

[0150] As used herein, the phrase “non-enteral administration” refers to a method of drug administration other than enteral and local administration, usually by injection, and including but not limited to intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intra-sacral, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0151] The phrases “systemic administration” and “administered systemically” used in this article refer to the administration of compounds, drugs or other substances other than directly to the central nervous system, so that they enter the patient’s system and undergo metabolism and other similar processes, such as subcutaneous administration.

[0152] The term "pure" means that a compound is substantially free of the associated non-target structure or chemical precursor (in the case of chemical synthesis). This property can be measured or expressed as "purity". In some embodiments, a target compound has less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, and 0.1% of the non-target structure or chemical precursor.

[0153] The term "carbohydrate" refers to a sugar or a polymer of sugars. The terms "sugar," "polysaccharide," "carbohydrate," and "oligosaccharide" are used interchangeably. Most carbohydrates are aldehydes or ketones with many hydroxyl groups, typically with one hydroxyl group on each carbon atom of the molecule. Carbohydrates typically have the molecular formula CnH2nOn. Carbohydrates can be monosaccharides, disaccharides, trisaccharides, oligosaccharides, or polysaccharides. The most basic carbohydrates are monosaccharides, such as glucose, galactose, sucrose, ribose, mannose, arabinose, xylose, and fructose. Disaccharides are two linked monosaccharides. Exemplary disaccharides include sucrose, lactose, cellodisaccharide, and maltose. Typically, oligosaccharides consist of three to six monosaccharide units (e.g., raffinose, stachyose), while polysaccharides consist of six or more monosaccharide units. Exemplary polysaccharides include starch, glycogen, and cellulose. Carbohydrates can contain modified sugar units, such as 2'-deoxyribose (where the hydroxyl group is removed), 2'-fluororibose (where the hydroxyl group is replaced by fluorine), or N-acetylglucosamine (the nitrogenous form of glucose). Carbohydrates can exist in many different forms, such as conformational isomers, cyclic forms, acyclic forms, stereoisomers, tautomers, mutarotation isomers, and isomers.

[0154] The term "liposome" refers to a closed bilayer membrane containing an encapsulated aqueous volume. Liposomes can be characterized as monolayer vesicles with a single membrane bilayer or as multilayer vesicles with multiple membrane bilayers, each membrane bilayer being defined by an intermediate aqueous layer. The resulting membrane bilayer structure is such that the hydrophobic (nonpolar) tail of the lipid faces the center of the bilayer, while the hydrophilic (polar) head faces the aqueous phase. Liposomes are typically composed of a smectic mesophase and can be composed of phospholipids or nonphospholipid smectic mesophases.

[0155] GlaxoSmithKline previously disclosed an adjuvant formulation designated AS01 (also known as AS01B or AS01E). The lipid bilayer of AS01 consists of neutral lipids that remain non-crystalline at room temperature. In some respects, the lipid bilayer comprises dioleoyl phosphatidylcholine, cholesterol, monophospholipid A (MPLA), and QS-21. Further details of this formulation are provided in U.S. Patent Publication No. 2011 / 0206758. In the manufacture of AS01, small unilamellar liposomal vesicles (SUVs) are first formed, and then purified QS-21 is added to the SUVs. QS-21 exhibits unique properties through its interaction with liposomal cholesterol, enabling the formation of perforations or other permanent structural modifications within the liposomal body. For example, see Alving et al., Front Immunol, 2023, Vol. 14, Pages 1102524. It is speculated that a decrease in the concentration of free QS-21 can lead to a reduction in pain at the local injection site, which is usually associated with the presence of free QS-21.

[0156] In embodiments of the present invention, the adjuvant composition comprises a liposomal composition containing a TLR4 agonist and at least one Synsap, wherein the Synsap is insoluble in water and therefore needs to be encapsulated in the liposome or bound to the outside of the formulation, wherein the liposome comprises (i) a lipid bilayer containing phospholipids (e.g., 1,2-dimyristoyl-sn-glycero-3-phosphocholine, DMPC, dimyristoyl phosphatidylglycerol, DMPG, 1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC) and / or dioleoyl phosphatidylglycerol. (ii) phosphatidylglycerol (DOPG) and / or 1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP) and cholesterol. When Synsap is encapsulated in liposomes, its molar percentage concentration is less than about 9% (mol / mol), preferably about 2% to 5%, more preferably about 3.8%; however, when Synsap is added externally to liposomes, a solubilizer is required to dissolve it in aqueous solution. It is noteworthy that this solubilizer does not destroy the liposome formulation. Furthermore, unlike QS-21, Synsap does not cause hemolysis and is therefore very safe. At least these three characteristics differ from those of AS01 discussed above. The adjuvant composition may contain about 2 mg or less, about 1.5 mg or less, 1 mg or less, 0.9 mg or less, 0.8 mg or less, 0.7 mg or less, 0.6 mg or less per milliliter of liposome suspension. Synsap in doses of 0.5 mg or less, 0.4 mg or less, 0.3 mg or less, 0.2 mg or less, or 0.1 mg or less.

[0157] As used herein, unless the context clearly specifies otherwise, the singular forms "a" and "the" include the plural meaning. Unless otherwise specified, a numerical range (e.g., 10% to 11% of A) includes both an upper and a lower limit (i.e., 10% ≤ A ≤ 11%). If the numerical range does not define a lower limit (e.g., B is less than 0.2%, or B is below 0.2%), then the lower limit can be 0 (i.e., 0% ≤ B ≤ 0.2%). The foregoing terms are for the purpose of describing and illustrating this disclosure and should not be construed as restrictive.

[0158] A pharmaceutically acceptable carrier may include one or more reagents selected from the group consisting of solvents, stabilizers, emulsifiers, suspending agents, disintegrants, flavoring agents, binders, excipients, cosolvents, chelating agents, diluents, gelling agents, preservatives, lubricants and surfactants.

[0159] The term "application" of the composition to a subject means the direct application of the composition to the subject. The composition may be applied by a medical professional or by the subject himself.

[0160] The term “cancer” in this article includes the broader term “abnormal cell proliferation,” also known as “excessive cell proliferation” or “proliferative disorders.” Examples of diseases associated with abnormal cell proliferation include metastatic tumors, malignant tumors, benign tumors, cancer, precancerous lesions, hyperplasia, and polyps.

[0161] The term "treatment" refers to a method for obtaining a beneficial or desired outcome (including clinical outcomes). For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, reducing or improving one or more symptoms, reducing the severity of disease, stabilizing the disease state (i.e., not worsening), preventing the spread of disease, delaying or slowing the progression of disease, and improving, reducing, and alleviating (partially or completely alleviating) the disease state.

[0162] The term “treatment” can also refer to extended survival compared to expected survival without treatment.

[0163] For the following experiments, unless otherwise specified, the experimental methods shall be selected according to the conventional methods and conditions, or according to the instructions of the kit.

[0164] Abbreviations: Synsap: Synthetic saponin. DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine): 1,2-dioleoyl-sn-glycero-3-phosphocholine. DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine): Myristyl phosphatidylcholine. DOPG (dioleoyl phosphatidylglycerol): Dioleoyl phosphatidylglycerol. DMPG (dimyristoyl phosphatidylglycerol): Dimyristoyl phosphatidylglycerol. DOTAP (1,2-dioleoyloxy-3-(trimethylammonium)propane): 1,2-dioleoyloxy-3-(trimethylammonium)propane. Chol: Cholesterol.

[0165] Materials and Manufacturing Methods

[0166] Preparation of liposomes

[0167] The lipids were dissolved in solvents such as methanol, ethanol, or chloroform. The solvent was evaporated under vacuum to form a lipid film. PBS solution was added to the mixture, followed by pre-homogenization and then high-pressure homogenization at 20,000 psi. The resulting liposomes were aseptically filtered through a 0.22 mm membrane and aliquoted into glass containers. Dynamic light scattering (DLS) analysis showed that the liposomes had high homogeneity (PdI < 0.3) and a particle size range of 50–200 nm. When Synsap was added additionally to the liposome solution, 2 mg of Synsap was dissolved in 3 mg of TPGS (D-α-tocopherol polyethylene glycol succinate), 45 mg of propylene glycol, and 45 mg of ethanol, and then diluted with 2 mL of PBS solution (pH 7.2).

[0168] Preparation of oil / water emulsions

[0169] 49 mg of polysorbate 80 was dissolved in PBS. 5.0 mg of Synsap, 0.5 mg of the TLR4 agonist, and 119 mg of α-tocopherol were dissolved in 107 mg of squalene, respectively. The two solutions were then mixed, pre-homogenized, and then subjected to high-pressure homogenization at 20,000 psi for approximately 10 to 15 cycles. The resulting liposomes were sterilely filtered through a 0.22 μm membrane and aliquoted into glass containers. Dynamic light scattering (DLS) analysis showed that the liposomes exhibited high homogeneity (PdI < 0.3) and a particle size range of 50 to 200 nm.

[0170] Immunoassay

[0171] On days 0 and 14, mice were intramuscularly injected (right quadriceps femoris muscle) with a 30G syringe (BD Ultra-Fine Insulin Syringes 30G) containing 100 μL of vaccine. The adjuvant preparation was prepared in the same manner as in Examples 1-5. H3N2 antigen was purchased from Adimmune and was mixed with the adjuvant preparation before being injected into the mice. Body weight was measured before administration (D0), D1, D2, D14, D15, D16, and D21.

[0172] Biological Sampling

[0173] Blood was collected via submandibular bleeding at the specific time points mentioned in the embodiments. Terminal blood was collected into a 1 ml 27G syringe via cardiac puncture. Then, it was centrifuged at 10,000 g for 5 minutes at 25°C. The supernatant was aliquoted into microcentrifuge tubes and stored in a -20°C freezer.

[0174] IgG ELISA

[0175] The serum anti-H3N2 IgG antibody titer was determined using ELISA. Maxisorp plates were coated with 100 mM carbonate buffer at a concentration of 2 mcg / mL onto the entire inactivated H3N2 antigen and incubated overnight at 4°C. The plates were blocked with blocking buffer (1% BSA in PBS) at 25°C for at least 1 hour, then washed with 0.05% PBST. A 10-fold diluted serum sample was added to the plates. After 1 hour, the plates were washed with 0.05% PBST and incubated with a 10-fold diluted mouse serum. After 1 hour, the plates were washed with 0.05% PBST Tween 20 and secondary antibody was added. All peroxide-labeled goat anti-mouse IgG (1:5000, Southernbiotech), goat anti-mouse IgG1 (1:5000, Southernbiotech), and goat anti-mouse IgG2c (1:5000, Southernbiotech) diluted in 1% BSA-PBS were added for 1 hour. The culture dishes were washed with 0.05% PBST and developed with TMB chromogen solution (Invitrogen, Cat. no. 00-2023) for 15 minutes, followed by development with stop solution (1 N HCl). OD450 values ​​were read using a Spectramax 190 (Molecular Devices).

[0176] Hemagglutination inhibition test

[0177] Also based on the WHO standard protocol with slight modifications, the presence of functional antibodies in serum was detected by hemagglutination inhibition (HAI). Serum was diluted 1:5 in ROE (ROE(II)) and then incubated overnight (18 to 20 hours) in a 37°C water bath. Serum was then inactivated by heating at 56°C for 30 minutes. It was then diluted 1:2 with PBS, resulting in a final serum dilution of 1:10. Guinea pig red blood cells (GRBCs) were prepared by mixing 0.75% GRBCs with PBS / 0.75% BSA. 25 µL of PBS was added to each well of a 96-well U-bottom assay pan. Serum was added to the top row and then diluted 2-fold along the column.

[0178] Each sample was tested twice. The second-to-last row contains the positive control serum, and the last row contains the negative control (PBS) and viral back titration. Except for the last row, 25 µL of virus was added to each well. The culture dish was stirred and incubated at room temperature for 1 hour. Then, 50 µL of GRBC was added to each well, followed by 1 hour of incubation. The hemagglutination pattern was then read by observing the "halo" or circles of precipitated cells at the bottom of the well.

[0179] Intracellular staining flow cytometer

[0180] Splenic cells were seeded at 2,000,000 cells / 0.2 mL in 96-well plates. Stimulated with or without antigen (0.2 mcg H3N2 / well), the spleen cells were cultured in a CO2 incubator for 2 hours and then treated with the protein transport inhibitor Brefeldin (Invitrogen, Cat. no. 00-4506-51) at 37°C for 12 hours. Cells were then collected, washed twice with PBS, and then stained at 4°C for 30 minutes with reactive staining agent (eBioscience, catalog number 65-0866-14) and surface CD3 (BioLegend, catalog number 100240), CD4 (BioLegend, Cat. 100540), CD8 (eBioscience, catalog number 11-0081-82), CD44 (BioLegend, Cat. 103007), and CD62L (BioLegend, catalog number 104412). After washing, cells were fixed for 30 minutes at room temperature using IC fixation buffer (Invitrogen, catalog number 00-8222-49). Cells were then washed in permeabilization buffer (Invitrogen, catalog number 00-8333-56) and stained with IFNγ antibody (BioLegend, catalog number 5058524) and IL-2 antibody (eBioscience, catalog number 25-7021-82). Cells were obtained from an Attune Nxt (Thermo Fisher Scientific) and analyzed using Flowjo software (v 10).

[0181] Enzyme-linked immunosorbent assay for IFNγ and IL-4

[0182] Spleen tissue was isolated from mice and processed into a single-cell suspension using a microcentrifuge sample pestle. Spleen cells were seeded at a density of 2,000,000 / 0.2 mL in 96-well plates, stimulated with or without 2 mcg / mL H3N2, and cultured in a CO2 incubator at 37°C for 40 hours. To detect IFNγ and IL-4, the culture supernatant from the 96-well microplates was collected, and cytokine concentrations were analyzed by ELISA using ELISA MAX™ Deluxe Set Mouse IFNγ (Biolegend, catalog number 430816) and IL-4 (Biolegend, catalog number 431104). OD450 values ​​were read using a Spectramax 190 (Molecular Devices).

[0183] Statistical Analysis

[0184] Statistical analysis was performed using GraphPad Prism. Data are presented as mean ± standard error (SEM). For antibody subtype quantification, sigmoidal 4-parameter nonlinear regression was used for curve fitting. Statistical comparisons between multiple groups were analyzed using the student's t-test or one-way ANOVA test, followed by Tukey's multiple comparison test. A p-value < 0.05 was considered statistically significant.

[0185] Example 1: Preparation of vaccine composition (Synsap / TLR4 agonist)

[0186] [Examples 1-1 to Examples 1-4]

[0187] DOPC, cholesterol, PHAD, and Synsap 3 were mixed and dissolved in methanol. Liposomes were then prepared according to the liposome preparation section described above. A vaccine composition was prepared by combining 5 mcg of PHAD (from Avanti) with 5 mcg, 50 mcg, 75 mcg, or 100 mcg of Synsap 3 (as an adjuvant) and 0.3 mcg of H3N2.

[0188] [Examples 1-5 to Examples 1-8]

[0189] DOPC, cholesterol, 3D-PHAD, and Synsap 3 were mixed and dissolved in methanol. Liposomes were then prepared according to the liposome preparation section described above. A vaccine composition was prepared by combining 5 mcg of 3D-PHAD (from Avanti) with 5 mcg, 50 mcg, 75 mcg, or 100 mcg of Synsap 3 (as an adjuvant) and 0.3 mcg of H3N2.

[0190] [Examples 1-9 to Examples 1-13]

[0191] DOPC, cholesterol, 3D-6A-PHAD, and Synsap 3 were mixed and dissolved in methanol. Liposomes were then prepared according to the liposome preparation section described above. A vaccine composition was prepared by combining 1 mcg of 3D-6A-PHAD (from Avanti) with 5 mcg, 50 mcg, 75 mcg, or 100 mcg of Synsap 3 (as adjuvant) and 0.3 mcg of H3N2. Another vaccine composition was prepared by combining 5 mcg of 3D-6A-PHAD (from Avanti) with 50 mcg of Synsap 3 (as adjuvant) and 0.3 mcg of H3N2.

[0192] [Examples 1-14 to Examples 1-18]

[0193] DOPC, cholesterol, EcML, and Synsap 3 were mixed and dissolved in methanol. Liposomes were then prepared according to the liposome preparation section described above. A vaccine composition was prepared by combining 1 mcg of EcML with 5 mcg, 50 mcg, 75 mcg, or 100 mcg of Synsap 3 (as an adjuvant) and 0.3 mcg of H3N2. Another vaccine composition was prepared by combining 5 mcg of EcML with 50 mcg of Synsap 3 (as an adjuvant) and 0.3 mcg of H3N2.

[0194] [Examples 1-19 to Examples 1-23]

[0195] DOPC, cholesterol, TLR-4-1, and Synsap 3 were mixed and dissolved in methanol. Liposomes were then prepared according to the liposome preparation section described above. A vaccine composition was prepared by combining 10 mcg of TLR-4-1 with 5 mcg, 50 mcg, 75 mcg, or 100 mcg of Synsap 3 (as an adjuvant) and 0.3 mcg of H3N2. Another vaccine composition was prepared by combining 5 mcg of TLR-4-1 with 50 mcg of Synsap 3 (as an adjuvant) and 0.3 mcg of H3N2.

[0196] [Examples 1-24 to Examples 1-25]

[0197] An oil / water emulsion was prepared according to the oil-in-water emulsion preparation method described above. A vaccine composition was prepared by combining 5 mcg of CRX-527 (from InvivoGen) with 50 mcg or 100 mcg of Synsap 3 (as an adjuvant) and 0.3 mcg of H3N2.

[0198] [Examples 1-26 to Examples 1-30]

[0199] DOPC, cholesterol, TLR-4-1, and Synsap 1 were mixed and dissolved in methanol. Liposomes were then prepared according to the liposome preparation section described above. A vaccine composition was prepared by combining 10 mcg of TLR-4-1 (an undisclosed TLR4-1) with 5 mcg, 50 mcg, 75 mcg, or 100 mcg of Synsap 1 (as an adjuvant) and 0.3 mcg of H3N2. Another vaccine composition was prepared by combining 5 mcg of TLR4-1 with 50 mcg of Synsap 1 (as an adjuvant) and 0.3 mcg of H3N2.

[0200] Example 2: Preparation of vaccine composition (Synsap / TLR2 agonist)

[0201] [Examples 2-1 to 2-3]

[0202] A vaccine composition was prepared by dissolving 10 mcg of Pam3CSK4 (from InvivoGen), 1, 10, and 100 mcg of Synsap 5 (as adjuvant), and TPGS (D-α-tocopherol polyethylene glycol succinate) in propylene glycol and ethanol, and then diluting with PBS solution. Subsequently, 0.3 mg of H3N2 was added to the mixture.

[0203] Example 3: Preparation of vaccine composition (Synsap / TLR7 / 8 agonist)

[0204] [Examples 3-1 to Examples 3-4]

[0205] A vaccine composition was prepared by dissolving 10 mcg of R848 (from InvivoGen) with 2, 10, 50, and 100 mcg of Synsap 14 (as an adjuvant) and TPGS (D-α-tocopherol polyethylene glycol succinate) in propylene glycol and ethanol, and then diluting with PBS solution. Subsequently, 0.3 mg of H3N2 was added to the mixture.

[0206] Example 4: Preparation of vaccine composition (Synsap / TLR9 agonist)

[0207] [Examples 4-1 to 4-6]

[0208] A vaccine composition was prepared by combining 10 mcg of CpG 1018 (from InvivoGen) with 10 mcg, 50 mcg, 75 mcg or 100 mcg of Synsap 17 (as an adjuvant), or by combining 50 mcg of CpG 1018 with 10 mcg or 50 mcg of Synsap 17 (as an adjuvant).

[0209] [Examples 4-7 to 4-11]

[0210] A vaccine composition was prepared by combining 10 mcg of ODN 7909 (from InvivoGen) with 10 mcg, 50 mcg, 75 mcg or 100 mcg of Synsap 3 (as an adjuvant), or by combining 50 mcg of ODN 7909 with 10 mcg of Synsap 3 (as an adjuvant).

[0211] Example 5: Preparation of vaccine composition (Synsap / TLR3 agonist)

[0212] [Examples 5-1 to 5-6]

[0213] A vaccine composition was prepared by combining 25 mcg of Poly(I:C) (from InvivoGen) with 2.5, 5, 25, 50, 100, or 250 mcg of Synsap 21 (as an adjuvant) and 0.3 mcg of H3N2.

[0214] [Examples 5-7]

[0215] A vaccine composition was prepared by combining 25 mcg of TLR3-1 with 50 mcg of Synsap 21 (as an adjuvant) and 0.3 mcg of H3N2.

[0216] Example A: Immunogenicity of Synsap with different ratios of TLR agonist candidates in B57CL / 6

[0217] Examples A-1 to A-4

[0218] Mice were administered 0.3 mcg of H3N2 alone or via intramuscular injection (right quadriceps femoris muscle) at doses of 5 mcg of the TLR-4 agonist 3D-PHAD conjugated to 5 mcg, 50 mcg, 75 mcg, or 100 mcg of Synsap3 on days 0 and 14. GSK adjuvant AS01B was used as a reference standard in this study. The vaccine formulation was prepared as described in Example 1. Blood samples were collected at weeks 2 (day 14) and 4 (day 28) to monitor H3N2-specific IgG antibody subtypes and HI titers. Additionally, spleens were collected from mice at week 4 to monitor Ag-specific multifunctional CD4+ T cells and the secretion of IL-4 and IFN-γ.

[0219] Result

[0220] Referring to Table 1, after receiving two doses of the adjuvanted H3N2 vaccine, two out of five mice in the AS01B adjuvanted H3N2 group lost more than 10% of their body weight one day after each vaccination, but the average weight loss was less than 10%. However, the body weight of all vaccinated mice recovered within four days after vaccination.

[0221] Table 1: Average weight change (%) Group After the first dose After the second dose D1 D2 D14 D15 D16 D21 H3N2 0.515 -0.064 6.111 3.153 5.322 4.979 1:1 3D-PHAD:Synsap 3 -6.070 -2.480 8.879 -3.407 0.886 4.724 1:10 3D-PHAD:Synsap 3 -7.569 -8.302 6.718 -6.946 -5.785 0.851 1:15 3D-PHAD:Synsap 3 -8.821 -8.805 5.653 -7.167 -7.988 -0.059 1:20 3D-PHAD:Synsap 3 -6.071 -6.199 6.660 -6.668 -8.891 -0.985 AS01B -8.173 -3.997 9.481 -9.599 -5.213 2.076 50 mcg Synsap 3 2.176 2.174 6.946 -0.739 1.208 2.329

[0222] Figure 1A shows the anti-H3N2 IgG antibody titers and subtypes on day 28 (after the second vaccination). The total IgG titer induced by the adjuvanted H3N2 vaccine in mice was approximately 1 to 2.5 log higher than that induced by the unadjuvanted H3N2 vaccine. The combination of 3D-PHAD and Synsap 3 did not show a statistically significant difference in IgG induction compared to AS01B, but it increased it by 4 to 6 times compared to the antigen alone. See Figure 1B. Similar results were observed in IgG2c titers, showing that SynSap induces a Th1-biased response in a dose-dependent manner. Please refer to Table 2 below. Consistent with the anti-H3N2 antibody response, all adjuvanted groups showed a higher HA neutralizing antibody response after booster immunization (day 28) compared to the control non-adjuvanted group, with 3D-PHAD and SynSap 3 ratios of 1:15 and 1:20, respectively, showing a higher response than AS01B.

[0223] Table 2: HI titers and comparisons Group GMT average Multiple (compared to H3N2 alone) Multiples (compared to AS01B) H3N2 160 1.0 0.125 1:1 3D-PHAD: Synsap 3 970 6.1 0.8 1:10 3D-PHAD: Synsap 3 1280 8 1 1:15 3D-PHAD: Synsap 3 3378 twenty one 2.6 1:20 3D-PHAD: Synsap 3 1689 10 1.3 AS01B 1280 8 1

[0224] To elucidate the cellular immune response induced by vaccination, groups including H3N2 alone, AS01B, 1:10, 1:15, and 1:20 of 3D-PHAD and Synsap 3 were included. Splenic cells were isolated and restimulated in vitro with H3N2 antigen, followed by intracellular staining to detect the production of IFNγ and IL-2. Analysis of H3N2-specific CD4+ T cell subsets in immunized mice was performed by flow cytometry (Figure 2). The H3N2 group without adjuvant did not induce significant cytokine production in CD4+ T cells. Conversely, compared with the AS01B group, 1:15 and 1:20 of 3D-PHAD and Synsap 3 significantly induced total CD4+ T cell cytokine production 2 weeks after booster immunization (p values ​​were 0.0016 and <0.0001, respectively). Furthermore, although not statistically significant, cytokine concentrations were trending higher in mice treated with 1:10 3D-PHAD and Synsap 3 adjuvants compared to the AS01B group in all mice. Additionally, compared to H3N2, 1:10, 1:15, and 1:20 3D-PHAD and Synsap 3 induced significant cytokine production from total CD4+ T cells 2 weeks after booster immunization (p values ​​were 0.0101, <0.0001, and <0.0001, respectively).

[0225] The production of antigen-specific cytokines was assessed in spleen cells. Secreted cytokines were detected by ELISA after H3N2 stimulation (Figures 3 and 4). Compared to the H3N2 and AS01B groups, the combination of 3D-PHAD and Synsap in all three groups showed a significant increase in IFNγ production, but less detectable IL-4 response, consistent with the Th1-biased profile of Synsap 3. The addition of Synsap 3 to the TLR4 formulation appears to modulate the immune response, possibly by triggering two Th1 pathways, as evidenced by the enhanced IFNγ secretion.

[0226] Examples A-5 to A-10

[0227] Mice were immunized intramuscularly (right quadriceps femoris) on days 0 and 14 with 0.3 mcg of H3N2 alone or 50 mcg of Synsap 3 or Synsap 1 with a series of TLR4 agonists (including PHAD, 3D-PHAD, 3D-6A-PHAD, CRX-527, EcML, and TLR4-1) at doses of 5 mcg. PHAD, 3D-PHAD, and 3D-6A-PHAD were obtained from Avanti. EcML was obtained from EuBiologics. GSK's adjuvant AS01B was used as a reference standard in this study. The vaccine formulation was prepared as described in Example 1. Blood samples were collected at weeks 2 and 4 to monitor H3N2-specific IgG antibody subtypes and HI titers. In addition, spleens were collected from mice at week 4 to monitor Ag-specific multifunctional CD4+ T cells and the secretion of IL-4 and IFN-γ.

[0228] Result

[0229] The ability of Synsap 3 in combination with other TLR4 agonists and H3N2 to induce humoral and cellular immune responses was evaluated in C57BL / 6 mice. See Table 3. During the study period, mice in the AS01B and TLR4 / Synsap groups showed a mean weight change of less than or approximately 10% one day after vaccination. However, all vaccinated mice regained their body weight within 4 days post-vaccination.

[0230] Table 3: Average weight change (%) Group After the first dose After the second dose D1 D2 D14 D15 D16 D21 0.3 mcg H3N2 0.515 -0.064 6.272 3.153 5.322 5.513 5 mcg 3D-6A-PHAD -5.362 -2.900 6.215 -1.147 0.463 2.331 5 mcg EcML -3.607 -1.832 7.914 -3.002 -0.735 1.468 10 mcg TLR4-1 -8.384 -4.650 4.990 -7.086 -8.750 -1.984 5 / 50 mcg TLR4-1 / Synsap 1 -3.290 -2.100 7.246 -3.9565 -3.8723 0.260 5 / 50 mcg TLR4-1 / Synsap 3 -5.653 -3.804 8.772 -9.683 -9.694 -6.052 5 / 50 mcg EcML / Synsap 1 -4.860 -3.643 8.756 -8.599 -10.324 -3.715 5 / 50 mcg CRX-527 / Synsap 3 -4.126 0.447 10.867 -6.815 -4.991 -0.584 5 / 50 mcg PHAD / Synsap 3 -5.441 -2.717 7.820 -9.229 -9.827 -0.541 5 / 50 mcg 3D-PHAD / Synsap 3 0.009 3.000 8.775 -7.397 -7.114 0.946 5 / 50 mcg 3D-6A-PHAD / Synsap 3 -3.217 -0.085 7.709 -9.626 -9.315 -1.644 5 / 5 mcg AS01B -8.173 -3.997 9.005 -9.599 -5.213 1.550 50 mcg Synsap 1 -1.096 -1.736 3.753 2.417 -1.393 6.148 50 mcg Synsap 3 2.176 2.174 6.946 -0.739 1.208 2.329

[0231] Antigen-specific antibody titers were determined by ELISA. Anti-H3N2 antibody titers were determined on day 28 and expressed as serum dilution at 450 nm, corresponding to half-maximal binding (optical density: 50%). Total IgG (Fig. 5A), IgG2c (Fig. 5B), and IgG1 (Fig. 5C) were measured in immunized mice on day 28.

[0232] IgG2c and IgG1 titers are associated with Th1 and Th2 responses, respectively. Refer to Figure 5A. Anti-H3N2 IgG antibody titers and subtypes were analyzed on day 28 post-immunization. The total IgG titer of the adjuvanted H3N2 vaccine was approximately 1 to 2.5 log units higher than that of the unadjuvanted H3N2 mice. Furthermore, the combination of adjuvant and AS01B enhanced IgG titers compared to groups using TLR4 agonists alone and groups using SynSap 3 adjuvants alone. Refer to Figure 5B. After booster doses, the anti-H3N2 IgG2c titers of the TLR4-1, 3D-PHAD, 3D-6A-PHAD, and EcML-bound Synsap 3 groups were statistically significantly different from those of the adjuvanted AS01B group, and the titers in all adjuvanted groups were significantly higher than those in the unadjuvanted groups. These results with bound adjuvants indicate a higher antibody response after immunization and elicit a Th1-biased response. As shown in Table 4, consistent with the anti-H3N2 antibody response, all adjuvanted groups exhibited higher HA neutralizing antibody responses after booster immunization (day 28) compared to the non-adjuvanted control group. Notably, the titers in the TLR4-1 / Synsap 3 and 3D-6A-PHAD / Synsap 3 groups increased at least twofold compared to AS01B.

[0233] To assess the cellular immune response induced by vaccination, spleen cells were isolated 14 days after a booster dose and restimulated in vitro with the H3N2 antigen. Intracellular staining was then performed to detect the production of IFNγ and IL-2, thereby enabling the assessment of multifunctional CD4 T cells. H3N2-specific CD4+ T cell subtypes in immunized mice were analyzed by flow cytometry. (See Figure 6). Compared to the group immunized with AS01B, mouse CD4 T cells immunized with a combination of Synsap 3 (50 mcg) and the TLR4 agonist EcML (5 mcg), PHAD (5 mcg), and 3D-6A-PHAD (5 mcg) showed significantly increased production of multiple cytokines (p values ​​0.0002, 0.009, and 0.0081, respectively). Compared with the group immunized with H3N2 alone, mice immunized with Synsap in combination with TLR4 agonists EcML, PHAD, 3D-PHAD, 3D-6A-PHAD and TLR4-1, and mice immunized with Synsap 1 (50 mcg) in combination with TLR4-1, showed a significant increase in the production of multiple cytokines of CD4 T cells, with p values ​​of <0.0001, <0.0001, 0.0115, <0.0001, 0.084 and 0.0139, respectively.

[0234] Table 4: HI titers and comparisons Group GMT average Multiples (compared to H3N2) Multiples (compared to AS01B) H3N2 160 1.0 0.1 3D-6A-PHAD 806.3 5.0 0.6 TLR4-1 403.2 2.5 0.3 EcML 403.2 2.5 0.3 TLR4-1 / Synsap 1 2229 14 1.7 TLR4-1 / Synsap 3 3378 twenty one 2.6 CRX-527 / Synsap 3 1280 8 1 PHAD / Synsap 3 1280 8 1 3D-PHAD / Synsap 3 1470 9.2 1.1 3D-6A-PHAD / Synsap 3 2560 16 2 EcML / Synsap 3 1689 10.6 1.3 AS01B 1280 8 1 Synsap 1 320 2 0.25 Synsap 3 192 1.2 0.15

[0235] Example B: Immunogenicity of Synsap 5+TLR2 agonist in B57CL / 6

[0236] Mice were immunized intramuscularly (right quadriceps femoris) on days 0 and 14 with either 0.3 mcg of H3N2 alone or 100 mcg of Synsap 5 in combination with 10 mcg of the TLR2 agonist Pam3CSK4. Pam3CSK4 was derived from InvivoGen. The vaccine formulation was prepared as described in Example 2. Blood samples were collected at weeks 2 and 4 to monitor H3N2-specific IgG antibody subtypes and HI titers.

[0237] Result

[0238] The ability of the combination of Synsap 5 and the TLR2 agonist adjuvant H3N2 to induce humoral immune responses was evaluated in C57BL / 6 mice. See Table 5. During the experiment, no severe weight loss (i.e., weight loss exceeding 10%) was observed after vaccination.

[0239] Table 5: Weight Change (%) Group After the first dose After the second dose D1 D2 D14 D15 D16 D21 H3N2 0.515 -0.064 6.272 3.153 5.322 5.513 Pam3CSK4 -0.996 2.635 6.308 -0.998 0.169 4.551 10:1 Synsap 5: Pam3CSK4 -4.402 0.799 4.817 -0.500 2.257 7.314 Synsap 5 -3.166 1.523 4.909 -0.174 3.144 7.000

[0240] Antigen-specific antibody titers were determined by ELISA. Anti-H3N2 antibody titers were measured on day 28 and expressed as serum dilutions corresponding to half-maximal binding (optical density: 50%) at 450 nm absorbance. Total IgG, IgG2c, and IgG1 were measured in immunized mice on day 28. IgG2c and IgG1 titers were associated with Th1 and Th2 responses, respectively. Anti-H3N2 IgG antibody titers and subtypes on day 14 (after the first dose) and day 28 (after the second dose) are shown in Figures 7A to 7C. The total IgG titer yield induced by the adjuvanted H3N2 vaccine was significantly higher than that in mice vaccinated with the unadjuvanted H3N2 vaccine. The combination of Synsap 5 and a TLR2 agonist significantly increased total IgG by 2 logs compared to the unadjuvanted control group, and differences were shown between the Synsap 5 and Pam3CSK4 groups. Referring to Table 6, consistent with the anti-H3N2 antibody response, all adjuvanted groups showed higher hemagglutination inhibition (HI) neutralizing antibody responses after booster immunization (day 28) compared to the unadjuvanted control group. Overall, the combination of Pam3CSK4o and Synsap 5 demonstrated a synergistic effect on humoral immunity.

[0241] Table 6: HI titers and comparisons Group GMT average Multiple (compared to H3N2 alone) Multiple (compared to CpG1018) Multiples (compared to Synsap 17) H3N2 160 1.0 0.1 0.1 Pam3CSK4 2176 14 1.0 1.1 10:1 Synsap 5: Pam3CSK4 2560 16.0 1.2 1.3 Synsap 5 2048 13 0.9 1.0

[0242] Example C: Immunogenicity of Synsap 14+TLR7 / 8 agonist in B57CL / 6

[0243] Mice were immunized intramuscularly (right quadriceps femoris) on days 0 and 14 with either 0.3 mcg of H3N2 alone or 50 mcg of Synsap 14 in combination with 10 mcg of a TLR7 / 8 agonist (Resiquimod, R848). R848 was derived from InvivoGen. The vaccine formulation was prepared as described in Example 3. Blood samples were collected at weeks 2 and 4 to monitor H3N2-specific IgG antibody subtypes and HI titers. Additionally, spleens were collected from mice at week 4 to monitor the secretion of Ag-specific multifunctional CD4 T cells, as well as IL-4 and IFNγ.

[0244] Result

[0245] Referring to Table 7, no severe weight loss (more than 10%) occurred after vaccination during the experiment. The ability of the Synsap 14 and R848 adjuvant H3N2 combination to induce humoral and cellular immune responses was evaluated in C57BL / 6 mice.

[0246] Table 7: Weight Change (%) Group After the first dose After the second dose D1 D2 D14 D15 D16 D21 H3N2 -1.153 1.480 7.352 -1.900 -1.542 -1.481 R848 -3.012 -0.416 5.224 -4.078 -0.804 1.856 Synsap 14 -1.769 -0.704 3.586 -0.277 -0.654 3.064 1:1 R848 / Synsap 14 -2.079 1.645 8.520 -3.125 0.812 1.701 1:5 R848 / Synsap 14 -2.019 1.362 10.692 -2.733 -0.206 0.173 1:10 R848 / Synsap 14 -2.044 0.606 8.691 -3.368 -0.938 2.902 5:1 R848 / Synsap 14 -4.941 -2.019 7.677 -4.567 -1.218 0.631

[0247] Antigen-specific antibody titers were determined by ELISA. Anti-H3N2 antibody titers were measured on day 28 and expressed as serum dilutions corresponding to 50% of the maximum response, based on absorbance at 450 nm. Total IgG, IgG2c, and IgG1 were measured in immunized mice on day 28. IgG2c and IgG1 titers were associated with Th1 and Th2 responses, respectively. Refer to Figures 8A to 8C. On day 28 post-immunization, anti-H3N2 IgG antibody titers and subtypes were analyzed. The total IgG titer of the adjuvanted H3N2 vaccine was approximately 0.5 to 2 log units higher than that of the unadjuvanted H3N2 mice. Furthermore, in all mice, those treated with the combination of Synsap 14 and R848 as adjuvants showed increased IgG titers compared to R848 alone, and the Synsap 14 and R848 adjuvant groups, when added at a 1:5 ratio, showed statistically significant differences in anti-H3N2 IgG and IgG2c titers (p < 0.0001). These results with the combined adjuvants indicate a higher antibody response after immunization and elicit a Th1-biased response. Consistent with the anti-H3N2 antibody response, all adjuvant groups showed a higher HA neutralizing antibody response after booster immunization (day 28) compared to the non-adjuvant control group (Table 8). Notably, all Synsap 14 and R848 combination groups showed at least a 1.5-fold increase in titers compared to R848 alone or Synsap 14 alone.

[0248] To assess the cellular immune response induced by vaccination, spleen cells were isolated 14 days after a booster dose and restimulated in vitro with the H3N2 antigen. Intracellular staining was then performed to detect the production of IFNγ and IL-2, thereby enabling the assessment of multifunctional CD4 T cells. Secreted cytokines were detected by ELISA following H3N2 stimulation. Refer to Figures 9A to 9B. Compared to groups immunized with AS01B and H3N2 alone, mouse CD4 T cells immunized with the combined Synsap 14 and R848 showed significantly increased production of multiple cytokines. To further characterize the Th1 / Th2 / Th17 response, the production of antigen-specific cytokines in spleen cells was assessed. Secreted cytokines were detected by ELISA following H3N2 stimulation. Refer to Figures 9 to 11. Compared to the Synsap 14 and R848 groups, the H3N2 group using the combined adjuvant of R848 and Synsap 14 showed a trend towards increased production of IFNγ and IL-17A. This indicates that this combination enhances immune activation and promotes a shift towards Th1 / Th17 immune responses, which may be beneficial for bacterial / protozoan vaccines. Overall, the combination of R848 and Synsap14 exhibits a synergistic effect on humoral and cellular immunity.

[0249] Table 8: HI titers and comparisons Group GMT average Multiple (compared to H3N2 alone) Multiplier (compared to the R848 treatment group) Multiple (compared to the Synsap 14 treatment group) H3N2 160 1.0 0.9 0.8 R848 183.8 1.1 1.0 1.0 SynSap 14 192 1.2 1.0 1.0 1:1 R848 / Synsap 14 278.6 1.7 1.5 1.5 1:5 R848 / Synsap 14 735.2 4.6 4.0 3.8 1:10 R848 / Synsap 14 367.6 2.3 2.0 1.9 5:1 R848 / Synsap 14 320 2.0 1.7 1.7

[0250] Example D: Immunogenicity of Synsap 17+TLR9 candidate in B57CL / 6

[0251] Mice were immunized intramuscularly (right quadriceps femoris) on days 0 and 14 with 0.3 mcg of H3N2 alone, a combination of 10 mcg of the TLR 9 agonist CpG 1018 with 10 mcg, 50 mcg, 75 mcg, and 100 mcg of Synsap 17, or a combination of 50 mcg CpG 1018 with 10 mcg Synsap 17. CpG1018 was obtained from InvivoGen. The vaccine formulation was prepared as described in Example 4. Blood samples were collected at weeks 2 and 4 to monitor H3N2-specific IgG antibody subtypes and HI titers. In addition, spleens were collected from mice at week 4 to monitor the secretion of Ag-specific multifunctional CD4 T cells and IL-4 and IFN-γ.

[0252] Result

[0253] The ability of the combination of Synsap 17 and the TLR9 agonist adjuvant H3N2 to induce humoral and cellular immune responses was evaluated in C57BL / 6 mice. See Table 9. No mice experienced a weight loss of more than 10% after vaccination.

[0254] Table 9: Weight Change (%) Group After the first dose After the second dose D1 D2 D14 D15 D16 D21 H3N2 -1.153 1.480 7.352 -1.900 -1.542 -0.820 CpG 1018 -5.463 -7.285 4.568 -2.546 -7.323 1.850 Synsap 17 -1.769 -0.704 3.586 -0.277 -0.654 2.921 1:1 CpG 1018:Synsap 17 -3.500 -6.986 0.255 0.944 -3.841 3.191 1:5 CpG 1018 : Synsap 17 -0.385 -1.953 3.447 -0.254 -1.145 5.159 1:7.5 CpG 1018:Synsap 17 -1.765 -1.808 3.327 -0.201 -0.719 6.178 1:10 CpG 1018:Synsap 17 -4.675 -2.366 1.477 -1.193 -1.716 4.577 5:1 CpG 1018:Synsap 17 -7.035 -7.286 1.598 -1.900 -6.982 2.319

[0255] Antigen-specific antibody titers were determined by ELISA. Anti-H3N2 antibody titers were determined on day 28 and expressed as serum dilutions corresponding to 50% of the maximum response (optical density: 50%) based on absorbance at 450 nm. Total IgG, IgG2c, and IgG1 were measured in immunized mice on day 28. IgG2c and IgG1 titers were associated with Th1 and Th2 responses, respectively. Figures 12A to 12C show the anti-H3N2 IgG antibody titers and subtypes in each group on days 14 and 28 (after the first and second doses). After the first dose, the total IgG titer induced by the H3N2 vaccine with the Synsap 17 and TLR9 agonist combination as adjuvants was approximately 0.5 to 1.5 log higher than that of the unadjuvanted, Synsap 17, and CpG 1018 groups. Increasing the dose of Synsap 17 in the combination therapy improved total IgG titers and IgG2c. Consistent with the anti-H3N2 antibody response, all adjuvanted groups showed higher hemagglutination inhibition neutralizing antibody responses after booster immunization (day 28) compared to the unadjuvanted control group, with the 1:10 Synsap 17 and CpG 1018 groups showing significantly higher responses (Table 10).

[0256] Table 10: HI titers and comparisons Group GMT average Multiples (compared to H3N2) Multiples (compared to CpG1018) Multiples (compared to Synsap 17) H3N2 160 1.0 0.5 0.9 CpG 1018 320 2.0 1.0 1.7 Synsap 17 183.8 1.1 0.6 1.0 1:1 CpG 1018: Synsap 17 367.6 2.3 1.1 2.0 1:5 CpG 1018: Synsap 17 970.1 6.1 3.0 5.3 1:7.5 CpG 1018:Synsap 17 1114 7.0 3.5 6.1 1:10 CpG 1018: Synsap 17 2560 16 8.0 14 5:1 CpG 1018: Synsap 17 640 4.0 2.0 3.5

[0257] To investigate cellular immune responses, spleen cells were isolated from mice immunized with H3N2 alone, combinations of CpG 1018 and Synsap 17 at ratios of 1:5, 1:7.5, and 1:10, and Synsap 17 alone and CpG 1018 alone. Spleen cells were restimulated with H3N2 antigen, followed by intracellular cytokine staining to assess the production of IFNγ and IL-2 (Figure 13). Although the unadjuvanted H3N2 group did not induce significant cytokine production from CD4 T cells, the Synsap 17 and CpG 1018 combinations at ratios of 1:5, 1:7.5, and 1:10 induced significant cytokine production from total CD4 cells compared to the unadjuvanted and Synsap 17 and CpG 1018 groups. Specifically, the Synsap 17 and CpG 1018 combinations at ratios of 1:5, 1:7.5, and 1:10, compared to H3N2, showed a significant increase in cytokine production (p values ​​of 0.0001, 0.0001, and <0.0001, respectively).

[0258] To further characterize the Th1 / Th2 response, the production of antigen-specific cytokines in spleen cells was assessed. Secreted cytokines were measured by ELISA after H3N2 stimulation. See Figures 14 and 15. Compared to the Synsap 17 and CpG 1018 groups, the H3N2 group, with the combination of CpG 1018 and Synsap 17 as adjuvants, showed a trend toward increased IFN-γ production. The CpG adjuvant group lacked a detectable IL-4 response, consistent with the Th1 bias profile. The addition of Synsap 17 to the CpG formulation appears to modulate the immune response, possibly by triggering both Th1 and Th2 pathways, as evidenced by enhanced IFN-γ and IL-4 secretion.

[0259] Example E: Immunogenicity of Synsap 21+TLR3 agonist in B57CL / 6

[0260] Mice were immunized on days 0 and 14 by intramuscular injection (right quadriceps) of 0.3 mcg H3N2 alone or in combination with 5 mcg, 25 mcg, 50 mcg, and 100 mcg of Synsap 21 in combination with a 25 mcg dose of a TLR3 agonist (containing Polyg(I:C) and TLR3). Polyg(I:C) was derived from InvivoGen, while TLR3-1 is an undisclosed TLR3 compound. The vaccine formulation was prepared as described in Example 5. Blood samples were collected at weeks 2 and 4 to monitor H3N2-specific IgG antibody subsets and HI titers. In addition, spleens were collected from mice at week 4 to monitor Ag-specific multifunctional CD4 T cells and the secretion of IL-4 and IFN-γ.

[0261] Result

[0262] The ability of the combination of Synsap 21 and the TLR3 agonist adjuvant H3N2 to induce humoral and cellular immune responses was evaluated in C57BL / 6 mice. Referring to Table 11, no severe weight loss (more than 10%) was observed after vaccination during the experiment.

[0263] Table 11: Average weight loss Group After the first dose After the second dose D1 D2 D14 D15 D16 D21 H3N2 alone -1.153 1.480 7.565 -1.900 -1.542 -0.820 TLR3-1 -4.647 0.769 6.060 -5.203 -2.131 -0.204 1:5 Synsap 21:TLR3-1 -4.527 1.505 7.444 -1.900 -1.542 -0.820 1:1 Synsap 21:TLR3-1 -4.764 -1.090 8.265 -6.418 -1.209 2.807 2:1 Synsap 21:TLR3-1 -5.886 -1.211 10.902 -8.842 -4.266 0.496 4:1 Synsap 21:TLR3-1 -6.864 -3.310 9.327 -8.637 -6.051 0.185 Poly(I:C) -4.287 1.634 9.773 -5.203 -2.131 -0.204 2:1 Synsap 21:Poly(I:C) -4.724 -0.453 8.007 -5.150 -1.825 0.022 SynSap 21 2.176 2.174 6.946 -0.739 1.208 0.149

[0264] Antigen-specific antibody titers were tested by ELISA. Anti-H3N2 antibody titers were determined on day 28 and expressed as serum dilutions corresponding to 50% of the maximum response (50% optical density) based on absorbance at 450 nm. Total IgG, IgG2c, and IgG1 were measured in immunized mice on day 28. IgG2c and IgG1 titers were associated with Th1 and Th2 responses, respectively. Anti-H3N2 IgG antibody titers and subtypes on day 28 (after the second dose) are shown in Figure 16A. The total IgG titer yield induced by the adjuvanted H3N2 vaccine was significantly higher than that in mice vaccinated with the unadjuvanted H3N2 vaccine. There were no significant differences in results between the two different TLR3 agonists. Compared to the unadjuvanted control group, all combinations of Synsap 21 and TLR3 agonists significantly increased total IgG and IgG2c by 1.5 to 2 log units, showing differences among the Synsap 21, TLR3-1, and Poly(I:C) groups (Figures 16B and 16C). Consistent with the anti-H3N2 antibody response, all adjuvanted groups showed higher hemagglutination inhibition neutralizing antibody responses after booster immunization (day 28) compared to the unadjuvanted control group (Table 12). Overall, the binding of TLR3-1 or Poly(I:C) to Synsap 21 had a synergistic effect on IgG and IgG2c titers, as well as HI titers. Furthermore, the binding of TLR3-1 or Poly(I:C) to Synsap 21 favored promoting the Th1 response.

[0265] Table 12: HI titer and comparison Group GMT average Multiples (compared to H3N2) Multiple (compared to TLR3) Multiples (compared to Synsap 21) H3N2 160 3.03125 1 1.515625 TLR3-1 485 2 0.659794 1 1:5 Synsap 21:TLR3-1 557.2 5.278125 1.741237 2.639063 1:1 Synsap 21:TLR3-1 844.5 6.063125 2.000206 3.031563 2:1 Synsap 21:TLR3-1 970.1 8 2.639175 4 4:1 Synsap 21:TLR3-1 1280 4.595 1.14875 2.2975 Poly(I:C) 640 1 0.329897 0.5 2:1 Synsap 21:Poly(I:C) 735.2 4 1 2 Synsap 21 320 3.4825 1.148866 1.74125

[0266] The data in Figure 17 clearly show that all tested adjuvants activated the secretion of multifunctional CD4+ and H3N2-specific IFNγ. Specifically, TLR3-1 and Synsap 21 in ratios of 2:1 and 4:1, respectively, and Poly(I:C) and Synsap 21 in a ratio of 2:1 induced a significant increase in splenic CD4+ Th1 cells and the secretion of antigen-specific IFNγ (Figure 18). These combinations showed increased IL-4 production (Figure 19), but this was not statistically significant, indicating a Th1-biased trend.

[0267] Although preferred embodiments of the present disclosure have been described above, it should be recognized and understood that various changes and modifications may be made, and the appended claims are intended to cover all such changes and modifications that may fall within the spirit and scope of the present disclosure. [Simplified Explanation of the Diagram]

[0040] To better illustrate the above-mentioned content of this disclosure, the preferred embodiments are described in detail below with reference to the accompanying drawings: Figures 1A to 1C show the anti-H3N2 antibody titers measured on day 28 (Figure 1A: total IgG, Figure 1B: IgG2c, Figure 1C: IgG1), the serum dilution corresponding to the half-maximal binding strength of the antibody-antigen (e.g., absorbance at 450 nm) reaching 50% of the maximum response, and represent the results from Example 1; Figure 2 shows the analysis of H3N2-specific CD4+ T cell subsets of each group discussed in Example 1; Figure 3 shows the IFN-γ concentrations of each group discussed in Example 1 measured by ELISA after H3N2 stimulation; Figure 4 shows the IL-4 concentrations of each group discussed in Example 1 measured by ELISA after H3N2 stimulation; Figures 5A to 5C show the anti-H3N2 antibody titers measured on day 28 (Figure 5A: total IgG, Figure 5B: IgG2c, Figure 5C: IgG1), the serum dilutions corresponding to half-maximal binding when the antibody-antigen binding strength (e.g., absorbance at 450 nm) reaches 50% of the maximum response, and represent the results from Example 1; Figure 6 shows the analysis of H3N2-specific CD4+ T cell subsets in each group discussed in Example 1; Figures 7A to 7C show the anti-H3N2 antibody titers measured on day 28 (Figure 7A: total IgG, Figure 7B: IgG2c, Figure 7C: IgG1), the serum dilutions corresponding to half-maximal binding when the antibody-antigen binding strength (e.g., absorbance at 450 nm) reaches 50% of the maximum response, and represent the results from Example 2; Figures 8A to 8C show the anti-H3N2 antibody titers (Figure 8A: total IgG, Figure 8B: IgG2c, Figure 8C: IgG1) measured on day 28, the serum dilutions corresponding to the half-maximal binding strength of the antibody-antigen combination (e.g., absorbance at 450 nm), and represent results from Example 3; Figure 9 shows the IFNγ concentrations of the groups discussed in Example 3, measured by ELISA after H3N2 stimulation; Figure 10 shows the IL-4 concentrations of the groups discussed in Example 3, measured by ELISA after H3N2 stimulation; Figure 11 shows the IL-17A concentrations of the groups discussed in Example 3, measured by ELISA after H3N2 stimulation.Figures 12A to 12C show the anti-H3N2 antibody titers measured on day 28 (Figure 12A: total IgG, Figure 12B: IgG2c, Figure 12C: IgG1), the serum dilutions corresponding to the half-maximal binding strength of the antibody and antigen (e.g., absorbance at 450 nm), and represent results from Example 4; Figure 13 shows the analysis of H3N2-specific CD4+ T cell subsets in each group discussed in Example 4; Figure 14 shows the IFN-γ concentrations measured by ELISA in each group discussed in Example 4 after H3N2 stimulation; Figure 15 shows the IL-4 concentrations measured by ELISA in each group discussed in Example 4 after H3N2 stimulation; Figures 16A to 16C show the anti-H3N2 antibody titers measured on day 28 (Figure 16A: total IgG, Figure 16B: IgG2c, Figure 16C: IgG1), the antibody and antigen binding strength (e.g., absorbance at 450 nm), and the serum dilutions corresponding to the half-maximal binding strength of the antibody and antigen (e.g., absorbance at 450 nm). Figure 17 shows the serum dilution corresponding to half-maximal binding when the absorbance at nm reaches 50% of the maximum response, representing the results from Example 5; Figure 18 shows the IFN-γ concentration measured by ELISA after H3N2 stimulation in each group discussed in Example 5; and Figure 19 shows the IL-4 concentration measured by ELISA in each group discussed in Example 5 after H3N2 stimulation.

Claims

1. An adjuvant composition comprising: (i) a saponin conjugate or a pharmaceutically acceptable salt or hydrate thereof as shown in formula (I): (I), wherein is a single or double bond; W is Me, —CHO, —CH2ORx or —C(O)Ry; wherein Rx is independently hydrogen or an oxygen-protecting group selected from the group consisting of alkyl ethers, allyl ethers, benzyl ethers, silyl ethers, acetals, ketals, esters, carbamates and carbonates, and Ry is selected from the group consisting of alkyl, allyl, benzyl, silyl, alkoxy and alkyl carboxylates; V is H or —OH; Y is CH2, —O—, —S—, —NR— or —NH—; Q is CH2, C=O, C=N—OH or C=N—OMe; X Is CH2, —O—, —NH—, —NH—(C=O)—, —(C=O)—NH—, —S—, —(C=O)—O— or —O—(C=O)—; R is a portion selected from the group consisting of aryl, arylaliphatic, cycloaliphatic, heteroarylaliphatic, alkoxy-aliphatic, and aryloxy-aliphatic, or a portion selected from the group consisting of: unsubstituted 5-10 arylaliphatic, substituted 5-10 arylaliphatic, wherein the substituted 5-10 arylaliphatic is substituted by at least one group selected from the group consisting of: alkyl, alkoxy, aryloxy, halogen, halogenated alkyl, hydroxyl, saturated heterocyclic group having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, sulfur, and combinations thereof; and 4-10 heteroarylaliphatic having 1-4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, sulfur, and combinations thereof, or having the following structure: , , or, where Rz is alkyl, Xa is NH or S, and n = 1-20; R1 is independently hydrogen, an oxygen-protecting group selected from the group consisting of alkyl ethers, allyl ethers, benzyl ethers, silyl ethers, acetals, ketals, esters, carbamates, and carbonates, or a carbohydrate having a monosaccharide structure; and Z is hydrogen, or a straight-chain or branched oligosaccharide selectively substituted with at least one group selected from the group consisting of amines, amides, amides, aralkyls, aryls, heteroaryls, aliphatic, heteroaliphatic, cycloaliphatic, and heterocyclic rings; and (ii) a Toll-like receptor (TLR) agonist.

2. The adjuvant composition as claimed in claim 1, wherein Z is a linear tetrasaccharide or trisaccharide, and the first sugar residue is directly linked to Y.

3. The adjuvant composition as claimed in claim 1, wherein Q is C=O and X is —NH—.

4. The adjuvant composition as claimed in claim 1, wherein W is -CHO and V is -OH.

5. The adjuvant composition as claimed in claim 1, wherein R1 is -H.

6. The adjuvant composition as claimed in claim 1, wherein R is selected from the group consisting of: , wherein Rz is an alkyl group.

7. The adjuvant composition as claimed in claim 1, wherein the saponin conjugate is selected from the group consisting of: (i) compounds represented by formula I-1: (I-1). Where G1 is 10-((2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-((8-(4-methoxyphenyl)octyl)aminomethyl)tetrahydro-2H-pyran-2-yl), 10-((2R,3R,4S,5S,6S)-6-((8-(4-fluorophenyl)octyl)aminomethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl), 10-((2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-((8-(4-phenoxyphenyl)octyl)aminomethyl)tetrahydro-2H-pyran-2-yl), 10-((2R,3R,4S,5S,6S)-6-((8-(4-(4-fluorophenoxy)phenyl)octyl)aminomethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl), 10-((2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-((8-(4-morpholinophenyl)octyl)aminomethyl)tetrahydro-2H-pyran-2-yl) or 10-((2R,3R,4S,5S,6S)-6-((8-(furan-2-yl)octyl)aminomethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl); (ii) Compounds represented by formula I-2: (I-2), Where G2 is 10-((2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-((8-(4-methoxyphenyl)octyl)aminomethyl)tetrahydro-2H-pyran-2-yl), 10-((2R,3R,4S,5S,6S)-6-((8-(4-fluorophenyl)octyl)aminomethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl), 1 0-((2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-((8-(4-phenoxyphenyl)octyl)aminomethyl)tetrahydro-2H-pyran-2-yl) or 10-((2R,3R,4S,5S,6S)-6-((8-(4-(4-fluorophenoxyphenyl)octyl)aminomethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl);(iii) Compounds represented by formula I-3: (I-3), wherein G3 is 10-((2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-((8-(4-methoxyphenyl)octyl)aminomethyl)tetrahydro-2H-pyran-2-yl), 10-((2R,3R,4S,5S,6S)-6-((8-(4-fluorophenyl)octyl)aminomethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl), 1 0-((2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-((8-(4-phenoxyphenyl)octyl)aminomethyl)tetrahydro-2H-pyran-2-yl) or 10-((2R,3R,4S,5S,6S)-6-((8-(4-(4-fluorophenoxyphenyl)octyl)aminomethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl); (iv) Compounds represented by formula I-4: (I-4), Where G4 is 10-((2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-((8-(4-methoxyphenyl)octyl)aminomethyl)tetrahydro-2H-pyran-2-yl), 10-((2R,3R,4S,5S,6S)-6-((8-(4-fluorophenyl)octyl)aminomethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl), 10-(2R,3R ... (4S,5S,6S)-3,4,5-trihydroxy-6-((8-(4-phenoxyphenyl)octyl)aminomethyl)tetrahydro-2H-pyran-2-yl) or 10-((2R,3R,4S,5S,6S)-6-((8-(4-(4-fluorophenoxyphenyl)octyl)aminomethyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl); and (v) compounds represented by formula I-5: (I-5), wherein Rg is 4-methoxyphenyloctyl, 4-fluorophenyloctyl, 4-phenoxyphenyloctyl or (4-fluorophenoxy)phenyl)octyl.

8. The adjuvant composition as claimed in claim 1, wherein the Toll-like receptor (TLR) agonist is selected from the group consisting of TLR2 agonists, TLR3 agonists, TLR4 agonists, TLR7 agonists, TLR8 agonists and TLR9 agonists.

9. The adjuvant composition as claimed in claim 1, wherein the Toll-like receptor (TLR) agonist is a TLR4 agonist selected from the group consisting of PHAD, MPLA, 3D-PHAD, 3D-6A-PHAD, EcML, CRX-527, GSK1795091, E6020, GLA, SLA, and combinations thereof.

10. The adjuvant composition as claimed in claim 1 further comprises a liposome-forming compound or an emulsion-containing compound.

11. The adjuvant composition as claimed in claim 10, wherein the liposome-forming compound comprises phospholipids, cholesterol, and combinations thereof.

12. The adjuvant composition as claimed in claim 10, wherein the emulsion is selected from compounds in the group consisting of polysorbate, α-tocopherol, Span 85, glycerol, poloxamer 188 and carboxymethyl cellulose.

13. The adjuvant composition as claimed in claim 1, wherein the adjuvant composition is an aqueous solution, an oil / water emulsion, or encapsulated in liposomes.

14. The adjuvant composition as claimed in claim 1, wherein the particle size of the adjuvant composition is less than 250 nanometers.

15. The adjuvant composition as claimed in claim 1 further comprises squalene.

16. A pharmaceutical composition comprising: an antigen and an adjuvant composition as claimed in claim 1.

17. The pharmaceutical composition of claim 16, wherein the antigen is selected from one or more of the group consisting of bacteria, viruses, protozoa and tumor-associated antigens.

18. Use of a pharmaceutical composition as described in claim 16 or 17 for the preparation of a medicament for the prevention or treatment of diseases caused by viral infections, bacterial infections, protozoan infections or cancer.

Images